APPARATUS FOR DIAGNOSING AND/OR TREATING MALARIA

A malaria diagnosis and/or treatment apparatus can include an optical source and an acoustic detector in a single probe (sensor). The optical source can provide optical energy configured to produce transient vapor nanobubbles around malaria-specific nanoparticles, such as hemozoin in skin, blood and other tissues infected with malaria, but not in uninfected tissues. The acoustic detector can detect pressure pulses generated by the transient vapor nanobubbles. A malaria diagnosis and/or screening process can be based on using several metrics of the detector signal output, which include the time and amplitude parameters of such signal. This metrics characterize both active and residual forms of malaria disease and can be used in the clinical diagnostics of malaria and in mass screening of the malaria transmission.

FIELD

The present application relates generally to the fields of detection and elimination of malaria parasites in a patient's body, in particular, with the use of laser-induced transient vapor nanobubbles.

BACKGROUND

Malaria is a widespread and infectious disease that can cause serious illness and death in humans. A patient can be infected when a malaria parasite infects cells of the patient, also known as a host. The parasite can produce hemozoin (HZ), which are nanocrystals formed when the parasite digests the hemoglobin in the host's red blood cells. Malaria-infected red blood cells or other body tissue infected by malaria parasites contain HZ (hemozoin) nanocrystals.

Current malaria diagnosis techniques include, for example, rapid diagnostic tests (RDTs), microscopy, and polymerase chain reaction (PCR). These diagnosis techniques analyze a patient's blood samples. RDT analyzes the proteins in the blood to look for presence of malaria parasites and is approved by the World Health Organization (WHO). Microscopy uses stain of a thick blood slide, such as with a 200 to 500 white blood cell count, to determine malaria parasite density and gametocyte counts. Microscopy is also WHO-approved for malaria diagnosis. PCR analyzes DNAs in the blood to determine presence of malaria parasites.

Malaria can be treated and/or prevented by administration of antimalarial drugs, such as quinine, chloroquine, atovaquone/proguanil, and others.

SUMMARY

Current malaria diagnosis generally employ invasive techniques which are costly, time-consuming and have low accuracy. The diagnosis and treatment of malaria can require separate procedures.

Antimalarial drugs have several disadvantages. Malaria parasites can develop drug resistance to the antimalarial drugs. The drugs can also be ineffective against malaria parasites that escape from the blood vessels into the tissue and/or skin of the patient through micro-capillaries in the tissue, also known as tissue-sequestered parasites. Tissue-sequestered parasites can cause lethal complications in the patient after treatments with antimalarial drugs, such as when the patient has low blood levels of parasites, and/or can cause relapses in patients treated with antimalarial drugs. Current malaria diagnosis techniques, such as RDTs, microscopy, and PCR, are not able to detect tissue-sequestered malaria parasites as these techniques rely on analyzing the patient's peripheral blood samples.

Current malaria diagnosis techniques also may not detect the HZ (hemozoin) nanocrystals without an active and/or live malaria parasite. However, detecting the HZ (hemozoin) nanocrystals without an active or live malaria parasite can provide valuable information for determining recent or past presence of malaria infection, the data important in screening and understanding the malaria transmission.

Laser-induced transient vapor nanobubbles can be used to diagnose and/or treat malaria in a noninvasive, efficient, and reproducible manner. The transient vapor nanobubbles can be generated around one or more malaria-specific nanoparticles (such as one or more HZ (hemozoin) nanocrystals (with or without an active malaria parasite) or malaria-specific nanoparticles introduced into the host red blood cells) when laser pulses are applied to the nanoparticles. The laser pulses can cause rapid heating of the malaria-specific nanoparticles, but not of uninfected red blood cells or other host tissues. Liquid (such as water) around the malaria-specific nanoparticles can rapidly evaporate, leading to the generation of a transient vapor nanobubble. The generation of transient vapor nanobubbles can be detected by acoustic detectors. In some embodiments, the transient nanobubble-based malaria detection mechanism can detect a single hemozoin nanoparticle. The transient nanobubble-based malaria detection mechanism disclosed herein can be advantageous over the bulk photoacoustic mechanism, which requires a large number of hemozoin nanoparticles to produce a detectable malaria-positive signal.

As the transient vapor nanobubble size increases with increasing energy level of the laser pulses, in some instances, the energy level of the laser pulses can be high enough to generate transient vapor nanobubbles that can cause mechanical damage to the HZ (hemozoin) nanocrystal host, the malaria parasite, the malaria-infected red blood cell, or a combination thereof. Additional details of employing transient vapor nanobubbles to detect and/or treat malaria-infected red blood cells are described in International Application No. WO2013/109722, filed Jan. 17, 2013 and titled “Theranostic methods and systems for diagnosis and treatment of malaria,” attached as Appendix A, the entirety of which is incorporated herein by reference and should be considered a part of the specification.

The vapor nanobubbles, also referred to as HZ (hemozoin)-generated vapor nanobubbles (HVNB) or nanobubbles, can be generated on a liquid sample test (such as blood, in particular peripheral blood, or urine), or on a patient's skin using a sensor that optically excites the malaria-specific nanoparticle in the skin to generate hemozoin-generated vapor nanobubbles.

In order to use transient vapor nanobubbles for detecting and/or treating malaria noninvasively, the laser pulses must penetrate a patient's skin and reach the malaria-specific nanoparticles despite attenuation of the laser pulses by the patient's body tissue as the laser pulses travel deeper under the skin. The optical delivery part of the malaria sensor, such as an optical fiber, needs to be brought as close as possible to the malaria-specific nanoparticles or malaria parasite. The nanobubble-generated pressure pulses reaching the surface of the acoustic detector also need to be strong enough for an acoustic (ultrasound) signal of that pressure pulse to be detected. Challenges in improving the sensitivity and/or specificity of the acoustic detector can include reducing a distance between the malaria-specific nanoparticles and the acoustic detector, and/or reducing a distance between an optical source and the acoustic detector.

In some instances, a malaria diagnosis and/or treatment apparatus can combine an optical source and an acoustic detector in a single probe (also referred to as a malaria sensor). The single probe can bring the acoustic detector closer to the source of the acoustic pulse, which is the transient vapor nanobubble, than having separate optical source and acoustic detector probes. However, the distance between the acoustic detector surface and the malaria-specific nanoparticle in the known single probes can still be too large. This can be due to the use of a spherical acoustic detector for detecting the transient vapor nanobubble, as the transient vapor nanobubble is considered a point source. The spherical acoustic detector can also have a large surface area, which weakens the signal output from the spherical detector. Other concerns with using a spherical acoustic detector can include high cost and structural complexity, which can make it infeasible to mass-produce the single probe.

The malaria probe according to the present disclosure can include the optical source and the acoustic detector in a single probe, and can diagnose and/or treat malaria. The malaria probe according to the present disclosure can increase the sensitivity and specificity of the malaria detection by having one or more small and substantially flat acoustic detectors placed in close proximity to the optical source, which can include one or more optical fibers, and to a probe tip surface. Sensitivity can be indicative of the probe's ability to correctly detect malaria-positive cases. Specificity can be indicative of the probe's ability to avoid false positive and false negative detections. In some embodiments, the malaria probe is able to detect tissue-sequestered malaria parasites. Embodiments of the malaria probe can also be immune to resistance from the malaria parasite, efficient, and/or safe for the patient. In some embodiments, the malaria probe can be cheap to build and/or economically feasible for mass production.

An apparatus configured for diagnosing malaria noninvasively can comprise: a sensor probe having a probe body terminating at a probe tip surface, the probe tip surface configured to be placed into contact with a predetermined detection location, the predetermined detection location being in vivo on a patient's skin or ex vivo in a patient's body fluid sample; an optical source configured to generate a plurality of laser pulses of at least one predetermined energy level or at least one predetermined wavelength, the optical source terminating at or near the probe tip surface, the laser pulses configured to cause generation of one or more transient vapor nanobubbles around malaria-specific nanoparticles at the predetermined detection location; and one or more acoustic detectors configured to detect acoustic pulses generated by the one or more transient vapor nanobubbles and output one or more signals indicative of the detected acoustic pulses to at least one processor, the one or more acoustic detectors comprising a piezo element and being flat, wherein a distance between an outer wall of the optical source and a radially inner edge of the one or more acoustic detectors, R1, can be 0.01 mm to 0.03 mm so as to improve a signal strength of the acoustic pulses striking a flat surface of the one or more acoustic detectors, wherein the R1 can be 0.01 mm to 0.03 mm such that the acoustic pulses strike the flat surface of the one or more acoustic detectors at an angle of incidence, a, of less than 45°, wherein the optical source and the one or more acoustic detectors can be enclosed within the probe body.

In a configuration, the optical source can comprise one or more optical fibers.

In a configuration, the one or more optical fibers can each have a core diameter of about 100 μm.

In a configuration, the piezo element can comprise a navy type II or type VI material or a composite material.

In a configuration, a tissue-facing surface of the one or more acoustic detectors can be 0.1 mm to 0.3 mm recessed from the probe tip surface.

In a configuration, the apparatus can further comprise a front layer between the probe tip surface and a tissue-facing surface of the one or more acoustic detectors.

In a configuration, an outer surface of the one or more optical fibers can be separated from a radially outer edge of the one or more acoustic detectors by 0.3 mm to 1.5 mm.

In a configuration, the malaria-specific nanoparticles can be located within an optical penetration depth beneath the predetermination location.

In a configuration, an outer diameter of the one or more acoustic detectors can be 0.2 mm to 3 mm such that the acoustic pulses strike the flat surface of the one or more acoustic detectors at a same or similar angle of incidence to reduce an effect of de-phasing.

An apparatus configured for diagnosing malaria noninvasively can comprise: a sensor portion, the sensor portion including an optical source configured to generate laser pulses of at least one predetermined energy level, the optical source comprising an optical fiber terminating at or near a distal end of the sensor portion, the laser pulses configured to cause generation of one or more nanobubbles around malaria-specific nanoparticles at a predetermined location, and one or more acoustic detectors configured to detect acoustic pulses generated by the one or more nanobubbles and output one or more signals indicative of the detected acoustic pulses to at least one processor, the one or more acoustic detectors comprising a piezo element and being flat, wherein, at the distal end of the sensor portion, a distance between an outer wall of the optical fiber and a radially inner edge of the one or more acoustic detectors, R1, can be 0.01 mm to 0.03 mm so as to improve a signal strength of the acoustic pulses striking a flat surface of the one or more acoustic detectors, wherein the R1 can be 0.01 mm to 0.03 mm such that the acoustic pulses strike the flat surface of the one or more acoustic detectors at an angle of incidence, a, of less than 45°; a housing, wherein the sensor portion can be at least partially disposed within the housing; and a spring disposed between a proximal end of the housing and the proximal end of the sensor portion, the spring biasing the sensor portion toward a distal end of the housing.

In a configuration, the spring can be configured to be compressed when the apparatus is applied to a measurement site, the compressed spring forcing the distal end of the sensor portion into contact with the measurement site.

In a configuration, the housing can comprise a patient interface at the distal end, the apparatus further comprising a liner covering the patient interface when the apparatus is not in use.

In a configuration, the patient interface can comprise an adhesive layer and/or a gel layer.

In a configuration, an outer diameter of the one or more acoustic detectors can be 0.2 mm to 3 mm such that the acoustic pulses strike the flat surface of the one or more acoustic detectors at a same or similar angle of incidence to reduce an effect of de-phasing.

An apparatus for diagnosing and/or treating malaria in a patient noninvasively can comprise a sensor probe having a probe body terminating at a probe tip surface, the probe tip surface configured to be placed into contact with a predetermined detection location; an optical source configured to generate a plurality of laser pulses of at least one predetermined energy level and/or at least one predetermined wavelength, the optical source terminating at or near the probe tip surface, the laser pulses configured to cause generation of one or more transient vapor nanobubbles around malaria-specific nanoparticles at the predetermined location; and one or more acoustic detectors configured to detect acoustic pulses generated by the one or more transient vapor nanobubbles and output one or more signals indicative of the detected acoustic pulses to at least one processor, the one or more acoustic detectors being substantially flat and in close proximity with the optical source, wherein the optical source and the acoustic detector can be enclosed within the probe body. The optical source can comprise one or more optical fibers. The apparatus can comprise two or more optical fibers, wherein each of the two or more optical fibers can be located between two acoustic detectors The optical fiber can have a core diameter of about 50 μm to about 200 μm, or about 100 μm. The optical source can further comprise a laser pulse generator coupled to the one or more optical fibers. The acoustic detector can comprise a piezo element. The apparatus can comprise two or more piezo elements configured to detect signals of the same or different frequency spectra. The piezo element can comprise a navy type II or type VI material, or a composite material. The acoustic detector can comprise a substantially centrally located opening sized to accommodate the optical fiber. The acoustic detector can comprise a substantially flat disc, or two or more substantially flat discs or elements of other shape and acoustic properties. A tissue-facing surface of the acoustic detector can be about 0.1 mm to about 0.3 mm recessed from the probe tip surface. The sensor probe can further comprise a front layer between the probe tip surface and a tissue-facing surface of the acoustic detector. An outer wall of the optical fiber can be separated from a radially inner edge of the acoustic detector by about 0.01 mm to about 0.03 mm. An outer surface of the optical source can be separated from a radially outer edge of the acoustic detector by about 1.0 mm to about 1.5 mm. The sensor probe can further comprise a disposable cap. The laser pulses can be configured to cause generation of transient vapor nanobubbles around malaria-specific nanoparticles in blood and/or tissue. The predetermined detection location can be a patient's skin at the patient's wrist, ankle, lip, or tongue base or other locations. The predetermined detection location can be a surface of a flow cuvette with a flow path for a patient's blood or urine or other biological fluid sample. The probe tip surface can be configured to be covered with a layer of gel before being placed into contact with the predetermined detection location. The malaria-specific nanoparticles are located within the optical penetration depth beneath such location. The apparatus can further comprise a housing, wherein the probe body can be at least partially disposed within the housing; and a spring disposed between a proximal end of the housing and the proximal end of the probe body, the spring biasing the probe body toward a distal end of the housing.

An apparatus configured for diagnosing and/or treating malaria noninvasively can comprise a sensor portion, the sensor portion including: an optical source configured to generate laser pulses of at least one predetermined energy level, the optical source comprising an optical fiber terminating at or near a distal end of the sensor portion, the laser pulses configured to cause generation of one or more transient vapor nanobubbles around malaria-specific nanoparticles at the predetermined location; and one or more acoustic detectors configured to detect acoustic pulses generated by the one or more transient vapor nanobubbles and output one or more signals indicative of the detected acoustic pulses to at least one processor, the acoustic detector being substantially flat and in close proximity with the optical fiber at the distal end of the sensor portion; a housing, wherein the sensor portion can be at least partially disposed within the housing; and a spring disposed between a proximal end of the housing and the proximal end of the sensor portion, the spring biasing the sensor portion toward a distal end of the housing. The spring can be configured to be compressed when the apparatus is applied to a measurement site, the compressed spring forcing the distal end of the sensor portion into contact with the measurement site. The housing can comprise a patient interface at the distal end, the apparatus further comprising a liner substantially covering the patient interface when the apparatus is not in use. The patient interface can comprise an adhesive layer and/or gel layer.

A method of detecting malaria using any of the apparatuses disclosed herein can comprise instructing a laser pulse source to apply one or more laser pulses to a measurement site, any of the apparatuses disclosed herein being applied to the measurement site; receiving one or more signals from the acoustic detector of the apparatus, the one or more signals indicative of acoustic pulses detected by the acoustic detector upon the application of the one or more laser pulses to the measurement site; determining whether the measurement site is malaria-positive by: determining electronically a peak time of the one or more signals; comparing the peak time with a predetermined diagnostic threshold; and outputting a malaria-positive message if the peak time exceeds the predetermined diagnostic threshold, and outputting a malaria-negative message if the peak time does not exceed the predetermined diagnostic threshold. Determining whether the measurement site is malaria-positive can further comprise determining parameters from an amplitude, phase and/or shape of the signal. The method can also include using any of the apparatuses disclosed herein to scan a plurality of close locations to probe a sufficient volume of skin so as to improve detection of low level of malaria parasite density in the skin.

A method of detecting malaria using any of the apparatuses disclosed herein can comprise instructing a laser pulse generator to apply one or more laser pulses to a measurement site, the apparatus being applied to the measurement site; receiving one or more signals from the one or more acoustic detectors of the apparatus, the one or more signals indicative of acoustic pulses detected by the acoustic detector upon the application of the one or more laser pulses to the measurement site; and determining whether the measurement site is malaria-positive based on parameters from an amplitude, phase, shape, and/or a peak time delay of the one or more signals. Instructing can comprise instructing a laser pulse generator to apply one or more laser pulses of the same or different energy levels and/or wavelengths. Instructing can comprise instructing a laser pulse generator to route the laser pulses sequentially to a plurality of optical fibers. Receiving can comprise receiving a signal from a high-frequency one of the one or more acoustic detectors and a signal from a low-frequency one of the one or more acoustic detectors.

DETAILED DESCRIPTION

Although certain embodiments and examples are described below, this disclosure extends beyond the specifically disclosed embodiments and/or uses and obvious modifications and equivalents thereof. Thus, it is intended that the scope of this disclosure should not be limited by any particular embodiments described below.

The test parameters and experimental data provided in the present disclosure show preliminary results collected from in-field testing in early-stage human studies. They are disclosed herein as the evidence and examples of the first non-invasive detection of malaria in humans. They also provide insight into the principle of operation of laser-induced transient vapor nanobubbles for detecting malaria in human patients in non-invasively and/or minimally invasive manners. The sensor prototypes, experimental hardware and software are in early stages of development and the results obtained are thus process-dependent. The results and the processes used for collecting the results have not been scientifically peer-reviewed. However, the results were obtained under a controlled environment in blinded studies. That is, the personnel responsible for signal collection did not know the malaria status of patients at the time of signal collection. Further, human data were obtained and documented under an Institutional Review Board (TRB)-approved protocol and by using WHO-approved reference methods for determining the malaria status of each subject studied. Accordingly, it may be that the protocols, data acquisition and validation procedures, and scientific results and conclusions discussed herein could be subject to further analysis and future study.

Overview of Example Systems and Processes of Diagnosing and/or Treating Malaria with Transient Vapor Nanobubbles (“Nanobubbles”)

FIG.1Aillustrates schematically a system10for noninvasively diagnosing and/or treating malaria. The system10can have an optical source for providing laser pulses to a test subject20. Although a patient is illustrated inFIG.1Aas the test subject20, a person skilled in the art will appreciate from the disclosure herein that the test subject20can include any other test subjects that may have been infected by malaria parasites, including but not limited to a blood and/or tissue sample.

The optical source can include a laser pulse generator102and one or more optical fibers104. The laser pulse generator102can generate optical energy, which can be laser pulses of predetermined energy and/or fluence levels. The optical fiber(s)104can deliver the generated laser pulses to a location26on the test subject20. Using the patient as an example, the optical fiber(s)104can direct laser pulses to any suitable locations on the patient, such as on the digits, hand, wrist, ankle (such as shown inFIG.1A), neck, earlobes, lips, under the patient's tongue (tongue base), or other locations.

If the test subject20has been infected by malaria parasites, the test subject20can contain malaria-specific nanoparticles, such as HZ (hemozoin) nanocrystals. HZ (hemozoin) nanocrystals have a significantly higher optical absorbance than that of an uninfected red blood cell, uninfected hemoglobin, or major proteins in the red blood cell. The malaria-specific nanoparticles can likely be present at the location26as malaria parasites can travel to various locations in the patient by blood.

If the malaria-specific nanoparticles are located within a depth from a surface of the location26that can be penetrated by the laser pulses, laser-induced transient vapor nanobubbles (“nanobubbles”) can be generated at the location26underneath the surface of the location26. Nanobubbles are a transient phenomenon. The generation of nanobubbles can produce sound waves.

The system10can have one or more acoustic detectors106configured for detecting the sound waves or an acoustic pulse of the nanobubbles generation. Close proximity of the acoustic detector(s)106and the acoustic pulse source, which is/are the nanobubble(s), can enhance the sensitivity and specificity of the acoustic detector(s)106. This can be due to the acoustic pulse being stronger near the source than further away from the source, in particular for a point source. A nanobubble is a point source that generates spherical wavefronts. If the acoustic detector (s)106is(are) too far away from the source, the signal reaching a surface of the acoustic detector(s)106may be too weak to be detected. A commonly used acoustic detector can be a piezo element. The piezo element can generate an electrical charge in response to vibrations caused by the pressure wave.

In some embodiments of the present disclosure, the optical fiber(s)104and the acoustic detector(s)106are located in a single sensor probe108. The single sensor probe configuration can be easier to use than having two separate probes for an optical fiber and an acoustic detector. The single probe configuration can also allow the acoustic detector(s)106to be closer to the nanobubble and to the optical fiber(s)104(which can reduce and/or minimize the angle of acoustic incidence) than if the optical fiber(s)104and the acoustic detector(s)106are in separate probes.

The system10can have one or more signal processors and/or controller110in electrical communication with the laser pulse generator102and/or the acoustic detector(s)106. The one or more signal processors110can process the signals from the acoustic detector(s)106to determine if the signals are indicative of nanobubble(s) generation and thus for the presence of malaria-specific nanoparticles. In some embodiments, the signals from the acoustic detector(s)106can be amplified before being processed by the one or more signal processors110. The one or more signal processors110can cause the processed signals and/or the detection or non-detection of nanobubble(s) generation to be displayed on a display device112.

In some embodiments, the one or more processors110of system10can instruct the laser pulse generator102to emit a plurality of laser pulses at the location26to determine if there is nanobubble(s) generation at the location26. As will be described in greater detail below, the laser pulses can have the same or different wavelengths and/or energy level. In some embodiments, the one or more processors110of the system10can instruct that the sensor probe108to expose to the laser pulse to different areas at the location26. This can be achieved by mechanically scanning the surface with one optical fiber or by using multiple optical fibers (seeFIG.7E) and routing the laser pulse sequentially though various optical fibers. In some embodiments, the one or more processors110of the system10can instruct that the sensor probe108be moved to a second location of the test subject20different from the location26to determine if malaria-specific nanoparticles can be detected at the second location. In some embodiments, more than two fiber-acoustic detector combinations can be used (seeFIG.7E). The more than two fiber-acoustic detector combinations can be mounted in one sensor probe. Each of the more than two fiber-acoustic detector combinations can collect signals from a different area within the same or different location(s).

In some embodiments, such as illustrated inFIG.1B, a system12having the same or similar features as the system10can include a second sensor probe109comprising a second optical fiber (or more than one optical fiber)105and a second acoustic detector (or more than one acoustic detector)107. Features of the system10inFIG.1Aand the system12inFIG.1Bcan be incorporated into one another. As shown inFIG.1B, the first sensor probe108can be applied to the first location26and the second sensor probe109can be applied to a second location28of the test subject20. The one or more processors110can be in electrical communication with the laser pulse generator102and both of the acoustic detectors106,107. The laser generator102can provide optical energy, such as one or more laser pulses to both of the optical fibers104,105. Laser pulse(s) can be directed to the first and second location26,28substantially simultaneously or in succession. The one or more processors110can receive outputs from both of the acoustic detectors106,107to determine if nanobubble generation can be detected at either or both locations. The one or more processors110can have a multiplex unit configured to instruct the laser pulse generator102to send pulses (for example, to the plurality of optical fibers in the sensor probe such as illustrated inFIG.7E), and/or to collect signals from the acoustic detectors106,107. The system can also have more than two sensor probes, such as three, four, five, or six sensor probes, which can be coupled to the laser pulse generator102and the processors110. The display112can display a single outcome of whether malaria-specific nanoparticles have been detected in the test subject20, and/or an outcome for each location.

FIG.2illustrates an exemplary process200of using the system for noninvasively diagnosing and/or treating malaria, such as the system10,12ofFIGS.1A and1B. At step202, the one or more signal processors of the system can instruct a user, such as a clinician or a health worker, to apply the malaria probe having both the optical fiber(s) and the acoustic detector(s) to a target location. The target location can be any of the test subject described above.

The step202can include applying a layer of optically transparent ultrasound gel (or any other material to act as an optical and acoustic coupling media between the probe and the tissue) to a probe tip surface before applying the probe to the target location. Applying the probe to the target location can include pressing the probe tip surface and/or the layer of gel firmly into contact with a surface of the target location. The layer of gel can act as an optical and/or acoustic coupler by expelling air between the probe tip surface and the surface of the target location. In some embodiment, applying the probe to the target location can also include keeping a longitudinal axis of the probe generally perpendicular to the surface of the target location. The generally perpendicular probe can prevent air from entering between the probe tip surface and the surface of the target location, which can improve optical and/or acoustic coupling of the probe and the target location.

At step204, the one or more signal processors can set an energy level of the laser pulse generator to a first predetermined level, E1. In some embodiments, E1 can be sufficient for generating nanobubbles around malaria-specific nanoparticles up to about 0.5 mm underneath a surface of the target location. In some embodiments, E1 can have an energy level of about 1 μJ to about 50 μJ, or about 10 μJ to about 15 μJ. In some embodiment, E1 can have a pulse rate of about 1 HZ (hemozoin) to about 100 HZ (hemozoin), or about 20 HZ (hemozoin) to about 50 HZ (hemozoin), or about 20 HZ (hemozoin), or about 50 HZ (hemozoin). The one or more signal processors can set more than one energy levels, such as two different energy levels. The one or more signal processors can also set one or more than one wavelengths for the laser pulses.

At step206, the one or more signal processors can cause the laser pulse generator to apply one or more laser pulses having an energy level of E1. The number of pulses to be applied at a location can be predetermined, manually configured, and/or determined by the one or more processors based on certain algorithms. For example, the processors can stop additional pulses as soon as nanobubble generation has been detected, or continue instructing that additional pulses be applied until a predetermined number of pulses have been applied at the location. At decision block208, the one or more signal processors can determine based on the signals outputted by the acoustic detector if one or more nanobubbles have been generated.

If the signal is not indicative of nanobubble generation, the one or more processors can optionally determine at decision block210if the pulse(s) applied at the step206include the last or final pulse to be applied to the patient for malaria detection. The number of pulses to be applied to each patient can be predetermined, manually configured, and/or determined by the one or more processors according to certain algorithms. The pulses can be applied to one or more measurement locations on the patient. The locations can be predetermined, manually selected by a user such as a clinician, and/or determined by the one or more processors according to certain algorithms. The one or more signal processors can instruct that the same or different numbers of pulses be applied to each location.

If the pulse(s) applied at the step206include the last or final pulse of the process200, the one or more signal processors can output a message that no malaria parasite is detected at step212. The message can be an audio signal, an optical signal, a text and/or symbol displayed on a display device, or a combination thereof. If the pulse(s) applied at step206do not include the last or final pulse of the process200, the one or more processors can instruct that the probe be applied to another location at step214. Pulse(s) can be applied to the new location to determine if nanobubble(s) generation can be detected at the new location. The energy level can be the same or different for each location.

If the signal is indicative of nanobubble generation, the one or more signal processor can also optionally determine at decision block216if the pulse(s) applied at the step206include the last or final pulse to be applied to the patient for malaria detection. If the pulse(s) applied at step206do not include the last or final pulse of the process200, the one or more processors can instruct that the probe be applied to another location at step218. Whether the signal is indicative of nanobubble generation can be determined by parameters derived from an amplitude, phase and/or shape of the signal.

If the pulse(s) applied at the step206include the last or final pulse of the process200, the one or more signal processors can output a message that one or more malaria parasites are detected at step220. In some embodiments, a signal indicative of nanobubble generation can include at least one (such as, two) spikes on a time-response trace (signal) received from the acoustic detector. Time taken for detecting the spike can be used to estimate a depth of the malaria-specific nanoparticles and/or malaria parasites. Time between the two spikes can characterize the maximal size of a detected vapor nanobubble.

At the step206, if malaria-specific nanoparticles are present at the location, the laser pulse(s) applied to the location may also be sufficient for generating nanobubbles of a size that can cause mechanical damage to the malaria parasites. In some embodiments, the nanobubbles cause mechanical damage and/or destruction of the malaria parasites without harming uninfected blood cells and/or tissues.

If some individual signals obtained from healthy tissue look similar to nanobubble-specific signals associated with malaria disease and presence of HZ (HEMOZOIN) in the laser-exposed volume, groups of N signals (N ranges from 1 to 10,000), each in response to the corresponding laser pulse, can be analyzed statistically. Examples of statistical analyses can include using signal amplitude-derived diagnostic parameters, such as the normalized positive count, N, and the hemozoin index, HI, and a user-defined diagnostic threshold for the N and HI parameters. The normalized positive count, N, can be calculated using the formula N=Np/Nt, where Ntis the total number of the collected signals, Npis the number of signals with the peak-to-peak amplitude above a threshold T. The hemozoin index, HI, can be calculated using the formula HI=<A>−T*NNPCT, where <A> is an average peak-to-peak amplitude of the signals above T. Parameters above the diagnostic threshold would be indicative of malaria disease and parameters below the threshold would be indicative of healthy condition. In some embodiments, the hemozoin index of a malaria-positive signal can be about one order of magnitude greater than the hemozoin index of a malaria-negative signal. Examples of statistical analyses can also include a peak time-delay parameter and a user-defined diagnostic threshold for the time-delay. More details of the statistical analyses are described further below.

Additional diagnostic combinations in addition to applying one level of the laser pulse energy, one laser wavelength, and/or one type of the acoustic detector can be used to further improve the sensitivity and specificity of the detection of malaria-specific signal in the background of the bulk signal associated with healthy (malaria-negative) tissue.

In some embodiments, the process200can be repeated with a different laser wavelength than the wavelength used in the previous process200. One of the two different wavelengths can be associated with a maximal optical absorption by malaria-specific nanoparticles.

In some embodiments, the process200can be repeated with a different laser pulse energy level than the laser pulse energy level used in the previous process200. The difference in the nanobubble signals detected in the two processes200can be different due to the different number of bulk signals produced by the different energy levels.

The process200can be repeated with two different laser wavelengths and/or two different laser pulse energies, using the same sensor, or different optical fiber-acoustic detector combinations in the same sensor (which will be described below with reference toFIG.7E).

The process200can also additionally and/or alternatively be performed with a sensor have two or more different acoustic detectors (described below with reference toFIGS.7D and7E). The different acoustic detectors can have different acoustic properties, which can enable detection of the acoustic signal in different frequency domains and can improve the identification of the malaria-specific signal and/or nanobubble generation.

As the malaria diagnosis and/or treatment apparatuses and processes disclosed herein are based on photo-excitation of the malaria-specific nanoparticle, the process200can be reproducible and free from parasite resistance. As the generation of laser pulses and generation of nanobubbles take seconds, or at most several minutes, the treatment process200can be more efficient than traditional malaria diagnosis and/or treatment procedures.

Examples of a Malaria Probe

Examples of the malaria diagnosis and/or treatment probe will now be described. As shown inFIGS.3A-3C, the probe300can have a sensor portion310and a body portion305. The sensor portion310can have a proximal end312and a distal end314. The proximal end312of the sensor portion310can be coupled to the body portion305. The sensor portion310can extend distally from the body portion305. The sensor portion310can have a probe housing315enclosing the optical fiber and the acoustic detector. The body portion305can include circuitry and/or processors for driving the optical fiber and/or the acoustic detector. InFIGS.3B and3C, the distal end314of the sensor portion310is covered by a protective cap301when the probe300is not in use.FIG.3Dillustrates the distal end of the sensor portion with the protective cap removed.

FIGS.3E and3Fillustrate the sensor portion310of the probe300with the protective cap301removed. As shown inFIGS.3E and3F, the distal end314of the sensor portion310can have a sensor tip cover317extending distally from the probe housing315. The sensor tip cover317can have a smaller outer diameter than the probe housing315. The sensor tip cover317can include a generally centrally located opening337. The optical fiber and the acoustic detector can be located generally concentrically with the opening317. The probe300can also include electrical connections, such as lead wire360, for connecting the acoustic detector330with a signal processor. The sensor design shown inFIGS.3A,3E, and3F may be more prone to detecting false-positive signals, reduced sensitivity to the detection of true-positive signals, which may be ameliorated in future sensor designs.

Before describing the sensor portion310in greater detail, a configuration of the malaria probe sensor portion410known in the art is described with reference to the schematic drawing inFIG.4A. The sensor portion410can have an optical fiber420and a spherical acoustic detector430enclosed by the probe housing415and/or the sensor tip cover such as shown inFIGS.3D and3E. The optical fiber420can extend substantially along a central longitudinal axis of the sensor portion410.

The acoustic detector430can be generally a hemisphere with a substantially centrally located opening436to accommodate the optical fiber420. The spherical acoustic detector430can circumferentially surround the optical fiber420. In some instances, the spherical acoustic detector430can have a diameter D of about 4 mm. When a malaria-specific nanoparticles-generated nanobubble22(“nanobubble”) is generated under the surface of the target location20, a distance d between the spherical acoustic detector430and the nanobubble22can be between about 2 mm to about 3 mm, or greater.

The spherical acoustic detector430can have some disadvantages. The pressure amplitude for the spherical pulse drops inversely proportional to the square of the distance from the source, as described above. As a result, the distance d can be large enough to result in substantial loss of the pressure between the nanobubble22and the acoustic detector430and hence in the reduced sensitivity of such sensor for detecting nanobubbles.

The acoustic detector430can be one or more flat piezo elements, which can be cheaper than spherical piezo elements. If the flat piezo elements are located in the same distance as the spherical piezo elements as shown inFIG.4A, it may be necessary to use a flat piezo element with a large surface area to detect spherical pulses with reduced pressure amplitude at the distance d. However, a flat piezo element having a large detector surface area can result in a weaker signal output. As shown inFIG.4B, due to the large surface area of the acoustic detector430, the pressure pulse24generated by the nanobubble22can strike the surface of the acoustic detector430at different angles of incidence and be in different phases. Some of the pressure pulses, such as the pressure pulses hitting the surface of the piezo element at an angle of incidence a1, can generate a positive electrical charge. Some of the pressure pulses, such as the pressure pulses hitting the surface of the piezo element at an angle of incidence a2, can generate a negative electrical charge. Such dephasing of piezo-effect in piezo element can result in the loss of the sensitivity because positive and negative charges can cancel each other. As a result, a greater surface area of the piezo element can reduce, not increase, the sensor sensitivity.

Further challenges of using the spherical piezo element430can include the high cost of spherical piezo elements, and/or the complexity of incorporating the spherical piezo element430and the optical fiber420into the single probe. In some instances, a separate holder is required to hold the optical fiber420in the opening436of the spherical piezo element430. The cost and complexity can make it difficult to mass-produce or commercialize a probe having a sensor portion410.

FIG.5Aillustrates schematically an example malaria probe sensor portion510that overcomes some challenges of using the spherical and/or flat acoustic detector430, and/or other challenges. The sensor portion510can have an optical fiber520and a substantially flat acoustic detector530enclosed by the probe housing515and/or the sensor tip cover such as shown inFIGS.3E and3F. As noted above, this sensor design may be more prone to detecting false-positive signals, reduced sensitivity to the detection of true-positive signals, which may be ameliorated in future sensor designs.

The optical fiber520can extend substantially along a central longitudinal axis of the sensor portion510. The optical fiber520can have a core diameter, or optical aperture, in the range of about 50 μm to about 200 μm, or about 80 μm to about 150 μm, or about 100 μm. The core diameter of the optical fiber520is small enough to reduce background bulk optical absorbance in order to maintain sufficient signal-to-noise ratio, and is not too small so it can easily miss the malaria parasite or malaria-specific nanoparticle.

The substantially flat acoustic detector530can form a disc with a substantially centrally located opening536to accommodate the optical fiber520. The flat acoustic detector530can circumferentially surround the optical fiber520. In some embodiments, the flat acoustic detector530can hold the optical fiber520in place without a separate holder. The flat acoustic detector530can be a piezo element. The piezo element can comprise a navy type II material, navy type VI material, any other piezo materials, or any combination of piezo and other materials.

FIG.5Billustrates a longitudinal cross-section of a distal part of the sensor portion310of the probe300inFIG.3F, which incorporates the optical fiber320circumferentially surrounded by a flat acoustic detector330. Accordingly, the sensor portion310can be an example of the sensor portion510inFIG.5A. As shown inFIG.5B, a proximal portion of the sensor tip cover317can be received in and/or securely attached (for example, by an interference fit, adhesives, welding, and the like) to a lumen of the probe housing315. A proximal end of the sensor tip cover317can terminate near or abut a shoulder352(seeFIG.5E) of a fiber housing350.

As shown inFIG.5E, the fiber housing350can have a distal portion351transitioning to a proximal portion353the shoulder352. The distal portion351can have a first external diameter that is smaller than a second external diameter of the proximal portion353. The second external diameter can be sized to allow the proximal portion351to be slidably received in the lumen of the probe housing315. As shown inFIG.5B, gap between an inner wall of the probe housing315and an outer wall of the distal portion351of the fiber housing350can accommodate a wall thickness of the sensor tip cover317.

Also as shown inFIG.5E, the fiber housing350can have a lumen with varying internal diameters. The internal diameters of the lumen proximal353and distal355to a neck354can be greater the internal diameter of the neck354. The lumen of the fiber housing350can accommodate and/or support the optical fiber320(seeFIG.5F), which can also have varying diameters. The variation in the outer diameter of the optical fiber320can be due to a varying thickness in a coating or protective layer(s) circumferentially surrounding the optical fiber520.

As shown inFIG.5B, the internal diameter of the neck354can reduce movements of the optical fiber320within the housing315. As shown inFIGS.5B and5C, the lumen distal355to the neck354can also accommodate a support body or ferrule340. The support body340can include a lumen342to accommodate the optical fiber320. The internal diameter of the lumen342can reduce movements of the optical fiber320within the housing315and/or the sensor tip cover317. The support body340can also improve rigidity of the distal end of the sensor portion310. The lumen342can terminate at a bore344, which can have a reduced internal diameter compared to the internal diameter of the lumen342. A distal portion of the optical fiber320that is not covered by coating or protective layer(s) (seeFIG.5F) can extend through the bore344.

As shown inFIGS.5B and5D, the flat acoustic detector330can be located distal of the support body340and enclosed by the sensor tip cover317. The acoustic detector330can have a generally centrally located opening336for accommodating and/or supporting a distal end of the optical fiber320.

As shown inFIG.5B, the sensor portion310can also include a front layer318. The front layer318can be substantially flush with a distal surface of the sensor tip cover317to define a probe tip surface316. The front layer318can improve acoustic coupling between the acoustic impedance of the tissue of the target location and the acoustic detector330, to optimize response of the acoustic detector330, to protect the acoustic detector330from backscattered laser radiation, and/or to form a durable contact surface which protects the acoustic detector330from mechanical damage, such as scratches. The front player318can include a material having suitable speed of sound, optical transparency, and/or optical absorbance or scattering properties. The speed of sound in the material can be similar to that in water. The material may not be transparent as a transparent layer can deliver a portion of the backscattered laser pulses into the acoustic detector330and thus create a noise in the signal. The material also may have minimal optical absorbance to avoid a photoacoustic effect in the front layer caused by the backscattered laser pulses. In some embodiments, the front layer318can have acoustic epoxy and metal particles that scatter backscattered laser radiation and thus shield piezo element from such backscattered laser radiation.

Although the acoustic detectors330,530are illustrated as a single flat element, the acoustic detector can also include two flat discs530A,530B, located on substantially opposite sides of the optical fiber520, such as shown inFIG.7D. The two flat discs530A,530B can have the same or different acoustic properties, such as the frequency spectrum. For example, the disc530A can have a high-frequency piezo material and the disc530B can have a low-frequency piezo material. The discs530A,530B can each detect amplitude and time information of the acoustic signal in different frequency spectra. The low frequency disc can operate in the range of about 0.01 MHZ (hemozoin) to about 4 MHZ (hemozoin). The high frequency disc530B can operate in the range of about 4 MHZ (hemozoin) to about 100 MHZ (hemozoin).

FIG.6Aillustrates a comparison of an acoustic detector530(which can also be the acoustic detector330) at a location535that is closer to the nanobubble22than an acoustic detector430at a location435. As shown inFIG.6A, a spherical pressure pulse can arrive at the acoustic detector430and the acoustic detector530at different angles of incidence. As the amplitude of spherical pulse is inversely proportional to the square of the distance from the source, the acoustic detector530can detect a stronger acoustic pulse from generation of the nanobubble22than the detector430. Similarly, as shown inFIG.6B, the flat acoustic detector530can be brought closer to the nanobubble22than the spherical acoustic detector430. In some embodiments, the distance d′ between the flat acoustic detector530and the nanobubble22can be as small as about 0.1 mm or about 0.2 mm, compared to about 2 mm to about 6 mm between the spherical acoustic detector430and the nanobubble22. The flat acoustic detector530can also be smaller than the spherical acoustic detector520. In some embodiments, such as shown inFIGS.7A and7B, the flat acoustic detector530can have an external diameter, or acoustic aperture, of about 0.2 mm to about 3 mm, or about 1 mm to about 2 mm, compared to about 4 mm to 8 mm for the spherical acoustic detector430. A distance R2 between the outer wall of the optical fiber520and a radially external edge534of the acoustic detector530can be no greater than about 0.3 mm to about 1.5 mm. As described above, the small R2 can contribute to reduction of de-phasing of the pressure-to-charge conversion process in a piezo element.

As shown inFIGS.7A-7C(which illustrate a preferred sensor probe configuration), a gap R1 between an outer wall of the optical fiber520and a radially internal edge532of the acoustic detector530can be about 0.01 mm to about 0.03 mm, or about 0.02 mm. A small gap R1 can bring the acoustic detector530closer to a position with the minimal angle of acoustic incidence and hence the maximal sensitivity of the sensor. This is because pressure pulses striking the surface of an acoustic detector, such as a piezo element, at a smaller angle of incidence can generate stronger signals than pressure pulses striking the surface of the acoustic detector at a larger angle of incidence. The angle of incidence, a, at which the pressure pulses strikes the surface of the acoustic detector can be less than about 45°. This can be due to flat piezo elements usually having relatively narrow pointing diagram, which can become narrower with the increasing size of piezo element. The optical fiber520can have a diameter in the range of about 10 μm to about 400 μm, or about 50 μm to about 200 μm, or about 80 m to about 150 μm, or about 100 μm.

Although a flat acoustic detector can be inferior, in theory, to a spherical detector for detecting the spherical pressure pulses from the nanobubble, the flat acoustic detector530is small in its size and in close proximity to the nanobubble such that the effect of the flat shape on the signal can be small. As a result, the flat acoustic detector530can improve the sensitivity and selectivity of malaria detection.

Moreover, the flat acoustic detector530does not have some of the above-described disadvantages associated with the spherical acoustic detector430. When spherical pressure pulses arrive at the acoustic detector530having a small surface area and located in close proximity to the point source, the pressure pulses can arrive at the surface of the acoustic detector530at substantially the same or similar angles of incidence. As a result, it is less likely that opposite electrical charges are produced by the pressure pulses and the effect of de-phasing can be reduced and/or minimized. The flat acoustic detector530can thus improve the peak-to-peak amplitude of the spike. Larger peak-to-peak amplitude of the spike can improve the sensitivity and/or specificity of the sensor portion510.

Incorporating the flat acoustic detector530and the optical fiber520into a single probe can be less complex than incorporating the spherical acoustic detector430and the optical fiber420into a single probe. As described above, the flat acoustic detector530can directly hold the optical fiber520without a separate holder. A flat acoustic detector, such as a flat piezo element, can also be significantly cheaper than a spherical acoustic detector, such as a spherical piezo element. As a result, the sensor portion510can be more suitable for mass production than the sensor portion410.

With continued reference toFIG.7A, the optical fiber520can terminate at or substantially at a probe tip surface516so that a distance Hf between the tip of the optical fiber520and the probe tip surface516can be substantially zero. Any material between the optical fiber tip and the surface of the target location can cause a background residual due to residual optical absorption, even if the material is transparent and the residual optical absorption is minor. Having the optical fiber502terminating at the probe tip surface516can reduce background residual and/or bring the optical source as close to the nanobubble as possible. As shown inFIG.7C, having the optical fiber5terminating at the probe tip surface can bring the optical source closer to the malaria-specific nanoparticle. A distance between a distal end of the optical fiber520and the malaria-specific nanoparticle can be between about 0.05 mm to about 0.5 mm.

As shown inFIGS.7A and7C, the acoustic detector530can be recessed from the probe tip surface516by a gap Hp. Hp can be from about 0.02 mm to about 4 mm, about 0.02 mm to about 1 mm, or from about 0.1 mm to about 0.2 mm. A distal portion of the acoustic detector530and the optical fiber520can be embedded in a front layer518. The front layer518can improve acoustic coupling between the acoustic impedance of the tissue of the target location and the acoustic detector530, to optimize response of the acoustic detector530, to protect the acoustic detector530from backscattered laser radiation, and/or to form a durable contact surface which protects the acoustic detector530from mechanical damage, such as scratches. The front player518can comprise a material having suitable speed of sound, optical transparency, and/or optical absorbance or scattering properties. The speed of sound in the material can be similar to that in water. The material may not be transparent as a transparent layer can deliver a portion of the backscattered laser pulses into the acoustic detector530and thus create a noise in the signal. The material also may have minimal optical absorbance to avoid a photoacoustic effect in the front layer caused by the backscattered laser pulses. In some embodiments, the front layer518, such as shown inFIG.7C, can comprise acoustic epoxy. The front layer518can also include metal particles in the acoustic epoxy to scatter backscattered laser radiation and thus shield piezo element from such backscattered laser radiation.

FIGS.7D and7Eillustrate schematically malaria sensor probe portions510A,510B, which can have any of features of the malaria sensor probe portions described with reference toFIGS.7A-7C, including but not limited to the dimensions and relative positions of the optical fiber, acoustic detector, and/or probe tip surface. As described above,FIG.7Dillustrates a malaria probe sensor portion510A with an optical fiber flanged by two flat acoustic detectors530A,530B. As shown inFIG.7E, more than two optical fiber-acoustic detector combinations can be mounted in a malaria probe sensor portion510B. The sensor portion510B can include a plurality of optical fibers520A,520B,520C,520D, and a plurality of acoustic detectors530A,530B,530C,530D. Each one of the plurality of optical fibers520A,520B,520C,520D can be located between two of the plurality of acoustic detectors530A,530B,530C,530D. Example combinations of optical fiber-acoustic detector can include the combination of optical fiber520A and acoustic detectors530A,530B, the combination of optical fiber520B and acoustic detectors530B,530C, the combination of optical fiber520C and acoustic detectors530C,530D, and the combination of optical fiber520D and acoustic detectors530A,530D. Each of the optical fiber-acoustic detector combinations can collect signals from a different area within the same or different measurement location. The plurality of acoustic detectors can have the same or different acoustic properties as disclosed herein. As noted above, this sensor design may be more prone to detecting false-positive signals, reduced sensitivity to the detection of true-positive signals, which may be ameliorated in future sensor designs.

In some embodiments, the sensor portion510can be reusable. A user can wipe the probe tip with alcohol to sterilize or disinfect the probe tip after each patient. In some embodiments, a disposable cap made of a thin film can be applied to the probe tip for each patient. The thin film can have little impact on the optical and acoustic contact between the sensor and the target location. The disposable cap can save a user's time for sterilizing or disinfecting the probe tip after each patient. In some embodiments, the film can have a thickness of about 1 μm to about 50 μm, or about 1 μm to about 20 m. The film may be covered with ultrasound gel or other biologically safe and optically- and acoustically-coupling material. The sensor probes described herein and a predetermined number of caps can also be provided in a kit. The number of caps can be sufficient for the life time and/or anticipated life time of the sensor probe in the kit.

FIGS.10A-13illustrate an example malaria probe1000. This sensor design may be more prone to detecting false-positive signals, reduced sensitivity to the detection of true-positive signals, which may be ameliorated in future sensor designs. The probe1000can have the same or similar features as the probe500. Features of the probe500and the probe1000can be incorporated into one another.

The probe1000can have a sensor portion1010and a body portion1005. The sensor portion1010can be coupled to the body portion1005and can extend distally from the body portion1005. The sensor portion1010can have a smaller outer dimension than the body portion1005. The probe1000can have a probe housing1015enclosing an optical fiber1020and a substantially flat acoustic detector1030.

The optical fiber1020can extend substantially along a central longitudinal axis of the sensor portion1010. The substantially acoustic detector1030can comprise two or more flat elements (such as discs) placed next to each other with the optical fiber1020running through a gap between the elements. As described above with reference toFIG.7D, the two or more flat elements can have the same or different acoustic properties. The acoustic detector1030can be held in place inside the sensor portion1010by a support body1040. The support body1040can have a lumen to accommodate the optical fiber1020. The probe1000can also include electrical connections1060for connecting the acoustic detector1030with a signal processor.

FIGS.14A-14Dillustrate an example malaria probe1400. The probe1400can have any of features of the probe500,1000. Accordingly, features of the probe500, features of the probe1000, and features of the probe1400can be incorporated into one another. Although the probe1400is illustrated as having a generally cylindrical shape, the shape of the probe1400is not limiting. Other physical specifications of the probe1400, such as size and materials, are also not limiting.

The probe1400can have a sensor portion1410and a housing1415. The sensor portion1410can include an optical source, such as an optical fiber (or more than one optical fiber) coupled to a laser pulse generator, and an acoustic detector (or more than one acoustic detector), such as a piezo element. The optical source can be configured to generate laser pulses of at least one (one, two, or more) predetermined energy levels and/or wavelengths. The optical fiber can terminate at or near the patient interface1442. The laser pulses can be configured to generate one or more nanobubbles around malaria-specific nanoparticles that are under a surface of a measurement site. The measurement site can be on a patient's body, or a flow cuvette described below. The acoustic detector can be configured to detect acoustic pulses generated by the one or more nanobubbles and output a signal indicative of the detected acoustic pulses to at least one signal processor. The acoustic detector can be substantially flat and in close proximity with the optical source.

The sensor portion1410can be located at least partially within the housing1415. The housing1415can include a distal end1440and a proximal end1444. At the distal end1440, the housing1415can include a mounting component1442. The mounting component1442can provide a greater footprint of the probe1400on the measurement site, such as the patient's skin, than the contact between the sensor portion1410and the patient's skin. In the illustrated embodiment, the mounting component1442can be generally ring-shaped to accommodate the sensor portion1410located generally at the center of the patient interface1442.

The mounting component1442can include a patient attachment mechanism, which can have an adhesive layer, a gel layer, and/or otherwise. The mounting component1442can also include an adhesive layer and/or gel layer that is covered by a liner1441when the probe1400is not in use. When in use, such as shown inFIG.14C, the liner1441can be removed to expose the adhesive layer on the mounting component1442. The adhesive layer can improve and/or maintain contact between the probe1400and the measurement site. A firmer contact between the sensor portion1410of the probe1400and the measurement site can improve optical and acoustic coupling between the sensor portion1410and the patient's tissue, and/or detection of the acoustic pulses by the acoustic detector of the probe1400.

The probe1400can be coupled electrically to the laser pulse generator, the signal processor, and/or the display that are disclosed herein. The housing1415of the probe1400can have an opening1445(shown inFIG.14C) at the proximal end1444. The sensor portion1410can establish communication with the laser pulse generator, the signal processor, and/or the display, via one or more cables extending into the housing1415through the opening1445.

As shown inFIGS.14B and14D, the housing1415can have a length that is greater than a length of the sensor portion1410. A spring1450can be positioned between the proximal end1440of the housing and a proximal end of the sensor portion1410. The spring1450can be configured to bias the sensor portion1410distally such that a distal end of the sensor portion1410can be at or near the distal end1440of the housing1415. The distal end of the sensor portion1410can optionally be recessed from the distal end of the housing1415when the sensor portion1410is biased to a distalmost position by the spring1450. The recess can allow the housing1415to shield the sensor portion1410from mechanical impacts, such as during a fall. When the measurement site is on the patient's body, the skin at the measurement site can be deformed to make contact with the recessed sensor portion distal end when the probe1400is applied to the measurement site.

As shown inFIG.14D, when a compressive force is applied to the distal end of the sensor portion1410, such as when the probe1400is applied and/or attached to a measurement site, the spring1450can be compressed to retract the sensor portion1410proximally. The compressed spring1450can exert a distal force on the sensor portion1410to force the sensor portion1410into contact with the measurement site. The distally directed force of the compressed spring1450can improve contact between the sensor portion1410and the measurement site. The compressed spring1450can provide a predetermined pressure at the skin-sensor interface to as to improve optical and acoustic coupling between the sensor and the skin.

Example Applications of Malaria Probe Embodiments

Embodiments of the malaria probe disclosed herein can be used in various applications to diagnose malaria. For example, a malaria probe, such as one having the sensor portion510ofFIG.5or any other embodiments of a malaria probe described herein, can be applied to a patient's skin at various parts of the patient. Suitable locations on the patient for a transdermal application can include the digits, hand, wrist, ankle, neck, earlobes, lips, tongue base, or others.

As shown inFIGS.8A-8C, malaria parasites804can escape from the blood802to the tissue and/or skin800in a process called sequestration. While antimalarial drugs may be effective against the malaria parasites804in the blood802, the drugs may not be effective against the malaria parasites804that hide in the tissue800. As shown inFIG.8C, some parasites804can survive in the tissue and/or skin800after the parasites804in the blood802have been killed. Whereas the parasites that circulated in the blood stream can usually be in young and unpigmented (ring) forms, the parasites that accumulated in the small blood vessels (sequestered) were mature, with the elevated level of hemozoin. The accumulation of hemozoin (in parasites and parasite-free tissue) in subcutaneous layers of skin within the depth of about 100-500 um may include the following hypothetical mechanism:Sequestration of mature parasites (including gematocytes) and/or hemozoin from peripheral blood into sub-cutaneous microvasculature. This may be typical for late-stage parasites with the highest level of hemozoin. Due to adhesion mechanisms, such parasites attach to wals of subcutaneous micro-vessels and, eventually, becomes immobilized in upper skin layers that contain such micro-vessels. Thus hemozoin accumulates in skin more than in blood.In skin, the sequestered parasites and hemozoin can persist for months while they clear from peripheral blood in days.Sequestered parasites (including gematocytes) are responsible for lethal complications in the clinic, uncontrollable transmission, and relapse, and represent a hidden pool of malaria.

The transdermal (skin) application can be effective not only in detecting and/or killing malaria parasites in the patient's blood. The malaria probe disclosed herein can have sensitivity and/or specificity to detect tissue-sequestered malaria parasites and/or hemozoin nanoparticles by generating nanobubbles around the malaria-specific nanoparticles in the micro-capillaries in the tissue.

The malaria probe disclosed herein not only has in vivo applications, but can also be used to detect malaria parasites ex vivo, to analyze blood, urine or other body fluids.FIGS.9A-9Cillustrate an example flow cuvette900that can be used with a malaria probe. The flow cuvette900can include a glass-silicon cuvette902sandwiched between a metal base904and a glass cover906. The cuvette902can contain a flow path908. The flow path908can be coupled to a tube910at each end of the flow path908. In some embodiments, the malaria probe can direct one or more laser pulses to a patient's blood or urine sample flowing through the flow path908to detect malaria parasites in the sample, such as by using the process200shown inFIG.2. The blood or urine sample can have a flow rate of about 5 mm/s. The flow rate can be controlled by a syringe pump or any other pumps.

The application of the probe on the liquid sample (for example, such as the peripheral blood) and on the skin stem from different biological mechanisms: active disease (peripheral blood) vs transmission (skin). Hence the peripheral blood cannot be analyzed “through the skin” and parasites or hemozoin properties do not correlate in peripheral blood and skin. Also, these applications have opposite scientific back-ups: well-established for blood and thus may provide an additional test if conventional blood test described above for malaria turns out to be inconclusive, and almost non-existent science for malaria in skin.

It is the skin rather than the blood that may be more challenging in the eradication of malaria. Malaria transmission can start with mosquitoes picking up parasites from the skin of an infected patient, specifically, the gametocytes from the subcutaneous layers of skin of the infected patient. Among all blood stages of parasites, these are the gametocytes that can deliver the maximal nanobubble signal because they have the highest quantity and size of hemozoin particles. The gametocytes, after becoming biologically inert, may last for many months in the skin. A transmission of malaria requires gametocytes be available for mosquito bites, that is, in the subcutaneous upper skin layer, which can be less than 1 mm in thickness. This biological mechanism delivers hemozoin to where it can meet the laser pulses emitted from the malaria probe examples disclosed herein (for example, at about 0.1 mm to about 0.5 mm optical penetration depth). The higher level of hemozoin in gametocytes, compared to other stages of parasites, can result in the higher nanobubble signal as found both in the laboratory and human studies disclosed herein.

The systems for noninvasively diagnosing and/or treating malaria can have multiple applications. The systems can have a diagnostic application for determining if a patient carries malaria-specific nanoparticles, hemozoin. If nanobubble(s) generation is detected using the processes, parameters and thresholds described herein, the patient can be diagnosed as malaria positive, including the residual malaria without active disease symptoms. If the nanobubble(s) generation is not detected, the patient can be diagnosed as malaria negative. The malaria-positive patients may have been treated when the nanobubble generations have caused mechanical destruction of the malaria parasites. The malaria-positive patients may additionally or alternatively be treated with anti-malarial drugs.

The systems can also have an epidemiological application for determining a malaria-infected region and/or the foci of malaria transmission. Caregivers typically provide anti-malarial drugs to a large population when there is an outbreak of malaria in a region. Regulators such as the WHO also issue rules and reports on the administration of anti-malaria drugs to various populations. As current malaria detection methods have difficulty detecting asymptomatic, sub-potent, past malaria infections, especially when peripheral blood is malaria-free, and are thus limited in locating where malaria originated, providing treatments to a large population, such as across an entire country, may be the only way to prevent transmission of malaria within that population. Anti-malarial drugs can have undesirable side effects, such as causing miscarriage in pregnant women, heart failures and other dangerous conditions. It is therefore desirable to narrow down the malaria-infected region/populations as much as possible to avoid administering anti-malarial drugs to people who may not need the drugs and/or may be harmed by the drugs.

The systems and processes described herein provide ways to narrow down the malaria-infected region. The human body carries malaria-specific nanoparticles, such as the HZ (hemozoin) nanocrystals, so long as the body was once a host of active malaria parasites. Even when the body no longer carries active malaria parasites, for example, if the malaria parasites went dormant and/or if the body had been infected by malaria parasites in the past, the HZ (hemozoin) nanocrystals can still be present in the body, especially in the skin. The ability of the system to detect the HZ (hemozoin) nanocrystals with or without an active malaria parasite using laser-induced nanobubbles can allow detection and/or narrowing down of geographical regions and population where malaria is endemic, and/or where malaria originated. Caregivers can treat a smaller population in the narrowed-down region, such as a small village, with anti-malarial drugs or other malaria treatments in order to contain the spread of malaria. The more localized and/or targeted administration of anti-malarial drugs can be effective in malaria eradication without unnecessarily dosing a large population with the drugs.

For geographical mapping of the malaria transmission through the mass screening of the population, the confirmed geographical location of human subjects can be mapped in a way such that the mapping shows the relative level of malaria positive (hemozoin-positive) signals for specific areas where the screening (collection of the signals) is performed. This can be achieved by calculating the relative level of malaria- or hemozoin-positive subject normalized by the total number of screened subject per specific area (for example, a village, a border checkpoint, a clinic, or other specific geographical location). Such an approach would be more efficient for a settled “stationary” population. The mapping of dynamic “transit” population (such as at a border checkpoint or an airport) would require additional information on the geographic origin of human subject. In either case, the absolute number of positive detections can be considered also after being normalized by the total number of subjects screened from the area in question.

FIGS.33A and33Billustrate examples of tracking malaria and/or its transmission. As shown inFIG.33A, at step3302, a controller of a malaria detection system can detect a GPS location of the malaria probe. The controller can apply laser pulses to a human subject via the probe at step3304. At decision step3306, the controller can analyze acoustic signals received from the probe to determine whether the subject is malaria positive. If malaria parasites or HZ (hemozoin) crystals are detected in the subject, at step3308, the controller can output an indication of malaria detection. If malaria parasites or HZ (hemozoin) crystals are not detected in the subject, at step3310, the controller can output an indication of no malaria detection. At step3312, the controller can upload the outcome of the malaria detection procedure and the location of the probe to a server, such as a cloud server. In some embodiments, the GPS may only be activated upon having determined that the subject is malaria positive by the sensor, and/or for a subject that is local (as opposed to being transient) the location to be tracked by the GPS.

The mapping of the malaria transmission may additionally involve the analysis of local demographics so the signals are analyzed not just as a function of the coordinates of the screened area but also of the demographic, climate and medical parameters of the screened area (age, gender, ethnicity, income, presence of other diseases, time of the year, stage of malaria transmission season, etc.) and other factors which are related to the transmission of malaria. Such analysis can be performed locally or remotely by uploading the signals and other area-specific data to the remote server. Such analysis would result in an epidemiological “maps” of malaria transmission which may be very useful for the treatment, elimination and prevention of malaria. As shown inFIG.33B, the server can receive GPS locations from a plurality of malaria probes at step3320. At step3322, the server can receive malaria detection information of one or more human subjects at the plurality of locations from the probes. At step3324, the server can also optionally receive demographic data at the plurality of locations (for example, via government databases). At step3326, a processor at the server can plot a malaria transmission data at least in part based on the information received from the malaria probes.

Collection and analysis of malaria population data will now be described in more detail. It is often reported that there is insufficient information with which to deploy the available resources for controlling the transmission malaria. Many malaria-endemic countries still suffer from weak health-management information systems and often lack vital registration. At a global level, only around 10% of estimated malaria cases are detected. Many patients with suspected infections receive empiric antimicrobial therapy rather than appropriate therapy dictated by the rapid identification of the infectious agent. The result is overuse of a small inventory of effective antimicrobials, whose numbers continue to dwindle due to increasing levels of antimicrobial resistance. As shown inFIG.33C, a device and data acquisition global system can solve both of these challenges to malaria elimination in less developed nations. Due to its digital signal capture, it has the ability to change the paradigm for screening, case identification and population monitoring in both endemic and malaria-free countries. The system can establish a global “nanobubble platform” for real-time epidemiologic data capture and analysis to guide a more efficient allocation of scarce health resources, and to monitor the residual malaria in treated patients to determine therapeutic (including experimental vaccines) efficacy.

In geo-tagging of malaria, primary data can be further combined with fingerprinting device incorporated with the malaria sensors for reliable identification of all screened subjects. With a properly designed and connected global data base, such information may be used by border and passport control authorities to track the malaria carriers and thus to contain the malaria infection and prevent its spread.

The table below describes the clinical and mass-screening applications of the malaria diagnostics disclosed herein.

TechnologyNon-invasive mass screeningClinical analysis of bloodApplicationEpidemiological: Monitoring andClinical: Confirmation of the malariascreening of the malariadiagnosis (positive or negative) intransmissionclinic for patients with unclear RDTand microscopy dataMethod andNon-invasive detection of skinExpress analysis of blood sample,samplessignals for parasites andincluding venous blood (currentlyhemozoin in skinused for high-sensitivity PCR)HemozoinEstimate: 1-100.1-0.5detectionthreshold,analog of par/uLPotential UsersHealth workers, boarder andClinics, hospitals, research labs andepidemiological administration,manufacturers of malaria drugs andgovernments and NGOvaccinesDesignPortable, simple and rugged inRugged, to be used by microscope andrequirementsuse by non-medical staff,PCR (medical) users, can use externalinexpensive, internal powerpowerMarket statusNo current analogs, will solve aWill improve the clinical use of RDTproblem of monitoring malaria,and microscopy where these methodstransmission, which currently hasare inconclusive and ultra-sensitiveno solution in global scalePCR is not available

Example Methods of Malaria Detection Using Peak Time-Delay

As described above, in addition and/or alternative to monitoring the parameters derived from the amplitude of acoustic signal when one or more predetermined laser pulses are applied to a measurement site, the systems described herein can independently detect nanobubbles generated around malaria-specific nanoparticles in a tissue based at least in part on a signal peak time-delay. The signal peak time-delay can be used as an additional diagnostic metric, which is independent of the metrics derived from the signal amplitude, such as the normalized positive count, N, and the hemozoin index, HI described above. The hemozoin index can represent a relative signal amplitude above an amplitude threshold. The normalized positive count can represent relative number of signals above the amplitude threshold. The peak timing metric can be used together with the signal amplitude metrics (N and HI) to increase the diagnostic sensitivity and specificity of the clinical diagnostics and/or mass screening.

FIGS.15A and15Billustrate schematically acoustic pulses from healthy tissues and from malaria-infected (or malaria-positive) tissues when one or more predetermined laser pulses are applied to the tissues. As shown inFIG.15A, heat generated by the bulk residual optical absorption of the laser pulse by components of healthy tissue (proteins, melanin, blood components) can produce a thermos-elastic stress in the patient's skin regardless of whether the patient is infected with malaria. This thermos-elastic stress results in a background acoustic signal. In malaria-negative (healthy) tissues, the acoustic pulses are the strongest at the skin surface. This is because the source of the acoustic pulses is the skin that absorbs the heat from the laser pulse(s), with the maximal optical fluence being at the skin surface. The deeper skin attenuates the light exponentially with the depth due to optical scattering and absorption. Due to the light attenuation in malaria-negative tissues, the thermo-elastic background signal decreases with the depth from the skin surface.

When the tissue is infected with malaria, the source of the acoustic pulses also includes nanobubbles generated around malaria-specific nanoparticles. The acoustic signal from nanobubbles can be different from the background acoustic signal from the skin. The acoustic signals from nanobubbles can be delayed by the time determined by the depth of nanobubble and the speed of sound.

Tissues with subcutaneous malaria-specific nanoparticles result in the maximal nanobubble-emitted pressure pulse, which is at some depth from the skin surface. The distance traveled by the pressure pulse to the sensor and from subcutaneous parasites, including the malaria-specific nanoparticles, can be higher (such as slightly higher) than the distance a thermos-elastic wave travels from the skin surface. The distance traveled by the pressure pulse from nanobubbles can be determined by the depth of the malaria-specific nanoparticles, such as HZ (hemozoin) nanocrystals. The malaria-specific nanoparticles can be located at the depth from about 10 μm to about 400 μm, or to about 500 μm below the skin surface. About 500 μm below the skin surface is not the location limit of the malaria-specific nanoparticles, but can be the maximal depth of the optical penetration of the laser pulse which still can generate a nanobubble around malaria-specific nanoparticles. Due to the additional travel distance, the acoustic pulses from the nanobubble generation also arrive at the sensor probe with a time delay compared to the background acoustic pulses from the skin surface. The time-delay due to the additional travel by the acoustic pulses can be determined by the speed of sound in tissue and the depth of HZ (HEMOZOIN) location. The time-delay can be about from 40 ns to about 300 ns, or to about 333 ns (assuming that sound travels at 1600-1800 m/s in the tissues).

When comparing the acoustic signals from malaria-negative (healthy) tissues and malaria-positive tissues, such as shown inFIG.16, there can be a time-shift1606to the right of the highest signal peak1602in the malaria-positive signal1601(solid line) compared to the highest signal peak1604in the malaria-negative signal1603(dashed line). The signal peaks1602,1604can have a negative value due to the negative charges formed upon the acoustic pulse reaching the piezo element.

The depth of the malaria-specific nanoparticle can be calculated by multiplying the speed of sound in the tissue and the time delay. As shown inFIG.16, the malaria-positive signal has a time-delay1606of about 120 ns. The malaria-specific nanoparticle can thus be located about 0.3 mm deeper than the skin surface (in the illustrated example).

The time-delay in the malaria-positive signal and the background signal can be detected with an ultrasound detector with sufficient temporal resolution, such as the acoustic detector described herein. The ultrasonic detector can have a frequency of at least about 4 MHZ (hemozoin) or more to detect a signal peak time from the malaria-positive and malaria-negative signals.

A peak timing-delay diagnostic threshold can be used as an additional and/or independent malaria diagnostic criterion. Signals with the peak timing below the diagnostic threshold can be assumed to indicate a malaria-negative status, and signals with the peak timing above the diagnostic threshold can be assumed to indicate a malaria-positive status. The diagnostic threshold can be predetermined, for example, using empirical data. As will be described below, the threshold can be determined based on the peak timing-derived time histograms.

FIG.15Cillustrates an example process for detecting malaria based at least in part on the peak time-derived diagnostic threshold. At step1502, one or more signal processors of a malaria diagnosis system can instruct a user, such as a clinician, to apply the malaria probe, such as any probe disclosed herein, to a target location. The target location can be any of the test subject described above, including but not limited to the ankle, base of the tongue, hand, described herein. The step1502can include applying a layer of gel to a probe tip surface before applying the probe to the target location. The layer of gel can improve acoustic and/or optical coupling between the malaria probe and the patient's skin. At step1504, the one or more signal processors can set an energy level of a laser pulse generator to a first predetermined level, E1. E1 can be sufficient for generating nanobubbles around malaria-specific nanoparticles up to about 0.5 mm underneath a surface of the target location. At step1506, the one or more signal processors can cause the laser pulse generator to apply one or more laser pulses having an energy level of E1 to the target location. As described above, the one or more laser pulses can have the same or different energy levels and/or wavelengths.

At step1508, the one or more signal processors can receive an acoustic signal from the acoustic detector of the probe. The signal can be indicative of the background acoustic pulses generated by the skin, and/or the acoustic pulses from the generation of nanobubbles (if malaria-specific nanoparticles are present at the target location). At step1510, the one or more signal processors can determine a peak time of the signal. At decision block1512, the one or more signal processors can determine whether the peak time of the signal exceeds a predetermined diagnostic threshold. The diagnostic threshold can vary depending on the type of sensor, the type of target location or measurement site, the depth of the malaria-specific nanoparticles, and/or the species of the malaria parasite.

If the peak time does not exceed the diagnostic threshold, the one or more signal processors can output a message that no malaria is detected at step1514. If the peak time exceeds the diagnostic threshold, the one or more signal processors can output a message that malaria is detected. At decision block1512, the one or more signal processors can also combine the determination based on the peak time with the signal amplitude parameters, such as the N and HI values. For example, if the signal peak time does not exceed the diagnostic threshold, but the signal amplitude parameters are highly indicative of a malaria-positive status, the one or more signal processors can output a malaria-positive status message. At the decision block1512, the one or more signal processors can also compare the signal amplitude parameters with a signal amplitude parameter threshold instead of comparing the peak time of the signal with the peak time threshold.

At step1518, the one or more signal processors can also optionally determine the depth of the malaria-specific nanoparticle based at least in part on the peak time of the malaria-positive signal.

FIG.17Aillustrates a peak timing-derived time histogram of an example application of the time-delay analysis on 22 malaria-positive subjects and 20 malaria-negative subjects in non-invasive human study with the blinded data collection. The study was targeted at the malaria parasiteP. Falciparum. Each subjects received 3 sets of 60 signals on the ankle. The diagnostic threshold1702is set to be 0.925 s. Most of the observed peak time-locations for the malaria-negative signals are below 0.925 s. Most of observed peak time-locations for the malaria-positive signals are above 0.925 s.

As shown inFIG.17A, the shape of the peak timing-derived time histogram formalaria-negative signals matches the exponential nature of the signal attenuation with the tissue depth. In contrast, the peak timing-derived time histogram for malaria-positive signals shows an increase of the signal peak with the tissue depth, up to the penetration range of the laser beam.

Table 1 below illustrates an example two-sample t-test performed on the results of the human study of the application illustrated inFIG.17A. This t-test finds two sub-populations, malaria-negative and positive (as independently was determined with the standard microscopy procedure) being significantly different

As shown in the table, there can be a 110/130 ns average/median delay in the timing of peaks of malaria-positive signals compared to malaria-negative signals. The mean time-delays can be statistically significant, regardless of whether equal variance is assumed.

FIG.17Billustrates a peak time histogram for subjects suspected of infection by the malaria parasiteP. Vivax. The malaria probe of another design was applied to the ankle than the malaria probe used in the test illustrated inFIG.17A. The diagnostic threshold can be about 0.46 s for the parasiteVivax. Accordingly, the peak time-delay diagnostic threshold can vary based on the malaria parasite species and the sensor design.

The human subjects were also tested for malaria using standard microscopy and PCR tests of peripheral blood samples. As shown inFIGS.17A and17B, the diagnostic thresholds1702correspond substantially with the determination using microscopy and PCR test, with a sensitivity of 91% forP. Falciparumand a sensitivity of 95% forP. Vivax. For both malaria species, the difference in average time-delays (about 70 ns to about 120 ns) corresponds to about a 200 μm depth of the malaria parasite and/or the malaria-specific nanoparticles from the skin surface.

FIGS.17A and17Billustrate clinical applications of the malaria detection sensor probes disclosed herein. In a clinical application, the subjects visit a healthcare facility, for example, to seek medical assistance.FIG.17Cillustrates using the malaria-detection sensors disclosed herein for mass screening (which usually assumes an asymptomatic infection). Mass screening can be performed by healthcare professionals taking the malaria detection sensors to a group of people, for example, all residents of a remote village, and using the sensors on the entire group of people. Mass screening using the malaria detection sensors disclosed herein can allow detection and/or narrowing down of geographical regions where access to healthcare facilities and/or personnel is difficult or infeasible, malaria is endemic, and/or where malaria originated, as described above. Current malaria detection methods, such as described in the Background section of the present disclosure, may not be suitable for mass screening. Compared to the nanobubble-based malaria detection methods disclosed herein, those methods are more time-consuming and/or demanding on equipment that may not be designed to function in a rugged environment. Current methods rely on the presence of malaria in a peripheral blood, but parasites can often escape to tissues though the mechanism known as a sequestration.

FIG.17Cillustrates a peak timing-derived time histogram for a mass screening study on 145 subjects with the ankle as the measurement site. The diagnostic threshold1702was pre-set to be 0.925 s. The subjects were also tested for malaria using microscopy and PCR tests of the subjects' peripheral blood samples. Compared to the determinations using microscopy and PCR tests of peripheral blood samples, the peak time-based test had a sensitivity of about 86%.

As also shown inFIG.17C, results of some malaria-negative subjects as determined from peripheral blood tests are above the peak time diagnostic threshold. This can be due to the tissue-sequestered hemozoin described above, leaving the peripheral blood of those subjects free of hemozoin. As discussed above, the nanobubble based malaria detection technology as disclosed herein can detect tissue-sequestered malaria infection when microscopy and PCR tests of peripheral blood samples indicate that the subject is malaria-negative.

In another example study of nanobubble based malaria detection, different types of sensor probes were used on the patient's ankle, back of hand, blood, and urine samples respectively and different diagnostic parameters were used. Data was collected from clinical studies using the malaria sensor described herein in various stages.

In addition to peak time as the diagnostic parameter, the signal amplitude-derived diagnostic parameters were used for some of the tests on the ankle. The subjects were tested for malaria using a signal amplitude-based diagnostic parameter (N-HI) for the rest of the tests.FIGS.18A-18Dillustrate the N-HI plots and the amplitude parameter-based diagnostic thresholds.FIG.18Aillustrates an N-HI diagnostic threshold1802when the test is performed on the ankle.FIG.18Billustrates an N-HI diagnostic threshold1804when the test is performed on the back of the hand.FIG.18Cillustrates an N-HI diagnostic threshold1806when the test is performed on the blood sample.FIG.18Dillustrates an N-HI diagnostic threshold1808when the test is performed on the urine sample.

The subjects were also tested for malaria using current diagnostic tools such as the RDT, PCR and microscopy. InFIGS.18A-18D, the malaria-positive subjects as determined by RDT, PCR and microscopy are shown as red (or darker) dots1810. The malaria-negative subjects as determined by RDT and microscopy are shown as grey (or lighter) dots1812. In case the RDT, PCR and microscopy data did not match, up to three microscopy reading were done independently and the microscopy data determined the malaria status.

Table 2 below summarizes the sensitivity and accuracy (as defined above) of the various tests in the example study.

TABLE 2AnkleBack ofBloodEnrollmentLocationTypeTypeTypeHandTypeUrine(malaria/Sensor112Type 13Type 3healthy)DiagnosticPeakN-HIN-HIN-HIN-HIN-HIparameterstimeStage 1B: Blinded (all settings and thresholds24 (12/12)were pre-set and fixed)Sensitivity0.861.01.00.861.00.71Accuracy0.881.00.880.920.790.75Stage 1: Total55 (30/25)Sensitivity0.91*1.00.940.870.970.68Accuracy0.91*0.960.870.850.760.84*based on the total count of 43 (22/21) because peak time was not collected for all subjects

Additional Example Human Study Data

Using the sensor probe such as shown inFIGS.3A-3D, a skin volume of a patient was probed noninvasively. The skin volume probed had a diameter of approximately 100 um and a depth of 400-500 um.3tests were performed at 3 seconds each, collecting a total of 180 signals (60 pulses per test). Test locations included the tongue base, inner lip, ear lobe, wrist, hand, and ankle. The sensor probe was placed at several close and random areas within one location, via ultrasound gel.

FIGS.19A-19Dillustrate two groups of 60 signals non-invasively collected from healthy (19A-19B) and positive (19C-19D) human subjects at the ankle. The healthy subjects did not have malaria within the past 1-3 years, were not from areas with high transmission, and were tested negative with microscopy. The negative subjects were from areas with high transmission, tested negative with microscopy, and often with confirmed history of malaria. Positive subjects were from areas with high transmission and tested positive with the standard malaria microscopy test.

FIG.19Eillustrates the diagnostic procedure performed. At step1902, the malaria probe can be gently pressed to the skin. At step1904, a plurality of, or N, laser pulses (for example, 60) can be applied while the user holds the malaria probe still for about 3 seconds. Signals were collected and inputted into a processor. At step1906, the processor can measure the peak-to-peak amplitude A for each signal. At step1908, the processor can obtain an A log for the N signals. At step1910, the processor can calculate the diagnostic metrics disclosed herein and save the metrics, for example, to a memory device. At step1912, the procedure can be repeated2more times in the same test location near the previous test site. The processor can calculate and save an average of the diagnostic metrics.

In addition to the skin tests, blood and urine tests were performed.3flow tests were performed with 1000 signals each. Capillary (peripheral) blood was taken with a finger prick. Reference methods, microscopy using the peripheral blood, RDT, and PCR, were also performed using peripheral (blood) samples.

The signal analysis can use the following equations for calculating the signal amplitude metrics in the liquid (urine and blood) sample and non-invasive skin tests. The normalized positive count Nnormcan be calculated as

where Nposis the number of signals above a threshold, and Ntotalis the actual number of laser pulses. The normalized amplitude above the amplitude threshold, HI, can be calculated as

where T is the threshold signal amplitude and A is the peak-to-peak amplitude for the signals equal to or above T.

This diagnostic procedure was found to be safe and without any detectable damage to human skin.FIGS.19F and19Gare images of human skin before and after the application of diagnostic laser pulses. As shown, no harm and no noticeable changes were can be observed on laser-probed skin, which was exposed to 180 pulses at 15 uJ per pulse. No discomfort or delayed complaints were observed in in more than 400 people that received the diagnostic procedure.

Table 3 below summarizes the sensitivity and accuracy (as defined above) of the various test in the example study. As shown, the worst signals (lowest amplitude, minimal separation of negative and positive components) were observed in the studies for an inner lip and a tongue base.

TABLE 3TongueLocationAnkleHandWristLipBaseSensitivity1.00.870.830.800.87Specificity0.920.840.840.870.73Separation of positive andHighHighMediumLowLownegative metrics HI and N

Data was collected in two studies from subjects in Sumatra and The Gambia. In Sumatra, the collected data was related primarily to the malaria speciesP. Vivax. In The Gambia, the collected data was related primarily to the malaria speciesP. Falciparum. Each study included two stages: a clinical application stage (Stage 1) and a mass screening stage (Stage 2). The clinical stage was further divided into Stage 1A and 1B. Additional details of the studies are provided in Table 4 below.

As determined in the validation stage (Stage 1B, blinded) and shown in Table 5 below, when the malaria status of the blood and skin correlate, such as when malaria parasite and/or hemozoin are present in both the blood and in the skin, the sensitivity and specificity of the method of malaria detection using the sensors disclosed herein can be high. Throughout this disclosure, “healthy” and “negative” both denote subjects without malaria infection.

For both types of malaria, in the above shown two independent blinded studies, a good separation of data for positive and healthy subjects for suspected clinical cases were found. As shown inFIG.20C, the skin tone did not influence malaria metrics. A skin tone was measured with the skin tone device in Sumatra (medium tone skin). In The Gambia study ofPlasmodium Falciparummalaria, the skin tone device returned saturated signals as all skin was too dark for the skin tone device.

As determined in the mas screening stage (Stage 2) and shown in Table 6 below, when the malaria status of the skin and the blood differ, such as when there is no malaria parasite and/or hemozoin in the blood but sequestered malaria parasite and/or hemozoin in the skin, the method of malaria detection using the sensors disclosed herein cannot be benchmarked against the standard methods on peripheral blood. Another skin-based reference method is required to validate the method using the sensors disclosed herein.

In the study of both clinical and asymptomatic cases, the malaria status was also determined through the microscopy and PCR analyses of peripheral blood samples. For clinical cases that are associated with the acute disease, which in turn develops in the peripheral blood, there was a good correlation between the blood malaria status and hemozoin-generated vapor nanobubble data as found both forPlasmodium VivaxandPlasmodium Falciparumtypes of malaria parasites. As determined by comparing the data from The Gambia in the clinical application stage (Stage 1, seeFIG.21A) and the mass screening stage (Stage 2, seeFIG.21B), the signals of these two subject groups with malaria infection in a highly endemic area were similar. The situation, however, has significantly changed for asymptomatic cases: a high level of blood-negative local subjects yielded hemozoin-generated vapor nanobubble-positive signals. As indicated by the negative group-average arrows inFIGS.21A and21B, the signals of healthy subjects differed from the blood-negative local subjects in a highly endemic area. According to the studies described herein, many local blood-negative subjects were not healthy subjects, they might have unknown malaria history and resided in highly endemic area. Their difference from healthy subjects can be seen from the comparison of the hemozoin-generated vapor nanobubble data. Table 7 below lists the ratios for average Stage 2 metric values to average Stage 1 metric values in the data from The Gambia, where ankle was the testing site.

As described above, the biological mechanisms causing the difference in the group average values could be at least the sequestration of parasites and hemozoin from the peripheral blood into subcutaneous microvasculature. As shown in Table 8 below, it can be typical for patients having late-stage malaria parasite infections, which would have produced a high level of hemozoin, to have more hemozoin accumulated in the skin than in the blood, or even to have malaria-free peripheral blood but still have parasites and/or hemozoin in their skin. In skin, the sequestered parasites and/or hemozoin can persist for months, whereas the parasites and/or hemozoin can clear from the peripheral blood in days. Sequestered parasites and/or hemozoin can be responsible for lethal complications in the clinic, such as uncontrollable transmission and relapses. Sequestered parasites and/or hemozoin can represent a hidden source of malaria injections. As described above, no standard method based on blood testing can detect the tissue-sequestered malaria parasites and/or hemozoin. Skin can also be a better source for malaria screening than standard methods using peripheral blood.

TABLE 8Latent,sub-potent orasymptomaticDisease stage (duration)Acute (days)(months to years)Parasites in bloodYesNo*Parasites in skin (sequestered is sub-YesYescutaneous microvasculature)Transmission capacity (mosquitoesYesYesbite skin)Detectability with laser nanobubbleYesYestechnology (in skin)Detectability with RDT, microscopyYesNo**and PCR (in peripheral blood)ApplicationClinicalMassdiagnosticsscreening*residual parasites may present in blood in a very low density**a high sensitivity PCR (<1 par/uL) may potentially detect residual parasites in blood when most of them are sequestered in tissue microvasculature

The differences observed above indicate that many local subjects from endemic area with malaria-negative blood still had skin which delivered hemozoin-generated vapor nanobubble-positive signals and hence potentially had parasites/hemozoin not found in their peripheral blood. The current malaria science suggests that there is no correlation between the malaria status of the peripheral blood and malaria transmission (which is related to the skin level of parasites, especially, gametocytes). Therefore, the asymptomatic data may support the hypothesis about high level of sequestered parasites and hemozoin in skin. Such cases may not represent clinical concerns, but they may represent transmission concerns even though such human subjects are not detected as malaria-positive with current standard malaria tests. That standard PCR analysis of most of such hemozoin-generated vapor nanobubble-positive subjects came out as negative. The non-invasive skin data suggest a possibility to use the hemozoin-generated vapor nanobubble method for the detection (and screening) of malaria transmission status of human subjects through a rapid and non-invasive hemozoin-generated vapor nanobubble test. The clinical studies suggest that a skin may be a better source for malaria detection and screening compared to the current standard, a peripheral blood, for the screening of malaria transmission. As shown inFIGS.22A and22B, an in-house in vitro blood model study (such as described above with reference toFIGS.9A-9C) onP. Falciparumshowed a parasite density detection threshold of at least 0.1 parasite/μL. The reproducibility of the test was approximately 10%.

Comparison of Nanobubble Signals with Bulk Thermal Response Signals

As described above, examples of the malaria sensor disclosed herein can deliver laser pulses into the skin of a test subject (such as a patient) through an optical fiber. In some embodiments, the optical fiber can be a multi-mode optical fiber. The sensor can collect a spherical pressure pulse from a subcutaneous source, which can be the generation of a nanobubble around a malaria-specific nanoparticle, such as a hemozoin nanoparticle. The sensor can also amplify the electrical signal indicative of the pressure pulse to a level that can be analyzed.

The nanobubble-based malaria detection mechanism is different than the photoacoustic mechanism described above with reference toFIGS.15A and15B. While the nanobubble-based mechanism can produce a pressure pulse due to expansion and collapse of as few as a single transient vapor nanobubble, the photoacoustic mechanism produces weaker pressure pulses due to the thermos-elastic effect of tissue and/or blood in a bulk volume. The pressure pulse from the photoacoustic mechanism has lower amplitude than the nanobubble-generated pressure pulse. Although the photoacoustic mechanism may produce different thermal response signals for malaria-infected red blood cells and healthy red blood cells, the diagnosis of a malaria-positive status based on the differences in thermal response signals requires the presence of a larger number (at least more than one) of hemozoin nanocrystals. Therefore, the nanobubble-based mechanism can be better at reporting a malaria-positive status than the photoacoustic mechanism, although the latter may still be useful for reporting malaria-negative status.

Signals from experimental models and human subjects described below show that the transient vapor nanobubbles were directly detected in the skin during a non-invasive test and that the nanobubble signals correlate to the malaria-positive status.

In an experiment using gold nanofim as a source of vapor nanobubbles as a reference model, such as shown inFIGS.23A-23C, a gold nano-film of about 50 nm thick was deposited on a microscopic slide glass. The gold film model was used with three acoustic media on top of the gold film: water (speed of sound in water 1480 m/s), ultrasound gel, and dark skin with an ultrasound gel on top of the dark skin. The sample has been scanned to expose an intact gold surface to each next laser pulse. As shown inFIG.23C, the malaria probe can be the probe shown inFIGS.3A-3D. The setup as shown inFIGS.23A-23C, coupled with the high stability and precision of the laser pulse delivery as disclosed herein, can ensure repeatability of photothermal effects in the uniform layer of gold film.

From the bottom of the glass slide, a single short 532 nm laser pulse was focused onto the gold film through the glass. This resulted in the generation of semi-spherical transient vapor nanobubbles in water and near the gold/glass surface. The maximal size (hence the lifetime) of a nanobubble was controlled through the energy of the laser pulse (through the amplification settings in a laser remote control). In this model, a vapor nanobubble acts as a point source which generates a pressure pulse with spherical wave front.

A calibrated reference hydrophone HNC 1000 or HNC 0400 (which may be the default choice) may be mounted, for example, at a slight angle above the gold surface. The hydrophone has a round element with the diameter 0.4 mm and the spectrum shown inFIG.23D(which was obtained from third-party data). Its electric output was connected to a 20 db voltage gain 25 MHZ (hemozoin) band pre-amplifier, which was in turn connected to a 50 Ohm input of digital oscilloscope (which may be LeCroy X42 or a part of the malaria system disclosed herein). The height of the hydrophone was adjusted so the signal arrived in water in 0.3 us (1480 m/s×0.3 us=0.45 mm).

FIGS.23E and23Fillustrate a returned signal in the gold-water model of vapor nanobubbles. Each division of the x-axis is 0.5 us. Each division of the y-axis is 2 mV. InFIG.23E, the nanobubble has a lifetime of about 1.6 us and an energy level of about 23 mV. A transient vapor nanobubble assumes that a nanobubble expands and collapses in elastic medium, such as water. While mechanisms of the expansion and collapse are hydrodynamically similar, these stages are driven by different sources. An expansion stage is driven by the optically absorbed energy which, converted into a potential energy of a vapor, determines the maximal diameter of a nanobubble and the amplitude of a first spike in its signal, such as shown inFIGS.23E-23F. A collapse stage is driven by a nanobubble environment, such as the pressure of a surface tension of the surrounding medium. This process results in accumulation of some energy which is released as a second pressure pulse (second spike) as shown inFIGS.23E-23F. As shown inFIG.23F, the smallest nanobubbles detected with this set-up revealed a lifetime of 0.35 us. A signal to background ratio can be estimated using the background peak-to-peak amplitude of 0.3 mV. The signal to background ratio was around 5 for the smallest nanobubbles detected.

As shown inFIG.23G, the dependence of the positive component of the spike amplitude upon the nanobubble lifetime (measured as the time interval between the first and second spikes) showed (1) quasi-linear performance, in line with the theory for elastic medium, (2) close similarity of the amplitude of the second and first spikes, in line with the minimal losses of the nanobubble energy in the medium (the collapse signal was close to the expansion signal), and (3) lack of negative components in first and second spikes, in line with the theory for the medium with low elastic modulus and without generation of tensile stress. In addition, moving the hydrophone away from the source to 2 mm distance significantly reduced the signal amplitude.

FIG.23H-Jillustrates multiple traces of the generation of transient vapor nanobubbles using a calibrated 20 MHZ (hemozoin) hydrophone with a piezo element having a diameter of about 1 mm and at a hydrophone distance of 2 mm from the source. As shown inFIG.23H, the transient vapor nanobubble typically results in a two-spike signal. The first spike2302is indicative of the nanobubble expansion and the second spike2304is indicative of the collapse of the nanobubble.FIG.23Iillustrates a relationship between the pulse energy and the first peak amplitude.FIG.23Jillustrates a relationship between the first peak amplitude and the time between the first and second spikes. The spike-to-spike time dt correlates to the laser pulse energy, which can be variable in some embodiments, and therefore, to the maximal size of the nanobubble. The results were similar to those shown inFIGS.23D and23E. However, the two-spike nanobubble signal may not be identifiable against the background of the thermal response bulk signals and/or the baseline signal, which may include distortions due to the internal functions of the malaria sensor. The 1 mm hydrophone was thus less sensitive in the detection of small nanobubble, probably due to the distance-induced attenuation of the signal as the pressure from the source in this model decreases inversely proportionally to the square of the distance to the hydrophone.

A ratio of the size of the piezo element in the hydrophone to the hydrophone-to-target distance can characterize a detection regime of the hydrophone as a far field (<1) and near field (≥1). In the far field, the sensor detects almost a flat wave so the detection conditions (angle of incidence and phase of the pressure pulse front) remain substantially equal across the whole surface of the sensor. In the near field, the sensor detects a spherical wave and the detection conditions significantly vary across the sensor surface. Further, any additional lateral shift of the sensor off the center axis (a factor that is unavoidable when the optical fiber is placed in the center and the piezo element has some lateral shift by definition) adds to the effective size of the sensor geometry. In the far field inFIG.23H, the nanobubble signal correctly shows the pressure pulse shape and the timing: dual—spike signal as the nanobubble signature, and the time-delay between the two spikes quantifies the maximal diameter of the nanobubble. The spike half-width is about 40 ns (close to the speed limit of the sensor used). This narrow spike shows a typical pressure pulse, not a wave, both at the expansion and collapse stages of the nanobubble lifespan in water.

FIG.23Killustrates a near field pattern with a hydrophone of 200 um in height and 1 mm in the detection regime. The signal inFIG.23Killustrates broadening of both the first and second spikes. The expected increase in the first spike relative to the second spike amplitude is not shown (possibly due to dephasing) compared to a hydrophone of a 1.5 mm detection regime. Decreasing the sensor-to-target distance does not increase the signal amplitude because the angle of incidence increases with a relatively narrow pointing diagram for the piezo element. This reduces the acoustic sensitivity. The echo signals, caused by reflection of pressure pulse from the sensor surface and back from the gold surface (the sensor-to-target distance×2) shift to the main spikes as the sensor-to-target distance decreases.

In the near field, the nanobubble signal becomes distorted due to several reasons: (1) the angle of acoustic incidence becomes too high, which reduces the sensor sensitivity and the signal does not increase despite shorter distance to the target; (2) the piezo element becomes too large in size, causing significant dephasing (mismatch) in the pressure-to-charge conversion, which broadens the signal spike and further reduces its amplitude. The lateral shift of the piezo element relative to the pulse source (a feature generally unavoidable for the malaria sensor with the optical fiber in the center, as described above) causes further broadening of the signal spikes and the decrease in the amplitude of the signal spike. Those effects can be explained by the increasing angle of acoustic incidence and the resulting dephasing of the piezo-effect in a piezo element, especially if its size is relatively large compared to the sensor-to-target distance. Further, significant lateral shift reduces the difference between the nanobubble-specific and bulk background signals (which will be described in greater detail below), making it harder to differentiate those two signals.

FIG.24Aillustrates an experimental model using a hydrophone2430placed in close proximity to an optical fiber2420. As shown by the dash-dotted line inFIG.24A, the hydrophone2430can be oriented to face a tip of the optical fiber2420. The tip of the optical fiber2420can be directed at a hemozoin nanoparticle. The optical fiber2420can deliver pulsed laser energy into the air at about 16 J at a wavelength of about 671 nm, which is suitable for generating a vapor nanobubble around a hemozoin nanoparticle located no more than about 0.5 mm below the skin surface of a human subject. As described above, the ratio of the size of the piezo element in the hydrophone to the piezo-to-target distance can characterize a detection regime of the hydrophone as a far field (<1) and near field (>1). In the illustrated model ofFIG.24A, the distance between the hydrophone tip, where the piezo element is located, and the tip of the optical fiber can be about 2 mm. The ratio of the size of the piezo element to the piezo-to-target distance is about 0.5, which characterizes the hydrophone as having a far field detection regime.

InFIG.24A, the tips of the optical fiber2420and the hydrophone2430can also be submersed in a liquid. As shown inFIG.24A, the liquid can be an absorbing liquid2410having a brown color. The absorbing liquid can be a solution having iodine, potassium iodine, ethanol, and/or water. The absorbing liquid can have an absorption coefficient of about 1.9 cm−1. The absorbing liquid can simulate skin pigmentation in a human subject.

Three testing models can be constructed using the optical fiber-hydrophone arrangement illustrated inFIG.24Aand/or an example malaria sensor disclosed herein. The optical fiber-hydrophone arrangement and/or the malaria sensor can be applied to (1) a hemozoin nanoparticle without the absorbing liquid to detect a pulse due to generation of a vapor nanobubble only (“nanobubble model”); (2) the absorbing liquid without a hemozoin nanoparticle to detect a pulse due to photoacoustic mechanism only (“bulk model”); and (3) a combination of the hemozoin nanoparticle and the absorbing liquid to detect pulses due to both the vapor nanobubble generation and the photoacoustic mechanism (nanobubble and bulk model”).

FIGS.24B and24Cillustrate example nanobubble signals received by the hydrophone in the nanobubble model. Similar to the signal inFIG.23J, the nanobubble signals have two spikes corresponding to the expansion2502and collapse2504of the nanobubble. However, as described below, the two-spike nanobubble signal may be not identifiable at the background of a bulk signal due to the thermal response of the tissue of the human subject.

For the water model of vapor nanobubbles, the transient vapor nanobubbles in water can produce a two-spike signal with the corrected spike amplitude in 2-3 mV range (with a 10-fold amplification of the hydrophone) for a nanobubble of 1 microsecond lifetime, which corresponds to the maximal diameter of 10-20 um approximately. The nanobubble expansion and collapse are nearly symmetrical, the second spike generated by the collapse can be close in amplitude to the first spike caused by expansion and such nanobubbles create no tensile stress in water.

A nanobubble in skin can be modeled with a human skin sample surrounding the nanobubble generated by the gold-water model described above. The test sample included a gold nano-film deposited on the microscope slide glass and the human skin sample on top of the gold film, in physiological solution or in water. The skin sample was dark human skin, 250 um thick, and including epidermis and dermis. A drop of water was deposited on the sample to couple the sample to the hydrophone. In this model, a nanobubble formed by optical excitation pulse as described above with reference toFIGS.23A-Gexpanded and collapsed in the skin, specifically, the dermis, its pressure pulse propagated through the skin towards a hydrophone at a speed of sound of 1620 m/s. The acoustic detection using a hydrophone was also as described above, except that there was a need for a ×0.5 correction of the spike amplitude to compensate for a dual pressure pulse.

After a sample of dark human skin of 250 um thickness was placed on top of the glass, the energy deposition and vaporization processes have not changed because the laser-target interaction and target properties have not changed. However, as shown inFIG.25A, under identical optical energies as shown inFIG.23E, the signals have changed dramatically. The nanobubble lifetime has decreased. The first spike amplitude decreased slightly. The second spike amplitude, shape and time position revealed a significant decrease which has varied in a broad range. The amplitude of the second spike amplitude and lifetime have decreased, and the spike width has increased.

FIG.25Billustrates a signal from a quantitative study of the skin vs. water, which included the analysis of the first spike shape, amplitude and life, and the second spike amplitude and shape. Tensile stress was observed as the negative component in the spikes in signals with significant damping of the second spike caused by the collapse of the nanobubble, such as shown inFIG.25A. This tensile (negative) component was much smaller in signals with a good second spike, that is, without significant damping, such as shown inFIG.25B. The skin has a much higher elastic modulus (much stiffer) than water and can develop a tensile stress during the propagation of the pressure pulse. A tensile stress may be one of the main causes of the structural damage for a skin at microscale.

FIG.25Cillustrates the dependence of the positive amplitude of the first spike (without correction) upon the nanobubble lifetime. Numbers in legend indicate a laser pulse energy. As shown, a much higher spread of signals can be observed after skin was added to the model.FIGS.25D-25Eillustrate histograms of a nanobubble life for water and skin under identical conditions.

The nanobubble energy is dissipated during the expansion stage in the skin model. As shown inFIGS.25C-25E, the amplitude of the first spike and the lifetime have decreased in the skin sample compared to those in water. Such energy loss was not observed for water. This energy loss can be caused by the plastic deformation in skin (vs elastic in water), formation of cracks, and viscous losses, all of which can be caused by the mechanical response of the skin to the nanobubble expansion. This energy loss has influenced the first spike, and caused the deviation of the nanobubble signal parameters from those in water.

The skin also has significantly influenced the collapse of a nanobubble (the second spike), sometimes up to its full damping sometimes as shown inFIG.25F. The disappearance of the second spike may mean the nanobubble did not collapse normally. Such severe damping of the collapse may be caused by plastic (vs elastic in water) deformation of skin during the nanobubble expansion so quasi-permanent air voids and cracks emerged in skin where a nanobubble was generated.

FIG.25Gillustrates dependence of the second spike to first strike amplitude ratio on the lifetime of a nanobubble generated in the gold-water and gold-water-skin models. The quantitative analysis of the compromised collapse of nanobubbles in skin can be seen for three laser energy levels: the second spike to first spike amplitude ratio, A2/A1, for water did not depend upon the nanobubble lifetime and can be highly reproducible in the range 85-100%. In skin, the ratio has dropped significantly and was very unstable (due to variation of skin properties from location to location since new location was used for each next signal). In addition, the detection of the second spike could be compromised due to its broadening. This effect was caused by heterogeneities in the skin, such as the cracks that could have been induced by the expanding nanobubble.

Table 9 below includes statistics for signals in skin and water under identical laser excitation and acoustic detection.

It is possible to expect irreversible local structural changes in the exposed skin after each nanobubble. A small air bubble may remain in skin for up to 30 seconds. Such changes may cause (1) delivery of new parasites into the exposed volume after the local implosion of the skin when the void created by a nanobubble finally collapses, and (2) a change of the local optical scattering properties of the skin (so the detection of “after-bubble” may be an option detection of a parasite).

It is also possible that at a smaller scale of hemozoin-generated vapor nanobubbles (the maximal diameter 10 um or less), the plastic deformation and associated damping of the nanobubble collapse may be the minimal and thus the quality of the second spikes may improve. However, such second spikes may be located close in time to the background spike (<300 ns interval), and may have a small amplitude. Such second spikes may require more precise detection.

Accordingly, in skin (unlike water), a more reliable signal for a transient nanobubble may be the first spike associated with its expansion. The second (collapse) spike has strongly damped behavior and appears to be unstable due to the plastic deformation of skin and viscous damping. Further, the simultaneous generation of several closely located nanobubbles of different maximal size (and hence lifetime) may amplify the first spike but may broaden/diffuse the signal of the second spikes (since individual nanobubbles collapse in different time).

FIG.26Aillustrates optical excitation and acoustic detection of parasites through the skin via a schematic representation of the test model. The sample included several layers of dark (African type) skin, such as shown inFIG.26D, with the high level of melanin and gel or water as an optical and acoustic coupling media. Intact skin samples had no parasites. Parasite-positive samples had culturedPlasmodium Falciparumhuman malaria parasites (gametocytes), which were deposited onto the dermis layer and then were covered with a sample of upper skin (of epidermis and dermis, 250 um total). The deposition process and the sample preparation in general were optimized to achieve single residual parasites located between two layers of dermis and within the laser-exposed skin volume. Ultrasonic gel or water was used to prevent sample from drying during the experiment.

FIGS.26B and26Cillustrate positions of the optical fiber and a tilted hydrophone in the experimental setup for studying optical excitation and acoustic detection of parasites through the skin. Optical excitation was performed via the multi-mode optical fiber with a 105 um core diameter, 2 m length. The fiber was brought in contact with the skin surface. Laser pulse (671 nm, 220 ps pulse duration, energy 15 uJ) was applied. Three to four laser pulses were applied to each skin location. Next, the sample was scanned in order to expose a new location to the next set of identical laser pulses. In this model, the laser pulse propagated through the skin surface and through the layer of melanin and deeper into dermis where parasites were placed.

Acoustic detection was performed using a HNC 0400 (0.4 mm) reference hydrophone (FIGS.26A-C) which has been brought as close as possible to the wall of the optical fiber and to the top of skin surface. A parasite-to-hydrophone distance was approximately 0.4-0.5 mm. A sufficient acoustically similar material was placed underneath the skin sample, 2-3 mm of skin dermis and 6 mm of acoustically similar rubber, in order to prevent acoustic reflections and minimize echo signals.

As shown inFIGS.26E and26F, the intact skin sample revealed a background signal: a single high amplitude bipolar spike caused mainly by melanin. The amplitude of the background at the time >0.5 us was 1-2 mV. The presence of a negative component indicated both a tensile stress being induced by melanin and a higher stiffness (higher elastic modulus) of the skin compared to water. The positive spike had a single maximum. The amplitude of positive component of the spike was almost one order of magnitude higher than that of the first spike of a single nanobubble, such as shown above inFIG.25A. Due to the high amplitude and the negative component after the spike, the baseline after the spike had a “ripple” extending to about 600-700 ns after the peak of the spike position. A slight decay of the amplitude of the background spike was observed at the same location for the second and next laser pulses (FIGS.26G and26H; Table 10 below). As shown in FIGS.26G and26H, both the positive and negative components decrease for the 2nd laser pulse. The signal was reproducible.

As shown inFIGS.261and26J, signals of skin withPlasmodium Falciparumparasites revealed several features different from signals of intact skin: (1) the second spike at the time interval typical for a collapse of transient nanobubble (FIGS.261and26J), (2) disappearance of the second (collapse) spike upon exposure of the same skin location with next laser pulses at the same time position where the initial second spike was observed (FIG.26J) and (3) doubling of the first spike in many (but not all) locations (FIGS.26K and26L). Signals obtained from parasite skin had components typical for vapor nanobubble signals in skin such as described above. After 3-4 laser pulses to the same location, the collapse spikes disappeared and the signal became similar to that of an intact skin. At the same time, a significant decrease in the amplitude of the first spike was observed (see Table 10 below). The first spikes included both the background and a nanobubble component. The dual first spike might have been caused by a depth difference for melanin and parasites (although the location of melanin is not a plane layer at specific skin depth). With the 0.4 mm aperture of the detector located at 0.3 mm from the skin surface, and with the aperture of the source of the background signal being approximately 0.1 mm, the background spike would naturally broaden. This, in turn, would decrease the time interval for signals from the melanin layer and parasites (located 100 um deeper). Hence the time interval observed between two peaks in the dual first spike was only 25-35 ns and did not allow for monitoring the depth of parasites. The negative component of the first spike was observed and it was quite strong, thus indicating a tensile stress in a relatively stiff (high elastic modulus) medium compared to water. The sample preparation could not guarantee that each laser pulse (each new location) would include malaria parasites. As a result, sometimes signals similar to those for intact skin were observed. In general, a shape of signals with a second spike was similar to that of signals of nanobubbles in gold-skin model (FIGS.261and26Jvs.FIG.25A, Table 10 below) and hence the signals detected were attributed to vapor nanobubbles.

The following metrics were observed for the signals in the model shown inFIGS.26A-26L. The first spike has a permanent time position (begins at around 300 ns). This spike had a component caused by the expansion of vapor nanobubble and hence such component was determined by the laser fluence, size of hemozoin and other laser- and parasite-related properties. Several typical features were observed. The positive component decreased after each pulse. That is, the amplitude decreases (the decay effect). The negative component did not change, its amplitude remaining constant (the tensile stress is thus present). The positive component doubled in this model (a possible separation of the background signal and the nanobubble expansion signal).

For the second spike, the time interval (lifetime) observed was in the range of 0.8-2.8 us. The second spikes were not observed below 0.7 us, maybe due to the ripple in the baseline. This spike was caused by the collapse of a vapor nanobubble and hence was determined by the mechanical properties of the skin. The skin influence on the second spike in parasite-treated skin appeared to be very similar to that observed in the gold-skin model. Several typical features were observed. In the initial time position, the amplitude of the signal decreases to 0% (the spike disappeared) when the same location is exposed to the second or next laser pulses. Additional spikes were observed at shorter time intervals (the “move-in” effect), which was associated with the generation of smaller vapor nanobubbles as consecutive laser pulses were applied to the same skin location. To quantify parasite-specific features, eight metrics were introduced:A1: Positive amplitude of the first spike, individual signals;D1: Decay of the first spike, the level of the residual amplitude in response to the second laser pulse vs the first laser pulse, two signals; Incidence of the dual 1st spike, %: group of signals;T: Time interval for the 2nd spike, measured between the second (largest if several) and first spikes,A2: Amplitude of the largest second spike, individual signals;D2: Decay of the second spike, the level of the residual amplitude in response to the second laser pulse vs the first laser pulse, two consecutive signals;N2: Incidence rate of the second spike (threshold peak-to-peak amplitude of the baseline in intact skin), %, group of signals; Move-in effect of the second spike(s), %, group of signals.

These metrics were analyzed for intact and parasite-treated dark skin of 250 um thickness, all in response to 671 nm 15 uJ laser pulse delivered via the optical fiber with a 105 um core. Table 10 illustrates a comparison of intact and parasite-treated skin samples, dark, 250 um thick.

The detailed analysis of the first spike (background signal and the effect of the expansion of a nanobubble) is provided herein. Regardless the shape of the peak (a single or dual peak), its amplitude in response to the first laser pulse and the decay were different in intact and parasite-treated skin (FIGS.26M-260). For the first laser pulse, the positive amplitude of the first spike increased in parasite-treated skin while the residual level during the second laser pulse (decay metric) decreased. Up to 6 signals in the parasite-treated group did not show second spikes and might not generate any nanobubbles (these locations may have been parasite-free). The first spike data suggests that the pressure pulse of expanding nanobubble adds to the background pressure by increasing the amplitude of the first spike and, in slower detectors, causing a spike time-shift to the right. During the next laser pulse, nanobubbles were smaller or disappeared resulting in a significant decrease in the positive component of the amplitude of the first spike (see the decay effect shown inFIG.26M).

The detailed analysis of the second spike (the effect of a nanobubble collapse) is provided herein. InFIG.26P, the amplitude measured during the first laser pulse was plotted vs the time interval. Amplitudes for the gold model were corrected with the coefficient 0.5 to account for the detection of doubled pressure pulses due to the echo from the glass in this model. The signals were obtained with a 0.4 mm hydrophone. The deviation from the nanobubble water model might have been caused by the effect of skin on (1) the nanobubble collapse and (2) propagation of the pressure pulse through the skin. For the interval of 1.6-1.8 us, the gold model signal amplitude was 4.6 mV (corrected) while the average (over 11 signals in this interval range) was 2.6 mV (57% of the amplitude in water, see Table 10).

The main features ofFIG.26P(also, compared toFIG.25Cfor the gold-water and -skin model) include the following. Signals in skin for nanobubbles (the gold model) and parasites (skin model) show similar trend, in line with that for a nanobubble collapse in a medium with plastic deformation. The increase of an amplitude with the lifetime of a nanobubble was observed but it was less reproducible compared to the data for the water sample. Signals in skin (in both models) show similar amplitudes to signals in water model of an “ideal” nanobubble. Compared to water, signals in both skin models show poor reproducibility of the spike amplitude.

FIGS.26Q and26Rillustrate the decay of the second spike amplitude after the second laser pulse is applied to the same skin location. The signals were obtained with 0.4 mm hydrophone. The second spike disappears in response to the next laser pulse or moves closer to the first spike (at short time interval from the first spike) for the parasite-treated skin while a similar part of a signal of intact skin experiences no changes (remaining, in fact, a baseline). This effect is in line with the nature of hemozoin-generated vapor nanobubbles. The cluster of hemozoin crystal is disintegrated by the impact of the first nanobubble so the second laser pulse produces much smaller or no vapor nanobubble from the same hemozoin target, which is no longer as large and clustered as before the first laser pulse.

In summary, signals obtained from a human skin sample with residual parasites had shape and metrics similar to those of the gold-skin nanobubble model. This similarity appears to validate the signals detected in a parasite-treated skin as signals of vapor nanobubbles. The properties of two main nanobubble signal components, the first and second spikes in the human skin sample are summarized in Table 11A.

TABLE 11ANanobubble signalcomponents1stspike2ndspikeRelation to a transientExpansionCollapsenanobubbleNanobubble vs backgroundAlmost coincides in timeWell-separated in time forsignal of melanin in dark skinIts amplitude is 5-20% of thelarge nanobubbles withbackground signallifetime >0.8 us (maxdiameter about 10 um)Acoustic detectabilityPoor direct detection -Poor with the current sensor-disguised by a much strongerunstable in the skin unless abackground signalsuperior sensor with the highDirect detection requiresacoustic sensitivity is usedbetter temporal resolutionIndirect detection ispossible through a time-shiftand the amplitude decay ofone integrated peak

As shown in Table 11A, vapor nanobubbles are generated in skin aroundPlasmodium Falciparumhuman malaria parasites in the human dark skin. Plastic and viscous properties of the skin can dampen the collapse stage of the nanobubble, the effect resulting in an unstable second spike associated with the dampened collapse. The acoustic detection of parasite-generated nanobubbles disclosed herein may be more effective for relatively large nanobubbles with the lifetime above 0.8 us, which corresponds to the maximal diameter of a nanobubble about 10 um. The background signal due to optoacoustic emission by melanin can be high and create one of major challenges in the generation and detection of nanobubbles around malaria parasites in a dark skin.

Several options are available for improving the nanobubble method disclosed herein. It may be possible to modify optical excitation in the way its fluence at the melanin depth is reduced (for example, via side launch of the focused launch of a pump laser pulse as shown inFIG.26S). The focused excitation beam reduces the background and improves the parasite signal. The shift of the background source (melanin) from the detection axis (and, generally, from the angle of acceptance) of ultrasound sensor reduces the background signal. As the melanin requires a lower fluence than the malaria parasites for optical excitation, having acoustic pulses from the melanin and the parasite arriving at the acoustic detector or sensor at different angles may suppress the melanin signal.

The propagation of the excitation laser beam and its absorption in melanin-rich dark human skin was modeled with a computer program. The skin was modeled through 7 layers to describe stratum cornea, epidermis with the layer of melanin and upper layers of dermis. Optical scattering of the excitation beam by each skin layer and its optical absorption were analyzed for specific wavelength of the excitation laser pulse. The program and the model were used to compare the propagation of the excitation laser beam in water and in human dark skin (FIG.34A-34B). The side- and focused launch resulted in an axial position of the focal point in skin shifting by +50, and a diameter of the focal zone for the NB generation becoming ten times larger in skin than in water.

Next, the monte-Carlo simulation method has been applied to analyze the propagation of individual photons through the skin under direct delivery from the optical fiber and the focused deliver from the top and from the side (see Table 11B below).

As can be seen from the results of the computational modeling, the background signal amplitude-driving laser fluence at the level of melanin in skin decreases in both cases of the focused beam compared to that for the standard launch with an optical fiber. Further, the fluence of the excitation beam at the level of possible location of malaria parasites increases (compared to that for the standard launch with an optical fiber) in both cases of the focused launch. This computational model has been used to design the experimental model with dark human skin and malaria parasites in the skin, and to compare the standard launch of the excitation laser beam and the side-focused launch.

The side launch of the excitation laser pulse spatially decouples the source of the background signal (melanin in human dark skin) from the source of malaria signal by shifting the source of the background pressure pulse out of the optimal detection angle of acceptance of the acoustic detector (FIG.39A).FIG.39Bshows that the release of the energy absorbed by melanin is shifted to the left relative to the array of acoustic detectors which are tuned into the area in the focus of the excitation laser beam (the focused beam).

The goal of the delivery of the pump beam is to minimize the optical fluence at and associated thermal impact of the background skin (especially, upper level-located melanin) and to maximize the optical fluence at the depth of parasites. This is achieved by focusing the probe beam and by launching it as some angle so the background skin volume is spatially decoupled from the skin volume with hemozoin and or parasites.FIG.40illustrates an example experimental example of such spatial decoupling. The experimental prototype inFIG.40was designed to side-launch and focus the excitation laser beam. Although the melanin layer produces some photothermal and photoacoustic background signals, the source of such signal is shifted away from the axis of the detection of the hemozoin-generated vapor nanobubble signal and the amplitude of the background signal is reduced due to much lower optical fluence of the excitation laser beam at the level of the background (melanin, usually located in the upper skin layer while parasites are located 100-300 um deeper in skin).FIGS.41A-41Care images of the excitation beam as it was launched into water from the right side show the intensity profile at the different depths.

Such spatial decoupling of the target (hemozoin) and background (melanin) was validated in dark human skin (rich with melanin) and human parasites placed at the depth of 250-300 um below the skin surface. Ultrasonic signals were compared for the background and hemozoin-generated vapor nanobubble to those obtained under the regular optical delivery through the flat 105 um core optical fiber.

Compared to a standard or direct optical fiber launch in intact dark skin (FIG.26U) and parasite-infected dark skin (FIG.26W), the signals from a side focused launch in the intact dark skin (FIG.26T) and the parasite-infected dark skin (FIG.26V) showed a 3-4 fold suppression of the background signal due to the melanin in the skin and a 2-fold increase in the hemozoin-generated vapor nanobubble signal amplitude. It may be possible to improve the acoustic sensitivity and temporal resolution of acoustic detector in order to differentiate the nanobubble signals from those of melanin. It may be more desirable to detect a nanobubble or/and its pressure pulse as close to its origin as possible. It may be possible to increase the statistics of the skin locations probed. Additional detail is provided in Table 11C.

Additional improvement of the optical delivery was achieved by suppression so called “hot spots” in the pump laser beam (which cause additional false-positive signals generated by melanin). Such suppression was achieved by homogenizing the laser beam intensity inside multi-mode optical fiber by increasing its lens from 1-2 μm to 12 m (see Table 11D).

A comparison of the skin model and the field human data will be described below.FIG.27Aillustrates schematically an experimental setup for detection of malaria parasites in skin with a slow-speed detector, such as the malaria probe examples disclosed herein (for example, the probe inFIGS.3A-3D). The skin model sample can include dark skin of 250 um thickness, facing down, withPlasmodium Falciparumparasites on the top or the bottom of the skin. A 1.0 mm reference hydrophone was placed on the top of the model at 2 mm distance in order to co-register signals with the malaria probe. In the human studies, the samples can include dark skin withPlasmodium Falciparummalaria infection and medium dark skin withPlasmodium Vivaxmalaria infection. The same malaria probe as the skin model was used.

The propagation of acoustic pulse from the VNB through the skin to the acoustic detector located at the skin surface is modeled. For the focused beam of 300 um diameter at the skin surface, a propagation and detection of a single acoustic pulse emitted was estimated, for example, during the expansion of a vapor nanobubble around malaria parasite as shown inFIGS.38A and38B.

Optical excitation used the same laser pulses as shown with reference toFIGS.23A-23C. Single laser pulses of the energy 15 uJ were applied. The skin sample was scanned across the sensor surface in order to avoid double laser exposure of the same location in the sample. The conditions were similar to those in human field studies.

The malaria sensor probe can include a low-speed sensor with a relatively poorer temporal resolution and poorer acoustic damping, compared to those of the 0.4 mm hydrophone). Co-registration was achieved with the 12 MHZ (hemozoin) reference hydrophone (1.0 mm element, Onda HNC1000) on top of the sample. Due to acoustic reflections from a hard surface of the malaria probe, the reference hydrophone detected both direct signals and their echoes. The only echo-less signals were those originated from the surface of the malaria probe. For the malaria probe, the detection conditions were similar to those in human field studies.

FIG.27Billustrates example bulk signals from the hydrophone in the bulk model. The signal represented by the solid line is from the hydrophone and the signal represented by the broken line is from the example malaria sensor. The sensor signal can have a single spike2601followed by distortions or non-flat regions2605in the baseline due to internal functions of the sensor. The single spike2601is due to the thermal response of the absorbing liquid to the laser pulses. The hydrophone signal can have a single spike2603that is later than the spike2601in the sensor signal. The delay can be due to the hydrophone being further away from the tip of the optical fiber than the distance from the acoustic detector to the tip of the optical fiber in the malaria sensor. The single spike2603in the hydrophone signal is followed by a generally flat baseline.

Validation of nanobubble detection with the malaria probe (such as shown inFIG.27F) was completed by simultaneous co-registration of single nanobubbles in the optically-absorbing water model with the malaria probe and the reference 1.0 mm hydrophone. In this model, iodine was added to water to increase optical absorbance to the level to produce a background signal (FIG.27B) and, with undiluted iodine nanoparticles in solution, to generate vapor nanobubbles (FIG.27F). The expansion (first) spikes of nanobubbles2601,2701were not temporally resolved and merged with the background spike. However, the nanobubble caused a slight shift of the background signal to the right. The nanobubble collapse stage (second spikes)2704were identified through the reference signal of the hydrophone. The malaria probe signal delivered the second spikes as small bumps over a distorted baseline. The distorted, non-flat baseline coupled with the limited temporal resolution appears to be an acoustic limitation/difference of the malaria probe compared to a reference hydrophone.

FIGS.27C-27Eillustrate example bulk signals from the example malaria sensor in healthy human subjects. The bulk signals are signals due to the thermal bulk response of the human tissue to the laser pulses. The bulk signals measured in healthy human subjects are consistent with the sensor signals in the bulk model as illustrated inFIG.27B. That is, the signals inFIGS.27C-27Ealso include a non-flat tail baseline2605after the spike2601correlating to the thermal response of the human tissue to the laser pulses. As described above, the non-flat tail baseline2605can be due to the internal functions of the sensor. InFIG.27E, the bulk signal can have a peak-to-peak that is large enough to be falsely interpreted as a malaria-positive status under the N and HI (described in greater detail below) amplitude-derived malaria diagnostic metrics. Accordingly, the peak-to-peak amplitude-derived metrics may need to be cross-checked with other types of diagnostic metrics described herein, such as the peak-time delay metric described above, and the metrics based on the second spike described below.

FIG.27Fillustrates example combined nanobubble and bulk signals from the hydrophone and the sensor, respectively, in the nanobubble and bulk model. The signal represented by the solid line is from the hydrophone and the signal represented by the broken line is from the example malaria sensor. In both the hydrophone signal and the sensor signal, the second spike2704of the nanobubble signal, correlating the collapse of the nanobubble, and/or a plurality of tail spikes2708can be identified against the background of the baseline. However, the first spike correlating to the expansion of the nanobubble may not be identifiable against the bulk signal spike2701,2703. For the sensor signal, the non-flat portion of the baseline can also make it difficult to identify the first spike of the nanobubble signal.

The sensor signals in the human skin model were obtained without and after adding parasites to 250 um thick dark skin (FIGS.27G and27H). An intact dark skin returned signals with the background first spike (as shown by the arrow inFIG.27G) and non-flat baseline. A second nanobubble spike was shown and indicated by the arrow inFIG.27H. The sensor signals were co-registered with the reference hydrophone signals (FIGS.271and27J), which included echo signals (as indicated by the double arrows) whose time position matches the position of melanin at the skin depth in the range 50-100 um (FIG.27I, for intact skin). The hydrophone signal of the parasite-treated human skin returned four to five spikes (FIG.27J). The outer pair of spikes was associated with the parasites, and the “inner” pair of spikes was associated with the melanin (with time position and interval similar to those observed for intact skin inFIG.27I). A difference between the intact and parasite-treated hydrophone signals was an increase in the time interval between the outer spikes.

Statistical analysis of the malaria sensor and hydrophone signals is described below. There was an increase in the peak-to-peak amplitude of the first spike and the time-shift in the signals from the parasite-treated skin. As shown inFIGS.27K and27Lwith 20 signals, adding parasites has shifted the spike to the right (see arrow inFIG.27L) and has increased its peak-to-peak amplitude. There were frequent appearances of additional spikes in the time window of 1.6-2.6 us in the signals from the parasite-treated skin. The echo pattern in the parasite-treated skin has changed from a single spike to the dual spike. The N-HI metrics show statistical difference between intact and parasite-treated skin samples.

Statistical analysis of the time-shift and amplitude of the first spike as described with reference toFIGS.27K and27Lwas performed by using N-HI amplitude metrics in specific time windows (as in non-invasive field human studies in The Gambia) and with another program that detected the time position of spikes (see Table 12 below). Both programs revealed the difference in signals of intact skin and the same skin sample after adding parasites in residual concentration.

TABLE 12Metrics of malaria sensor signals for intact andparasite-treated dark skin samples, 3 experimentsExperiment 1(1stspikeExperiment 3Experiment datecapped)Experiment 2**(new setup)**1.Time position of the 1stspikePeak 1, us, intactNot measured0.33 std 0.030.29 std 0.01Peak 1, us, parasitesNot measured0.48* std 0.010.36* std 0.02Difference (time-Not measured0.150.07shift) us,2.N-HI metrics, 0.38-1.0 us gate, Tha = 0.16 V (1stspike window)N, intact,Not measured00N, parasitesNot measured0.450.58HI, intact,Not measured00HI, parasitesNot measured0.140.183.N-HI metrics, full time range Tha = 0.16 V (similar to humanstudies)***N, intact,Not measured0.150.25N, parasitesNot measured0.450.68HI, intact,Not measured0.010.08HI, parasitesNot measured0.140.214.N-HI metrics, range 2.0-2.6 us, Tha = 0.01 V (2ndspike window)***N, intact,0.480.450.1N, parasites0.720.850.3HI, intact,0.140.50.06HI, parasites0.942.420.23*significantly different from the intact sample according to a two-sample t-test, p < 0.001 (this data is not available for N-HI)**the sample preparation has been changed from leaving the stock suspension of parasites on top of the skin (as was done on Experiment 1) to leaving only residual parasites on top of the back skin surface by staining and then washing the stock suspension. The presence of many red blood cells (without parasites) on top of the skin might have influenced the acoustic signal. Thus the experiments on Experiment 2 and Experiment 3 may have a higher fidelity.

The corresponding hydrophone data revealed a similar time-shift trend (Table 13). Average values for the first spike position and interval for three independent experiments with black skin sample of 200-300 um thickness (every day a new sample was prepared, and the skin thickness variability may exceed 50 um) were calculated. The number of signals in each group was 16-20.

TABLE 13Statistics for the hydrophone signals co-registeredwith malaria sensor signals, skin modelExperimentExperimentExperiment 3Experiment date12**(new setup)**Peak 1, us, intact1.30 std 0.021.24 std 0.061.45 std 0.01Peak 1, us, parasites1.21 std 0.07*1.11 std 0.04*1.33 std 0.04 *Difference us,0.090.120.12Interval, us, intactNot measured0.23 std 0.080.11 std 0.02Interval, us, parasitesNot measured0.44 std 0.06*0.34 std 0.07*Difference, us,Not measured0.210.23*significantly different from the intact sample according to a two-sample t-test, p < 0.001**the sample preparation has been changed from leaving the stock suspension of parasites on top of the skin (as was done on Experiment 1) to leaving only residual parasites on top of the back skin surface by staining and then washing the stock suspension. The presence of many RBCs (without parasites) on top of the skin might have influenced the acoustic signal. Thus the experiments on Experiment 2 and Experiment 3 may have a higher fidelity.

The observed influence of parasites on the first spike was further analyzed by using HI-N signal amplitude metrics and by varying the time window where they were calculated for. In the full time range (similar to how it has been done in human studies, with the similar amplitude threshold, see Table 13), adding parasites has increased values of both N and HI by 2.5-14 fold. The values of N and HI were close to those obtained above for the analysis of the first spike only. This may indicate a visual fact that, in the full time range mode, the first spike dominates the signal. Further, in the time range from 2.0 us (where nanobubble signals were observed for the same sensor in the experimental water model of nanobubbles and where nanobubble-like signals were observed in the non-invasive human study in The Gambia) to 2.6 us (the max time range to avoid any echo signals), a 2-6 fold increase in both metrics was observed after parasites were added.

The time-shift observed for the first spike after parasites were added matches the hydrophone data for the same samples. A similar direction and value of the time shift was observed with the hydrophone for the first spike (note an opposite location of the hydrophone hence an opposite time-shift). However, the malaria sensor may not resolve melanin and parasite spikes as the hydrophone did. Instead, it appears that sensor has integrated closely located spikes into one spike with varying amplitude as adding parasites has increased the peak-to-peak amplitude of the first spike detected with the sensor but did not increase the amplitude of any spike in the hydrophone signal. These differences in signal parameters could be caused by a narrow acoustic frequency bandwidth of the sensor compared to that of the hydrophone. Nevertheless, the optimization of the time window range and the amplitude threshold allows for distinguishing intact and parasite skin even with a slow-speed sensor (see, e.g.,FIG.27M).

In the field human studies the malaria sensor returned typical signals for healthy (n=25) (FIG.27N) and malaria-infected (n=30) subjects (FIG.27O).FIG.27Nillustrates the malaria probe signals obtained from healthy subjects.FIGS.27O and27Pillustrate example combined nanobubble and bulk signals in malaria-positive human subjects using the malaria sensor. In malaria-positive subjects, the peak-to-peak amplitude of the first spike increased while the peak position shifted slightly to the right, by about +50±47 ns (statistically significant, p=0.0001), as indicated by a horizontal arrow. The analysis was based on the Gambia2017data for African type of dark skin andPlasmodium Falciparumtype of malaria parasites. Similar to the sensor signal in the nanobubble and bulk model, a plurality of small second spikes or tail spikes2708correlating to nanobubble generation can be observed. In the illustrated example, the tail spikes2708can be observed in the time window about 2 s after the delivery of the laser pulse. Similarly, the first spike of the nanobubble signal inFIGS.27O and27Pcoincides with the spike2701of the bulk signal and may not be identifiable against the background of the bulk signal and/or the non-flat baseline after the spike. However, the tails spikes2708are not accounted for in the peak-to-peak amplitude-derived diagnostic metrics because the amplitudes of the tail spikes2708are lower than the maximum peak-to-peak amplitude, which can depend on the amplitude of the spike2701of the bulk signal. Statistical analysis of the above differences used the signal amplitude HI and N signal metrics (Table 15 andFIGS.28C-28Ebelow).

Accordingly, new diagnostic metrics that can account for the second spike correlating to the collapse of the nanobubble may be needed.FIG.28Aillustrates the new metrics. N nb is the number of second spikes per run of N (for example, 60) signals. N nb can represent the number of transient vapor nanobubbles detected in a specific time window. The time window can be from about 2 s to about 5 s. T nb is the time interval between the first and second spikes in the combined signal. T nb can represent or approximate the lifetime of a nanobubble and can be a measure of the maximal size of a nanobubble. Table 14A below summarizes HZ (HEMOZOIN) (hemozoin-positive) signals in blood samples of culturedP. Falciparumparasites. The results are also shown inFIG.28B. In the bloodP. Falciparummodel in flow test, HI increases with the size of HZ (HEMOZOIN) nanoparticle cluster (in a parasite or if the blood is free from parasites. Gametocytes generate larger nanobubbles since their level of HZ (HEMOZOIN) is higher. Table 14B summarizes HZ (HEMOZOIN) (hemozoin-positive) signals in blood samples of positive human subjects. The presence of hemozoin in skin can be observed by hemozoin-generated vapor nanobubble signals in human skin and malaria-positive mosquitoes fed from human blood

TABLE 14BHZ (hemozoin) signals in blood samplesof positive human subjectsGametocyte count in blood0-1>30Blood HI0.332.24

The HI-N amplitudes inFIGS.28C-28Eand Table 15 below were analyzed for the time window covering the whole signal and only the first spike and the time window for the second spike.

The N-HI diagram (for all subjects, single data point corresponds to a single subject) shows the following statistical properties of signals. The metrics were calculated for the similar settings for the time window to include both the first spike and secondary spikes and an amplitude threshold of 0.16 V. Table 15 shows group-averaged values (N=30 for malaria, N=25 for healthy). For human field data, one specific location, an ankle, was analyzed. When assuming a time-shift to the right of the first spike and setting the time-window at 1.02 us to 5.0 us, the amplitude threshold unchanged Tha=0.16 V, the separation of healthy and malaria data improves, their group averages increasing by more than one order of magnitude and two groups being statistically significantly different (p<0.001). Metrics of the first spike, with the time window optimized for the time-shift observed, provided the best diagnostic separation for signals obtained with the slow-speed sensor.

When applied to the time window associated with the second spike of a nanobubble collapse, at time window 2.0-5.0 us, only peak-to-peak amplitudes (without subtracting a non-flat baseline) were analyzed. This limitation of the analysis decreased the separation between healthy and malaria-positives but still indicated some statistically significant difference between these subject groups. The distribution of HI-N data in the model (FIG.27M) and humans (FIGS.28C-28E) follows the same trend.

In the skin model experiment, the incidence of the second spike was lower than in field human data. This can be explained by two factors: (1) mechanical properties of live (humans) skin are more favorable for the collapse of nanobubble compared to properties of dead skin (the model), and (2) real human skin had more parasites and they also were closer to the surface compared to the condition of residual single parasites at 250 um depth in the model.

FIG.29Aillustrates the T nb-N nb diagram for thePlasmodium Falciparummalaria using the signal obtained from the Stage 1 study in The Gambia, where N is number of second spikes nanobubbles observed, T is time interval between the first and second spikes (the nanobubble lifetime, which is proportional to the maximal diameter of the nanobubble). The N nb and Tnbvalues are subject-averaged per a test run of 60 laser pulses. The diagonal line represents a malaria diagnostic threshold, with the T nb-N nb value above the line being interpreted as a malaria-positive status and the T nb-N nb values below the line being interpreted as a malaria-negative status. The incidence and interval for the secondary spike was additionally analyzed with time-based metrics and the probability of observation of the spike (FIG.29A). Compared to the amplitude analysis of the second spikes (FIG.28C), the time-based approach inFIG.29Acan be more efficient. Compared to the first spike, not all malaria-positive signals included the second spikes. This deficiency of the second spike was similar to that observed in the human skin model and may be associated with the mechanical properties of the skin, plastic deformation during the expansion of nanobubble and viscous losses that reduces the nanobubble energy and prevent its collapse as it occurs in the water, a fully elastic medium without plastic deformations. Nevertheless, the second spike data from the two field studies of two malaria strains,Plasmodium FalciparumandPlasmodium Vivax, in two groups, clinical and asymptomatic (the latter was studied via the mass screening) show the diagnostic significance of the second spikes in signals even obtained with a slow-speed sensor.

FIG.29Billustrates the N nb histogram for thePlasmodium Falciparummalaria using the signals obtained in the study groups in The Gambia.FIG.29Cillustrates the N nb histogram for thePlasmodium Vivaxmalaria using the signals obtained in the study group in Sumatra. The vertical line represents the diagnostic threshold, which can be set at an Nnb value of 5 per a test run of 60 laser pulses.

Table 16 below summarizes the group-averaged N nb values and additional details ofFIGS.29B and29C.

Several factors influenced the occurrence of the second spike: (1) not all nanobubbles collapsed, not all collapsed nanobubbles were detected, only those with the lifetime above 1 us were detected, because the second spikes of smaller nanobubbles were obscured by a non-flat baseline of the signal output of malaria sensor. The occurrence of collapse spikes (2ndspikes) was random through the test run from 1st to 60st signals. They were not linked to specific, for example, initial laser pulses like they were in the skin model where a strong signal decay was observed. This difference between the dead and live skin suggests that the laser-exposed volume in live skin does not remain static during the test (which can be about 3 seconds) and new hemozoin targets can enter the volume during the test. This may be the result of plastic deformation of skin or cracks induced by nanobubbles and the resulting “mixing” of the skin volume.

In summary, in several independent experiments, adding parasites (including the residual levels of parasites) to the bottom of the human dark skin sample of 200-300 um thickness has caused changes in the time position and peak-to-peak amplitude of the first spike and the appearance of irregular second spikes in signals of the slow-speed sensor. Co-registration of acoustic signals in the skin sample with two sensors, the reference hydrophone and the malaria sensor, presented independently obtained evidence of the signals associated only with parasites in skin. Unlike the hydrophone, the malaria sensor does not resolve signal spikes which are close in time. Instead, it reveals some effects of the integration of close spikes. These effects influence time and amplitude parameters of the first, largest, spike in the signal, whose amplitude and time position correlate to parasites in the skin. The similarity of signal shapes and signal metrics observed in three different studies-non-invasive human studies, water model of nanobubbles, and the skin model with human parasites —suggests that the non-invasive skin signals detected in the field in malaria-positive subjects were caused by vapor nanobubbles generated around parasites in the skin.

Metrics N nb and T nb are independent of the amplitude derived metrics described above, and can be used in addition to and/or instead of the amplitude-derived metrics. In some embodiments, the malaria sensor can have reduced distortions of the tail baseline or a flat tail baseline to make it easier to detect the second and/or tail spikes.

Optical excitation and acoustic detection of parasites in a liquid sample of whole blood will now be described. The liquid sample included water, human blood (whole) and human blood with 50Plasmodium Falciparumparasites per microliter (the lower limit of the microscopy detection of parasites in blood). Static and flowing samples were studied in an Eppendorf tube. The flow was achieved by the pipette-induced mixing of the tube content during the signal collection.

The same laser pulse as above (220 ps, 671 nm, 15 μJ) was delivered via the optical fiber (with 105 or 50 μm fiber core) as a 60-pulse train. The 50-um fiber was used with a reduced laser pulse energy of 3.6 uJ in order to maintain the same optical fluence as the one at the exit of the 105-um fiber. Acoustic detection was performed using a reference hydrophone of 1.0 mm diameter (with a bandwidth of 10-12 MHZ (hemozoin), and a sensitivity of 0.5 V/MPa at 5 MHZ (hemozoin)). The hydrophone tip was located at 2 mm (approximately) distance from the source.

The water and blood signals were detected as a reference (FIGS.30C and30D). As shown inFIG.30D, residual absorbance by whole blood components (such as hemoglobin and other proteins) resulted in highly reproducible single bipolar spike. This spike reported both a compression and tensile pressure components and represented typical thermo-elastic signal in response of optically absorbing volume as determined by the laser beam aperture and penetration depth to a single laser pulse. The reproducibility and noise floor can be seen in the overlay of 60 signals obtained in one test. The flow of the blood did not seem to induce detectable changes into the intact blood signal, which has been considered as the background (seeFIG.30E). Both in static and flowing blood samples, a second spike was occasionally observed (a small signal in the time window 2-4 us inFIGS.30D and30E) with the probability of less than 1%. This second spike may not indicate a nanobubble because a nanobubble of such a large lifetime would have produced a second spike of a much higher amplitude.

As shown inFIGS.30F and30G, addingPlasmodium Falciparummalaria parasites has returned signals with typical vapor nanobubble signals with second spikes and sometimes time-resolved first spikes as compared to nanobubbles in the water model disclosed herein. The signals are different from the background spike of the whole blood. As shown inFIG.30G, the incidence of nanobubble signals has increased in the flowing sample. The amplitude of the second spike has increased with the nanobubble lifetime, which was observed in the range from 0.5 us (smallest nanobubbles detected) to 2.7 us. Both the first and second nanobubble spikes had mainly positive components, without pronounced negative components. This may indicate (1) a low elastic modulus of the sample (in line with water-like mechanical properties of the blood), and (2) absence of significant tensile stress compared to those observed in the skin model. Under an identical parasite density, the static sample (FIG.30F) returned fewer and smaller sized nanobubbles than the flowing sample (FIG.30G) in response to a 60 laser pulse train. Static sample generated nanobubbles in response to only 1-2 first laser pulses, thus confirming the destruction of HZ (hemozoin) cluster after it generated initial nanobubble(s). Since the size of HZ (hemozoin) cluster varies from parasite to parasite, mixing the laser-exposed volume brought more parasites for the optical excitation and hence resulted in an increase in the number of nanobubble signals and in their lifetime. A time interval between the background and the first nanobubble spikes was often observed inFIG.30G. The background signal was technically generated at the hydrophone-blood interface while the nanobubbles were generated at the whole laser pulse penetration depth and thus might have emerged at some distance from the hydrophone tip. The maximum time-interval observed corresponded to the distance of about 300-350 um. The amplitude of the second spike was close to that of the first spike (when the first spike was time-resolved), which may be another piece of evidence of an unrestricted collapse of a nanobubble in the whole blood.

As shown inFIGS.30H and301, reducing the laser-exposed volume by replacing the 105 um core fiber with the 50 um core fiber has resulted in a much lower background signal (FIG.30H). At the same time, fewer nanobubbles were generated/detected (FIG.32, right) compared to the excitation via the 105 um core fiber (see also Table 17 with statistical analysis).

Comparing the signals inFIGS.30F and30Gto skin signals obtained under similar conditions, parasites in the whole blood apparently do not restrict the nanobubble expansion and do not dampen the nanobubble collapse the way it was observed in skin. This is in line with much higher elasticity and lower viscosity of the blood than the skin. Thus, the conditions of blood (micro) vessels are more favorable for the detection of parasites compared to those when HZ (hemozoin) or the parasite is surrounded by the skin only and without any adjacent liquid.

In the flowing sample, the amplitude HI-N metrics, coupled with tailored time-window (1.25us) which excluded the background signal, and the amplitude threshold of 4 mV, resulted in zero HI and N values for intact blood and 0.29 and 0.24, respectively, for blood withPlasmodium Falciparumparasites at the density 50 p/uL.

The volume exposed to the laser pulse may influence the background signal metrics. A smaller fiber has resulted in a reduction of 4 to 12-fold in the amplitude of the background signal (see, e.g.,FIGS.30H and30I). Further, the ratio of the amplitude of the first nanobubble spike to that of the background signal has increased. A smaller excitation volume improves the nanobubble detection but covers fewer parasites with a single laser pulse. The parameter N has dropped by 4-fold so 4-5 times more locations/volumes must be probed with the small diameter laser pulse in order to achieve the same number of parasites detected as by a laser pulse delivered via the 105 um core fiber. However, an advantage of a smaller excitation volume is a lower laser energy required, 4-fold lower in this case.

In these experiments, one parasite density has been used, 50 p/uL. For water-diluted blood samples (with a factor of 20:1), the threshold of detection of the samePlasmodium Falciparumparasites was 0.01 parasite per microliter of suspension. This is equal to 0.2 p/uL in the whole blood, and is still below the detection threshold of regular PCR, microscopy, and RDT, the three standard methods for detecting parasites in blood in the clinical and laboratory settings.

Compared to the skin model, the human whole blood seems to better support the generation of vapor nanobubbles without damping their expansion and collapse, similar to those in water. Thus, non-invasive detection of parasites in skin may benefit from the presence of blood around the subcutaneous parasites, which is the case for substantially all micro-vessels in dermis, the smallest of which are 5-10 um in diameter. The smallest nanobubbles detected in the human whole blood had a lifetime of around 500 ns, which is close to the detection threshold of 300-400 ns lifetime in the water model. Reducing the laser-probed (exposed to the pump laser pulse) volume may improve the signal-to-background ratio in detecting parasite-generated nanobubbles, but may require increasing the number of probed (scanned) locations and hence the diagnostic time. In addition, diluted blood samples returned more nanobubbles than the whole blood due to deeper laser penetration.

A chicken breast model was used to analyze parasites in melanin-free tissue. The sample was prepared from manufactured chicken breast meat (which remained visually wet). To model the malaria infection, blood with parasites was injected with a needle to the depth of 0.5-1.0 mm (as was verified later by measuring a cross-section of the sample as shown inFIG.31A). A thick layer of meat underneath served as acoustic damping medium. The local density of parasites in tissue was estimated to be in the range 10-100 p/uL based upon the initial injection concentration of 250 p/uL, with an assumption of limited diffusion into the volume 1.5 to 3 times larger than the injected volume. Optical excitation and acoustic detection were identical to those in the whole blood model and used 1.0 mm hydrophone at 2 mm distance from the tissue surface (FIG.31B). 60 laser pulses were applied to each location and ultrasonic gel was used as a coupling medium.

As shown inFIG.31C, signals observed revealed none or very small background (bulk) signal in intact tissue. This was different from both the blood (with hemoglobin creating the volume background opto-acoustic signal) and dark skin (with melanin creating the volume background opto-acoustic signal) models. Adding the parasites to the depth of 0.5-1.0 mm resulted in two-spike signals typical for vapor nanobubbles and similar to those detected in blood or water (FIG.31C). These signals had no negative components, thus indicating no tensile stress unlike signals detected in the human skin model. The second spikes were always observed when the first spikes were detected. The amplitude of the second spike was close to that of the first spike. These features indicated the generation of nanobubbles in an elastic medium without plastic deformation, without viscous losses to nanobubbles during their expansion, and without damping of the nanobubble collapse. This result is in contrast to the nanobubble generation in the human skin model where viscous losses during the expansion and damping of the collapse were usually observed.

The other difference to the tissue model describe above was that nanobubble signals in this model were not always observed in response to the first laser pulse and sometimes were detected in the same location after tens of laser pulses (FIG.31D). This effect was similar to that observed in the flowing blood sample described above. It appears that the probed volume in the tissue included some liquid component which has been actively mixed during the laser exposure. Such mixing was not caused by the first nanobubble but might have been caused by the ongoing re-distribution of the injected parasite blood. Statistical signal amplitude metrics N and HI were obtained to quantify the difference between the samples, as shown in Table 18 below.

TABLE 18The signal amplitude metrics N and HI obtained for the fulltime window for intact and parasite-injected tissueSampleNHITissue + Blood (n = 8)0.0150.001Tissue + Plasmodium0.15*0.16*Falciparum − Blood (n = 10)*the group was found to be statistically significantly different from the control in two-sample t-test

Despite a much higher tissue depth, 500-1000 um in the chicken breast model compared to 250 um in the human dark skin model, nanobubble signals were stably detected in melanin-free chicken breast tissue. Therefore, the presence of melanin and associated opto-acoustic background appears to be a limiting factor in the detection of parasite-generated vapor nanobubbles in human skin. As shown inFIG.31C, another and independent feature of this model was the absence of tensile stress, plastic deformation and high elastic modulus of the tissue (compared to those of the skin model) resulting in the unrestricted expansion and collapse of vapor nanobubbles around malaria parasites. Absence of the high background signal coupled with unrestricted nanobubble behavior provided strong signal with better (10 to 100-fold) separation of the signal amplitude metrics of intact and “infected” tissues (Table 18), compared to the human skin model (Table 10). The result for this model suggests that properties of the tissue, for example, stiffness (elastic modulus), plastic deformation, and viscosity may influence the diagnostic sensitivity of the method, which appears to be higher in tissue with properties similar to those of water, that is, lower stiffness, viscosity and plastic deformation.

In the tissue model with chicken breast and the injected human blood, malaria parasites generated vapor nanobubbles similar to those generated in the gold-water model, with symmetrical expansion and collapse and without damping effects observed in the human skin sample. Nanobubble signals were observed at parasites at a tissue depth of up to 1.0 mm. The difference between the chicken breast and human skin models can be explained by the differences in the mechanical properties of two tissue samples and, additionally, by the higher volume of liquid (blood around parasites) in the chicken breast model compared to that in human skin model.

FIGS.32A-32Cillustrate additional ultrasonic signals from a single transient vapor nanobubble in the gold-water model of a single vapor nanobubble (32A), in the gold—human skin model (32B), and around malaria parasites in human skin (32C). In the gold-water model, the acoustic signals report an unrestricted and symmetrical expansion and collapse of a transient vapor nanobubble. Symmetry is seen through similar amplitudes of the first (expansion) and second (collapse) spikes. In the gold-skin model, the acoustic signals report much faster expansion (although restricted, the amplitudes of both acoustic and optical signals are lower than those in water) and delayed (compromised) collapse, which results in a lower relative amplitude of the second spike. This delayed collapse is caused by plastic (vs elastic in water) deformations and possible formation of micro-voids. In the skin with parasite model, the acoustic signals appear to be similar to the previous case of vapor nanobubble in skin, with restricted expansion and delayed collapse of the nanobubble. The damping effect of skin can also be seen inFIG.26P. The comparison of the three different cases studied with two different methods shows a good agreement in skin signals for nanobubbles and parasites, and thus the vapor nanobubble nature of the parasite signals in skin.

In summary, malaria parasites were detected through the acoustic response of vapor nanobubbles (defined earlier as hemozoin-generated vapor nanobubble). Hemozoin-generated vapor nanobubbles were generated and detected in whole blood, human dark skin and chicken breast tissue models, and in human subjects withPlasmodium falciparumandPlasmodium vivaxparasite strains in the field studies. The result was achieved through comparative studies in six different experimental systems (in addition to two previously studied systems, individual infected human red blood cells and infected animals).

For the liquid sample, the method detects down to 0.01Plasmodium Falciparumparasite (gametocyte) per microliter of solution and 0.1Plasmodium Falciparumparasite (trophozoa) per microliter of solution. For the dark human skin, the method detected residual singlePlasmodium Falciparumparasites (gametocytes). For the non-invasive model with a dark skin sample, the method might not detect all parasites present in the skin because it could detect only relatively large nanobubbles with the lifetime above 0.7 us. Such a high detection threshold suggests that smaller vapor nanobubbles are being generated in the skin but cannot be detected with the current excitation and detection setups.

The human field and two skin model studies revealed the influence of the mechanical properties of skin, such as the high elastic modulus and viscosity, and plastic deformation, on the generation and collapse, and hence the detectability of (relatively large, around 10 um max diameter) vapor nanobubbles. Smaller nanobubbles may be less influenced by the above mentioned properties of the skin and hence may improve the detection of parasites.

In human dark skin, the background signal of melanin may have a 10-fold higher amplitude than that of a parasite-generated nanobubble. The suppression of the melanin signal and its separation (decoupling) from the nanobubble signal improve the diagnostic performance of non-invasive skin-based device.

Another approach to improve the diagnostic performance is to use a minimally invasive skin probing device with the optical fiber penetrating 200 um of the upper skin may suppress the melanin background and improve the optical excitation of nanobubbles.

Terminology

Terms of orientation used herein, such as “proximal,” “distal,” “radial,” “central,” “longitudinal,” and “end” are used in the context of the illustrated embodiment. However, the present disclosure should not be limited to the illustrated orientation. Indeed, other orientations are possible and are within the scope of this disclosure. Terms relating to circular shapes as used herein, such as diameter or radius, should be understood not to require perfect circular structures, but rather should be applied to any suitable structure with a cross-sectional region that can be measured from side-to-side. Terms relating to shapes generally, such as “circular” or “spherical” or “semi-circular” or “hemisphere” or any related or similar terms, are not required to conform strictly to the mathematical definitions of circles or spheres or other structures, but can encompass structures that are reasonably close approximations.

While a number of variations of the disclosure have been shown and described in detail, other modifications, which are within the scope of this disclosure, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the disclosure. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed.

Features, materials, characteristics, or groups described in conjunction with a particular aspect, embodiment, or example are to be understood to be applicable to any other aspect, embodiment or example described in this section or elsewhere in this specification unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings) may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The protection is not restricted to the details of any foregoing embodiments. The protection extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination so disclosed.