Patent Description:
The present disclosure relates generally to a method and apparatus to analyze components in a sample and in particular to determining an individual's level of exposure to radiation.

The ability to determine an individual's level of exposure to ionizing radiation (bio-dosimetry) due to a nuclear event is critical in the performing of triaging and determining the appropriate medical treatment of the exposed individuals. The following is a review of the current art for bio-dosimetry.

Molecular responses to ionizing radiation exposure fall in three broad categories according to the type of biomolecule chosen for analysis. These responses involve DNA, RNA, and protein.

Of these three, DNA responses occur first in the cell, i.e. DNA damage by radiation occurs instantly upon exposure. However, repair of the damage can take hours or even days to weeks, depending on the cell type and the nature of the damage. DNA damage occurs in several broad categories, including chromosomal breaks and rearrangements that are visible through the microscope, and point mutations including small (submicroscopic) deletions / insertions / rearrangements. Detection of chromosomal events is slow because the steps needed for their identification typically require cell culture followed by time-consuming analysis involving trained observers. The turn-around time is typically several days. Some chromosomal changes can be observed in cells that have not been cultured to metaphase, but these approaches require hybridization technologies followed by manual scoring involving trained observers.

Another type of cytogenetic response, that does not require cell culture, is the evaluation of micronuclei in erythrocytes. Micronuclei are formed by chromosomal fragments and by disruption of the mitotic spindle apparatus. Micronuclei are rare events and, in humans, erythrocytes bearing micronuclei are very efficiently filtered out by the spleen. In individuals who have had their spleen removed, micronucleated erythrocytes are not filtered out, and because the micronuclei are readily visible they can be efficiently counted by manual and by flow cytometric means. However, only about one adult per <NUM> has been splenectomized, effectively negating this assay from broad-based population monitoring.

Another flow-cytometry-based approach might involve the analysis of rearranged chromosomes, e.g. dicentrics and/or translocations. In this approach, chromosomes that have undergone radiation-induced rearrangements would be detected and enumerated. Rearranged chromosomes would appear to be bi-colored because they would have been hybridized with whole chromosome paints in a manner that is analogous to in situ hybridization. However, for such flow-based analyses the chromosomes would need to remain in solution, meaning that hybridizations would also have to occur in solution. These chemistries have not been widely adapted due to technical difficulties related to the separation of unbound probe from the chromosomes.

Another approach to biological dosimetry involves protein analyses. Of the three biological response systems (DNA, RNA, protein), protein is the least well-characterized. There are several reasons why proteins may not be the subject of much research effort. First, the number of proteins is very large, perhaps an order of magnitude greater than the number of genes due to alternative splicing of mRNA and post-translational modification such as phosphorylation. Second, proteins often occur in complexes, sometimes making it difficult to enumerate quantities of specific peptide sequences. Third, proteins are all-too-readily denatured, which can interfere with common analysis methods such as enzyme-linked immunosorbent assay (ELISA). Finally, efficient quantitative analysis of proteins requires the use of monoclonal antibodies, which are not available for every protein, not to mention the vast array of protein variants that result from alternative splicing of mRNA and post-translational modification. Numerous changes to protein occur following irradiation. Unfortunately, the analytical techniques available for protein analysis lag substantially behind those for nucleic acid. To date, there are no systems using proteins that offer hope of performing meaningful biological dosimetry.

The third category of biological response to radiation is RNA. Specific RNA sequences are known to respond to ionizing radiation. <CIT> and <CIT> describe microfluidic cartridges for polymerase chain reaction (PCR) according to the state of the art.

The cartridge of the invention is defined in claim <NUM>. Preferred embodiments are described in claims <NUM>-<NUM>.

RNA levels can be quantified quickly, easily, and inexpensively. Cell culture is not required, and every tissue contains RNA, so the analyses are not limited to a particular cell type. Significantly, RNA is readily reverse-transcribed into cDNA which can be amplified via the polymerase chain reaction (PCR) and quantified by real-time PCR. This means that very small amounts of tissue can be analyzed rapidly, efficiently, and quantitatively. Furthermore, RNA analyses take advantage of the very rapid response to ionizing radiation. Further, specific sequences of RNA undergo increased expression within an hour of exposure, while other genes continue to have elevated expression up to <NUM> hours, providing a flexible range of options for performing bio-dosimetry.

A portable bio-dosimetry system that can perform rapid and reliable testing of radiation exposure is needed. Disclosed is a handheld field deployable real-time quantitative polymerase chain reaction (qPCR) based system that can assess exposure of individuals to ionizing radiation based on the levels of specific mRNA sequences present in the leukocytes from a drop of blood. The drop of blood may be obtained, for example, by a finger stick in a field or triage setting following a nuclear event. The system can predict whether an individual has received a dose of ionizing radiation greater than or less than a predetermined threshold value to allow field medical personnel to identify individuals in urgent need of medical treatment and to determine exposure levels to aid in medical treatment.

The portable-dosimetry system includes sub-systems for receiving a blood sample from a person, lysing (breaking up of) blood cells from the blood sample into component parts including mRNA, binding the mRNA in a binding region for further processing while discarding waste from the lysing process, converting the bound mRNA to complimentary cDNA and performing quantitative polymerase chain reaction (qPCR) on the complimentary cDNA. The results of the qPCR analysis can provide an indication of exposure to radiation of the person.

<FIG> illustrate an example bio-dosimetry system <NUM>. The bio-dosimetry system <NUM> includes a bio-dosimetry device <NUM> and a cartridge <NUM> for processing a blood sample to determine the level of exposure to radiation of a respective person. As described in detail below, the bio-dosimetry device <NUM> may be a reusable, portable device that may be configured to receive one disposable cartridge <NUM> at a time, each cartridge <NUM> processing a blood sample of a respective person. The cartridge <NUM> is intended to be a single-use cartridge, used to measure a single sample. The bio-dosimetry device <NUM>, performs a number of functions on the sample, including pumping, lysing, binding, conversion and PCR analysis. Based on these operations, the bio-dosimetry device <NUM> can determine a level of exposure to radiation of the blood sample.

As an example, the bio-dosimetry system <NUM>, along with the various sub-systems and related processes, will be described in relation to analyzing a blood sample for exposure to radiation. It is envisioned within the context of this disclosure that the bio-dosimetry system <NUM> and/or some of the sub-systems, can be used for other applications such as analyzing cancer cells, types of cells (phenotypes) such as with use of flow cytometry, RNA viruses, types of bacteria, etc..

The bio-dosimetry device <NUM> comprises a housing <NUM>, a socket <NUM> for receiving the cartridge <NUM> and a door <NUM> coupled to the housing <NUM>. The door <NUM> encloses the socket <NUM> in a closed position (<FIG>) and provides access to the socket <NUM> in an open position (<FIG>). The housing <NUM> includes a reservoir <NUM> for receiving a blood sample and a cap <NUM> for enclosing the sample within the reservoir <NUM> (e.g., to avoid contamination). and a handle <NUM> for carrying the bio-dosimetry device <NUM>. The housing <NUM> further may include a display <NUM> for displaying information such as measurement data to a user, and a user input device <NUM> for receiving input from the user.

The door <NUM> may be coupled to the housing <NUM> with a hinge. When the door <NUM> is the opened position the socket <NUM> can receive the cartridge <NUM>. Upon receiving the cartridge <NUM> in the socket <NUM>, the door <NUM> can be closed. As described below, closing the door <NUM> can couple components in the bio-dosimetry device <NUM> to the cartridge <NUM>.

The bio-dosimetry device <NUM> includes a battery <NUM> which provides electrical energy to the bio-dosimetry device <NUM>. The battery <NUM> may be a standard battery such as a lead-acid, nickel-cadmium, lithium-ion battery etc. The battery <NUM> enables the bio-dosimetry device <NUM> to be portable and used in locations without access to a power grid.

The bio-dosimetry device <NUM> further include a computer <NUM> including one or more processors <NUM> and one or more memories <NUM>, the memories <NUM> including instructions for programming the one or more processors <NUM>. The computer <NUM> executes instructions which can be used carry out the processes for preparing and analyzing the sample for exposure to radiation as described herein. The computer <NUM> is communicatively coupled to components of the bio-dosimetry device <NUM> including the display <NUM>, the user input device <NUM>, sensors <NUM>, an illumination system <NUM>, optical detector <NUM>, heating systems <NUM>, pumps <NUM>, blister actuators <NUM> and a piezo electric element <NUM>.

The display <NUM> may be, for example, a liquid crystal display (LCD) or organic light emitting diode display (OLED) and can be used for outputting data, for example measurement data, to a user. The display <NUM> may further be a touch-screen device and used as an input device by the user.

The user input device <NUM> includes one or more input elements such as buttons, turn knobs, touch-screens, a microphone, etc. that permit a user to input data and/or instructions to the bio-dosimetry device <NUM>.

The sensors <NUM> can include, for example, temperature sensors, pressure sensors and contact sensors. The sensors <NUM> can receive instructions from and provide data to the computer <NUM> to enable the computer <NUM> to control the processes for preparing and analyzing the samples as described below.

The illumination system <NUM> provides light for performing PCR. The illumination system <NUM> is communicatively coupled to the computer <NUM> and includes a light source. The light source optimally emits light in a narrow band of wavelengths to stimulate the fluorescent tag. The fluorescent tag, in response, emits light at a different wavelength. The band of wavelengths radiated by the light source to stimulate the fluorescent tag needs to be limited to a range that does not overlap the light emitted by the fluorescent tag. There are many possible fluorescent tags (markers) and the wavelength of the light source needs to be matched to the tags. The light sources should be as monochromatic (narrow banded) as possible, such as a laser or a narrow emission light emitting diode or other light source with a narrow notch filter blocking wavelengths outside of the narrow band needed to stimulate the fluorescent tags.

The illumination system <NUM> may further include electronic circuitry to turn the light source on and off, bias the light source in an operating condition, control or limit the power supplied to the light source, etc. As described below, the illumination system <NUM> can radiate light on the cartridge <NUM> during analysis of material to be analyzed, based on commands from the computer <NUM>. Material to be analyzed as used herein is material that has been prepared to be analyzed based on components of the blood sample. The material to be analyzed may include fluorescent markers.

The optical detector <NUM> is communicatively coupled to the computer <NUM>, and can be, for example, a camera, a charge-coupled device (CCD), a light sensitive, CMOS array, etc. The optical detector <NUM> may include a filter to block the excitation wavelength (from the light source) used to stimulate a fluorescent tag and allow the fluorescent tag emission through for detection. Following or during illumination of the material to be analyzed by the illumination system <NUM>, the optical detector <NUM> receives light emitted by the material to be analyzed. The optical detector <NUM> can provide data to the computer <NUM> based on the received light. In an example, the material to be analyzed includes fluorescent markers. Following illumination, the fluorescent markers radiate light at a wavelength based on the markers and the optical detector <NUM> detects the light. In some cases, the optical detector <NUM> may be used to detect different fluorescent tags emitting light at different wavelengths. In these cases, the optical detector <NUM> may receive respective images of the two fluorescent tags by applying different filters for each of the respective fluorescent tags.

In at least one example, each of the heating system(s) <NUM> of the bio-dosimetry device <NUM> includes a heating element such as a resistance heater and may include electronic circuitry to turn the heating element on and off, bias the heating element in an operating condition, control or limit the power to the heating element, etc. The heating system <NUM> may further include a sensor <NUM>, such as a temperature sensor, that can be used to detect a temperature of the heating element and provide feedback to the heating system <NUM> such that the heating system <NUM> can regulate the temperature of the heating element. As will be explained in greater detail below, the heating system(s) <NUM> may be used to heat a portion(s) of the cartridge <NUM> during processing of the blood sample. Further, the heating elements included in the heating system <NUM> may in some cases be contained in the bio-dosimetry device <NUM> and in other cases included as part of the cartridge <NUM>.

In at least one example, the pump(s) <NUM> of the bio-dosimetry device <NUM> may be electrically coupled to and controlled by the computer <NUM> such that the computer <NUM> can turn them on and off. As described in additional detail below, when the cartridge <NUM> is inserted into the bio-dosimetry device <NUM>, and the door <NUM> is closed, the pumps <NUM> may further be in fluid communication with the cartridge <NUM> such that the pumps <NUM> can move fluids, e.g., from the reservoir <NUM> to the cartridge <NUM> or within the cartridge <NUM> from one region to another.

The bio-dosimetry device <NUM> may further include one or more blister actuators <NUM>. Each blister actuator <NUM> may be communicatively coupled with the computer <NUM> and include a plunger. The blister actuators <NUM> may be arranged within the bio-dosimetry device <NUM> such that, when the cartridge <NUM> is inserted in the bio-dosimetry device <NUM> and the door <NUM> is closed, the blister actuator <NUM> is within a range of elements on the cartridge <NUM> such that the plunger, in an extended state, extends into and compresses the elements on the cartridge <NUM> and releases fluids into a processing flow on the cartridge <NUM>.

The bio-dosimetry device <NUM> may further include a piezo electric element <NUM>. The piezo electric element <NUM> may be communicatively coupled with the computer <NUM> such that it can receive commands, for example, to turn the piezo electric element <NUM> on and off or adjust a power level of the piezo electric element <NUM>. When the piezo electric element <NUM> is turned on, it may vibrate at a frequency, for example <NUM>. When a cartridge <NUM> is inserted into the bio-dosimetry device <NUM> and the door <NUM> is closed, the piezo electric element <NUM> may be coupled, either directly (in physical contact), or indirectly (for example, via a small air space) with elements on the cartridge <NUM> and cause the elements to vibrate, transferring mechanical energy to the elements.

The cartridge <NUM> is a disposable cartridge that is intended to be used to analyze a single blood sample and is configured to be inserted into the bio-dosimetry device <NUM>. The cartridge <NUM> includes a cassette <NUM> and a substrate <NUM>. The cassette <NUM> is adapted to be inserted into the socket <NUM> of the bio-dosimetry device <NUM> and includes a recessed area <NUM> for receiving and supporting the substrate <NUM>. The substrate <NUM> has a first side <NUM> and a second side <NUM> and supports a plurality of sections configured to process and analyze a blood sample and determine a level of exposure to radiation. The cartridge <NUM> further includes a cover <NUM> having a first side <NUM> and a second side <NUM>. The second side <NUM> of the cover is attached to the first side <NUM> of the substrate <NUM> and encloses the processing regions formed in the substrate <NUM>. The substrate <NUM> and the cover <NUM> can be made of a material such as glass (e.g., Pyrex), silicon, a silicone-based material, sapphire, a polymer, or any transparent substrate.

The cartridge <NUM> contains a plurality of regions for processing and analyzing a blood sample to determine a level of exposure to radiation. An overview of the regions of the cartridge <NUM> will be presented in the next paragraph. Thereafter, the structure and features of different sections of the cartridge <NUM> will be described in additional detail.

The blood sample is pumped into a lysing region <NUM> on the cartridge <NUM>, by a micropump <NUM> or via a port <NUM>. In the lysing region <NUM>, the blood sample is broken into components parts including mRNA. After lysing, the blood sample including the mRNA, is transferred to the binding region <NUM>. In the binding region <NUM>, mRNA from the blood sample is bound (held). The remaining components are removed, by flushing. Thereafter, the bound mRNA in the binding region <NUM> is flushed with a material, which forms cDNA with the bound mRNA. The cDNA, formed in the binding region <NUM> is then transferred to a mixing region <NUM> where the cDNA is more evenly distributed within a reaction mixture. Next, the cDNA is transferred to a polymerase chain reaction (PCR) region <NUM> where the cDNA is analyzed for exposure to radiation of the mRNA with which the cDNA was previously formed (in the binding region <NUM>).

The micropump <NUM> may be built into and/or on the substrate <NUM> and based on a micro-electro-mechanic systems (MEMS) technology. When the cartridge <NUM> is inserted in the bio-dosimetry device <NUM> and the door <NUM> is closed, an input to the micropump <NUM> may be fluidly coupled to the reservoir <NUM> in the bio-dosimetry device <NUM> such that the micropump <NUM> can pump the sample into the cartridge <NUM> (for example, into the lysing region <NUM>).

The cartridge <NUM> may further include one or more ports <NUM>. The ports <NUM> may be openings formed in the cover <NUM> of the cartridge <NUM>. The ports <NUM> may be arranged to fluidly couple, for example, with an outlet of a pump <NUM> in the bio-dosimetry device <NUM> when the cartridge <NUM> is inserted in the bio-dosimetry device <NUM> and the door <NUM> is closed. For example, the outlet of the pump <NUM>, which may be a nozzle or a tube, may be inserted into the port <NUM> when the door <NUM> of the bio-dosimetry device <NUM> is closed, such that when the pump <NUM> is activated, the pump <NUM> can output a fluid into the port <NUM>.

To store and, at a proper time during the sample processing, inject liquid materials in the processing flow, the cartridge <NUM> may include one or more blisters <NUM> fluidly coupled to respective regions in the cartridge <NUM>. For example, one or more blisters 86a may be fluidly coupled to an inlet to the lysing region <NUM>. One or more blister 86b may be coupled to an inlet to the binding region <NUM> and one or more blister 86c may be coupled to an inlet to the mixing region <NUM>. The blisters <NUM> may be, for example, a flexible, polymer based, fluid container, that is formed, for example, on a first side <NUM> of the cover <NUM>.

Microchannels <NUM> may be used to fluidly couple regions and features of the cartridge <NUM>. For example, a microchannel 85a may couple one or more of the micropump <NUM>, one or more blisters 86a and one or more ports <NUM> to an inlet of the lysing region. A microchannel 85b may fluidly couple an outlet of the lysing region <NUM> and an outlet of one or more blisters 86b to an inlet of the binding region <NUM>. A microchannel 85c may couple an outlet of the mixing region <NUM> to an inlet of the PCR region <NUM>, etc. Each of the microchannels <NUM> may be formed in the substrate <NUM> and closed on a first side <NUM> of the substrate <NUM> by a second side <NUM> of the cover <NUM>.

An example blister <NUM> is shown in <FIG>. The blister <NUM> may be a hollow, flexible container made of a polymer that forms a cavity for containing a fluid. The blister <NUM> may have a flat portion <NUM> for attaching to a surface, for example the first side <NUM> of the cover <NUM>. The blister <NUM> may further have a dome-shaped structure <NUM> arranged on a side of the flat portion and forming the cavity. The dome shaped structure <NUM> may be, for example, round, or in the form of an elongated rectangle or oval. The blister <NUM> may have an outlet <NUM> which may be fluidly coupled, to one of the processing regions on the cartridge <NUM>. The blister <NUM> is configured such that, when pressure is applied to the dome shaped structure <NUM>, fluid contained in the blister <NUM> is expelled through the outlet <NUM>.

A blister <NUM> may be actuated by a blister actuator <NUM> on the bio-dosimetry device <NUM>. The blister actuator <NUM> may be arranged in the bio-dosimetry device <NUM> such that when the cartridge <NUM> is inserted in the socket <NUM>, the blister actuator <NUM> is in a range to actuate the blister <NUM>. At an appropriate time during a processing cycle, the computer <NUM> in the bio-dosimetry device <NUM> may instruct the blister actuator <NUM> to extend a plunger toward and apply pressure to the dome-shaped structure <NUM>, and expel the liquid contained in the blister <NUM> into the processing region to which the blister <NUM> is fluidly coupled. A size of each blister <NUM> in the cartridge <NUM> can be selected based on a volume of liquid required for the processing step in which the blister <NUM> participates.

An example lysing region <NUM> is illustrated in <FIG>. The lysing region lyses biological samples by applying ultrasonic stimulation. In an example, the sample to be lysed may be a drop of blood. In other examples, the sample may be any fluid with cells, for example waver with bacteria, cells in lymphatic fluid, cells in urine, etc. The lysing region <NUM> may also be used for other applications such as lysing microbes, animal and plant cells, fracturing bonds in DNA, RNA, protein aggregates and lipid aggregates. The lysing region <NUM> may be applied to prepare a sample for polymerase chain reaction (PCR) and rolling circle genetic amplification.

The lysing region <NUM> includes an inlet <NUM>, a distribution manifold <NUM>, a lysing array <NUM>, an outlet manifold <NUM>, and an outlet <NUM>. The lysing region <NUM> is configured to receive a sample via the inlet <NUM>, pass the sample through the distribution manifold <NUM>, lyse, by transfer of mechanical energy, the sample into component parts in the lysing array <NUM> and output the component parts through the outlet manifold <NUM> to the outlet <NUM>. Each of the inlet <NUM>, the distribution manifold <NUM>, the lysing array <NUM>, the outlet manifold <NUM> and the outlet <NUM> can be formed in the substrate <NUM> (<FIG>).

The inlet <NUM> can receive the sample and/or other input material from one or more of the micropump <NUM>, a blister <NUM> or a port <NUM>, and deliver the sample or input material to the lysing array <NUM> via the distribution manifold <NUM>. The distribution manifold <NUM> may be triangular shaped, with an apex of the triangle connected a microchannel 85a delivering the sample and/or input material and a base of the triangle connected to the lysing array <NUM>, such that the distribution manifold <NUM> broadens from the microchannel 85a to the lysing array <NUM> to distribute the material across a side of the lysing array <NUM>.

The lysing array <NUM> may be square and with a typical length of the side in a range from <NUM> to <NUM>. Other shapes are possible. For example, the lysing array <NUM> may be rectangular with either a short side of the rectangle of a long side of the rectangle connecting to the distribution manifold. Further, the size of the lysing array <NUM> is scalable. Increasing the size of the lysing array <NUM> increases a volume of material that can be lysed in a period of time. A smaller lysing array <NUM>, can be used, for example, to lyse viruses or other small components.

The outlet manifold <NUM> and outlet <NUM> output material from the lysing array <NUM> to a microchannel 85b. The outlet manifold <NUM> may be triangular shaped with a base of the triangle connected to the lysing array <NUM> and an apex connected to the microchannel 85b, such that the material output from the lysing array <NUM> is directed into the microchannel 85b.

The lysing array <NUM> includes a diaphragm <NUM> formed in the substrate <NUM>. Referring to <FIG>, the diaphragm <NUM> has a first side <NUM>, a second side <NUM> and a thickness t1. The thickness t1 can be in a range from <NUM> millimeters to <NUM> millimeter with a typical value of <NUM> millimeter. Referring to <FIG>, the first side <NUM> of the diaphragm <NUM>, together with the substrate <NUM> and the cover <NUM>, define a cavity <NUM>. The cavity <NUM> contains the sample to be lysed during lysing. The cavity <NUM> may have a typical volume of four microliters and a typical volume range from one to <NUM> microliters. This range is not intended to be limiting. In addition to cavity volumes within this range, a lysing array <NUM> according to this disclosure can also have volumes greater than or less than this range.

A plurality of micropillars <NUM> extend outwardly from the first side <NUM> of the diaphragm <NUM> into the cavity <NUM>. Each of the micropillars <NUM> has a top end <NUM>. In an example, the micropillars <NUM> are arranged in columns and rows and may be evenly spaced. As an example, the lysing array <NUM> may include <NUM> rows and <NUM> columns of micropillars <NUM>, for a total of <NUM> micropillars. The micropillars <NUM> may be formed, for example, by an embossing or etching process.

The lysing array <NUM> may include, coupled to the second side <NUM> of the diaphragm <NUM>, a piezo electric layer <NUM>. During lysing, the piezo electric layer <NUM> may be stimulated (i.e., an electric current may be applied). Based on the stimulation, the piezo electric layer <NUM> may apply an oscillating force to the diaphragm <NUM>, causing the diaphragm <NUM> to flex. At points towards a center of the lysing array <NUM>, the flexing of the diaphragm <NUM> may be substantially perpendicular to a plane of the diaphragm <NUM>. In an example, the flexing of the diaphragm can result in a displacement in a range from <NUM> to <NUM> microns at points towards the center of the lysing diaphragm. In other examples, the displacement can be larger, up to a range of <NUM> millimeter or more. The amount of displacement may vary with the frequency of stimulation as there is a relaxation time dependent on displacement. That is, a lower frequency of stimulation may result in a larger displacement. The flexing causes a lateral motion on the top ends <NUM> of the micropillars <NUM>. The lateral motion of the top ends <NUM> can create a liquid shock wave that can lyse the sample contained in the cavity <NUM>, breaking the sample into component parts. An energy level applied to the diaphragm can be controlled to allow for lysing, for example, of cell walls, without damaging internal organelles in the blood cells.

As an alternative to the piezo electric layer <NUM>, a piezo electric element <NUM> included in the bio-dosimetry device <NUM> can be used to stimulate the diaphragm of the lysing region <NUM>. This is illustrated in <FIG>. In this case, when the cartridge <NUM> is inserted in the bio-dosimetry device <NUM> and the door <NUM> closed, the piezo electric element <NUM> is arranged to physically couple with the diaphragm <NUM> such that when the piezo electric element <NUM> is turned on, ultrasonic waves radiated from the piezo electric element <NUM> cause the diaphragm <NUM> to flex.

<FIG> is a top view of a section of the lysing array <NUM>, with the cover <NUM> removed. The section shows two rows R1 and R2 and two columns C1 and C2. The micropillars <NUM> may be cylindrically shaped, and have a diameter d. The diameter d may, for example, be in a range from <NUM> millimeters to <NUM> millimeters with a typical value of <NUM> millimeters. The micropillars <NUM> may be evenly spaced and have a spacing W<NUM> between adjacent micropillars <NUM> in a row (e.g., the row R1) and evenly spaced and have a spacing W<NUM> between two micropillars <NUM> in a column (e.g., the column C1). In a typical example, W<NUM> may be substantially equal to W<NUM>. The spacings W<NUM> and W<NUM> may be in a range from <NUM> millimeters to <NUM> millimeters and have a typical value of <NUM> millimeters which is effective for most cells. The ranges of W<NUM> and W<NUM> may be selected based on a particle size of the sample to be lysed and would typically increase as the particle size to be lysed increases.

In another example, the lysing array <NUM> may be applied to open or break apart protein chains and viruses. In this case, the spacings W<NUM>, W<NUM> and the diameter d may be in a range of tens of nanometers.

<FIG> is a side view of the section of the lysing array <NUM> shown in <FIG>. The micropillars <NUM> can have a height h<NUM>. The height h<NUM> can be in a range from <NUM> millimeters to <NUM> millimeters and can depend on the diameter d chosen for the micropillars <NUM> and the sample to be lysed. The height h<NUM> and diameter d are typically selected such that an aspect ratio h<NUM>/d is <NUM>, with a range from <NUM> to <NUM>. For example, in the case that the diameter d is selected to be <NUM> millimeters, the height h<NUM> can be selected to be <NUM> millimeters.

The diaphragm <NUM> has a thickness t<NUM>. As noted above, the thickness t<NUM> can be in the range <NUM> millimeters to <NUM> millimeter with a typical value of <NUM> millimeters. The thickness of the piezo electric layer <NUM> (when present) may be in a range from <NUM> millimeters to <NUM> millimeters.

Other shapes may be used for the cross-section of the micropillars <NUM>. For example, the cross-section may be rectangular or triangular, the micropillars <NUM> may be conical with a base of the cone located at the first side <NUM> of the diaphragm <NUM>, etc..

<FIG> illustrate an example binding region <NUM>. The binding region <NUM> includes a microchannel <NUM> having an inlet <NUM> and an outlet <NUM>. During operation, material, for example, a lysed blood sample flows through the microchannel <NUM> in a direction <NUM>.

The microchannel <NUM> includes a stationary phase <NUM>. The stationary phase <NUM> includes a plurality of micropillars <NUM>. The micropillars <NUM> are formed in and extend outwardly from the substrate <NUM>. As shown in <FIG> and <FIG>, the micropillars <NUM> are arranged in rows, for example rows R3 and R4. Each row is offset from a previous row such that the micropillars <NUM> in the row R4 are arranged to correspond with an opening between micropillars <NUM> in the row R3.

As an example, as shown in <FIG>, the micropillars <NUM> may have a cross-section having a hexagonal shape. The micropillar <NUM> has first, second, third, fourth, fifth and sixth sides <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. First and fourth sides <NUM>, <NUM> are parallel, and have a length l<NUM>. Second and fifth sides <NUM>, <NUM> are parallel and have a length l<NUM>. Third and sixth sides <NUM>, <NUM> are parallel and have length l<NUM>. In an example, the micropillar <NUM> has a diameter d<NUM> and a spacing between the micropillars d<NUM>. The diameter d<NUM> may be in a range of two microns to <NUM>,<NUM> microns, with a typical value of <NUM> microns. The spacing may be in a range from one micron to <NUM>,<NUM> microns, with a typical value of <NUM> microns. The micropillars <NUM> have a height h<NUM>. The height h<NUM> may be in a range of <NUM> microns to <NUM> millimeters, with a typical height of one millimeter.

In some cases, the micropillars <NUM> may be coated with a binding material to bind target components of the sample. For example, in the case of a blood sample containing mRNA, the micropillars <NUM> may be coated with oligo dT25 to bind the mRNA in the blood sample to the micropillars <NUM>.

The binding region <NUM> further may include a heating element <NUM> having two contacts <NUM> on opposite ends. The heating element <NUM> may be supported on the cover <NUM>. The heating element <NUM> may be formed of a resistive material such that, when an electrical voltage is applied across the two contacts <NUM>, a current will flow through the heating element <NUM> and generate heat.

Additionally or alternatively, during operation of the bio-dosimetry system <NUM>, a heating system <NUM> in the bio-dosimetry device <NUM> may be arranged such that when the cartridge <NUM> is inserted into the socket <NUM> and the door <NUM> closed, the heating system <NUM> is thermally coupled to (i.e., can heat) the binding region <NUM>.

The binding region <NUM> can be used to bind mRNA in a blood sample to the stationary phase <NUM>. This is described below as the process <NUM> in reference to the <FIG>.

The binding region <NUM> can further be used to convert the mRNA, bound in the stationary phase, to cDNA. This is described below as the process <NUM> in reference to the <FIG>.

<FIG> illustrate the mixing region <NUM>. The mixing region <NUM> includes a microchannel <NUM> having an inlet <NUM> and an outlet <NUM>. The microchannel <NUM> may be in the form of a meander having straight portions coupled at angles of, for example, <NUM>° to one another. A length l<NUM> of a straight portion in the meander may have a typical value of <NUM> millimeter to <NUM> millimeters. The microchannel <NUM> is formed in the substrate <NUM> and has a width d<NUM>. The width d<NUM> can be in a range of <NUM> millimeters to <NUM> millimeters with a typical value of <NUM> millimeters. The depth U<NUM> of the microchannel 240can be in a range from <NUM> millimeters to <NUM> millimeter with a typical value in a range from <NUM> to <NUM> millimeters.

The microchannel <NUM> can be used to remove gradients of cDNA in reaction mixture. The reaction mixture, containing the cDNA may enter the mixing region <NUM> at the inlet <NUM>, pass through the microchannel <NUM>, continue through the outlet <NUM>. As the reaction mixture containing the cDNA passes through the microchannel <NUM>, the changes of directions as the microchannel <NUM> passes through the <NUM> passing through <NUM>° angles, may cause turbulence in the fluid flow, mixing the cDNA within the reaction mixture. Through the mixing process, the cDNA may be more evenly distributed, reducing or eliminating gradients in the cDNA within the reaction mixture.

The mixing region <NUM> may have other shapes or features. For example, the width d<NUM> of the microchannel <NUM> may be varied alternatively between a first width and a second width or the depth U<NUM> of the microchannel <NUM> varied alternatively between a first depth and a second depth to create turbulence in the reaction mixture flow. As another example, extensions or bumps may be formed, extending from a bottom of the microchannel <NUM> into the microchannel <NUM> to increase turbulence in the reaction mixture flow.

<FIG> is a diagram of an example PCR region <NUM>. The PCR region <NUM> receives material including cDNA from the mixing region <NUM>. The PCR region analyzes the cDNA for exposure to radiation of the mRNA from which the cDNA was generated. Analyzing the cDNA includes thermally cycling the material to be analyzed including the cDNA between two temperatures, as described below. The PCR region <NUM> receives the material via a microchannel 85c. The microchannel 85c couples the mixing region <NUM> (<FIG>) via four sub-microchannels 98a, 98b, 98c, 98d to four respective PCR channels 250a, 250b, 250c, 250d.

A PCR region <NUM> including four PCR channels 250a, 250b, 250c, 250d is only an example. The PCR region <NUM> may include any number of PCR channels <NUM>.

As shown in <FIG>, the first PCR channel 250a includes the pre-PCR zone 252a, the blister 86e and the PCR zone 254a, the second PCR channel 250b includes the pre-PCR zone 252b, the blister 86f and the PCR zone <NUM>, the third PCR channel 250c includes the pre-PCR zone 252c, the blister <NUM> and the PCR zone 254c and the fourth PCR channel 250d includes the pre-PCR zone 252d, the blister <NUM> and the PCR zone 254d. The four PCR channels 250a, 250b, 250c, 250d may be the same or similar, and will be described as an example PCR channel <NUM> including an example pre-PCR zone <NUM>, an example blister <NUM> and an example PCR zone <NUM> below.

<FIG> illustrate an example pre-PCR zone <NUM>. The pre-PCR zone <NUM> heats material prior to beginning analysis in the PCR zone <NUM>. The pre-PCR zone <NUM> includes a microchannel <NUM> including an inlet <NUM> and an outlet <NUM>. Material enters the microchannel <NUM> at the inlet <NUM> and flows towards the outlet <NUM>. The outlet <NUM> may be coupled, for example, to a sub-microchannel <NUM> such as the sub-microchannel 98a. The outlet <NUM> may be coupled to the PCR zone <NUM>.

The microchannel <NUM> may have a width d<NUM> and a depth U<NUM>. Adjacent parallel sections of the microchannel <NUM> may be spaced away from each other by a distance d<NUM>. The width d<NUM> may be in a range from <NUM> millimeters to <NUM> millimeters with a typical value of <NUM> millimeter. The depth U<NUM> of the microchannel <NUM> may be in a range from <NUM> millimeters to <NUM> millimeters with a typical value of <NUM> millimeter. The spacing d<NUM> between adjacent sections of the microchannel <NUM> may have a range from <NUM> millimeters to <NUM> millimeters with a typical value of <NUM> millimeters.

The pre-PCR zone <NUM> further may further include a heating element <NUM> including two contacts <NUM>. on opposite ends. The heating element <NUM> may be supported on the cover <NUM>. The heating element <NUM> may be formed of a resistive material such that, when an electrical voltage is applied across the two contacts <NUM>, a current will flow through the heating element <NUM> and generate heat.

Additionally or alternatively to including the heating element <NUM>, a heating system <NUM> in the bio-dosimetry device <NUM> may be arranged such that when the cartridge <NUM> is inserted into the socket <NUM> and the door <NUM> closed, the heating system <NUM> thermally couples to (i.e., can heat) the pre-PCR zone <NUM>.

<FIG> illustrate an example PCR zone <NUM>. The PCR zone <NUM> thermally cycles the material between a first temperature and a second temperature, and then images the material using fluorescent markers. The imaging provides data indicating a degree of exposure to radiation of the original sample.

The PCR zone <NUM> includes a microchannel <NUM>. As shown in <FIG>, the microchannel <NUM> meanders from an inlet <NUM> to an outlet <NUM>. In practice, material to be analyzed by the PCR zone <NUM> is pumped into the inlet <NUM> and flows through the microchannel <NUM> to the outlet <NUM>.

The PCR zone <NUM> includes a first heating region <NUM>, a detection region <NUM> and a second heating region <NUM>. One or more first heating elements <NUM> extend across the first heating region <NUM>. Similarly, one more second heating elements <NUM> extend across the second heating region <NUM>. The first and second heating elements <NUM>, <NUM> are arranged on and supported by the cover <NUM>.

The first and second heating regions <NUM>, <NUM> are spaced away one from the other, to eliminate or reduce thermal interference between the first and second heating regions <NUM>, <NUM>. That is, maintaining the first heating region <NUM> at a first temperature should not interfere with maintaining the second heating region <NUM> at a second temperature, and vice versa.

In an example, the detection region <NUM> is arranged between the first heating region <NUM> and the second heating region <NUM>. The detection region <NUM> is characterized in that there are no heating elements organized above the microchannel <NUM>. Other arrangements of the detection region <NUM>, are possible. However, placing the detection region <NUM> between the first and second heating regions <NUM>, <NUM> has the advantage of efficient use of the space required for separating the first and second heating regions <NUM>, <NUM>.

Additionally to the first and second heating elements <NUM>, <NUM>, a heating system <NUM> included in the bio-dosimetry device <NUM> can be used respectively to heat each of the first the second heating regions <NUM>, <NUM>.

The microchannel <NUM> is formed of a plurality of interconnected segments <NUM> (<FIG>) each segment <NUM> is arranged to perform one thermal cycle on the material flowing through the microchannel <NUM>. One thermal cycle is defined as passing through the first heating region <NUM> followed by passing through the second heating region <NUM>. Typically, in a PCR zone <NUM>, the microchannel <NUM> may include approximately <NUM> segments <NUM>. The number of segments <NUM> can be adjusted, based on the specific analysis to be performed in the PCR zone <NUM>.

<FIG> is an enlarged view of a section of the microchannel <NUM> illustrating an example segment 303a. The segment 303a has a first end <NUM> and a second end <NUM>. The segment 303a starts at the first end <NUM>, continues through the first heating region <NUM>, through the detection region <NUM>, through the second heating region <NUM> and back through the detector region to the second end <NUM>. The microchannel <NUM> then continues in a next segment 303b. As described below, when performing PCR, the first heating region <NUM> will be maintained at a first temperature and the second heating region <NUM> will be maintained at a second temperature, so that, as the material to be analyzed passes through the microchannel <NUM>, it is repeatedly cycled between the first temperature and the second temperature.

As shown in <FIG>, the microchannel <NUM> has a width d<NUM>. The width d<NUM> can be in a range from <NUM> millimeters to <NUM> millimeter with a typical value of <NUM> millimeters. The width d<NUM> can be selected depending on a volume of fluid to be passed through the microchannel <NUM>, a particle size of the fluid passing through the microchannel <NUM>, the manufacturing process used to create the microchannel <NUM> in the substrate <NUM>, etc..

The microchannel <NUM> further has a spacing d<NUM> between parallel portions of microchannel <NUM>. The spacing d<NUM> can be in a range from <NUM> millimeters to <NUM> millimeters with a typical value of <NUM> millimeter and can depend on factors such as the manufacturing process used to create microchannel <NUM>. To conserve space in the substrate, the spacing d<NUM> will typically be chosen to be close to or equal to a minimum feature size allowed by the manufacturing process used to form the microchannel <NUM> in the substrate <NUM>.

The microchannel <NUM> further has a height h<NUM>. The height h<NUM> is the distance, as measured along the axis Y-Y, from a furthest extension of the microchannel <NUM> within the first heating region <NUM>, for example the point 306a, to a furthest extension of the microchannel <NUM> within the second heating region <NUM>, for example the point 308a.

As further illustrated in <FIG>, the first heating region <NUM> has a height h<NUM>. The detection region <NUM> has a height h<NUM> and the third heating region <NUM> has a height h<NUM>. The height h<NUM> of the microchannel <NUM> is equal to the sum of the height h<NUM> of the first heating region <NUM>, the height h<NUM> of the detector region and the height h<NUM> of the second heating region <NUM>. That is, h3 = h<NUM> + h<NUM> + h<NUM>.

The height h<NUM> of the first heating region <NUM> can be determined, for example, by the area required for the first heating element <NUM>, based on the target first temperature of the first heating region <NUM>. Similarly, the height h<NUM> of the second heating region <NUM> can be determined by the area required for the second heating element <NUM>, based on the target second temperature for the second heating region. The height h<NUM> of the detection region <NUM> can be determined, for example, based on having a specifically defined region for detecting (counting) cDNA based on a tag emission. The sensitivity of the detection increases as an area of the detection region <NUM> increases. Further, as described above, in the case that the detection region <NUM> is located between the first heating region <NUM> and the second heating region <NUM>, the height h<NUM> can be determined based on a distance required to thermally isolate the first heating region <NUM> from the second heating region <NUM>.

<FIG> is a partial side view of a section of the PCR zone <NUM> shown in <FIG>, cut along the axis X-X. The microchannel <NUM> is formed in the substrate <NUM>. The cover <NUM> is attached to the substrate <NUM>, forming a top of the microchannel <NUM>. The microchannel <NUM> has a depth U<NUM>. The depth U<NUM> can be in a range from <NUM> microns to <NUM> microns, with a typical value of <NUM> microns.

<FIG> is a partial side view of a section of the PCR zone <NUM> shown in Figure 8C, cut along the axis Y-Y. As above, the microchannel <NUM> is formed in the substrate <NUM> and covered by the cover <NUM>. First and second heating elements <NUM>, <NUM> are arranged on and supported by the cover <NUM>.

<FIG> is a partial perspective view of the section of the PCR zone <NUM> shown in <FIG>. The microchannel <NUM> is shown extending through the second heating region <NUM> and back again with each segment of the microchannel <NUM>.

In an example, the pre-PCR zone <NUM> may be physically arranged on the substrate <NUM> near (e.g., adjacent) the PCR zone <NUM> in the same PCR channel <NUM>. More specifically, the pre-PCR zone <NUM> may be arranged near the first heating region <NUM> of the PCR zone <NUM>. This allows the pre-PCR zone <NUM> to be heated to a substantially same temperature as the temperature of the first heating region of the PCR zone <NUM>. The heating element <NUM> in the pre-PCR zone <NUM> may be electrically coupled to the first heating element <NUM> in the first heating region. In a case that the heating system <NUM> is used to heat the first heating region <NUM>, the same heating system <NUM> can also be used to heat the pre-PCR zone <NUM>.

<FIG> illustrates an example organization of a cartridge <NUM>. A cassette <NUM> supports a substrate <NUM> in a recessed area <NUM>. The substrate <NUM> has a first side <NUM> and a second side <NUM>. A micropump <NUM> receives a blood sample and pumps the blood sample to the lysing region <NUM>. After performing lysing on the blood sample, the lysing region <NUM> outputs the lysed blood sample to the binding region <NUM>. A blister 86b is arranged above the binding region <NUM>, which may be used to inject fluid, for example, a reaction material for conversion of the mRNA to cDNA in the binding region <NUM>. A single blister 86b is only an example. One or more blisters 86b may be used to inject fluid into the binding region <NUM>. The binding region <NUM> outputs material including cDNA to the mixing region <NUM>. In the mixing region <NUM>, gradients of the cDNA in the material are reduced or eliminated. The mixing region <NUM> outputs the material including the cDNA to four pre-PCR zones 252a, 252b, 252c, 252d. Four blisters 86e, 86f, <NUM>, <NUM> are arranged respectively above the four pre-PCR zones 252a, 252b, 252c, 252d. The four pre-PCR zones 252a. 252b, 252c, 252d output material respectively to the four PCR zones 254a, 254b, 254c, 254d. Additionally, the four blisters 86e, 86f, <NUM>, <NUM> can output primer including fluorescent markers to the material to be analyzed respectively in the four PCR zones 254a, 254b, 254c, 254d.

In an embodiment, DNA based cells (bacteria and other cells) can be analyzed. In this case, a DNA trapping region can be substituted for the RNA binding region. The trapped DNA can go directly to amplification in a PCR region <NUM>.

<FIG> is a diagram of an exemplary process <NUM> for performing bio-dosimetry on a sample with the bio-dosimetry system <NUM>. A sample size can be in a range of <NUM> to <NUM> microliters and can typically be approximately <NUM> microliters. An example sample is a drop of blood. The process <NUM> begins in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to initiate bio-dosimetry on a sample. The computer <NUM> may receive a trigger event, such as an input from the user via the user input device <NUM> to begin the bio-dosimetry process <NUM>, or inputs, for example from sensors <NUM>, that the sample is available in the reservoir <NUM> and a fresh cartridge <NUM> has been inserted into the bio-dosimetry device <NUM>. Based on the trigger event, the computer <NUM> may instruct the micropump <NUM> on the cartridge <NUM> to pump the sample from the reservoir <NUM> into the lysing region <NUM> on the cartridge <NUM>. Alternatively, in a case that the cartridge <NUM> does not include the micropump <NUM>, the computer <NUM> may instruct a pump <NUM> on the bio-dosimetry device <NUM> to pump the sample into the lysing region <NUM>. The process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to lyse the sample according to process <NUM>, described below. Lysing the sample is defined herein as breaking the sample into component parts. Upon lysing the sample, the process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to bind target components of the sample in the binding region <NUM> according to a process <NUM>, described below. Target components are components of the sample that the bio-dosimetry system <NUM> targets to remove from the lysed sample and contain in the binding region <NUM>. During the binding process, target components in the lysed sample are bound to the stationary phase <NUM> in the binding region <NUM>, and other components are discarded. The process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to convert target components from the sample bound in the binding region <NUM> to a material to be analyzed by PCR according to the process <NUM>, described below. For example, mRNA, bound in the binding region, can be converted to cDNA. Upon converting the bound target components into material to be analyzed by PCR, the process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to perform PCR analysis on the material to be analyzed according to the process <NUM> described below. The process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to store and/or output results of the PCR analysis. The computer <NUM> may output data on the display <NUM> on the bio-dosimetry device <NUM>. Additionally or alternatively, the computer <NUM> may transmit the data, for example via wireless communications to another computer. Still further, the computer <NUM> may be programmed to store the data from the PCR analysis for retrieval at a future time. Upon outputting and/or storing the data, the process <NUM> may end.

<FIG> is a diagram of an exemplary process <NUM> for lysing the sample received from the bio-dosimetry device <NUM> in an example lysing region <NUM>. The sample to be lysed may be a drop of blood. Alternatively, the lysing region may be used to lyse other biological samples such as gram positive bacteria, gram negative bacteria, and encapsulated bacteria such as Anthrax. The process <NUM> can be initiated by block <NUM> of process <NUM> and begins in the block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to pump the sample through the lysing region <NUM>. For example, the sample may be a drop of blood or other liquid with a size of <NUM> microliters. A micropump <NUM> on the cartridge <NUM> may perform the pumping (<FIG>). Alternatively, a pump <NUM> in the bio-dosimetry device <NUM> can perform the pumping. As an example, the sample can be pumped at a flow rate corresponding to a resonance time within the cavity <NUM> of the lysing array <NUM> in a range of <NUM> to <NUM> seconds. With an average resonance time of <NUM> seconds, and a cavity volume of four microliters, a sample can be lysed within a time of five minutes or less. The process <NUM> continues in a block <NUM>.

In the block <NUM>, which can occur concurrently with all or a portion the block <NUM>, the computer <NUM> is programmed to apply ultrasonic stimulation to the lysing array <NUM>. The lysing array <NUM> may include a piezo electric layer <NUM> as shown in <FIG>. Alternatively, the lysing array <NUM> may be coupled to a piezo electric element <NUM>, included in the bio-dosimetry device <NUM>, as shown in <FIG>. The computer <NUM> is programmed to activate the piezo electric layer <NUM> or piezo electric element <NUM> to induce flexing of the diaphragm <NUM> in the lysing array <NUM>. At points of maximum flexing, for example near a center of the diaphragm <NUM>, a displacement of the diaphragm <NUM> may be in a range from one to <NUM> microns, with a typical displacement of <NUM> microns, and may be in a direction substantially perpendicular to a plane of the diaphragm <NUM>. The induced movement can typically be at a frequency of <NUM>, with a range between <NUM> and <NUM>. The range is not intended to be limiting, and other frequencies above or below this range can also be used.

Based on the induced flexing of the diaphragm <NUM>, the micropillars <NUM> will be induced to move laterally, back and forth through their respective non-stimulated positions in the lysing array <NUM>. The induced lateral motion of the micropillars <NUM> can transfer mechanical energy to the sample, causing the sample to break into component parts. In an example, the sample includes human blood. Blood cells (leukocytes and/or erythrocytes) can be broken up to free mRNA from the cells. The process <NUM> continues in a block <NUM>.

In the block <NUM>, that can occur concurrently with all or a portion of the blocks <NUM> and <NUM>, the computer <NUM> is programmed to output the lysed sample to the binding region <NUM>. Based on pressure from one of the micropump <NUM> and the pump <NUM>, as described with regard to block <NUM>, the lysed sample passes through the outlet manifold <NUM>, through a microchannel formed in the substrate, and into the binding region <NUM>. The process <NUM> ends.

<FIG> is a diagram of an exemplary process <NUM> for binding components of the sample in the binding region <NUM>. The process <NUM> can be initiated by block <NUM> of process <NUM> and begins in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to pump the lysed sample through the binding region <NUM>. The block <NUM> can occur concurrently with all or a portion of the process <NUM>, as described above. That is, one of the micropump <NUM> or the pump <NUM> can pump the sample into the lysing region <NUM> and continue pumping such that the sample continues to flow into the binding region <NUM>. As described above, a volume of the sample in a typical case can be <NUM> microliters. The volume of the lysing array <NUM> can be in a range of four microliters. Accordingly, a portion of the sample can have exited the lysing region <NUM> and entered the binding region <NUM> while another portion of the sample has not yet entered the lysing region <NUM>, such that a continuous flow exists from the reservoir <NUM>, through the lysing region <NUM> and through the binding region <NUM>.

As the lysed sample flows through the binding region <NUM>, the binding region <NUM> binds to target components in the sample, containing the target components. As described above, the binding region <NUM> includes a stationary phase <NUM>. The stationary phase <NUM> includes a plurality of micropillars <NUM>. The micropillars <NUM> may be coated with a binding material that binds with target components in the lysed sample.

As the lysed sample flows through the binding region <NUM>, the target components bind with the binding material coated on the micropillars <NUM>. During the block <NUM>, a sufficient quantity of the lysed sample can be passed through the binding region <NUM> such that the binding material on the stationary phase <NUM> is saturated. That is, substantially every site available to bind with a target component is occupied by a target component. As an example, the target components may be mRNA cells. The binding material may be oligo dT25 that specifically binds to any mRNA present in the sample. An amount of lysed sample through the binding region <NUM> to saturate the stationary phase <NUM> for a set of target components can be determined empirically. For example, for the binding region <NUM> as described above, including a stationary phase <NUM> coated with oligo dT25, a quantity of lysed sample of <NUM> microliters was determined to be sufficient to saturate the stationary phase <NUM>.

As the sample continues to be pumped through the lysing region <NUM> and the binding region <NUM>, waste material, after passing through the binding region, can be discarded. The computer <NUM> can be further programmed to turn off the micropump <NUM> or pump <NUM> when sufficient material has been passed through the binding region to saturate the stationary phase <NUM>. The process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to rinse the binding region <NUM>. The computer <NUM> sends instructions to force a rinsing fluid through the binding region <NUM>. For example, the rinsing fluid may be contained in a blister <NUM> arranged at the inlet <NUM> of the binding region <NUM>. The computer <NUM> may instruct a blister actuator <NUM> in the bio-dosimetry device <NUM> to actuate the blister <NUM>. The blister actuator <NUM> may extend a plunger into the blister <NUM>, expelling contents of the blister <NUM> into the binding region <NUM>. The rinsing fluid may be selected to be a material that mixes with the non-bound material in the binding region but does not react with the target components. In this manner, the target components can remain bound in the binding region <NUM> after rinsing. The process <NUM> continues in a block <NUM>.

In the block <NUM>, which can occur during all or a portion of the block <NUM>, the non-bound material, mixed with the rinsing fluid is discarded. The process <NUM> ends.

<FIG> is a diagram of an exemplary process <NUM> for converting the target components of the sample, bound in the binding region <NUM>, to material for analysis. The process <NUM> can be initiated by block <NUM> of process <NUM> and begins in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to flush the binding region <NUM> with a reaction mixture. For example, the cartridge <NUM> can include a blister 86b containing the reaction mixture fluidly coupled to the inlet <NUM> of the binding region <NUM>. The computer <NUM> can instruct a blister actuator <NUM> to actuate the blister 86b such the reaction mixture is forced into and through the binding region <NUM>. The reaction mixture can include the components necessary to convert the bond target components in the binding region <NUM> to material to be analyzed.

In an example, the target components may be mRNA from the sample. The reaction mixture can include the components necessary to convert, through reverse transcription, the mRNA to cDNA which can then be subjected to PCR analysis. The process <NUM> continues in a block <NUM>.

In the block <NUM>, components in the reaction mixture react with the target components that are bound in the binding region <NUM>, forming material to be analyzed. In some cases, heat may be applied to the binding region <NUM> to cause/support the reaction. For example, in the case of forming cDNA from mRNA bound in the binding region <NUM>, the computer <NUM> cay be programmed to heat the binding region <NUM> to <NUM> for <NUM> minutes. The newly formed material may be bound to the stationary phase <NUM> of the binding region <NUM>. The process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to release the material to be analyzed for analysis. For example, in the case that the material to be analyzed is cDNA, the cDNA may be bound to the stationary phase <NUM>. In this case, the computer <NUM> can be programmed to heat the binding region to <NUM> for <NUM> minutes + sufficient time to release the cDNA. A time that is sufficient to release the cDNA can be determined empirically by performing tests with sample cartridges <NUM>. The process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to remove gradients of the material to be analyzed in the reaction mixture. The computer <NUM> sends commands to pump the reaction mixture through the mixing region <NUM>. As the reaction mixture flows through the mixing region <NUM>, the material to be analyzed, for example, cDNA, is distributed throughout the reaction mixture, reducing or eliminating gradients of the cDNA. The process <NUM> ends.

<FIG> is a diagram for a process <NUM> for performing qPCR analysis on the material to be analyzed. The process <NUM> includes thermally cycling the material to be analyzed between a first temperature and a second temperature, and then imaging the material to be analyzed using fluorescent markers. The process <NUM> can be initiated by block <NUM> of process <NUM> and begins in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to distribute the reaction mixture including the material to be analyzed to four PCR channels 250a, 250b, 250c, 250d for analysis. The four PCR channels 250a, 250b, 250c, 250d include respectively four pre-PCR zones, 252a, 252b, 252c, 252d. As shown in <FIG>, the four pre-PCR zones 252a, 252b, 252c, 252d (collectively pre-PCR zones <NUM>) may be associated respectively with the four PCR zones 254a, 254b, 254c, 254d. The computer <NUM> can activate a pump <NUM> to pump the reaction mixture into the pre-PCR zones 252a, 252b, 252c, 252d.

In an example, the microchannel <NUM> coupling the mixing region <NUM> to the four pre-PCR zones 252a. 252b, 252c, 252e can split into four sub-channels 98a, 98b, 98c, 98d. The reaction mixture including the material to be analyzed can be pumped through the microchannel, and divide between the four sub-channels 98a, 98b, 98c, 98d, under pressure from the pump <NUM>, such that a portion of the reaction mixture including the material to be analyzed is directed toward each of the four pre-PCR zones 252a, 252b, 252c, 252d.

The process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> may be programmed to heat the reaction mixture including the material to be analyzed in the pre-PCR zones 252a, 252b, 252c, 252d. For example, the material to be analyzed may include cDNA. The computer <NUM> may be programmed to heat the reaction mixture including the material to be analyzed, to separate the cDNA into single strands prior to adding the primers and markers. The computer <NUM> may be programmed to heat the reaction mixture to <NUM> for <NUM> minutes. Other temperatures and times can be used for different materials to be analyzed. The times and temperatures can be determined empirically. The process continues in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to add a set of markers and primers to the reaction mixture flowing respectively into each of the four PCR zones 254a, 254b, 254c, 254d. A different set of markers may be used respectively for each of the four PCR zones 254a, 254b, 254c, 254d. For example, as shown in <FIG>, a first blister 86e may be coupled to add markers and primers to a microchannel between the pre-PCR zone 252a and PCR zone 254a, a second blister 86f may be coupled to add markers and primers to a microchannel between the pre-PCR zone 252b and PCR zone 254b, a third blister <NUM> may be coupled to add markers and primers to a microchannel between the pre-PCR zone 252c and the PCR zone 254c and a fourth blister <NUM> may be coupled to add markers and primers to a microchannel between the pre-PCR zone 252d and the PCR zone 254d. At a time that the reaction mixture including the material to be analyzed is flowing from the pre-PCR zone 252a to PCR zone 254a, the computer <NUM> may actuate the first blister 86e. At a time that the reaction mixture including the material to be analyzed is flowing from the pre-PCR zone 252b to PCR zone 254b, the computer <NUM> may actuate a second blister 86f. Similarly, the computer <NUM> may actuate a third blister <NUM> at a time that the reaction mixture including the material to be analyzed is flowing from the pre-PCR zone 252c to PCR zone 254c and a fourth blister <NUM> at a time that the reaction mixture including the material to be analyzed is flowing from the pre-PCR zone 252d to PCR zone 254d. The times that the reaction mixture is flowing respectively from pre-PCR zone 252a to PCR zone 254a, from pre-PCR zone 252b to PCR zone 254b, from pre-PCR zone 252c to PCR zone 254c and from pre-PCR zone 252d to PCR zone 254d may be the same, partially overlapping, or different, depending on an organization of the microchannel and four sub-channels between the mixing region <NUM> and the four pre-PCR zones <NUM>. For example, each of the four sub-channels coupling the respective pre-PCR zones 252a, 252b, 252c, 252d to the microchannel coming from the mixing region <NUM>, may be a same length or a different length. The lengths of the sub-channels may impact a timing for adding the sets of markers to the reaction mixture for the respective PCR zones 254a, 254b, 254c, 254d.

The primers for each PCR zone <NUM> are selected to mark prepare a particular component or pair of components in the material to be analyzed for qPCR analysis. For example, for each of the four PCR zones 254a, 254b, 254c, 254d, respective primers containing fluorescent markers may be selected to allow two genes (or DNA sequences) to be evaluated simultaneously. One gene may be an endogenous control (EC) gene that is stable with regard to exposure to radiation. An example EC gene is RB1. A second gene may be a radiation response gene. Examples of radiation response genes include CDKN1A, ASTN2, and GDF15. The process continues in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to pump the reaction mixture, including the material to be analyzed and combined with the primers, into the respective PCR zones 254a, 254b, 254c, 254d. The blocks <NUM> through <NUM> will be described with respect to an example PCR zone <NUM>. It can be understood that the process of block <NUM> can be applied similarly to each of the PCR zones 254a, 254b, 254c, 254d.

The computer <NUM> may, for example, instruct a pump <NUM> in the bio-dosimetry device <NUM> to pump the reaction mixture including the material to be analyzed and the primers into the example PCR zone <NUM>. As described above, the PCR zone <NUM> includes a microchannel <NUM>. The microchannel <NUM> is formed of a plurality of segments. Each segment extends back and forth between a first heating region <NUM>, a detection region <NUM> and a second heating region <NUM>. In an example, the microchannel <NUM> includes <NUM> segments.

As an example, the material to be analyzed includes cDNA. The computer <NUM> may be programmed to maintain a first temperature of the first heating region at <NUM> and to maintain second temperature of the second heating region at <NUM>. As the reaction mixture together with the material to be analyzed and the primer passes through the first heating region <NUM>, the <NUM> temperature causes the cDNA to separate into single strands. As the reaction mixture together with the material to be analyzed and the primer passes through the second heating region <NUM>, the <NUM> temperature allows single stranded primers to attach to single stranded DNA to form complementary strands. As the reaction mixture continues to pass through the segments of the microchannel <NUM>, the material to be analyzed continues to thermally cycle, that is, to cycle between the first and second heating regions <NUM>, <NUM> at a rate of one cycle per segment, such that, with each consecutive segment, a greater number of strands of the cDNA form complementary strands with the primer.

The computer <NUM> can be programmed to continue to pump the reaction mixture in the PCR zone <NUM> until all or most of the microchannel <NUM> is filled with the reaction mixture. In this case, the farther through the microchannel <NUM> the reaction mixture has passed, the greater number of cycles of the first and second heating regions the reaction mixture will have experienced. The process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> is programmed to illuminate the microchannel <NUM> in the PCR zone <NUM>. The computer <NUM> may radiate the detection region <NUM> of the PCR zone <NUM> with light for a time. The time may be in a range of from <NUM> to <NUM> seconds, with a typical time of <NUM> seconds. The process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> receives images from the detection region <NUM> of the PCR zone <NUM>. The fluorescent markers that combined with the cDNA will fluoresce at wavelengths determined by the markers. The optical detector <NUM> in the bio-dosimetry device <NUM> receives the light fluoresced from the markers. Before being received by the optical detector <NUM>, a filter may be used to select which wavelength of light can be received by the optical detector <NUM>. As described above, in the case that different markers were used for two different components of the material to be analyzed (e.g., two different genes), a first image can be received from the detection region <NUM> with a first filter for a first range of wavelengths and a second image can be received from the detection region <NUM> with a second filter for a second range of wavelengths. The optical detector <NUM> can provide data representing the first and second images to the computer <NUM>. The process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> analyzes the data of the first and second images. For example, the computer <NUM> may determine the Ct of each of the genes combined with a respective fluorescent marker. The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to exceed background level. The number of cycles required for the fluorescent signal to exceed the background level can be determined by a position within the PCR detection region <NUM> along an axis of increasing cycles that shows a radiation of light at the wavelength of the particular fluorescent material that exceeds the radiation of the background. The position can be directly correlated to a number of cycles to which the material to be analyzed (i.e., the genes with the fluorescent material currently being measured) were exposed before their collective illumination exceeded the background illumination. A measurement taken with an example PCR zone is described below in reference to <FIG>. The process <NUM> continues in a block <NUM>.

In the block <NUM>, the computer <NUM> reports and/or stores the results of the PCR analysis. The computer <NUM> may report the results to another computer and/or on a display such as the display <NUM> on the bio-dosimetry device <NUM>. Further, the computer <NUM> may store the results, for example with a time stamp, or an identification of the cartridge that was measured, in memory associated with the computer <NUM>. The process <NUM> ends.

As used herein, the adverb "substantially" modifying an adjective means that a shape, structure, measurement, value, calculation, etc. may deviate from an exact described geometry, distance, measurement, value, calculation, etc., because of imperfections in materials, machining, manufacturing, data collector measurements, computations, processing time, communications time, etc..

As an example, the bio-dosimetry system <NUM> can be used to determine radiation exposure of an individual. A drop of blood from the individual can be evaluated to determine the radiation dose they received based on their gene expression levels. As evidence of efficacy ex vivo irradiation of human blood was utilized and the subsequent culturing of that blood for quantitative study of utilizing gene expression for human dosimetry.

Selection of valid control genes is critical to have a point of reference to evaluate radiation responsive genes. Several very stable endogenous control (EC) gene products were identified. As an example, <FIG> illustrates a fold increase in expression level as a function of dose for the gene RB1 at <NUM> hours (trace <NUM>), <NUM> hours (trace <NUM> and <NUM> hours (trace <NUM>). This EC gene is stable with respect to dose and time. It is also stable in its expression across all donors in our study.

Having selected a series of EC genes as controls, radiation responsive genes can be evaluated by examining the gene expression level of a potential response gene relative to that of a selected EC gene for exposed sample. A fold increase in expression levels as a function of dose and time following exposure can be determined by comparing that ratio to the population average of the non-exposed ratio of those same genes. The results for three potential radiation response genes (CDKN1A, ASTN2, and GDF15) that can be utilized as dosimetry biomarkers as shown respectively in <FIG>, <FIG> referenced to RB1.

<FIG> illustrates a fold increase of expression level as a function of dose for the gene CDKN1A at <NUM> hours (trace <NUM>), <NUM> hours (trace <NUM>) and <NUM> hours (trace <NUM>).

<FIG> illustrates a fold increase of expression level for the gene ASTN2 at <NUM> hours (trace <NUM>) <NUM> hours (trace <NUM>) and <NUM> hours (trace <NUM>).

<FIG> illustrates a fold increase of expression level as a function of dose for the gene GDF15 at <NUM> hours (trace <NUM>), <NUM> hours (trace <NUM>) and <NUM> hours (trace <NUM>).

For each of these genes CDKN1A, ASTN2 and GDF15, the curves show clear increases in expression for each of the times post irradiation examined. In a case of a nuclear event, the time of the nuclear event is known. A user can, in this case, follow the appropriate dosimetry curve for each gene product. All the gene products shown, and many others, exhibit significant increases in genes expression even at doses as low as <NUM> Gy. In the case of some genes, up to <NUM>-fold increases of expression can be observed at higher doses. Fold changes in gene expression of <NUM> or <NUM> are large can easily and accurately be detected by qPCR analysis, indicating that these changes will be readily detectable by a portable PCR system.

In the case of several radiation responsive genes such as CDKN1A (<FIG>) gene expression levels slowly increase with time post-exposure and were highest at the longest time point examined in our studies. While other gene products such as ASTN2 and GDF15 (<FIG>) rapidly increase at very short times post exposure and then decrease with time, they still maintain an easily-detectable increase in expression out to <NUM> hours post exposure. Also of note is that while a number of gene products such as CDKN1A and GDF15 (<FIG> and 14D) show a response to radiation over the entire range of doses examined, other gene products such as ASTN2 (<FIG>) show a relatively steep dose response over a short range of doses and plateau in their expression at higher doses. This wide range in dose and time responses observed in the radiation response genes identified provides the ability to select a series of genes that can be utilized and optimized for dosimetry at various exposure levels and times post exposure.

<FIG> illustrates a fold increase of expression of the gene CDKN1A v. dose for a in vivo radiation utilizing an animal model (C57BI/<NUM> male mice). Traces <NUM>, <NUM> and <NUM> show the fold increase in expression of CDKN1A v. dose respectively at <NUM> day, <NUM> days and <NUM> days. Although direct comparisons of gene expression levels between humans and mice cannot be made, the measurements indicate that fold increases in CDKN1A expression due to radiation exposure in vivo can be detected in the animal models.

<FIG> illustrates a comparison between human cancer patients irradiated in vivo and blood samples from these patients irradiated ex vivo. Sample of blood was drawn from the patients immediately prior to the patient receiving total body irradiation doses of between <NUM> and <NUM> Gy from a Cobalt <NUM> source A portion of the pre-radiation therapy blood sample was then irradiated with the same source and conditions as that received by the patient, and this blood was then cultured in the absence of mitogen for <NUM> hours. A second blood sample was drawn from the patient at approximately <NUM> hours post exposure. An in vivo / ex vivo exposure comparison for one of the genes examined (CDKN1A) was referenced to DPM1. Similar increase in gene expression can be observed in both the in vivo and ex vivo irradiated samples for each patient. In this study (#<NUM>, #<NUM>, #<NUM>) were given higher levels of chemotherapy while the later patients (#<NUM>, #<NUM>, #<NUM>, #<NUM>) were given lower doses of chemotherapy in their treatment. In these later patients (#<NUM>, #<NUM>, #<NUM>, #<NUM>), increases in gene expression from in vivo exposure closely match those seen in ex vivo exposure for CDKN1A shown as well as for other genes examined, and in several cases such as CDKN1A expression increases match those seen in samples from healthy donors. The fact that gene expression levels for several genes, which could be utilized for radiation dosimetry, remain viable as biomarkers even in cancer patients receiving light doses of chemotherapy is a good indication of the robustness of these markers and to their potential resistance to confounding factors in the general population.

Both the results from our mouse study and our evaluation of TBI patients support the validity of the use of ex vivo irradiated blood as a valid model for radiation exposure in humans for the study of gene expression from a blood sample. The animal study showed that comparable gene expression increases, resulting from radiation exposure, occur in an in vivo irradiation animal model as those seen with the ex vivo irradiation of cultured blood samples. The animal study further confirms that gene expression is a valid and feasible dosimetry method even at times of seven days post exposure and possible beyond. In vivo / ex vivo comparisons of gene expression levels in human cancer patients confirm that increases in gene expression due to radiation exposure for the genes examined in our study are not dependent on whether the radiation exposure was performed in vivo or ex vivo.

The results from individual genes as shown in <FIG> illustrate a time dose dependence to the genes expression levels. To accurately perform dosimetry based on data from these genes, a statistical model is required. To develop a statistical model, multiple predictive models to determine dosimetry were developed for the various time points post exposure. These regression models, a quadratic fit to the log of dose, were fit using forward stepwise regression. At each step a gene was added to the model based on the following: (a) the greatest contribution to increasing R-squared and (b) maintaining the available sample size. The statistical model for time point post exposure is shown in <FIG> where the actual radiation dose is plotted against the model's predicted dose. The genes utilized in this model were CDKN1A, ATM, ASTN2, and GDF15. <FIG> compare modeling results with ex vivo human data respectively for <NUM> hours, <NUM> hours and <NUM> hours post exposure.

While the modeling of the gene expression data is modeled with respect to the log of dose, due to the exponential nature of Ct values, this data is converted to a linear scale for the plots in <FIG>. It is this conversion from the native log scale of the model to a linear dose for plotting that makes the data at high doses in <FIG> appears to be more scattered then at the lower doses.

An example result is shown in <FIG> where a receiver operating characteristic (ROC) curve for a prediction of exposure of <NUM> Gy or greater is shown at <NUM> hours post exposure. For the data shown in <FIG>, the area under the ROC curve was <NUM> (value of <NUM> being perfect), which is very good for biomedical screening application. Similar ROC curves were obtained for the predictive models for the other post exposure time points examined in the ex vivo human study and for the data from the animal model. In order to further reduce any false negatives, sensitivity could be maximized at the cost specificity resulting in increased false positives.

Tests were performed on an example lysing device to estimate the lysing capability of the lysing region <NUM> described above. The example lysing device used for the test had included <NUM> micropillars arranged in a <NUM> by <NUM> lysing array. The micropillars were supported on a <NUM> x <NUM> x <NUM> thick diaphragm. The micropillars had a diameter of <NUM> and a height of <NUM>. The spacing between the micropillars was <NUM>. To estimate the effectiveness of the lysing region, PCR analysis was performed on four different samples exposed to varying degrees of lysing. The results are of the tests are shown in <FIG>, which illustrates example traces of cycle threshold (Ct) from PCR analysis of the four different blood samples. The Ct level indicates a number of remaining unlysed lymphocites in each blood sample.

Trace <NUM> in <FIG> is an example trace from PCR analysis of whole blood that is passed through an example lysing device without powering the lysing device.

Trace <NUM> is an example trace from PCR analysis of whole blood that was not passed through the lysing device (a sample representing the condition of the blood as input into the lysing device).

Trace <NUM> is an example trace from PCR analysis of a control_lynmphocyte GM40. The control lymphocyte is a set amount that is injected as a known control for reference.

Trace <NUM> is an example trace from PCR analysis of whole blood that is passed through the lysing device under power. That is, the diaphragm is actuated at <NUM> while the whole blood is passed through the lysing device.

<FIG> illustrates an example image of a PCR zone <NUM> containing material to be analyzed that has been illuminated and is fluorescing. As described above, the PCR zone <NUM> includes a microchannel <NUM> including a plurality of segments. Each segment cycles through a first heating region <NUM>, a detection region <NUM> and a second heating region <NUM>.

In the test, material to be analyzed is pumped into the microchannel <NUM> in the PCR zone <NUM>. In an example, sufficient material can be pumped into the microchannel <NUM> that all of the plurality of segments contain material. When, the microchannel <NUM> is filled in this manner, the detection region <NUM> in the PCR zone <NUM> can be illuminated. Following illumination, an image can be taken of the detection region <NUM>. The resulting image can show, by an intensity of fluorescent light at locations (i.e., in different segments) of the microchannel <NUM>, the cycle threshold (Ct) required to detect a component (for example, a gene) within the material to be analyzed. The cycle threshold (Ct) is defined as the number of cycles (through the first and second heating regions <NUM>, <NUM>) required for the fluorescent signal from the tag to exceed background level. The cycle threshold provides an indication of a degree of exposure to radiation of the original sample used to prepare the material to be analyzed.

In <FIG>, the number of cycles of the PCR to which the material to be analyzed has been exposed increases from left to right. Imaging is performed in the detection region <NUM> between the first and second heating regions <NUM>, <NUM> (see, for example, <FIG> and <FIG>). In the area labelled <NUM>, the fluorescing signal from the material to be analyzed begins to exceed the background level. This is indicated by the dark oval shaped areas <NUM> in the middle of the image. As can be seen, the intensity of the fluorescing signal continues to increase from left to right. This increasing discontinues at approximately a point <NUM>. This indicates that the material to be analyzed was only pumped into the PCR zone up to this point.

<FIG> shows an example amplification of two targeted genes with a PCR. The first target gene was cDNA made from human <NUM> rRNA. The sample was run through the sample PCR and yielded a Ct (PCR cycled threshold) value of <NUM> (trace <NUM>). The same sample was then diluted by a factor of <NUM> to <NUM> and run on the PCR. This dilution should correspond to a Ct shift of ~ <NUM> cycles. The resulting trace <NUM> corresponds well with this expected shift.

With regard to the media, processes, systems, methods, etc. described herein, it should be understood that, although the steps of such processes, etc. have been described as occurring according to a certain ordered sequence, such processes could be practiced with the described steps performed in an order other than the order described herein. It further should be understood that certain steps could be performed simultaneously, that other steps could be added, or that certain steps described herein could be omitted. In other words, the descriptions of systems and/or processes herein are provided for the purpose of illustrating certain embodiments and should in no way be construed so as to limit the disclosed subject matter.

Accordingly, it is to be understood that the present disclosure, including the above description and the accompanying figures is intended to be illustrative and not restrictive. Many embodiments and applications other than the examples provided would be apparent to those of skill in the art upon reading the above description. The scope of the invention should be determined, not with reference to the above description, but should instead be determined with reference to claims appended hereto.

Claim 1:
A cartridge (<NUM>) comprising:
a substrate (<NUM>) including a polymerase chain reaction (PCR) zone (<NUM>) including:
a first heating region (<NUM>);
a second heating region (<NUM>) spaced away from the first heating region (<NUM>);
a detection region (<NUM>); and
a microchannel (<NUM>) formed in the substrate (<NUM>), the microchannel (<NUM>) configured to receive a fluid flowing therethrough, the microchannel (<NUM>) passing through the first heating region (<NUM>) and second heating region (<NUM>) to thermally cycle the fluid, and the microchannel (<NUM>) passing through the detection region (<NUM>) after the fluid has been thermally cycled, the cartridge further comprising: a cover (<NUM>) having a first side (<NUM>) and second side (<NUM>), the second side (<NUM>) of the cover (<NUM>) attached to a first side (<NUM>) of the substrate (<NUM>) and enclosing the microchannel (<NUM>), the cartridge characterized by,
a first heating element (<NUM>) arranged on the first side (<NUM>) of the cover (<NUM>) in the first heating region (<NUM>); and
a second heating element (<NUM>) arranged on the first side (<NUM>) of the cover (<NUM>) in the second heating region (<NUM>).