Patent Description:
Metabolism is a vital cellular process, and its malfunction can be a major contributor to many diseases. Metabolites (i.e. substances involved in metabolism) can be good indicators of disease phenotype, and can serve as a metabolic disease biomarkers. Therefore quantification and analysis of metabolites can play a significant role in the study and early diagnosis (detection) of many diseases.

Metabolite biomarkers of different diseases are also becoming increasingly well understood which paves the way for developing new diagnostic systems. The importance of the link between metabolomics and a person's state of health is governing the need to look at both targeted and untargeted metabolites. A single metabolite can be a biomarker for several different diseases. In addition, multiple metabolites together can serve as a biomarker for a particular disease. It is therefore often necessary to detect and quantify the presence of multiple metabolites in order to accurately identify a disease. Metabolite biomarker profiling deals with the screening of a huge number of metabolites, and there are a particular panel of metabolite biomarkers which are good indicators of a person's state of health. In particular, multiplexed assaying, where multiple biomarkers are simultaneously measured in a single run, has the capacity to provide resource-rich information for decision making and prognosis, leading to the correct diagnosis and treatment for complex disease conditions such as stroke, cancer and cardiovascular diseases where directed therapy is important.

A commonly used technique for detecting and quantifying metabolites is mass spectrometry (MS). This involves ionising a chemical species and sorting the product ions based on their mass-to-charge ratios. Separation methods such as gas chromatography and liquid chromatography are often required prior to performing a mass spectrometry measurement. Nuclear magnetic resonance (NMR) spectroscopy is another technique which is used for metabolite studies. NMR can be used to detect, identify and quantify a wide range of metabolites without having to first separate them. However, both of these techniques require bulky and expensive equipment, which confines their use to hospitals and laboratories.

As an example, elevated cholesterol levels are well known for their association with an increased risk of coronary heart disease (angina or heart attack), narrowing of the arteries (atherosclerosis), stroke, peripheral heart disease and hypertension. Such conditions are often correlated with poor diet, an excessive fat intake, lack of exercise and other lifestyle choices. Measuring or therapeutic monitoring of cholesterol level in blood serum helps to assess susceptibility of the person to develop coronary artery diseases and hence is a good indicator of the state of health of a person. One of the diagnostic methods for quantifying cholesterol concentration depends on enzyme-based assays that require a spectrophotometer to measure changes in intensity of colour products from those enzyme reactions. A general purpose spectrophotometer would incorporate a sophisticated setup of a white light source, a monochromator containing a diffraction grating and a light transducer that converts light into electrical signal such as a charge-coupled device (CCD), a photodiode or a photomultiplier tube. The wide spectrum range of the spectrophotometer makes it bulky and power hungry which consequently confines its usefulness to laboratories and hospitals. Another method involves metabolites undergoing chemiluminescence reaction. This method requires a more specialised light detector such as a charge-coupled detector (CCD) to detect low light emission of luminol that is used for quantification of small analyte concentration.

More recently, use of a photodiode in a disposable sensing platform to measure change in colour of enzyme assay has been demonstrated as a means of detecting cholesterol by measuring intensity of transmitted light through assay solution [<NUM>]. The platform was based on a complementary metal oxide semiconductor (CMOS)-based photodiode array and an off-the-shelf light emitting diode (LED). The photodiode array is fabricated using commercial standard CMOS process, which is readily available for low-cost mass-production.

Photodiodes made in a CMOS process are generally sensitive to light in the <NUM> to <NUM> range, owing to the bandgap of silicon (<NUM> eV). This range makes them suitable for colorimetric enzyme assays that use visible light or fluorescent mediators, which often use wavelengths in the range <NUM> to <NUM>. A colour change within this range can be exploited for a range of enzyme assays, e.g. cholesterol ester hydrolase, cholesterol dehydrogenase, cholesterol esterase and cholesterol oxidase can be exploited to measure metabolites such as cholesterol. For metabolites with low concentrations, more sensitive CMOS compatible detector such as single photon avalanche diode (SPAD) can be integrated on the same chip and therefore, increase the dynamic detection range.

In other recent work, another type of CMOS-based chip fabricated with an integrated ion-sensitive field effect transistor (ISFET) array was used to measure glucose concentration in blood through the activity of hexokinase. The action of the hexokinase on the glucose releases hydrogen ions that are detected by the ISFET [<NUM>].

Point of care diagnostics are transforming the healthcare industry, by facilitating the use of home-testing to provide an early indication of potential illness and disease. The development of low-cost, rapid, specific and high sensitivity consumable biosensors are at the forefront of the research for user-orientated testing, driven in part by the need for rapid diagnosis and monitoring without overburdening the resources of the healthcare services. For example, glucose biosensors have become widespread in their use for managing diabetes. Liquid delivery is crucial for point of care (i.e. portable) diagnostic devices. A paper strip (e.g. chromatography paper or nitrocellulose membrane) has been proven to be an effective approach for delivering liquid samples. One well known example is the pregnancy test which measures the level of human chorionic gonadotropin (HCG) in human blood or urine to give an indication of pregnancy status.

<CIT> discloses a smartphone-based technique for imaging a colorimetric reaction of a target sample on a test strip.

<CIT> discloses an apparatus comprising an array of sample-retaining regions capable of retaining a chemical or biological sample. Each sample-retaining region may have a chemical field transistor associated with it and configured to generate at least one output signal related to a characteristic of the sample.

<CIT> discloses analytical systems, devices, and cartridges therefor, for detecting or quantifying at least two different analytes using at least two different techniques, in a single sample. The cartridges typically comprise at least two test sites and the location of at least one test site is not dependent on a corresponding measurement device. The systems generally comprise a device, memory, and a processing module. The device comprises a light source, an array detector, and a port configured to accept at least a portion of a cartridge. The processing module is configured to perform an image analysis of the cartridge. The methods comprise the steps of acquiring calibration information, acquiring an image of the cartridge, performing an image analysis, and cycling through specific detection or quantification techniques corresponding to the techniques required by the test sites. Computer readable media are also described.

<CIT> discloses an integrated test device for optical and electrochemical assays and, more particularly, test devices having the ability to perform optical and electrochemical assays and methods of performing optical and electrochemical assays using such test devices. Such devices may be particularly useful for performing immunoassays and/or electrochemical assays as the point-of-care.

<CIT> discloses a method of sensing an analyte that comprises: passing a fluid over a sensor array, the sensor array comprising at least one particle positioned within a cavity of a supporting member, wherein the particle comprises a polymeric resin and a receptor coupled to the polymeric resin; monitoring a spectroscopic change of the particles as the fluid is passed over the sensor array, wherein the spectroscopic change is caused by the interaction of the analyte with the particle. The receptor can be an enzyme substrate and the analyte an enzyme such as protease, nuclease or glycosidase.

At its most general, the present invention provides a CMOS-based chip having one or more sensing modalities that are able independently to detect multiple biomarkers (e.g. metabolites that acts as metabolic biomarkers) present in a sample. In particular, the invention relates to a scenario in which detection by the multiple sensing modalities occurs at different locations with respect to the chip, whereby the chip can simultaneously detect a plurality of metabolites by measuring behaviour of a test material in the different locations. With this technique, multiple metabolites may be measured in real time using a small scale point-of-care device.

According to first and second aspects of the invention, there is provided an apparatus for detecting biomarkers in a biological sample as set out in claims <NUM> and <NUM> respectively. The apparatus comprises: a sample receiving module arranged to receive the biological sample and transport it to a reaction zone for testing, wherein the reaction zone comprises a first testing region and a second testing region spatially separated from the first testing region, wherein properties of the first testing region and the second testing region are affected by the presence of metabolites to be detected; and a CMOS-based sensor unit disposed in relation to the reaction zone to detect independently the properties of the first testing region and the second testing region thereby to obtain separate signals indicative of the presence of metabolites in each of the first testing region and the second testing region. By probing different regions of a reaction zone, the invention effectively provides a multiplexed measurement system, where separate signals corresponding to different metabolites can be obtained from the same device. This is particularly useful where the first and second testing regions detected by the sensor unit are independently affected by one or more metabolites to be detected, i.e. there is substantially no cross-talk between the signals.

Herein the phrase "CMOS-based" may mean that the device is capable of fabrication using conventional semiconductor chip processes, e.g. comprising a series of depositing, masking and etching steps on a substrate. The sensor unit and its constituent components may thus be semiconductor components. This may enable the sensor unit to be mass-produced at low cost. The apparatus may thus be embodied as a compact hand-held device which is easily transportable, thus facilitating rapid point-of-care diagnostics. Compared with current analytical methods for metabolite detection and quantification, no expensive detection equipment is required.

The sensor unit itself may resemble a semiconductor chip, and may have mounted thereon or connectable thereto means for controlling and processing the chip functions. For example, the apparatus may comprise a controller, e.g. a microprocessor or the like, arranged to send and receive signals from the sensor unit. For example, the controller may be arranged to activate the sensor unit by applying an appropriate voltage.

The properties of the first testing region and the second testing region that are detected by the sensor unit may be physical or chemical. For example, the sensor unit may be arranged to detect changes in appearance, chemical composition, mass, temperature, etc..

The reaction zone may have more than two discrete testing regions. For example, there may be three, four, five or more testing regions, as space allows. References to the first and second testing region below should be understood as being equally relevant to examples with more than two testing regions.

In the first aspect of the invention, the first testing region and the second testing region are sensitive to different metabolites. That is, the first testing region is sensitive to a first metabolite, and the second testing region is sensitive to a second metabolite, whereby the separate signals are indicative of the presence of the first metabolite and second metabolite respectively. The reaction zone may include a control region that is not sensitive to the presence of metabolites to be detected, e.g. to provide a reference signal against which signals from the first testing region and second testing region can be compared.

The first testing region and the second testing region may be physically separated to prevent cross-talk therebetween. For example, a fluid flow barrier may separate the first testing region from the second testing region to inhibit or prevent transfer of the biological sample therebetween.

In the second aspect of the invention, the first testing region and the second testing region each comprise a respective micro-well, the micro-wells being separated from each other by a barrier portion. The barrier portion may be a raised part of the reaction zone between the micro-wells. It may be shallow, e.g. having a height relative to the base of the micro-wells in the range <NUM> to <NUM>, preferably <NUM> to <NUM>. The reaction zone may include four or more micro-wells. The micro-wells may be pre-loaded with an enzyme (and/or other reagents) in preparation to react with a substance in a sample.

The first testing region and/or the second testing region may comprise a test material arranged to support a metabolite-activated reaction upon receiving the biological sample. For example, each of the first testing region and/or the second testing region may each comprise an assay region, e.g. located within a microfluidic channel. The microfluidic channel may be pre-loaded with a test solution comprising one or more enzymes. Properties of the test solution may change due to a reaction between the enzymes and metabolites to be detected.

The biological sample is typically a liquid. In one example, the sample receiving module comprises a paper strip or other capillary structure for transporting the liquid biological sample to the reaction zone, e.g. by capillary action. Any suitable material that exhibit a wicking ability may be used for the paper strip. For example, chromatography paper or nitrocellulose membrane can be used. The paper strip may be disposed over the CMOS-based sensor unit, i.e. to carry the biological sample directly into or over the reaction zone.

In one example, the reaction zone includes microfluidic channels arranged to draw the biological sample away from the paper strip. In this case, the first testing region and the second testing region may be disposed on the CMOS-based sensor unit. However, in other examples the first testing region and the second testing region are integrally formed in the paper strip. This may enable the sensor unit to be used for different combinations of testing regions, and may enable the sensor unit to be used repeatedly without needing to clean or re-load the reaction zone with a test solution.

The CMOS-based sensor unit may comprise an optical sensor. The optical sensor may be arranged to detect changes in the appearance of the reaction zone, e.g. by capturing an image or determining a change in optical properties thereof. The apparatus may comprise an optical source (e.g. LED or the like) for illuminating the test material with optical radiation. In one example, the optical sensor may be a spectral absorption sensor, e.g. a photodiode or an array of photodiodes or/and a single photon avalanche diode (SPAD) to increase the detection dynamic range.

The CMOS-based sensor unit may have multiple sensing modalities. For example, it may comprise a substrate having a first sensing element and a second sensing element fabricated thereon. The first sensing element and the second sensing element may be arranged to detect simultaneously different properties of the reaction zone to enable simultaneous detection of a plurality of metabolites. The first sensing element may comprise an optical sensor (e.g. as described above). The second sensing element may detect a different property from the first sensing element. For example, the second sensing element may be a chemical sensor, e.g. arranged to determine a change in composition or chemistry within the reaction zone (or within one or more of the testing regions). In one example, the second sensing element is a pH sensor, e.g. comprising an ion sensitive field effect transistor (ISFET) having a gate electrode in contact with the test material. The apparatus may include a reference electrode arranged to apply a voltage to the reaction zone.

The apparatus may comprise an array of CMOS-based sensor units. Respective signals can be measured from each sensor unit. Alternatively, an average signal can be measured from the array of sensor units. Measuring an average signal can greatly reduce signal noise: according to Gaussian statistics, signal noise is reduced as a function of <MAT>, where N is the number of sensor units.

Each CMOS-based sensor unit in the array may be independently addressable to obtain signals corresponding to each of the testing regions.

The biological sample may be blood serum, but the invention can be used with any biological sample capable of communicating metabolites into the reaction zone.

In a further aspect, the invention may provide a method of detecting biomarkers in a biological sample as set out in claim <NUM>.

Embodiments of the present invention provide a metabolite detection device arranged to detect simultaneously multiple metabolites from a single biological sample. The device includes a reaction zone with spatially separated testing regions that have properties that are sensitive to the presence of different metabolites. The device comprises a single CMOS-based chip having one or more sensing modalities capable of detecting the properties of the separate testing region to determine the presence of multiple metabolites in the sample.

The one of more sensing modalities are provided by components fabricated on to the CMOS-based chip. In the examples discussed below, the sensing modalities include an optical sensor, e.g. for sensing optical radiation, and a pH sensor, e.g. for sensing a concentration of ionic species in a sample. However, it may be understood that the principles of the invention are applicable to any kind of sensor that can be fabricated or post processed on a CMOS chip and which is capable of detecting information indicative of the presence of a metabolite.

The sample may be a biological sample (e.g. fluid or tissue) obtained from a subject in any conventional manner. In the example discussed below the sample is blood serum, but it should be understood that the invention may encompass the use of other (or additional) sample types such as urine, sweat and swab from other body openings.

<FIG> shows a plan view of a complementary metal oxide semiconductor (CMOS) chip <NUM> having an array of sensors <NUM> across the surface of the chip. The chip <NUM> is typically a silicon integrated circuit (IC), and the sensors <NUM> may be photosensitive (e.g. photodiodes or/and single photon avalanche diodes (SPADs)) or chemical sensors (e.g. ion-sensitive field-effect transistors (ISFETs) or electrochemical electrodes) as will be explained in further detail below. Alternatively, in certain embodiments which are indicated below, each sensor <NUM> may include a pH sensor in addition to a photodiode.

The present invention relates to the use of a single chip of the kind shown in <FIG> to make multiple simultaneous measurements on a liquid sample (e.g. blood, blood serum, urine) by dividing the array of sensors <NUM> into multiple assay regions. Division of the sensors <NUM> can be done by physical separation of the assay regions on the chip itself or by providing discrete treatment zones (e.g. multiple microfluidic channels) on a paper strip which is used to introduce a liquid sample to the sensors <NUM>. Although the chip <NUM> shown is a <NUM> × <NUM> array of sensors <NUM>, the present invention may comprise a <NUM> × <NUM> array of sensors <NUM>.

<FIG> shows a schematic perspective view of a first multiplex assay apparatus for performing multiple simultaneous measurements on a liquid sample <NUM>. Close up views of the chip <NUM> and paper strip <NUM> used for the assay are shown in <FIG>, respectively. The assay apparatus comprises a chip carrier <NUM> for a CMOS chip <NUM>. The surface of the chip <NUM> has an array of photodetectors (e.g. photodiodes or/and SPADs) , in the manner shown in <FIG>. An epoxy layer <NUM> is provided on the chip carrier <NUM> to form a channel across the surface of the chip <NUM> for receiving a paper strip <NUM>. The epoxy layer <NUM> also protects wire bindings between the chip <NUM> and chip carrier <NUM>. An LED <NUM> is positioned above the chip <NUM> to illuminate the photodiodes such that they are able to detect a change in colour during the assay, as described below.

The chip <NUM> is shown in more detail in <FIG>. A series of physically discrete treatment zones are provided by microfluidic channels <NUM>, <NUM>, <NUM>. In this example, there are three channels. Two of the channels are activated to respond to substances to be detected, and the third channel is a control. The principles of the invention are not limited to this arrangement. There may be any number of channels activated in a manner to detect a plurality of different substances.

The channels are fabricated with a photoresist <NUM> on top of the chip <NUM>, which is glued and wire-bound to the chip carrier <NUM>. Microfluidic channel I <NUM> is coated with enzyme I <NUM>; microfluidic channel II <NUM> is coated with enzyme II <NUM>; and microfluidic channel III <NUM> is not coated with any enzyme so as to give a negative control channel. In this way, the photoresist physically separates assay regions on the chip <NUM> itself.

The paper strip <NUM> is shown in more detail in <FIG>. The paper strip <NUM> is sized to fit the channel formed in the epoxy layer <NUM> across the surface of the chip <NUM>. The paper strip <NUM> has a reaction zone that is arranged to fit over the chip in use. The reaction zone is modified using a hydrophobic polymer to form three microfluidic channels <NUM>, <NUM>, <NUM> which correspond respectively to the three microfluidic channels <NUM>, <NUM>, <NUM> on the surface of the chip <NUM>. The polymer confines the sample that is transported along the paper strip within the three channels <NUM>, <NUM>, <NUM>. This arrangement prevents cross-talking and cross-contamination.

To perform a multiplexed assay using the apparatus shown in <FIG>, the paper strip <NUM> is inserted into the channel formed in the epoxy layer <NUM> until the reaction zone is in contact with the surface of the chip <NUM>. A drop of analyte solution <NUM> is applied to one end of the paper strip <NUM>. The analyte solution contains (possible among other things) substances I and II, which may be different metabolites, wherein enzyme I <NUM> is specific for substance I and enzyme II <NUM> is specific for substance II. Due to capillary force, the analyte solution <NUM> flows along the paper strip <NUM> to the top of the chip <NUM>, where reactions takes place in the microfluidic channels. The enzyme reactions may generate colour changes on the paper strip <NUM>. Enzyme I <NUM> generates one colour change <NUM>, and enzyme II <NUM> generates a different colour change <NUM>. The negative control channel generates no colour change <NUM>. The colour changes are detected in real time by the photodiodes on the chip <NUM> under the illumination of LED <NUM>, the multiple colour changes producing multiple detections. The change in colour is detected as light absorption by the enzyme reaction products. In this way, the apparatus allows the detection of multiple metabolites in a single assay.

In the configuration depicted in <FIG>, microfluidic channels <NUM>-<NUM> are defined on the surface of the chip <NUM>. Each microfluidic channel is a distinct assay region which is physically separated from the other channels and which has its own photodiodes. The paper strip <NUM> wets the surface of the chip <NUM> such that the analyte solution <NUM> is drawn down into the microfluidic channels <NUM>-<NUM> on the chip surface. Optical cross-talk is limited as the colour change chemistry is in an immediately proximal channel, the channels being physically separated by the photoresist <NUM>. Cross-talk may also occur by chemical diffusion and capillary action from the microfluidic channels on the chip into the paper, and subsequent transfer across into an adjacent channel. Such cross-talk is minimised by ensuring that the distance between adjacent microfluidic channels is large enough, for example by thickening the walls of photoresist <NUM>.

By replacing the photosensitive sensors with chemical sensors, the apparatus described may be suitable for detecting reactions by a pH change of the solution. This is described in more detail below.

<FIG> shows a plan view of a second multiplex assay apparatus for performing multiple simultaneous measurements on a liquid sample <NUM>. A close up view of the paper strip <NUM> is shown in <FIG>. The assay apparatus comprises a chip carrier <NUM> for a CMOS chip <NUM>. The surface of the chip <NUM> has an array of photodiodes and/or single photon avalanche diodes, in the manner shown in <FIG>. An epoxy layer <NUM> is provided on the chip carrier <NUM> to form a channel across the surface of the chip <NUM> for receiving a paper strip <NUM>. The epoxy layer <NUM> also protects wire bindings between the chip <NUM> and the chip carrier <NUM>. An LED <NUM> having a known, specific wavelength is positioned above the chip <NUM> to illuminate the photodiodes such that they are able to detect a change in colour during the assay, as described below.

The paper strip <NUM> is shown in more detail in <FIG>. The paper strip <NUM> is sized to fit the channel formed in the epoxy layer <NUM> across the surface of the chip <NUM>. The paper strip is modified with a hydrophobic polymer <NUM> to form three microfluidic channels <NUM>, <NUM>, <NUM> which extend a substantial distance between a first end and a second end of the elongate paper strip <NUM>. The hydrophobic polymer <NUM> prevents cross talk and cross-contamination between the three microfluidic channels <NUM>, <NUM>, <NUM>. Microfluidic channel I <NUM> is coated with enzyme I <NUM>; microfluidic channel II <NUM> is coated with enzyme II <NUM>; and microfluidic channel III <NUM> is not coated with any enzyme so as to give a negative control channel. Different assay regions are thereby defined by the different channels on the paper strip <NUM>.

To perform a multiplexed assay using the apparatus shown in <FIG>, the paper strip <NUM> is inserted into the channel formed in the epoxy layer <NUM>. To prevent cross talk and cross-contamination the paper strip <NUM> should not come into contact with the surface of the chip <NUM>, but should instead rest a distance away from the chip <NUM>. A drop of analyte solution <NUM> is applied to one end of the paper strip <NUM>. The analyte solution contains substrates I and II, which may be different metabolites, wherein enzyme I <NUM> is specific for substrate I and enzyme II <NUM> is specific for substrate II. Due to capillary force, the analyte solution <NUM> flows along the paper strip <NUM> through the microfluidic channels <NUM>-<NUM>, where reactions with the enzymes <NUM>, <NUM> take place. The enzyme reactions produce colour changes on the paper strip <NUM>. Enzyme I <NUM> generates one colour change <NUM> in microfluidic channel I <NUM>, and enzyme II <NUM> generates another colour change <NUM> in microfluidic channel II <NUM>. Microfluidic channel III <NUM> for negative control generates no colour change <NUM>. The colour changes are detected in real time by the photodiodes on the chip <NUM> under the illumination of LED <NUM>, the multiple colour changes producing multiple detections. In this way, the apparatus allows the detection of multiple metabolites in a single assay.

In the configuration depicted in <FIG>, microfluidic channels <NUM>-<NUM> are defined on the paper strip <NUM>. Each microfluidic channel is a distinct assay region which is physically separated from the other channels. The paper strip <NUM> is in close proximity to the photodiodes on the surface of the chip <NUM>, but it is not in contact. Cross talk may occur by optical scattering of large angles through the paper casting light onto adjacent sensors. The distance between the chip <NUM> and the paper strip <NUM> is made small so as to limit or eliminate such optical cross talk. In addition, channel spacing on the paper strip <NUM> must be large enough to keep cross talk at a low level to allow independent measurement of the colour change occurring in each microfluidic channel. This is done by varying the thickness of the microfluidic channels and the hydrophobic polymer barriers separating them.

The embodiments described above each work by detecting a colour change, using photodiodes illuminated by an LED having a known, specific wavelength. However, for embodiments where the sample is brought into contact with the chip, the invention may alternatively or additionally make use of an array of chemical sensors on a chip. For example, the chemical sensors may be ion-sensitive field-effect transistors (ISFETs). An ISFET is a field-effect transistor in which a solution is used as the gate electrode. A change in pH (i.e. a change in concentration of H+ ions) of the solution causes a current running though the ISFET to change by a measurable amount.

<FIG> shows a schematic view of an ISFET <NUM> which may be used as a chemical sensor for detecting the pH of a solution <NUM> in the present invention. The ISFET <NUM> comprises a well for receiving solution <NUM> formed by an epoxy <NUM> on the surface of the ISFET <NUM>. The solution <NUM> contains a concentration of H+ ions to be detected. The well ensures that the solution <NUM> is in contact with a gate oxide layer <NUM>. A source <NUM> and a drain <NUM> are also provided in a bulk layer <NUM>, with both source <NUM> and drain <NUM> in contact with the gate oxide <NUM>, on a side which is opposite the solution <NUM>. The presence of H+ ions in the solution <NUM>, which are adsorbed onto the surface of the gate oxide <NUM>, causes migration of charge carriers to the upper surface of the bulk layer <NUM>. Current is thereby able to flow between the source <NUM> and the drain <NUM> through the bulk layer <NUM>. As the source/drain current is affected by the concentration of H+ ions, applying a known voltage to the reference electrode <NUM>, which is at least partially immersed in the solution <NUM>, allows the pH of the solution <NUM> to be determined. Alternatively, the source/drain current can be kept constant and the voltage change at the reference electrode <NUM> measured to determine the pH of the solution <NUM>.

<FIG> shows a plan view of a third multiplex assay apparatus for performing multiple simultaneous measurements on a liquid sample <NUM>. A close up view of the paper strip <NUM> used for the assay is shown in <FIG>. The assay apparatus comprises a chip carrier <NUM> for a CMOS chip <NUM>, similar to the embodiments shown above. However, in this third embodiment the surface of the chip <NUM> has an array of chemical sensors, in particular ISFETs, substantially as described above with reference to <FIG>. An epoxy layer <NUM> is provided on the chip carrier <NUM> to form a channel across the surface of the chip <NUM> for receiving a paper strip <NUM>. The epoxy layer <NUM> also protects wire bindings between the chip <NUM> and chip carrier <NUM>. An LED having a known, fixed wavelength may be provided where the sensors on the chip <NUM> also include a photodiode, as described herein.

The chip <NUM> is substantially identical to chip <NUM> shown in <FIG>. However, chip <NUM> comprises an array of chemical sensors. Chip <NUM> may, however, comprise a number of microfluidic channels which are coated with enzymes, and a negative control channel which is not coated with an enzyme, in the manner of chip <NUM> shown in <FIG>. The microfluidic channels provide multiple distinct assay regions.

The paper strip <NUM> is shown in more detail in <FIG>. The paper strip <NUM> is sized to fit the channel formed in the epoxy layer <NUM> across the surface of the chip <NUM>. The paper strip <NUM> is modified with a hydrophobic polymer to form three microfluidic channels <NUM>, <NUM>, <NUM> which correspond to microfluidic channels on the surface of chip <NUM>. This arrangement prevents cross-talking and cross-contamination. The paper strip <NUM> also comprises a metallised region <NUM>, which may be printed or impregnated with silver such that the metallised region <NUM> may be used as a reference electrode in the assay process as described below.

To perform a multiplexed assay using the apparatus shown in <FIG>, the paper strip <NUM> is inserted into the channel formed in the epoxy layer <NUM> until it is in contact with the surface of the chip <NUM>. A drop of analyte solution <NUM> is applied to one end of the paper strip <NUM>. The analyte solution contains multiple substrates, which may be different metabolites, wherein each enzyme in a microfluidic channel on the surface of the chip <NUM> is specific to a substrate, in a manner which has been described above with reference to first and second multiplex assay apparatus. Due to capillary force, the analyte solution <NUM> flows along the paper strip <NUM> to the top of the chip <NUM>, where reactions take place in the microfluidic channels. The enzyme reactions generate pH changes on the paper strip <NUM>; although there is no pH change in the negative control channel. The pH changes may be detected by applying a fixed reference voltage to the metallised region <NUM> of the paper strip <NUM>, and measuring the change in source to drain current through each of the ISFET chemical sensors on the surface of the chip <NUM>. To convert these current measurements to pH values, the measurements are compared with a source to drain current obtained with a solution of known pH and the same, fixed reference voltage. In this way, the apparatus allows the detection of multiple metabolites in a single assay.

Each microfluidic channel on the surface of the chip <NUM> is a distinct assay region which is physically separated from the other channels and which has its own chemical sensors. The paper strip <NUM> wets the surface of the chip <NUM> such that the analyte solution <NUM> is drawn down into the microfluidic channels on the chip surface. Cross-talk may also occur by chemical diffusion and capillary action from the microfluidic channels on the chip into the paper, and subsequent transfer across into an adjacent channel. Such cross-talk is minimised by ensuring that the distance between adjacent microfluidic channels is large enough, for example by thickening the walls of photoresist on the surface of the chip <NUM>.

In addition to pH sensitive detectors as described, the chip <NUM> may comprise an array of sensors which combine a photodiode and an ISFET. In this way, the multiplex assay apparatus of <FIG> may also be configured to perform assays by the light absorption method described above, using an LED having a known, specific wavelength to illuminate the paper strip <NUM> and the chip <NUM>. The pH change of the reactions in the microfluidic channels may also be measured at the same time as light absorption to carry out more in depth multiplex assays.

<FIG> shows a schematic view of an alternative paper strip <NUM> which may be used with the multiplex assay apparatus of <FIG>. The paper strip <NUM> comprises a blister pack <NUM> for containing a solution, such as an electrolyte of pH buffer solution, which is able to aid transport of an analyte sample along the paper strip <NUM>. The paper strip <NUM> also comprises a metallised region <NUM>, which may be printed or impregnated with silver, to be connected to a reference voltage <NUM> such that the metallised region <NUM> can act as a reference electrode in a manner as described above.

In use, an analyte sample may be spotted at <NUM>. The blister pack <NUM> can be squeezed or otherwise burst to release the solution within, and so aid transport of the analyte sample along the paper strip <NUM> for a multiplex assay.

<FIG> shows a set of graphs that illustrate the results of a test using a single paper strip having three channels on one single paper strip. The three channels were tested simultaneously by adding one drop of analyte from one side of the paper strip. The top image <NUM> in <FIG> is an intensity graph of the three channels (dark regions <NUM>) on the photodiode under the illumination of an LED. The middle channel was used as control (i.e. was not printed with an enzyme). The side channels were each printed with a respective enzyme (Enzyme <NUM> and <NUM>). The three lower images are graphs showing electrical signal amplitude from each of the channels with time. When the analyte reached the sensing region, there is a big step jump of the electrical signal in all channels due to the wetting of the paper. After around half minute, a decreasing signal can be seen from the side channels with enzymes. As a control, the middle channel gives nearly no signal change.

<FIG> shows a schematic view of a fourth multiplex apparatus <NUM> for performing multiple simultaneous measurements on a liquid sample. A close up view of the chip <NUM> used for the assay is shown in <FIG>. The assay apparatus comprises a chip carrier (not shown) for a CMOS chip <NUM>. Spaced away from the chip is an annular wall <NUM> which defines the outer edge of four reaction chambers, or quadrants, which are separated from each other by separation walls <NUM>. A different reaction may take place in each quadrant, as explained in more detail below. An epoxy layer <NUM> forms the base of the reaction chambers, and also protects wire bindings between the chip <NUM> and the chip carrier. However, the epoxy layer <NUM> does not extend over the surface of the chip <NUM>, which ensures that the surface of the chip <NUM> is open to be able to detect reaction parameters, such as a colour or pH change. An LED having a known, specific wavelength may be positioned above the chip <NUM> if the reactions are to be detected by light absorption.

The chip <NUM> is positioned at the meeting point between the separation walls <NUM> such that the chip <NUM> is divided into four distinct assay regions 605a, 605b, 605c, 605d. Each assay region <NUM> is defined by a micro-well in the epoxy layer <NUM>. When a liquid sample is deposited in each of the reaction chambers, the micro-wells are filled such that liquid is in contact with sensors <NUM> in each of the assay regions <NUM>. The assay regions <NUM> are separated from each other by sidewalls <NUM>, so that liquid cannot leak into an adjacent assay region. The sensors <NUM> may be photosensitive (e.g. photodiodes or single photon avalanche diodes) or chemical sensors (e.g. ISFETs or electrochemical electrodes). Alternatively, the chip <NUM> may comprise an array of sensors <NUM> which combine a photodiode and an ISFET. In this way, the multiplex assay apparatus may also be configured to perform assays by the light absorption method described above, using an LED having a known, specific wavelength to illuminate the reaction chambers and the chip <NUM>. The pH change of the reactions in the microfluidic channels may also be measured at the same time as light absorption to carry out more in depth multiplex assays.

In one example, the multiplex apparatus <NUM> is manufactured as follows. Each assay region <NUM> is protected by a polydimethylsiloxane (PDMS) block, which helps to shape an epoxy (preferably black epoxy), which is introduced in a succeeding step, and also protect the sensor array area of the chip <NUM> from damage during the manufacturing process. The use of black epoxy can prevent cross talk in a signal (either optical or electronic) from occurring between different assay regions (also referred to herein as micro-wells).

The PDMS blocks are positioned by microscope assisted translation on top of the chip <NUM>. The gap between them determines the separation distance between adjacent micro-wells, and the height of the blocks is an upper limit to the micro-well depth. Epoxy mixture is then carefully poured around the blocks, and over the surface of the chip carrier to form the epoxy layer <NUM>. The epoxy is left to cure and harden for around <NUM> hours. The annular wall <NUM> is then introduced to form the outer wall of the four reaction chambers. The annular wall <NUM> may be a ring made of plastics material, having a height of around <NUM>. The annular wall may be held in place with epoxy. PDMS blocks are then used to define the shape of the reaction chambers and separation walls <NUM>. They may be shaped and positioned manually, or the separation walls may be first made from a polystyrene sheets with the desired separation wall dimensions and PDMS poured into the cavities defined by the polystyrene and annular wall <NUM> to be cured. When the polystyrene sheets are removed, the sidewalls <NUM> can be properly formed from epoxy by filling the gaps between the PDMS blocks. After curing of the epoxy, the PDMS blocks may be removed, as well as the PDMS blocks defining the micro-wells <NUM>. The micro-wells thus have pipettable access and sidewalls <NUM> separate the liquid into individual reaction chambers to prevent mixing. This multilevel sequential die casting technique is compatible with CMOS processing since it can be used at room temperature. It may be expedited by raising temperature to <NUM>.

In this technique, the resulting micro-wells may exhibit a step profile opening into a wider area. This allows pipettable access to the micro-wells without requiring micro-tubing or fluid management pumps for sample delivery. In turn this can save on time required for the delivery of samples.

In another example, the reaction chambers (and micro-wells discussed below) are manufactured by mounting a preformed cartridge over the chip carrier. The cartridge may be formed from any suitable material, e.g. plastic. It may have a form similar to the annular wall <NUM> and separation walls <NUM> discussed above. The cartridge may be affixed to the chip carrier in any conventional manner, e.g. by screws or the like.

<FIG> is a schematic view of an optical alignment tool <NUM> may be used to align the cartridge <NUM> with the chip carrier <NUM> during the mounting process and to maintain alignment during the attachment (e.g. screwing) process. The optical alignment tool comprises a reciprocating movement mechanism <NUM> for bringing a movable holder <NUM> into contact with a platform <NUM>. The chip carrier <NUM> is mounted on the platform <NUM> via an alignment stage <NUM> that permits adjustment in two orthogonal linear dimensions (e.g. x- and y-dimensions) in the plane of the platform <NUM> and a rotation dimension (e.g. about an axis extending perpendicular to the platform <NUM>). A pivoting lever may be used to operate the linear motion of the reciprocating movement mechanism <NUM>.

A laser source <NUM> is mounted on the movable holder <NUM> to emit a laser beam towards the platform <NUM>. The cartridge <NUM> is mounted on the holder to partially block the laser beam, whereby a pattern of the separation walls is projected on to the chip carrier <NUM> on the platform <NUM>.

With this arrangement, the laser illuminates a spot on the chip with a pattern that is indicative of alignment with the cartridge.

The movable holder <NUM> may use suction or a magnetic retainer to hold the combination of laser source and cartridge. The alignment between the cartridge and chip carrier can be adjusted via the alignment stage <NUM> during linear movement of the cartridge and subsequent fixing thereof to the chip carrier.

In one example, the optical alignment tool may be fabricated as part of a microscope. Visual inspection of alignment with the chip itself can be carried out through the microscope while fixing the cartridge in place.

To perform a multiplexed assay using the apparatus shown in <FIG>, a different reaction enzyme is pipetted into each of three micro-wells 605a, 605b, 605c, leaving one micro-well 605d without an enzyme to act as a negative control. An analyte solution may then be introduced into each of the four reaction chambers. The analyte solution may contain at least three substrates, or metabolites, wherein each enzyme is specific to a substrate, as described above with reference to other embodiments of the invention. Reactions between the enzymes and substrates take place in each of the micro-wells 605a-c, where they are detected by sensors <NUM>. For example, the reaction may cause a colour change which may be detected by photodiodes and/or a pH change which may be detected by chemical sensors in a manner substantially as described above. In this way, the apparatus allows the detection of multiple metabolites in a single assay, using a single chip <NUM>.

In embodiments of the invention that use the micro-well arrangement discussed above, it may be desirable to have a minimum of <NUM>×<NUM> pixels per micro-well. The width of the wall separating adjacent micro-wells may be around <NUM>. This ensures that the separation between micro-wells consumes no more than one pixel row or column. The walls may seal against the sensor array area of the chip using a pressure sensitive adhesive. The adhesive may expand under applied pressure, so a thickness of the walls is set within a tolerance to ensure this expansion does not block additional pixels.

The micro-wells may have a height selected to be between an average height for a microchannel (e.g. -<NUM>) and a typical assay height (e.g. ~<NUM>) for <NUM> micro-well chip.

To facilitate rapid delivery of different analytes, the device may comprise a plurality of inlet ports for directing a fluid sample into a respective micro-well. A pitch of the inlet ports may be matched to a pitch of multi-channel micro-pipette to enable simultaneous delivery. To maintain the reagent fluidic volume and for easy passage of reagents by capillary forces, each micro-well may comprise an outlet. The inlet into each micro-well may include both a capillary conduit to enable liquid delivery through capillary action, as well as one or more reservoirs for reagent mixing before delivery to the micro-well.

In one example, a reference electrode for the micro-wells may be formed on or integrated with the walls that define the micro-wells. For example a <NUM> diameter Ag/AgCl electrode may be integrated into the micro-wells from the side of the micro-wells. This arrangement can provide an independent reference electrode for each micro-well, which in turn enables the ISFET function of chip to be used simultaneously in each micro-well.

If the chip is equipped with multiple sensing modalities, the number of analytes that can be assayed simultaneously can multiply by the number of micro-wells that are present. For example, in an arrangement with four micro-wells and two independent sensing modalities, one can assay eight analytes simultaneously in real time.

In one example, the delivery of fluid to the device may be controlled through a fluid management algorithm configured to effect sequential delivery of the reagents including any or all of the steps of (i) diluting of the analyte, (ii) introducing supporting reagents for the reaction, (iii) introducing the sample (e.g. human bodily fluid such as blood, serum, urine, etc.), and (iv) introducing the enzyme to initiate the reaction.

In each of the multiplex assay apparatus described above, the chip and chip carrier may be mounted on a printed circuit board (PCB), where the chip is integrated with a microcontroller to provide addressing signals and to acquire output readings from the array of sensors on the chip. The readings may then be transferred wirelessly or via universal serial bus (USB) to a computer based program (e.g. LabVIEW ®) or android based program in which the data may be processed and analysed.

Where an LED is used to perform the multiplex assays, the optical characteristics of the LED and the sensors on the chip must be evaluated prior to carrying out the assay to examine their spectral relationship. The LED should preferably be selected to emit light having a wavelength which is close to the peak sensitivity of the photosensitive sensors.

Claim 1:
An apparatus for detecting biomarkers in a biological sample, the apparatus comprising:
a sample receiving module arranged to receive the biological sample (<NUM>), the sample receiving module comprising a capillary transport structure configured to transport the biological sample to a reaction zone, positioned on either the capillary transport structure or on the surface of a CMOS-based sensor unit, for testing, wherein the reaction zone comprises a first testing region and a second testing region spatially separated from the first testing region, wherein properties of the first testing region and the second testing region are affected by the presence of biomarkers to be detected, the first testing region being sensitive to a first biomarker and the second testing region being sensitive to a second biomarker that is different from the first biomarker; and
wherein the CMOS-based sensor unit (<NUM>) is adapted to detect independently the properties of the first testing region and the second testing region, wherein the CMOS-based sensor unit is configured to generate separate electrical signals for each of the first testing region and the second testing region, and wherein a change over time in amplitude of the separate electrical signals is indicative of the presence of the first biomarker and second biomarker respectively,
wherein the first testing region and the second testing region each comprise a microfluidic channel formed on either the capillary transport structure or a surface of the CMOS-based sensor unit, wherein the microfluidic channel of the first testing region is activated to respond to the first biomarker and the microfluidic channel of the second testing region is activated to respond to the second biomarker,
wherein, when the microfluidic channels are formed on the capillary transport structure, the capillary transport structure is configured to not contact the surface of the CMOS-based sensor unit, and
wherein, when the microfluidic channels are formed on the surface of the CMOS-based sensor unit, the capillary transport structure includes corresponding microfluidic channels configured to contact a surface of the CMOS-based sensor unit.