FIELD OF THE INVENTION
[0001] The disclosure relates to microfluidic array devices and methods for analysis of glucose and other analyte concentration in a plurality of samples suitable for mass population screening.
DESCRIPTION OF THE RELATED ART [0002] Diabetes mellitus affects an estimated of 246 million people worldwide, which is expected to rise to 380 million by 2025. Every year about 7 million people develop diabetes and a staggering 3.8 million deaths per year are linked directly to diabetes-related causes including cardiovascular disease which is worsened by diabetes-related lipid disorders and hypertension.
[0003] Precise monitoring with stringent control of the physiological glucose levels is highly essential for the diagnosis and treatment of diabetes. The most common technology for blood glucose determination is an enzyme-based method. Commercially available glucose biosensors of enzyme-based method suffer from limitations which arise due to the enzyme activity dependency on temperature, humidity, interference due to high-applied potential and inhibition of electron transfer process by the enzyme layer. Enzymatic glucose biosensors have evolved through the generations and have achieved the most significant commercial success. However, these biosensors cannot be used for simultaneous measurements of multiple samples obtained from different patients which are an essential requirement for mass population screening.
[0004] Various glucose sensors are available based on different metal electrodes. The mechanism of direct electrooxidation of glucose depends on the catalyst used in the process. Recently, materials used are not simple metals or metal oxides but they are based

on composites or hybrids of the metals, because of the benefit of their integrated properties and the potential to overcome the drawbacks of the traditional noble metal electrodes. Metal and metal oxide based sensors are very sensitive, relatively inexpensive and have the advantage of rapid response associated with specific nanostructures such as nanowire, nanorod, nanotube, nanoparticle and nanofibre.
[0005] WO2012018777 discloses a non-enzymatic glucose sensor comprising an electrode, one or more metal oxide nanofibers, and an alkaline solid electrolyte. The metal oxide nanofibers and the alkaline solid electrolyte are disposed over the surface of the electrode and at least a portion of the alkaline solid electrolyte is in contact with the metal oxide nanofibers. The metal oxide nanofibers are each independently and optionally metal doped.
[0006] US7857760 discloses a system for measuring analyte concentration in a host with an enzymatic membrane disposed on an electro active portion of the electrode wherein the membrane is configured to control an influx of the analyte and the membrane comprises a substantially non-smooth outer surface. However, the enzymatic glucose sensors suffer from major problems that they depend on free oxygen as a catalytic mediator and they are sensitive to the presence of electro-active interfering species in the blood.

[0007] A microfluidic device and a method for analyzing glucose concentration in a plurality of samples are disclosed.
[0008] In one embodiment of the disclosure, the microfluidic device comprises a plurality of assembled units, each unit comprising a sample well to receive a sample, a reagent well to receive reagent from a common reagent reservoir, and a mixing well that receives reagent from the reagent well and sample from the sample well and mix the reagent with the sample to form corresponding a mixture. It further comprises a meander channel comprising a plurality of meanders with alternatively varying diameters, to receive the mixture and enhance the mixing of analyte and reagent within the mixture, thereby forming a uniform solution. A sensor chamber affixed with a sensor receives the uniform solution via the meander channel. Each of the sensors generates an output indicative of the glucose concentration for the individual sample.
[0009] In various embodiments, the sensor chambers are connected to a common outlet and a pump is mounted at the outlet to supply necessary negative pressure for fluid movement. In various embodiments, the assembled unit further comprises microchannels, which connect each of the reagent wells to the corresponding sample well at the inlet of the mixing well by means of T-inlets. In various embodiments, the device further comprises a display unit configured to display the glucose concentrations of the plurality of samples.
[0010] In various embodiments, the sensors are non-enzymatic glucose biosensors. In various embodiments, each sensor in the device is a three-electrode system with at least one active electrode modified with CuO nanoparticle coating, a carbon counter electrode and an Ag/AgCl reference electrode. In various embodiments, the substrate for the active electrode is one of polyethylene terephthalate, polycarbonate, polyethylene, polypropylene, polyvinyl chloride, polyamide, polymethyl methacrylate, or polysulfone.
[0011] In one embodiment, the working electrode has a diameter of 1.8-2.2 mm. In

another embodiment the working electrode is screen printed between the counter and the reference electrodes at a distance of 0.4-0.6 mm apart.
[0012] In various embodiments, the line length of a cut line drawn from the first meander to the outlet of the meander channel is between 15000-20000 µm. In one embodiment, the meander channels have alternately varying diameters of 150-250 µm and 350-450 µm, and a depth of 100-120 µm. In one embodiment, the meander channels of the device have alternately varying diameters of 150-250 µm and 350-450 µm.
[0013] The generated output may in some embodiments be a current or a voltage indicative of the glucose concentration in the sample. In various embodiments, the device is incorporated as a Lab-on-a-chip device wherein a potentiostatic meter module comprising a microcontroller integrated with the sensor chambers is configured to convert the output from the sensor device into the glucose concentrations and a display unit configured to display the glucose concentration for the plurality of samples.
[0014] In one embodiment, a method of determining glucose concentration in a plurality of analyte samples is disclosed. The method comprises loading the plurality of samples obtained from one or more subjects in an array device and mixing each of the analyte samples with reagent in the array device. The loaded samples are contacted with reagent to obtain a mixture. The mixture is passed through meander channels having alternately varying diameters thereby obtained enhanced mixing of the glucose and the reagent in the mixture. After mixing, each sample-reagent mixture contacts with a non-enzymatic biosensor array element comprising CuO nanoparticles in the working region to generate an output indicative of glucose concentration in the sample. The output is converted to determine glucose concentration for each sample. The generated output may be voltage indicative of the glucose concentration in the sample.
[0015] This and other aspects are disclosed herein.

[0020] The invention has other advantages and features which will be more readily apparent from the following detailed description of the invention and the appended claims, when taken in conjunction with the accompanying drawings, in which:
[0021] FIG. 1 illustrates the microfluidic device for glucose concentration analysis for a
plurality of analytes.
[0022] FIG. 2 shows the screens designed for plurality of electrode system.
[0023] FIG. 3 illustrates the mass transport through the reagent and the analyte stream
and the meander channel.
[0024] FIG. 4A shows the microfluidic device integrated as a Lab-on-a-chip device.
[0025] FIG. 4B shows the block diagram of electronic meter module.
[0026] FIG. 4C shows the system architecture of the electronic meter module.
[0027] FIG. 5 illustrates the method for analyzing glucose concentration in a plurality of
samples.
[0029] FIG. 6 represents the variation in concentration along the cut line through the
meander channel.
[0030] FIG. 7A shows the profilometer plot for the depth of the fabricated
microchannels.
[0031] FIG. 7B and FIG. 7C shows the profilometer plots for the dimensions of the
alternatively varying diameters in microchannels.
[0032] FIG. 8A shows linear sweep voltammograms results for the electrochemical
response of glucose on the modified electrode.
[0033] FIG. 8B shows the chronoamperometric results for the electrochemical response
of glucose on the modified electrode.
[0034] FIG. 9A shows the electrochemical response on ten different electrodes for 3mM
glucose concentration.

[0035] FIG. 9B shows the electrochemical response on ten different electrodes for 9mM
glucose concentration.
[0036] FIG. 10 shows the potentiostatic meter developed for the simultaneous detection
of plurality of analytes.
[0037] FIG. 11 shows the multi-potentiostat configuration of Analog Front End (AFEs).
[0038] FIG. 12A shows the meter result for the glucose concentration of 3mM for
different electrodes.
[0039] FIG. 12B shows the meter result for the glucose concentration of 9mM for
different electrodes.
[0040] Referring to the drawings, like numbers indicate like parts throughout the views.

[0041] While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.
[0042] Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on." Referring to the drawings, like numbers indicate like parts throughout the views. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.
[0043] The word “exemplary” is used herein to mean “serving as an example, instance, or illustration”. Any implementation described herein as “exemplary” is not necessarily to be construed as advantageous over other implementations.
[0044] The present disclosure provides for microfluidic array devices and methods for analysis of glucose and other analyte concentrations in a plurality of samples. The components of the device are integrated in a Lab-on-a-chip device which integrates the fluidics, electronics and various sensors. The disclosed devices and methods can be used for simultaneous measurements of samples from one or more patients and is suitable for mass population screening. The device is capable of analyzing biochemical liquid samples like solutions of metabolites, macromolecules, proteins, nucleic acids or cells and viruses. Also these devices facilitate fluidic transportation, sorting, mixing and separation of liquid samples. They have many advantages including compactness, accurate diagnosis, portability, modularity, re-configurability, embedded computing, automated sample handling, low electronic noise, limited power consumption and straight forward integration

of components. Also, since the volume of sample is very small, the Lab-on-a-Chip devices reduce analysis time.
[0045] The microfluidic device 100, as shown in FIG. 1, includes a plurality of assembled units. In one embodiment, provided herein is a plurality of sample wells 101, each sample well 101-1, 101-2….101-n configured to receive and store a sample from a plurality of subjects. In some embodiments, a plurality of reagent wells 102 is provided, each reagent well 102-1, 102-2….102-n configured to receive a reagent from a common reagent reservoir 103. In some embodiments, the device 100 includes a plurality of mixing wells 104, each mixing well 104-1, 104-2….104-n configured to receive the reagent from the corresponding reagent well 102-1, 102-2….102-n and sample from the corresponding sample well 101-1, 101-2….101-n and mix the reagent with each sample to form a reagent-sample mixture. In one embodiment, the device 100 includes a plurality of meander channels 105, each meander channels 105-1, 105-2….105-n configured to receive the reagent-sample mixture from the corresponding mixing well 104-1, 104-2….104-n. In some embodiments, the meander channels 105 are provided with a plurality of meanders 106 to enhance the mixing of an analyte and reagent within the mixtures, thereby forming an uniform solution. In one embodiment, the analyte is glucose or an analog thereof. In one embodiment, the device 100 includes a plurality of sensor chambers 107 connected to the outlet of the meander channels 105 and housing a sensor device 108, each sensor chamber 107-1, 107-2….107-n configured to receive the uniform solution from the corresponding meander channel 105-1, 105-2….105-n. In some embodiments, each sensor device 108-1, 108-2….108-n is configured to generate a value in glucose concentration for the sample in the sample well 101-1, 101-2….101-n.
[0046] In one embodiment, the device 100 may include 5 to 200 of sample wells 101, reagent wells 102, mixing wells 104, meander channels 105, and sensor chambers 107. In one embodiment, n is 10 as shown in FIG. 1.

[0047] In one embodiment, the sensor wells 107 are connected to a common outlet 109, as shown in FIG. 1, on to which a syringe 110 or pump 111 is mounted to supply the necessary negative pressure for the fluid flow.
[0048] In one embodiment of the invention, the meander channels 105 have alternately varying diameters for improving chaotic advections. In some embodiments, the meander channels 105 enhance the mixing of the analyte and reagent within the mixtures thereby forming a uniform solution.
[0049] In one embodiment, the device further comprises a display unit 113 to display the glucose concentration of the plurality of samples.
[0050] In one embodiment of the invention, the sensor device 108 comprises a non-enzymatic glucose biosensor 200 for detecting blood glucose level, as shown in FIG. 2. In some embodiments, the glucose biosensor 200 is a three electrodes system, with at least one active electrode 201, a carbon counter electrode 202 and Ag/AgCl reference electrode 203.
[0051] In one embodiment, the substrate for the active electrode 201 is selected from polyethylene terephthalate, polycarbonate, polyethylene, polypropylene, polyvinyl chloride, polyamide, polymethyl methacrylate, or polysulfone.
[0052] In some embodiments, the working electrode region of the non-enzymatic glucose biosensor 200 is fabricated using screen printing technology. In another embodiment, the non-enzymatic glucose biosensor 200 comprises a coating of CuO nanoparticles 204 in the working electrode region. In yet another embodiment, the non-enzymatic glucose biosensor 200 demonstrates excellent electro catalytic activity of CuO towards glucose oxidation.
[0053] In one embodiment, the diameter of the working electrode region is 1.8-2.2 mm. In another embodiment, the working electrode is screen printed between the counter and the reference electrodes at a distance of 0.3-0.7 mm apart, as shown in FIG. 2. In one

embodiment, polyethylene terephthalate (PET) sheets of 0.4-0.8 mm thickness are used as the substrate for screen printing.
[0054] In one embodiment, silver, AgCl and carbon inks are sequentially printed and cured on the PET substrate to obtain the screen printed biosensor 200. In another embodiment, a catalytic ink formulation is prepared by dispersing 30-40 wt% CuO nanoparticles in a medical sensor grade conductive carbon ink and used for printing the working electrode of the biosensor 200.
[0055] In one embodiment, the microfluidic devices 100 are morphologically characterized using a profilometer and electrochemically characterized for its glucose sensing capabilities. In another embodiment, the mixing efficiency of the microfluidic device 100 is evaluated by computational simulation study using computational fluid dynamics (CFD) tool.
[0056] In one embodiment of the invention, the meander channel 105, as shown in FIG. 3, is fabricated using polydimethylsiloxane (PDMS) by soft lithography technique. In one embodiment, the line length of a cut line drawn from the first meander 106 to the outlet of the meander channel 105 is between 15000-20000 µm. In another embodiment, the concentration of the mass transported across the channel 105 is between 0.1-2 mol/m3. In another embodiment, the dimensions of the fabricated channel are studied using profilometer. In one embodiment, the depth of the individual meander 106 is 100-120 µm. In another embodiment, the alternatively varying diameter of the meander channels is 150-250 µm in the X-direction and 350-450 µm in the Y-direction.
[0057] In one embodiment of the invention, a plurality of microchannels 312 connects each of the sample well 101 to the corresponding reagent well 102. In one embodiment, the sample streams are connected to the reagent stream at the inlet of the mixing well 304 by T- inlets 314.
[0058] In one embodiment of the invention, the device 100 is tested for varying concentrations of glucose by linear sweep voltammetry, where the current at the working

electrode is measured while the potential between the working electrode and the reference electrode is swept linearly in time. In one embodiment, the oxidation of glucose is registered as a peak in the current signal for the applied potential and the oxidation current increases linearly with the glucose between 0-30 mM. In one embodiment, the highest peak potential is found to be 0.5 to 0.8V from the linear sweep voltammetry (LSV).
[0059] The electrochemical characteristics of the device are studied using chronoamperometry in which the potential of the working electrode is stepped and the resulting current at the electrode is studied as a function of time. In one embodiment of the invention, the chronoamperometric experiments are carried out for an applied potential of 0 to 1V. In yet another embodiment the sampling time is fixed between 10-15 seconds as the current decreases with time and becomes stable around this time.
[0060] In one embodiment of the invention, blood serum samples are tested for different glucose concentrations and the results obtained are found to be comparable to the results of the commercially available glucose sensor strips. In one embodiment, the variation in response current obtained for different electrode is found to be around 0 to 5%.
[0061] In one embodiment of the invention, the microfluidic device is incorporated in a Lab-on-a-chip (LOC) device 400, as shown in FIG. 4A. In certain embodiments, the device 400 includes a portable electronic meter module system 401 that is used for the detection of glucose in multiple patient samples which is sent to the output unit 405. In some embodiments, the electronic meter module system 401 works based on principles of amperometry.
[0062] In some embodiments, the electronic module 401 includes a microcontroller 402 interfaced to all the individual modules as shown in FIG. 4B. The meter module 401 includes a multi potentiostat circuit made from a plurality of configurable potentiostats 412 that maintains a constant required potential across the working and counter electrode, with reference to the reference electrodes of all the sensors. The current produced from the electrochemical reaction is converted into voltage and fed to the microcontroller 402. The

microcontroller 402 converts this voltage to corresponding glucose concentrations. The obtained voltage is converted to the corresponding glucose concentrations with the help of the calibration plot programmed into the microcontroller 402 and the results are displayed on the display unit 413. In one embodiment, the meter 400 is used for determining differences in glucose concentrations obtained from different electrodes.
[0063] In some embodiments, the microcontroller 402 is connected to the plurality of sensors 108 through a plurality of AFEs (analog front end) 403-1,…403-n. The microcontroller 402 is connected to a plurality of analog front ends 403 and each of the analog front ends 403 is connected to the corresponding sensor device 108. The output voltage from each AFE 403 in turn is fed to separate analog to digital converter 407-1,…407-n (ADC) channels of the microcontroller 402. In some embodiments, a real time clock (RTC) 406 is connected to the microcontroller to time the operation. A power management IC 409 manages the power requirement of the entire system with the help of a rechargeable cell 410. The power management IC 409 supplies a voltage of 3.3V to all individual modules 401.
[0064] The microcontroller 402, through executable computer program instructions stored in a non-transitory computer-readable storage medium, converts the obtained voltage into the corresponding glucose concentrations and sends the result to the an output unit 405. The output unit 405 includes a graphical Liquid Crystal Display (LCD) unit 413 or a Bluetooth module 408 for wireless transmission of the output, or both. In some embodiments, a real time clock (RTC) 406 is connected to the microcontroller to time the operation.
[0065] The system architecture of the developed meter module 401 is shown in FIG. 4C. The flow chart of the controller program starts with the initialization of serial peripheral interface (SPI) communication which is used by the controller 402 for communicating with the LCD 413. Upon initialization, commands are provided to the LCD module 413 for the desired operation, such as displaying a message. I2C communication 411 is used by

RTC 406 and AFE 403 connected to the controller 402. After AFE initialization, ADC module 407 is enabled for converting the analog into its corresponding digital value. After all the different modules are initialized, the system waits for the sample to reach the analysis wells by continuously monitoring the output values of all AFEs. Once the sample reaches the analysis well, a change in current is detected. Once this change is detected, the microcontroller measures the voltage sequentially from the AFEs after predetermined time duration. The voltage is then converted to corresponding analyte concentration and displayed on the LCD 413, the Bluetooth enabled device 408, or both.
[0066] In one embodiment, a method 500 for analyzing glucose concentration in a plurality of samples is disclosed, as shown in FIG. 5. In step 501, the plurality of samples obtained one or more subjects are loaded in the array device. In step 502, pressure is applied to allow each of the samples to be mixed with a reagent. In step 503, the mixture is allowed to pass through meander channels for complete mixing to obtain an uniform solution. In step 504, each sample mixture is contacted with a CuO-based non-enzymatic biosensor array element to determine a voltage proportional to current generated from glucose oxidation. In step 505, the voltage is converted to the corresponding glucose concentration and the glucose concentration of the sample is displayed.
[0067] While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope. Further, the examples to follow are not to be construed as limiting the scope of the invention which will be as delineated in the claims appended here.

[0069] An exemplary device according to the various embodiments of the invention as illustrated in FIG. 1 was prepared. The experiment was conducted with Glucose (Glucose, reagent), ascorbic acid (AA, reagent grade), dopamine (DA), uric acid (UA, ≥ 99.0% crystalline), acetamidophenol (AP, 98% analytical standard). Conducting inks of carbon, silver and Ag/AgCl were used for the preparation of electrodes. A silicone elastomer kit and Polydimethylsiloxane (PDMS) were mixed in the ratio of 10:1 prior to use. Negative photoresist and developer were used in the fabrication of microchannels. [0070] The experiment was carried out with a three electrode cell. In the case of screen printed electrodes AgCl ink printed electrode was used as the pseudo reference electrode. The modified electrodes were used as working electrodes and carbon as the counter electrode. Surface morphology of the modified electrodes was studied using atomic force microscope in non-contact mode. A direct laser write lithography instrument was used for patterning the negative photoresist. Blood serum samples were successfully tested and the results obtained were found to be close to the results of commercially available glucose sensor strips.
[0071] Example 2: Synthesis of CuO nanoparticles
[0072] Considering the excellent electrocatalytic activity of CuO towards glucose oxidation, CuO based nano slurry was developed and was employed with screen printing technology for the fabrication of the glucose sensors. The mechanism of direct electrooxidation of glucose depends on the catalyst used. Cu based materials have shown better catalytic effect towards electrooxidation of glucose due to the redox couples mediated by Cu(OH)2/CuOOH. CuO, being a p-type semiconductor, shows excellent electrochemical activity which led to its immense use in the development of gas and glucose sensors. The mechanism of oxidation of glucose on Cu and CuO modified electrodes involves the complete cleavage of C-C bonds resulting in greater sensitivity of the non-enzymatic sensors.
[0073] CuO nanoparticles were synthesized by adding 26 ml of ammonia drop wise to 700 ml 0.05 M CuSO4 under constant stirring till the solution turns to dark blue color. 150

ml of 1 M NaOH solution was added drop wise which resulted in the formation of a light blue colored precipitate of [Cu(OH)4]2- as the pH reaches 14. This precipitate was filtered and washed with distilled water several times and calcined at 400°C for 3 hours.
[0074] Example 3: Design and development of screen printed electrodes [0075] Screen printing technology was used for the fabrication of the sensor electrodes. The design for the different screens used for sequential printing is shown in FIG. 2. The working electrode of the three electrode system was designed with a 2 mm diameter working electrode placed between the counter and the reference electrodes. The working and the counter electrodes were placed at a distance of 0.5 mm apart. Since the fabrication of the screen printed electrodes requires sequential printing of silver, AgCl and carbon inks, three different designs were created.
[0076] Polyethylene terephthalate (PET) sheets of 0.6 mm thickness were used as the substrate for screen printing. Prior to screen printing, the PET sheet was cleaned with acetone and was pre-heated at 90°C for 12 hours. The first layer to be screen printed on the PET substrate was the silver conducting layer. The silver layer is then coated with conductive carbon ink to prevent it from oxidization. Ag/AgCl is then applied to the tip of the reference electrode.
[0077] Thermal curing at 65°C for 15 min was carried out after printing of each layer. For printing the working electrode, a catalytic ink formulation was developed by dispersing the CuO nanoparticles in a medical sensor grade conductive carbon ink. The optimized ink formulation which is prepared with 33% CuO nanoparticles in carbon ink was screen-printed onto the working electrode region and dried at 45°C for 2 hours. The reduced temperature and slow drying process helped to avoid the formation of cracks on the printed region during drying.
[0078] Example 4: Fabrication of microfluidic channels
[0079] The PDMS based microfluidic channels were fabricated by soft lithography. Initially, a glass substrate of 3 inches diameter was cleaned thoroughly in piranha solution (H2SO4 and H2O2 in the ratio 5:1) for 15 minutes and thoroughly washed with distilled

water. The photoresist was spin coated at 1000 rpm for 100s followed by a spread cycle of 1400 rpm for 10s and prebaked at 120°C for 30 minutes. After cooling, the pattern was exposed using a direct laser write pattern generator with an ultraviolet (UV) dose of 56 mW and energy intensity of 80%. Post exposure baking was carried out at 95°C for 90 min followed by developing in developer solution for 7 minutes.
[0080] The developer removed the areas that had not been exposed to UV, resulting in the formation of the negative (master) of the microstructure pattern. Silicon elastomer was used for the micro fabrication of the stamp. The silicon encapsulant contains two components, base and curing agent that were thoroughly mixed in the ratio 10:1 and dispensed over the master placed in a petri dish with the features facing upward. The air bubbles in the solution were removed by degassing using a vacuum pump. Following this, PDMS was cured at 100°C for 35 minutes and peeled off from the surface of the master to obtain the PDMS stamp.
[0081] Example 5: Computational Fluid Dynamics (CFD) Simulation [0082] To evaluate the mixing efficiency of the proposed design, computational simulation was carried out. The inlet was given an atmospheric pressure of 101325 Pa. No-slip boundary condition was assumed along the walls of the microchannel. The outlet boundary condition was specified to be laminar outflow with a zero static pressure. Two model fluids, water in the analyte stream and ethanol in the reagent stream were fed in to the device for analysis. The model fluid system considered was assumed to have a low diffusivity value (1.2 x 10-9 m2 s-1). The Navier-Stokes equation was solved for obtaining the velocity profile through the microchannels while Fick’s law of diffusion was used for studying mass transport within the microchannels.
[0083] The concentration plot obtained for the mass transport between the two streams is shown in FIG. 6. A cut line was drawn from the first meander to the outlet of the channels to understand the variation of concentration along the microchannels. The variation in concentration along the cut line through the channel is represented in FIG. 6. The design was modeled in such a way that the primary inlet was assigned a concentration value of 0

(blue color) and the secondary channel had an initial concentration of 0 (red color). On the completion of mixing, a uniform green color was obtained at the outlet of the channel. From the simulation results, it is evident that the mixing is complete as the cross-sectional concentration at the outlet is uniform with a value of 0.5. The presence of meander channels leads to chaotic advections within the microchannel, which improves mixing efficiency.
[0084] Example 6: Study of fabricated microchannels using profilometer [0085] The microfluidic channels were morphologically characterized using a profilometer and electrochemically characterized for its glucose sensing capabilities. Blood serum samples were successfully tested. The dimensions of the fabricated master were studied using profilometer. FIG. 7A shows the profilometer plots for the depth of the fabricated microchannels. It was evident that the fabricated micro channels have a depth of 110 µm. The dimensions of the alternatively varying diameters were also confirmed. FIG. 7B and Fig. 7C show that the dimensions of the micro channels were 200 µm in the X and 400 µm in the Y direction.
[0086] Example 7: Electrochemical characterization of the LOC [0087] FIG. 8A represents the linear sweep voltammograms recorded on the Lab-on-a-Chip with varying concentrations of glucose. The oxidation current increases linearly with glucose concentration up to 27 mM. There are two linear ranges, the first range is up to 15 mM with a linear regression equation (µA), Ip = 195.1 + 32.67 C (mM) where regression coefficient, r = 0.9944. The second range is from 15 mM to 27 mM with Ip = 441.6 + 18.64 C (mM) where r = 0.9919. A shift in peak potential is observed with increasing glucose concentration. This can be attributed to the slow kinetics of electrooxidation of glucose on the electrode surface. From the linear sweep voltammetry (LSV), it is found that the highest peak potential observed is 0.6 V.
[0088] Hence all amperometric experiments were carried at an applied potential of 0.6 V. The mechanism of glucose oxidation on copper electrode in alkaline medium shows that CuO is responsible for the direct electrooxidation of glucose through six catalytic cycles

each involving two-electron transfers leading to the formation of formic acid. The formation of Cu (II)/Cu (III) states and the oxidation peak of glucose occur at around +0.40 to +0.80 V. Hence the Cu (III) species is acting as a mediator for the electrocatalytic oxidation of glucose. This is distinctly different from that of chemical and biochemical glucose oxidation that involves gluconic acid intermediate formation. [0089] The results of chronoamperometric experiments carried out at 0.6 V is shown in FIG. 8B. The steady current response was recorded for 20 seconds. From the graph, it is observed that current decays with time obeying Cottrell equation, which describes the change in electric current with respect to time in a controlled potential experiment. It is also observed that the current decreases and becomes stable at around 12 seconds and hence the sampling time was fixed as 12 seconds. FIG. 8B shows two linear ranges of current response with concentration. For glucose concentration up to 15 mM, the linear regression equation is Ip (µA) = 34.82 + 19.02 C (mM) with regression coefficient, r = 0.9979. The other linear range is from 15 mM to 27 mM with Ip (µA) = 261.4 + 32.5 C (mM) with r = 0.9905.
[0090] Example 8: Reproducibility, repeatability and storage stability of the LOC [0091] The developed LOC is intended for but not limited to community screening of blood glucose. It is essential that the fabrication process be highly reproducible. For this purpose, LOCs were fabricated and the chronoamperometric response of these sensors towards 3 mM and 9 mM of glucose at 0.6 V was compared. FIG. 9A shows the electrochemical response for 3mM glucose concentration on ten different electrodes. FIG. 9B shows the electrochemical response for 9mM glucose concentration on ten different electrodes. The response current is obtained from the various chips and the variation was found to be less than 4%. This confirmed that the process used for the fabrication of the disposable strips is highly reproducible. An important criterion for the commercial viability of the LOC is their storage condition and shelf life. Sensors with minimum storage restrictions and prolonged shelf life are highly desired. In order to understand these two characteristics of the LOC, numerous electrodes were fabricated and

evaluated over a period of 4 months for their repeatability and reproducibility by testing them chronoamperometrically at +0.60 V with 9 mM of glucose. From the tests conducted, a variation of less than ±3% was observed over the entire testing period.
[0092] Example 9: Blood serum sample analysis in LOC
[0093] As the Lab-on-a chip device in intended for mass screening of patient samples, it is highly essential to validate the performance of the sensor with real samples. So, chronoamperometry was carried out with serum obtained from two volunteers which were mixed and tested at +0.60V. From Table 1 it is evident that the results obtained from the fabricated sensor are in line with the commercially available strips. Moreover, Table 1 also highlights the very high recovery rate of the CuO based glucose sensor along with acceptable relative standard deviation (RSD) values that fall within 3 to 10%.
Table 1: Determination of glucose concentrations in blood serum samples (n=4)
Commercial
Added glucose Recovered
Samples Glucometer LOC (mM)
(mM) %
(mmol L-1 )
1 5.44 5.69 9.0 92
2 5.11 4.82 9.0 94
*Each sample was tested four times
[0094] Example 10: Multi-potentiostat configuration
[0095] For developing the multi potentiostat circuit, ten different analog front ends were connected to SCL and SDA pins of the microcontroller. The Multi-potentiostat configuration of AFEs is shown in FIG. 11A. The SCL and SDA pins are common to the ten AFEs. Three different MENB ports of the microcontroller were connected to the three different AFEs. The microcontroller was programmed to activate the AFE sequentially through the MENB. The communication between the AFE and microcontroller was carried out using Inter-Integrated circuit (I2C) protocol. I2C communication commonly uses two wires for data transfer - SCL and SDA. In the case of multi-potentiostat circuit, it uses 10

separate AFEs and all AFEs (LMP91000) were given the same fixed seven bit address (1001000). If the MENB pin of a particular AFE is at logic level low all the I2C communication was enabled to that AFE and communication was disabled if MENB was at high logic The AFE is configured as 3-lead amperometric set up using the MODECN register. Suitable bias voltage, bias sign and internal zero values were set using REFCN register. Rload value and TIA gain were set using TIACN register. The potentiostat maintained a constant potential and electrochemical reaction current was converted to voltage and the change in voltage was observed.
[0096] Example 11: Working and integration of the potentiostatic meter module with the array device
[0097] The potentiostatic meter module was tested for the variation in glucose concentration values in a plurality of electrodes with the known glucose concentration of 3mM and 9mM. When the meter is turned on, the bias current is set to ‘0’ volts so that no current spike occurs due to incomplete covering of the sensor electrodes present in the analysis wells. Also, since the potential is 0 V, no electrochemical reaction occurs during the filling up of the analysis wells. After the different analysis wells are completely filled, the meter provides the preprogrammed potential of 0.6 V to the working electrode of all the sensor electrodes. This result in the oxidation of glucose on the working electrode and a voltage proportional to the reaction current is made available to the microcontroller from each analog front end (AFE).
[0098] From the electrochemical characterization of the LOC, it was observed that a sampling time of 12 seconds was required for the chronoamperogram to become stable. Hence the microcontroller was programmed to measure the voltage from the AFE’s after a time interval of 12 seconds after application of 0.6 V. With the help of the calibration plot programmed into the microcontroller, the obtained voltage is converted to the corresponding glucose concentrations and the results are displayed on the graphical LCD screen. The pictorial representation of printed circuit board (PCB) of meter is shown in FIG.10. The test results obtained using the developed meter were compared for the ten

electrodes and is shown in FIG. 12A for 3mM glucose concentration and for 9mM glucose concentration in FIG. 12B. It is seen that the variation of glucose concentration for plurality of electrodes was less than 3 %.

WE CLAIM:
1. A microfluidic array device (100) for determining glucose concentration in a
plurality of samples, comprising a plurality of assembled units, each unit comprising:
a) a sample well (101) configured to receive a sample;
b) a reagent well (102) configured to receive reagent from a common reagent reservoir (103);
c) a mixing well (104) configured to receive the sample from the sample well and reagent from the reagent well and form a mixture;
d) meander channel (105) comprising a plurality of meanders (106) fabricated with alternatively varying diameters and configured to receive the mixture from the mixing well and enhance the mixing of the mixture, thereby forming a uniform solution; and
e) sensor chamber (107) housing a sensor device (108) configured to receive the uniform solution from the meander channel and generate an output indicative of glucose concentration in the sample.

2. The device of claim 1, wherein the sensor chambers (107) of the plurality of assembled units are connected to a common outlet (109) mounted with a syringe (110) or a pump (111) to supply negative pressure thereon.
3. The device of claim 1, wherein each unit further comprises microchannels (312) connecting the reagent well to the corresponding sample well at the inlet of the mixing well configured as a T-inlet (314).
4. The device of claim 1, further comprising a display unit (113) to display the glucose concentration in the samples.
5. The device of claim 1, wherein the sensor device is a non-enzymatic glucose

biosensor (200).
6. The device of claim 5, wherein the sensor device comprises: a three-electrode system comprising at least one active electrode (201) modified with a CuO nanoparticle coating (204), a carbon counter electrode (202) and an Ag/AgCl reference electrode (203).
7. The device of claim 6, wherein the substrate for the active electrode is selected from polyethylene terephthalate, polycarbonate, polyethylene, polypropylene, polyvinyl chloride, polyamide, polymethyl methacrylate, or polysulfone.
8. The device of claim 6, wherein the working electrode has a diameter of 1.8-2.2 mm.
9. The device of claim 6, wherein the working electrode is screen printed between the counter and the reference electrodes at a distance of 0.4-0.6 mm apart.
10. The device of claim 1, wherein the line length of a cut line drawn from the first meander to the outlet of the meander channel is between 15000-20000 µm.
11. The device of claim 1, wherein the meander channel has an alternately varying diameters of 150-250 µm and 350-450 µm, and a depth of 100-120 µm.
12. The device of claim 1, wherein the generated output is voltage indicative of the glucose
concentration in the sample.
13. The device of claim 1, wherein the sensor chambers are integrated with a
potentiostatic meter module (401), the module comprising:
a plurality of analog front ends (403), wherein each of the analog front ends are

connected to the corresponding sensor units (108);
a microcontroller (402) connected to each of the analog front ends (403) and configured to interface with the sensor units and convert the output into glucose concentrations of the samples; and
an output unit (405), configured to transmit the glucose concentrations.
14. The device of claim 13, wherein the output unit comprises a graphical LCD display
unit (413) or a Bluetooth enabled device using a Bluetooth module (408).
15. A method of determining glucose concentration in a plurality of samples,
comprising:
a) loading a plurality of samples obtained from one or more subjects in an array device;
b) contacting the samples with a reagent, thereby obtaining a mixture;

c) passing the mixture through meander channels having alternately varying diameters, thereby obtaining enhanced mixing of the glucose and reagent as an uniform solution;
d) contacting the uniform solution with a non-enzymatic biosensor array element comprising CuO nanoparticle coating in the working region to obtain an output indicative of glucose concentration in the sample; and
e) converting the output to determine glucose concentration in the samples.
16. The method of claim 15, wherein the obtained output is voltage indicative of
glucose concentration in the sample.