Graphene-based BIO-sensors and method of fabrication of the same

Abstract

A bio-sensor that is sensitive to low levels of lactate and / or glucose can be formed by modifying a graphene surface to include –COOH or other oxygen-containing functional groups, forming amide bonds with the –COOH or other oxygen-containing functional groups and then forming amide bridges between the –COOH or other oxygen-containing functional groups and an enzyme sensitive and selective to lactate and / or glucose The bio-sensor can be used in non- invasive bio-sensors.

Description

GRAPHENE-BASED BIO-SENSORS AND METHOD OF FABRICATION OF THE SAMETECHNICAL FIELD

[0001] The current disclosure relates to bio-sensors and in particular to the fabrication of graphene-based bio-sensors.BACKGROUND

[0002] Bio-sensors are important for a range of applications in both medical and non-medical settings. They can be used to monitor bodily functions, detect diseases and infections, and track fitness levels, among other things. Non-invasive bio-sensors are particularly valuable because they do not require the insertion of a probe or device into the body, which can be uncomfortable and potentially harmful. Instead, non-invasive sensors can detect and measure bio-markers through the skin or other non-invasive means.

[0003] Sensors may be used to monitor various bio molecules such as lactate and glucose. Lactate is a molecule that can indicate muscle fatigue, tissue hypoxia, and metabolic disorders. Glucose is a sugar, a vital source of energy for the cells in our body. Monitoring glucose levels regularly is essential for diabetic individuals. For diabetic individuals, keeping glucose levels in check is especially significant as it can prevent hyperglycemia or hypoglycemia and consequent life-threatening complications.

[0004] An additional, alternative, and or improved bio-sensor capable of measuring the presence of molecules is desirable.SUMMARY

[0005] In accordance with the present disclosure there is provided a method of fabricating a bio-sensor capable of detecting a bio-molecule comprising: modifying a graphene surface to introduce -COOH or other oxygen-containing functional groups; forming amide bonds to the - COOH or other oxygen-containing functional groups; and forming an amide bridge between the -COOH or other oxygen-containing functional groups and an enzyme sensitive and selective to the bio-molecule of the bio-sensor.

[0006] In a further embodiment of the method, modifying a graphene surface comprises: treating the graphene surface with at least one of: a solution of graphene oxide solution inwater; and pyrrole-2-carboxylic acid solution; rinsing the graphene surface with deionized (DI) water; and drying the graphene surface.

[0007] In a further embodiment of the method, modifying a graphene surface comprises: treating the graphene surface with a modifying solution comprising at least one of: graphene oxide solution in water; reduced graphene oxide solution in isopropyl alcohol / water; edge functionalized graphene dispersions; quantum carbon dots dispersions; pyrrole-2-carboxylic acid solution; nafion / ethanol solution; chloroplatinic H2PtCle acid in water; and tetrachloroauric(lll) acid trihydrate HAuCl^xHzO; rinsing the graphene surface with deionized (DI) water; and drying the graphene surface.

[0008] In a further embodiment of the method, the modifying solution is drop casted onto the graphene surface.

[0009] In a further embodiment of the method, forming amide bonds comprises, after modifying the graphene surface: treating the graphene surface with an amide solution comprising at least one of: N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride ; N-Hydroxysuccinimide; and 2-(N-Morpholino)ethanesulfonic acid; rinsing the graphene surface with deionized (DI) water; and drying the graphene surface.

[0010] In a further embodiment of the method, the amide solution is drop casted onto the graphene surface.

[0011] In a further embodiment of the method, forming the amide bridge between the -COOH or other oxygen-containing functional groups and the enzyme sensitive and selective to the biomolecule comprises: treating the graphene surface with one or more of: lactate oxidase; and glucose oxidase; rinsing the graphene surface with deionized (DI) water; and drying the graphene surface.

[0012] In a further embodiment of the method, the enzyme is drop casted onto the graphene surface.

[0013] In a further embodiment of the method, the graphene surface comprises a plurality of graphene channels.

[0014] In a further embodiment of the method, the method further comprises: measuring conductivity characteristics of all graphene channels; and selecting graphene channels having measured resistance below a threshold as bio-sensor channels.

[0015] In a further embodiment of the method, the method further comprises annealing the graphene surface at a temperature of 30 - 100°C to remove moisture and / or absorbed gases on the graphene surface.

[0016] In a further embodiment of the method, the graphene surface is on a substrate comprising one of: rigid semiconductor substrates; rigid insulating polymer / glass substrates; and flexible insulating polymer / glass substrates.

[0017] In accordance with the present disclosure there is further provided a bio-sensor comprising: a bio-sensor formed according to any one of the methods described above; microfluidic channels for providing a sample to the bio-sensor; and electronics coupled to the biosensor for determining electrical characteristics of the bio-sensor and providing data indicative of the electrical characteristics to a computing device.

[0018] In a further embodiment of the bio-sensor device, the bio-sensor is formed by treating the graphene surface with lactate oxidase, and wherein bio-sensor device senses lactate.

[0019] In a further embodiment of the bio-sensor device, the bio-sensor is formed by treating the graphene surface with glucose oxidase, and wherein bio-sensor device senses glucose.

[0020] In a further embodiment of the bio-sensor device, the electronics comprise a communication radio for wirelessly transmitting the data indicative of the electrical characteristics to the computing device.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In the accompanying drawings, which illustrate one or more example embodiments:

[0022] FIG. 1 depicts a process for manufacturing a graphene-based bio-sensor and device incorporating the bio-sensor;

[0023] FIG. 2 depicts a system incorporating a bio-sensor device;

[0024] FIG. 3 depicts a method of manufacturing a graphene-based bio-sensor;

[0025] FIGs. 4A, 4B are optical images of an area of a graphene channel before and after modification;

[0026] FIGs. 5A - 5D depict Raman characterization of the area of the graphene channel before and after modification;

[0027] FIGs. 6A - 6H depict results of functionalized bio-sensors for the detection of lactate and glucose;

[0028] FIGs. 7A, 7B depict performance of the bio-sensor over a period of time;

[0029] FIGs. 8A, 8B depict sub-nanomolar level detection of lactate and glucose;

[0030] FIGs. 9A - 9D depict performance and calibration curves of the bio-sensors based on lactate concentration;

[0031] FIGs. 10A - 10E depict measurement of lactate from sweat samples and molecular biology grade water; and

[0032] FIG. 11 depicts detection of lactate concentrations in sweat as detected by the biosensor.DETAILED DESCRIPTION

[0033] Graphene based bio-sensors can be formed on a graphene surface that are highly sensitive to the molecule the bio-sensor is designed to detect. For example bio-sensors can be formed to detect bio-molecules such as lactate or glucose. A non-invasive graphene based bio-sensor is described further herein that is capable of sub-nanomolar sensitivity detection level of bio-markers, including electrolytes, such as lactate and / or glucose in fluids samples. The graphene-based bio-sensor is capable of the high sensitivity detection at room temperature and also has a long shelf lifetime.

[0034] Chemical vapor deposition graphene (CVD-g) is a well suited material for bio-sensors. CVD-g is a thin layer of graphene that is grown on a substrate using chemical vapor deposition techniques. The substrate may be rigid or flexible. The use of CVD-g has several advantages for bio-sensors, including its biocompatibility, electrical conductivity, and large surface area. CVD-g also has high mechanical strength, which makes it more durable and resistant to wearand tear compared to other sensor materials. The current process functionalizing CVD-g devices that allows them to detect ultra-low levels of bio-molecules or biomarkers such as lactate and glucose.

[0035] Lactate is a molecule that can indicate muscle fatigue, tissue hypoxia, and metabolic disorders. Glucose is a sugar, a vital source of energy for the cells in our body. Monitoring glucose levels regularly is essential for diabetic individuals. For diabetic individuals, keeping glucose levels in check is especially significant as it can prevent hyperglycemia or hypoglycemia and consequent life-threatening complications. Hyperglycemia can lead to a diabetic coma, the prevalence and mortality of certain malignancies, like breast, liver and colorectal cancers. Hypoglycemia can cause various symptoms such as blurred vision, difficulty concentrating, numbness, and drowsiness and some cases in seizures and coma.

[0036] The process described further herein involves modifying the surface of the CVD-g with functional groups that can bind to lactate or glucose molecules. This enables the sensors to detect lactate or glucose at sub-nanomolar levels, which is orders of magnitude more sensitive than previous sensors.

[0037] CVD-g bio-sensors can be used in a wide-ranging number of applications. In the fitness industry, functionalized CVD-g sensors can be used to monitor lactate levels during exercise, which can help athletes optimize training and performance. In the medical industry, functionalized CVD-g sensors can be used to diagnose and monitor a wide range of diseases, including sepsis, cancer, and metabolic disorders. CVD-g based sensors are also valuable for environmental monitoring and industrial applications, where they can be used to detect pollutants and monitor manufacturing processes. Bio-sensors formed using CVD-g can provide sensors that are highly sensitive, durable, and biocompatible.

[0038] The formation of bio-sensors is described further below with particular reference to detection of lactate and glucose, however it is possible to functionalize the graphene surface with other enzymes or molecules. The biosensors provided by the process described below can be used to detect lactate or glucose concentration at the sub-nanomolar level (i.e. < 1 nM). The developed biosensor to detect the extremely low concentration of lactate or glucose. The bio-sensor can be formed from CVD mono- or bilayer multilayer graphene as a sensing material with well-designed functionalization c developed for lactate or glucose detection andquantification. At this level of detection, fitness and medical testing could be made much easier via sweat as opposed to a more invasive method such as blood tests.

[0039] The bio-sensor may also be used for the detection of lactate or glucose mass down to 10-14grams. Small areas of a designed sensing element, such as from 90 microns to 1000 microns, are able to detect the extremely low volume of liquid starting from 0.5 pL with an ultralow concentration of the lactate or glucose such as less than 1 nM. Ultra-high precision in mass weighing is required in many fields of study including analytical chemistry, nano-technology, materials science, pharmaceutical industry and bio-medical research.

[0040] The formation of the bio-sensor uses functionalization procedures combining unique properties of mono- and / or bilayer or multilayer of graphene and a modification approach including enzymes that are sensitive to the biomarkers to be detected. The biosensors use CVD-g as an active sensing layer. CVD-g possesses a unique set of properties that are successfully used for the fabrication of biosensors. On the one hand, CVD-g has low electrical resistance and on another hand, CVD-g can be easily modified and functionalized. The proposed functionalization methodology produced highly effective biosensors for lactate or glucose detection in commercial samples of sweat as an example application.

[0041] The process described further herein provide bio-sensors having reproducible, high signal / noise and real-time responses to lactate or glucose biomarkers. The sensors can provide continuous non- invasive monitoring of biomarkers detection and long lifetime of biosensors. The sensing areas of the biosensors react immediately with targeted lactate or glucose showing a very high response to the biomarker. The response results from a combination of unique properties of CVD-g, i.e. low electrical resistance and easy surface modification, and specially modified surface.

[0042] Successful laboratory validation of bio-sensors for lactate and glucose detection capabilities are demonstrated using human full sweat from multiple donors. The developed biosensors were able to detect not only pure lactate or glucose from its solutions but also the lactate or glucose from commercial sweat samples from multiple donors. For the accurate quantification of the lactate or glucose, the biosensors should be calibrated accordingly.

[0043] The biosensors may be provided as disposable or replaceable sensing elements that can be easily changed in a sensor device. Replacing the sensing element can reduce costs and avoid possibly undesirable saturation of the sensors’ surface by the targeted liquid, which can provide accurate results.

[0044] The bio-sensors were tested on various samples of sweat, however the sensors can be used to detect lactate or glucose in saliva, tears blood and other human body fluids.

[0045] FIG. 1 depicts a process for manufacturing a graphene-based bio-sensor and device incorporating the bio-sensor. Single layer, double layer or multi layers of CVD-g were used as a platform for the fabrication of the biosensors in conjunction with well -designed functionalization procedures for lactate or glucose detection and its quantitative evaluation.

[0046] As depicted in FIG. 1 , a first substrate 102, such as Cu / Ni, is used to grow the graphene surface 104 using chemical vapour deposition. The CVD-g graphene surface 104 is transferred to a second substrate 106. The graphene surface may be graphene / graphene derivatives including for example, monolayer graphene, bi-layer graphene, tri-layer graphene, 4-10 layers of graphene, possibly including substrate doping using chemical and metal nano-particles doping the graphene or graphene derivatives. The second substrates 106 may include rigid semiconductor substrates such as SIO2 / SI, oxides on silicon, etc., as well as rigid or flexible insulating polymer / glass substrates such as substrates glass, PET, PI, PMMA, SU-8. The graphene surface can for formed on the rigid / flexible substrate. Additionally or alternatively, the graphene surface may be provided as a commercially available mGFET chips. Using graphene produced by the fully controlled CVD method allows creating the stable and highly sensing material with suitable constancy of certain parameters such as electrical resistances of the graphene channels of the devices and reproducibility. CVD-g is an ideal base for the creation of a general sensing platform that can be used for the creation of different types of biosensors for detection of the certain kinds of molecules. Electrical features such as contacts 108 can be printed onto the graphene surface using existing fabrication techniques and the graphene surface functionalized 110 for the detection of lactate or glucose. The functionalized CVD-g surface can be used as a bio-sensor 112 that can be packaged into a sensor device 114. The sensor device may include the bio-sensor, micro-fluidics 116 for transferring liquid to the bio-sensor. The sensor device 114 may also include electronics 118 providing an interface between the bio-sensor 112 and a computing device or other processing and possible displays.

[0047] FIG. 2 depicts a system incorporating a bio-sensor device. The system 200 comprises a bio-sensor device 202 comprising a bio-sensor 204 and electronics 206. Although not depicted in FIG. 2, the bio-sensor device 202 may include other components such as microfluidics and packaging, power sources, etc. The sensor device 202 may be interfaced with a computing device, depicted as a mobile device 208. The computing device 208 may include a processor 210, input / output (I / O) interface 212, sensors 214, communication radios 216, nonvolatile storage 218 and memory 220. The computing device may include a display 222 or other output device, or may be connectable to an external display or other output device. The memory 220 may store instructions which when executed by the processor configure the computing device 208 to provide various functionality including sensor communication functionality 224 that allows the computing device to communicate with the sensor device, over a wireless or wired communication channel. The instructions may further provide sensor data processing 226 that can process the data received from the bio-sensor device, and possibly other sources. The functionality may also include data display interface functionality 228 for displaying information to the user.

[0048] The computing device, whether a mobile device or other computing device, can include software that serves as an interface between the sensor and the user, allowing users to view and interpret the data generated by the sensor. Interactive software can display data from biosensors in real-time, allowing users to monitor their health, fitness, and other parameters continuously, or use the sensor data in other real-time or non-real-time applications.

[0049] The software may provide actionable items and feedback to the users based on the data collected by the bio-sensor. For example, a fitness tracker can recommend specific exercises based on the user's lactate levels or heart rate. Similarly, a medical bio-sensor can alert patients or healthcare providers if any biomarker levels exceed the safe range. The software can also help users interpret the data generated by the bio-sensor by providing context and insights into the data. This feedback can help users make more informed decisions about their health, fitness, and overall well-being. The software may combine the data from the sensor device 202 with data from other sensors and data sources.

[0050] FIG. 3 depicts a method of manufacturing a graphene-based bio-sensor. The method 300 modifies a graphene surface in order to make it highly sensitive to lactate or glucose. The modified graphene surface can be used in a sensor capable of detecting lactate or glucose.The graphene surface may be a commercially available graphene surface formed by chemical vapour deposition (CVD). The graphene surface may be provided as a field transistor made of graphene and graphene derivatives including monolayer graphene, bi-layer graphene, tri-layer graphene, 4-10 layers of graphene or monolayer, bi-layer, tri-layer, 4-10 layers of graphene doped with chemical and / or metal nano-particles. The graphene surface may be provided as a mGFET formed using CVD-g with metal contacts providing at least one channel. The mGFET can be provided on a rigid or flexible substrate.

[0051] The graphene surface is prepared (302), which may include removing moisture and absorbed gases from the surface by annealing at a temperature of between 30-100°C. N2 blowing may be used, before and / or after annealing in order to remove small particles of residue on the surface. The surface preparation may include testing the electrical properties of the graphene surface in order to ensure it will provide an effective base for the biosensor. The testing can also be used to provide a baseline resistance of the channels. For example, the graphene surface may have a plurality of different channels that can be tested for their electrical characteristics such as resistance, current and or voltage. The channels may be tested for a range of possible operating conditions. For example the resistance and current characteristics can be measured for a range of voltages such as from -1 V to 1 V. Channels that pass threshold values for the characteristics may be selected for use in the biosensor device, while those channels that do not pass threshold values may be not used in the biosensor device. The “good” and “bad” channels may be marked or identified in various ways including in software of the device.

[0052] The CVD-g graphene surface, on mGFET chips or on rigid or flexible substrates, can be modified in order to create biosensors that are sensitive and selective to lactate or glucose. The surface can be modified by specific enzymes for the biomarker to be sensed. For example the enzyme may be lactate oxidase for sensing lactate, or glucose oxidase for sensing glucose. In order to modify the graphene surface with the enzymes, it can first be modified to introduce -COOH or other oxygen-containing functional groups, which can then be bound to amide and subsequently form an amide bridge to the enzymes. To modify the final sensor, it is needed to form an “amide-bridge” between the required enzyme and graphene oxide flakes or -COOH groups on the CVD-g surface

[0053] Once the graphene surface is prepared, it is modified (304). The graphene surface modification comprises treatment of the graphene, or graphene derivatives, surface with chemical solutions in order to introduce -COOH or other oxygen-containing functional groups onto the graphene surface. The -COOH or other oxygen-containing functional groups can be introduced by treating, for example by drop casting or micro-pipetting or chemical dispensing system, the graphene surface with a solution of graphene oxide flakes solution in water (0.05- 4 mg / mL) or Pyrrole-2-carboxylic acid solution (0.1 - 10 mM) for a length of time such as 4 minutes. After treating the graphene surface with the solutions, the graphene surface may be rinsed using deionized water and blown dry possibly using low pressure N2. The rinsing process may be performed a number of times. The graphene surface may be inspected by optical microscopy to verify that the graphene surface is appropriately modified.

[0054] Once the -COOH or other oxygen-containing functional groups are introduced onto the graphene surface, amide bonds are formed (306) be treatment of the graphene surface by an amide solution of deionized water and one or more chemicals, including for example N-(3- Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 0.01 -0.5 M ), N- Hydroxysuccinimide (NHS, 0.01 -0.5 M), 2-(N-Morpholino)ethanesulfonic acid (MES, 0.01 -0.5 M). The amide solutions may be drop casted onto the channels of the graphene surface. The solution may be applied for a period of time sufficient to form an amide bond to the -COOH or other oxygen-containing functional groups, such as 2 hours. The graphene surface can then be rinsed using DI water and dried using low pressure N2.

[0055] Once the amide bonds are formed, the graphene surface is treated with enzymes (308) for sensitizing the graphene surface to the detection of the desired biomarker. The enzymes used to treat the graphene surface may include lactate oxidase, 1 -10 jiL for a lactate sensor and glucose oxidase, 1 -10 JLLL for a glucose sensor. The enzyme solutions were drop-casted onto the sensor channel using a micropipette or chemical dispensing system and the sensor left for between 24-48 hours at temperatures between 4-25°C. The graphene surface was again rinsed using DI water and dried using low pressure N2.

[0056] It is noted that graphene oxide material has a lot of functional oxygen groups on its surface and could be modified directly by the enzyme without amide bonding. Further, reducedgraphene oxide, quantum carbon dots, Nation solutions, chloroplatinic HzPtCle acid, tetrachloroauric(lll) acid trihydrate HAuCl^xHzO could be also used for modification of CVD-g.

[0057] After the enzyme treatment, the bio sensor may be verified (310), for example by various baseline tests. The baseline tests may measure the l-V characteristics of the selected channels in order to identify the new baseline electrical characteristics after the functionalization. The baseline characteristics may be stored in association with the bio-sensor.

[0058] In order to fabricate reliable and mass-scalable biosensors with sufficient efficiency and high yield for the detection of certain kinds of bio-molecules / biomarkers (e.g. lactate, glucose), it is desirable to have a less batch-to-batch variation of initial parameters of the CVD-g surface materials ensuring consistency in the graphene surface that can be subjected to further modification / functionalization. mGFET chips having channels with constant parameters (e.g. resistance) was successfully used to fabricate a working reliable and robust functionalized biosensors. The performance of the selected devices was demonstrated. These devices were functionalized using the above approach and tested to determine the detection ability. The general curves for the detection of lactate and glucose are below.

[0059] FIG. 4A depicts an optical image of a selected area of the initial 3 graphene channels before functionalization. FIG. 4B depicts an optical image of the selected area of the 3- graphene channel after modification with graphene oxide. FIGs. 5A and 5B depicts the Raman characterization of the selected area of 3 graphene channel before modification, that is the monolayer or bilayer of graphene. The Raman shift (cm-1) for SiOz is 519.8cm-1, 1596.5 cm-1for the G band and 2689.2 cm-1for the 2D band. FIGs. 5C and 5D depicts the Raman characterization of the selected area of 3 graphene channel after modification with graphene oxide. The Raman shift (cm-1) for SiOz is 519.8cm-1, 1339.3cm-1, 1596.5 cm-1for the G band and 2689.2 cm-1for the 2D band. The optical image of FIG. 4B show the arrangement of graphene oxide flakes covering the CVD-g surface after the electrochemical modification process. According to Raman spectroscopy depicted in FIG. 5A - 5D, the graphene areas are homogeneous and uniform before modification as well as for CVD-g covered by graphene oxide after electrochemical treatment (FWHM (full width half maximum) of D, G and 2D bands). The spectra obtained from different areas of graphene are similar for initial mGFET chips, G and 2D bands could be easily classified as mono and bilayer of graphene. Modification of CVD-g by graphene oxide gives homogeneous and uniform areas combining overlapping D, G, and2D bands belonging to graphene oxide and graphene. The typical spectra are presented in FIGs. 5B and 5D.

[0060] Biosensors were manufactured according to 3 procedures. The first procedure used an initial modification of the CVD-g surface with pyrrole-2carboxylic acid solution followed by treatment with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and then with the enzyme lactate oxidase. The second procedure used an initial modification of the CVD-g surface with graphene oxide flakes solution followed by treatment with N-(3- Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and then with the enzyme lactate oxidase. The third procedure used an initial modification of the CVD-g surface with graphene oxide flakes solution followed by treatment with N-(3-Dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride and then with the enzyme glucose oxidase.

[0061] FIGs 6A - 6H depict results of tests of biosensors formed according to procedures #1 , #2 and #3. For the tests, PBS (phosphate buffered saline) solution was drop casted onto the sensor channel using a micropipette. A potential of 1 V was applied across the two electrodes while monitoring the 2-probe current with a source meter unit. For lactate / glucose detections, a series of solutions were drop casted onto the sensing channel while continuously monitoring the current readings. After the current baseline was stabilized, the series of chemicals were drop casted using micropipette or chemical dispensing system. The tests performed comprised:• 0.1-1 .0 jiL of 1 mM Sodium (Na)-L-Lactate (or D-(+)Glucose);• After the current was stabilized again, 0.1 -1 p.L of 10mM Na-L-Lactate (or D-(+)- Glucose);• This was repeated again with 0.1 -1 |_iL of 20mM Na-L-Lactate (or D-(+)-Glucose) and;• For some cases 0.8)iL of 40mM Na-L-Lactate (or D(+)-Glucose) and 0.1 -1 JJL of 60 mM Na-L-Lactate (or D-(+)-Glucose) solution;• Negative control could be used after Na-L-Lactate (or D-(+)-Glucose) test: 1 L of 1 x PBS was added after the additional chosen concentration of Na-L-Lactate (if applicable).

[0062] To detect the concentration of lactate / glucose of an unknown sample, the response signal can be compared with a calibrated percentage change (I) vs concentration curve. For detection and quantification of the lactate or glucose in different body fluids (sweat, saliva, tears, etc.) 0.1 -1 |iL of body fluids should be drop casted onto the sensor channel using a micropipette or microfluidic system.

[0063] Na-L-Lactate and D-(+)-Glucose are typically used for the substrate for lactate oxidase and glucose oxidase. However, it is possible to use other forms of L-lactate and D(L)-glucose.

[0064] FIG. 6A depicts performance of a bio-sensor according to procedure #1 . FIG. 6C depicts performance of a bio-sensor according to procedure #2. FIG. 6F depicts performance of a biosensor according to procedure #3. FIG. 6B depict normalized response, or calibration curve, of a bio sensor according to procedure #1 depending on lactate concentration in the range of 1 - 20 mM of lactate. FIG. 6D depict normalized response of a bio sensor according to procedure #2 depending on lactate concentration in the range of 1 - 20 mM of lactate. FIG. 6G depict normalized response of a bio sensor according to procedure #3 depending on glucose concentration in the range of 1 - 20 mM of lactate. FIG. 6E depict normalized response of a bio sensor according to procedure #2 depending on lactate concentration in the range of 1 - 60 mM of lactate. FIG. 6H depict normalized response of a bio sensor according to procedure #3 depending on glucose concentration in the range of 1 - 60 mM of glucose.

[0065] The performance of the biosensor was demonstrated after 1 -month its fabrication. The device showed a stable initial current before the lactate or glucose tests. This particular case confirmed that such device is able to detect lactate in different concentrations accordingly changing the current. The biosensor is suitable for multiple usages and provides a long shelf life time. FIG. 7A depicts the results for the first usage of the bio-sensor for continuous detection of lactate in the range 1 - 40 mM of concentration. The first usage was performed 1 month after fabrication of the bio-sensor. FIG. 7B depicts the results of a further usage of the bio-sensor for continuous detection of lactate in the range 1 -40 mM of concentration.

[0066] The fabricated devices were found to successfully detect ultra-low concentrations of lactate or glucose, in the nano-molar regime (~1 nM). FIGs. 8A, 8B shows the graphs, depicting the extremely high sensitivity response of the biosensor to a concentration level of lactate or glucose lower or equal to 1 nM. FIG 8A depicts the sub nano-molar detection for lactate. FIG8B depicts the sub nano-molar detection for Glucose. The device demonstrates the detection of extremely low concentrations of lactate (9x10-14g).

[0067] Laboratory tests were performed to quantify the ability of biosensors to detect lactate present in full sweat from commercial sources. To this end, the developed functionalization process was used to produce a lactate-sensitive biosensor. After that, the human sweat sample (1 pL) was added to the top of functionalized chips, and the resistance was measured.

[0068] Firstly, the detection range of the freshly functionalized devices biosensor was tested using lactate solutions. The biosensors could consistently detect lactate at 1 , 10, and 20 mM concentrations as depicted in FIG. 9A and 9B. Due to these promising results, it was further tested whether the biosensor could detect lower concentrations. A positive response at 1 nM was achieved as depicted in FIGs. 9C and 9D, which is believed to be unparalleled in the biosensor literature. FIGs. 9A and 9C depict the performance of biosensors depending on the lactate concentrations. FIGs. 9B and 9D depict calibration curves for from 1 mM to 20mM range and from 1 nM to 1 mM range of lactate concentration.

[0069] Afterward, the new biosensors were used to detect lactate from human sweat obtained from four donors as depicted in FIGs. 10A - 10D. Each sweat sample was tested independently in the biosensor to prevent saturation of detection sites. To this end, the sensors were calibrated with pure lactate solutions at 1 nM and 1mM concentrations. The negative control was pure bio-grade water and lactate solutions depicted in FIG. 10E.

[0070] The signal response (Al / lo) obtained from the sweat, depicted by blue circles in FIG. 11 , was compared with the corresponding concentrations obtained from FIG. 9A. For these tests, the lactate levels measured in sweat samples fall in the range of (1 pM to 1 mM), in agreement with the values for lactate in sweat typically reported in the literature. It is worth highlighting the very high sensitivity of the biosensors, which detected a lactate level of only 0.12 nM from one of the sweat samples as depicted in FIG 11 .

[0071] A commercial application of bio-sensors for lactate detection includes fitness tracking. Bio-sensors for lactate detection can be used to monitor lactate levels during exercise, which can help athletes optimize their training and performance. Fitness wearables and smartwatches can integrate these sensors to give users real-time feedback on their lactate levels and tailortheir workouts accordingly. Biosensors for glucose detection could be very informative for sugar level checking and could be helpful to optimize training performance and to avoid undesirable health complications.

[0072] A further commercial application includes medical diagnosis. Lactate is a biomarker that can indicate several health conditions, including sepsis, cancer, cardiac arrest, liver diseases and other metabolic disorders. Bio-sensors for lactate detection can be used in hospitals and clinics to diagnose and monitor these conditions. The monitoring of glucose biomarker level is essential for diabetic individuals to prevent possible life-threatening complications and in other critical systemic conditions such as cystic fibrosis, gestational diabetes and pancreatic cancer.

[0073] A further commercial application includes sports medicine. In sports medicine, biosensors for lactate detection can be used to assess the performance of athletes and diagnose overtraining. This information can help coaches and trainers adjust training regimens to optimize performance, prevent injury and manage glucose levels in diabetic patients.

[0074] A further commercial application includes pharmaceutical development. Bio-sensors for lactate or glucose detection can be used in pharmaceutical development to assess the efficacy of drugs that affect lactate or glucose metabolism. These sensors can be used to monitor lactate or glucose levels in vitro and in vivo, providing valuable insights into drug mechanisms of action and toxicity.

[0075] A further commercial application includes environmental monitoring. Bio-sensors for lactate detection can also be used in environmental monitoring to detect lactate-producing microorganisms in soil and water samples. This information can be used to assess the health of ecosystems and detect pollution.

[0076] The process of functionalization is important for developing biosensors that can accurately detect and measure specific biomolecules. In this case, the aim is to make biosensors sensitive to either lactate or glucose, two important biomolecules in biological systems. To achieve this, specific functionalization procedures have been proposed. These procedures involve the modification of the biosensor's surface by using chemical or biological molecules that can specifically interact with lactate or glucose molecules. The modified biosensor can then detect and measure the concentration of the target biomolecule with highsensitivity and accuracy. The functionalization procedures developed for lactate or glucose biosensors may vary depending on the specific biosensor design and the type of biomolecule being detected. However, some approaches include immobilizing enzymes or antibodies on the biosensor surface, which can selectively bind to the target biomolecule. Other techniques involve modifying the surface with chemical groups that can specifically interact with the target biomolecule

[0077] It is contemplated that any part of any aspect or embodiment discussed in this specification can be implemented or combined with any part of any other aspect or embodiment discussed in this specification.

[0078] The scope of the claims should not be limited by the embodiments set forth in the above examples, but should be given the broadest interpretation consistent with the description as a whole.

[0079] It should be recognized that features and aspects of the various examples provided above can be combined into further examples that also fall within the scope of the present disclosure. In addition, the figures are not to scale and may have size and shape exaggerated for illustrative purposes.

Claims

CLAIMS1 . A method of fabricating a bio-sensor capable of detecting a bio-molecule comprising: modifying a graphene surface to introduce -COOH or other oxygen-containing functional groups; forming amide bonds to the -COOH or other oxygen-containing functional groups; and forming an amide bridge between the -COOH or other oxygen-containing functional groups and an enzyme sensitive and selective to the bio-molecule of the biosensor.

2. The method of claim 1 , wherein modifying a graphene surface comprises: treating the graphene surface with at least one of: a solution of graphene oxide solution in water; and pyrrole-2-carboxylic acid solution; rinsing the graphene surface with deionized (DI) water; and drying the graphene surface.

3. The method of claim 1 , wherein modifying a graphene surface comprises: treating the graphene surface with a modifying solution comprising at least one of: graphene oxide solution in water; reduced graphene oxide solution in isopropyl alcohol / water; edge functionalized graphene dispersions; quantum carbon dots dispersions; pyrrole-2-carboxylic acid solution; nafion / ethanol solution; chloroplati nic H2PtCle acid in water; and tetrachloroauric(lll) acid trihydrate HAuCl4*xH2O; rinsing the graphene surface with deionized (DI) water; and drying the graphene surface.

4. The method of claim 3, wherein the modifying solution is drop casted onto the graphene surface.

5. The method of any one of claims 1 to 4, wherein forming amide bonds comprises, after modifying the graphene surface: treating the graphene surface with an amide solution comprising at least one of:N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride ;N-Hydroxysuccinimide; and2-(N-Morpholino)ethanesulfonic acid; rinsing the graphene surface with deionized (DI) water; and drying the graphene surface.

6. The method of claim 5, wherein the amide solution is drop casted onto the graphene surface.

7. The method of any one of claims 1 to 6, wherein forming the amide bridge between the -COOH or other oxygen-containing functional groups and the enzyme sensitive and selective to the bio-molecule comprises: treating the graphene surface with one or more of: lactate oxidase; and glucose oxidase; rinsing the graphene surface with deionized (DI) water; and drying the graphene surface.

8. The method of claim 7, wherein the enzyme is drop casted onto the graphene surface.

9. The method of any one of claims 1 to 8, wherein the graphene surface comprises a plurality of graphene channels.

10. The method of claim 9, further comprising: measuring conductivity characteristics of all graphene channels; andselecting graphene channels having measured resistance below a threshold as biosensor channels.11 . The method of any one of claims 1 to 10, further comprising annealing the graphene surface at a temperature of 30 - 100°C to remove moisture and / or absorbed gases on the graphene surface.

12. The method of any one of claims 1 to 11 , wherein the graphene surface is on a substrate comprising one of: rigid semiconductor substrates; rigid insulating polymer / glass substrates; and flexible insulating polymer / glass substrates.

13. A bio-sensor device comprising: a bio-sensor formed according to any one of claims 1 to 12; micro-fluidic channels for providing a sample to the bio-sensor; and electronics coupled to the bio-sensor for determining electrical characteristics of the bio-sensor and providing data indicative of the electrical characteristics to a computing device.

14. The bio-sensor device of claim 13, wherein the bio-sensor is formed by treating the graphene surface with lactate oxidase, and wherein bio-sensor device senses lactate.

15. The bio-sensor device of claim 13, wherein the bio-sensor is formed by treating the graphene surface with glucose oxidase, and wherein bio-sensor device senses glucose.

16. The bio-sensor device of any one of claims 13 to 15, wherein the electronics comprise a communication radio for wirelessly transmitting the data indicative of the electrical characteristics to the computing device.

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