Non-enzymatic Electrochemical Salivary Urea Sensor Device for Point-of-Care Settings

Abstract

Abstract Title: Non-enzymatic Electrochemical Salivary Urea Sensor Device for Point-of-Care Settings The present invention relates development of sensor device intended for point-of-care diagnosis. Additionally, the present invention can also be utilized for personalized diagnosis and regular kidney health monitoring. The described sensor device offers a low cost, scalable, portable, smart-phone integrated, non-enzymatic electrochemical platform for the rapid detection of urea from non-centrifuged, minimally processed human saliva samples. More specifically the non-enzymatic sensor device consists of silver dendrite modified porous graphene electrode that circumvents the issues of oxygen evolution reactions and negates the requirement of sample centrifugation steps as required in the reported works, thus being readily translatable to point-of-care settings. Figure 1

Specification

Description:Field of the Invention

The present invention relates to the development of sensor device intended for point-of-care diagnosis. Additionally, the present invention can also be utilized for personalized diagnosis and regular kidney health monitoring. The described sensor device offers a low cost, scalable, portable, smart-phone integrated, non-enzymatic electrochemical platform for the rapid detection of urea from non-centrifuged, minimally processed human saliva samples. More specifically the non-enzymatic sensor device consists of silver dendrite modified porous graphene electrode that circumvents the issues of oxygen evolution reactions and negates the requirement of sample centrifugation steps as required in the reported works, thus being readily translatable to point-of-care settings.

Background of the Invention
Most of the existing commercial urea detection platforms utilize spectrophotometric laboratory-based procedure towards quantification of urea from bio fluid samples such as saliva, serum, plasma, and others. However, the use of bulky instruments, requirement of skilled personnel for equipment handling & data analysis, along with laborious sample processing, high investment, and operational costs limits their applicability for low resource settings and mass community screening programs. It should be noted that "while the access to science is universal, the access to technology is largely dictated by socio-economic factors." Hence, in resource-limited settings, there is a dire need for deployment of point-of-care sensor devices.

Additionally, samples which can be easily collected by non-skilled/untrained volunteers/personnel in a non-invasive manner is advantageous for mass screening programs and point-of-care settings. In this regard, detection of urea from saliva samples finds significance. Moreover, high positive correlation (r2 ~ 0.99) of urea levels between saliva and blood samples bolsters the quantification of salivary urea over blood urea nitrogen. However, most of the works report urea detection from serum/blood samples, which is an invasive procedure and, in some cases, require specialist such as phlebotomists for venous blood collection, which is followed as a standard protocol.

The reported works in this realm, popularly utilize colorimetric or electrochemical detection principle for urea quantification. While the works based on colorimetric principle mainly utilize enzymatic detection strategy, electrochemical transduction works utilize both enzymatic and non-enzymatic strategies for detection purposes. Moreover, electrochemical sensors confer improved sensitivity over the colorimetric alternative and are hence advantageous.

While electrochemical enzymatic biosensors employing urease enzyme has been utilized, they have issues pertaining to stability and demand complex fabrication procedures associated with enzyme immobilization, requiring polymeric matrix to enhance stability of coating and more. On the other hand, while the nanomaterial modified electrodes for non-enzymatic sensing addresses the issue of sensor stability, the reported works involve complex/laborious synthesis protocols, demonstration of proof-of-concept in lab-based setup and requirement of laborious sample processing procedure such as centrifugation, thereby indicating a significant dearth in device development for point-of-care settings.
Enzymatic electrochemical urea sensors utilize urease enzyme which mediates hydrolysis of urea into ammonia and carbon dioxide. The latter (either ammonia or CO2) is detected stoichiometrically via different electroanalytical techniques such as potentiometry, voltammetry and amperometry, and the measured values are indirectly used to assess the urea levels. Potentiometric techniques measure the interfacial potential changes between the enzyme modified electrodes and reference electrode. Voltammetry and amperometry method directly detect the enzyme hydrolysis products, and the output signal is enhanced using Nano interface such as metal nanoparticles and others modifications. On the other hand, some of the voltammetry and amperometry methods incorporate redox mediators in the biosensor device which shuttles electron transfer between enzyme and electrode and establishes an indirect approach to quantify urea levels.

However, each of these methods suffer from certain disadvantages. For instance, Ag NPs modified electrodes can facilitate the effective electro catalysis of NH3 only at higher temperature (> 60oC), thus necessitate a further heating step for detection. In a similar manner, the redox mediator-based approach involves separately adding a chemical substance, thus further complicating the detection procedure. Furthermore, there is a requirement of stable reference electrode in case of potentiometric methods which commonly suffers from issues of potential drift. Lastly, the use of enzyme confers stability and cost related issues and is influenced by temperature, relative humidity, and pH.

On the other hand, reports on electrochemical non-enzymatic sensing of urea have mainly featured nickel-based materials (Babu et al., 2017) for the electro catalytic oxidation of urea in alkaline medium. Zhao et al. reported a 3d printed setup consisting of NiS/GO modified carbon-paste electrode (CPE) for salivary urea detection. However, this work employed both FDM & DLP based printing for developing device, which is difficult to combine and presents issues in fabrication. Moreover, sensing was performed on centrifuged saliva samples and on a less-scalable CPE thus posing issues in their translation to point-of-care settings.

Moreover, nickel-based metals are prone to oxygen evolution reaction (OER) in alkaline medium which interferes with signals corresponding to urea sensing. While this is addressed by synthesizing composites/alloys of nickel with other metal/metal oxides such as cobalt (Amin et al., 2019) and others, such methods come associated with complexities in material synthesis, demanding extensive characterization, and poses challenges to scalability.

In this regard, works utilizing other metal NPs such as silver for urea electro oxidation have circumvented the OER issues (Kumar and Sundramoorthy, 2018; Liu et al., 2020; Manea et al., 2008). However, most of the non-enzymatic electrochemical urea sensing works reported in literature involve complex pre-processing steps such as centrifugation/performed in lab-based electrochemical setup, and show poor % recovery/performance in biofluid samples, and thus are not translatable for the point-of-care settings.

The prior art searches involved are as follows:
1. Babu, K. J., Senthilkumar, N., Kim, A. R. & kumar, G. G. Freestanding and binder free PVdF-HFP/Ni-Co nanofiber membrane as a versatile platform for the electrocatalytic oxidation and non-enzymatic detection of urea. Sensors Actuators, B Chem. 241, 541-551 (2017).
2. Amin, S. et al. A practical non-enzymatic urea sensor based on NiCo2O4 nanoneedles. RSC Adv. 9, 14443-14451 (2019).
3. Liu, J. et al. An AgNP-deposited commercial electrochemistry test strip as a platform for urea detection. Sci. Rep. 10, 1-11 (2020).
4. Kumar, T. H. V. & Sundramoorthy, A. K. Non-Enzymatic Electrochemical Detection of Urea on Silver Nanoparticles Anchored Nitrogen-Doped Single-Walled Carbon Nanotube Modified Electrode. J. Electrochem. Soc. 165, B3006-B3016 (2018).
5. Manea, F. et al. Voltammetric detection of urea on an Ag-modified zeolite-expanded graphite-Epoxy composite electrode., Sensors 8, 5806-5819 (2008).

Urea sensor devices have popularly used enzyme-based membranes coupled with ion selective electrodes (or) membranes attached to permeable polymeric membranes and diffusion barrier to confer stability, enhance shelf-life and service life (US20200199646A1). While such procedures are useful, they demand sequential and multiple modification steps. Works based on ion-selective electrode coupled with enzyme for a potentiometric point-of-care blood urea detection consisted of two ionophore membranes in a single device, wherein first ionophore membrane was utilized for calibration in standard buffer solutions, whose data formed the reference and was used in calculating the differential signal from second ionophore for urea quantification (CA3007872A1), thus conferring a multistep procedure. Similarly, a differential measurement involving a urease immobilized on chemiresistor arrangement for urea detection is reported (USOO5698O83A).

Urease enzyme immobilized over the pH sensitive metal oxides such as IrO2 which exhibits electrochromic changes on urea hydrolysis has been reported on solid state electrodes. However, the use of rare earth elements may hinder the large-scale production and contribute to carbon emissions (US005945343A).

Additionally, use of non-biological moieties such as nanomaterial as non-enzymatic sensing platform for urea detection addresses the issues of stability and shelf life. Works based on nickel nanomaterial have been reported (CN112538620A), wherein Graphene/NiO-PANI composite synthesized via chemically intensive Hummer's method and drop casted on GCE was utilized towards the electro oxidation and hence sensing of urea in alkaline medium (CN109239157A).

Similarly, nickel-cobalt oxide in 3D foam graphene also demonstrated electrochemical urea sensing activity in alkaline medium and was utilized to quantify urea from centrifuged human urine samples. However, nickel-based catalysts promote oxygen evolution reactions which interfere with electro oxidation of urea in alkaline medium. Moreover, Ni-Co electrodes experience structural deterioration/expansion and hence require structural carriers such as 3D foam graphene/CNT (KR20170064403A). However, graphene obtained in these works are synthesized via chemically intensive procedures and not environmentally benign. Moreover, demonstration of sensor performance in lab-based setup such as GCE is not readily translatable to portable setting. Additionally, processing of biofluids via centrifugation and other procedures, as reported in these works further hinders their point-of-care utilization prospects.

The related prior patents are as follows:

1. US20200199646A1
2. USOO5698O83A
3. CA3007872A1
4. US005945343A
5. CN109239157A
6. KR20170064403A

However, it was learned that the prior works are associated with several weaknesses as follows:
• Most of the existing urea detection methods involving spectrophotometric methods utilize blood samples, and require trained personnel for sample handling/collection and data analysis, thus rendering them unfeasible for point-of-care applications.
• Most of the reported urea sensing works involves use of enzyme, which suffers from issues of requiring multiple steps for sensor fabrication, and the existing non-enzymatic sensors, are performed on lab-based setup, suffer from oxygen evolution issues, and they require complex pre-processing steps such as centrifugation, thus impeding their translation to real-time point-of-care settings.
• Most of the small metabolite sensing works utilizing mass-producible, porous LIG electrodes fabricated via a single-step maskless strategy, unlike the existing screen-printed electrodes, involve a polymeric modification step to minimize capacitance/background currents and such modification decreases the inherent surface area offered by LIG electrodes and introduces additional step in sensor fabrication process.
• Furthermore, most of the works utilize one-parameter-at-a-time optimization methodology to improve the sensor/technique's performance, such methods fail to consider the interaction and combinatorial effects between factors
• While most of the works carry out peak identification via a cursor-pointer manual procedure, they are prone to manual error.

Henceforth to overcome the above mentioned weaknesses, there is a continuing need in the art to develop maskless, mass producible, portable strip-based electrodes integrated with mobile phone app for rapid non-invasive salivary urea detection addressing the cost related issues circumventing the issues of oxygen evolution reactions and negating the requirement of sample centrifugation steps as required in the reported works, thus being readily translatable to point-of-care settings

Objectives of the Invention

Primary objective of the present invention is to develop mass producible, portable strip-based electrodes integrated with mobile phone app for rapid salivary urea detection addressing the cost related issues circumventing the issues of oxygen evolution reactions and negating the requirement of sample centrifugation steps as required in the reported works, thus being readily translatable to point-of-care settings followed by its method of fabrication.

SUMMARY OF THE INVENTION
Thus, according to the basic aspect of the present invention, there is provided an electrochemical non-enzymatic analyte/urea sensor device suitable for Point-of-Care Settings comprising
Metallic dendrite modified porous graphene based electrode favouring metallic Nano interface including silver Nano interface for circumventing oxygen evolution reactions and adapted to generate metal oxide/silver oxide based soluble complex with hydrolysis products of urea to thereby expose underlying metal/silver for its related stoichiometric electrochemical oxidation based peak currents corresponding to analyte/urea levels in samples and their quantification.

According to another aspect of the present invention, the electrochemical non-enzymatic analyte/urea sensor device adapted as a portable, rapid, real time, salivary urea sensing and detection device wherein :
said electrode is configured as a three-electrode assembly based electrochemical sensor strip consisting of a working electrode (2) of silver dendrite modified graphene-electrode configured as a three-electrode assembly based electrochemical sensor strip capable of holding diluted non-centrifuged saliva samples as analyte for testing when drop casted on sample holding spot in said strip,
with said sensor strip for desired sensing is operationally connected to a portable potentiostat operable by a smart phone/laptop in connection having said stoichiometry supporting analytics for said real time sensing and electrochemical quantification of urea levels in saliva samples held in said strip.

Another aspect of the present invention, the electrochemical non-enzymatic analyte/urea sensor device wherein said silver dendrite modified porous graphene based electrode favouring silver Nano interface is laser induced graphene-based (LIG) electrodes fabricated from precursor polymer substrates including polyimide films having graphitic patterns thereon based on laser engraving/ ablation that is modified with metal-based electro-catalytic material including silver adapted for said metallic Nano interface to facilitate electro-oxidation of analytes including urea.

Another aspect of the present invention, the electrochemical non-enzymatic analyte/urea sensor device wherein:
said electrode as working electrode (2) and configured as a three electrode assembly (1) on said sensor strip includes 3 number of channel as electrodes and connecting track regions connecting said channels on said strip with the remaining regions of the strip (4) passivated with hydrophobic, non-conducting material including insulating tape/cello tape to ensure confinement of the diluted saliva sample within said 3-electrode channel regions of the sensor strip, said electrode channels and connecting tracks being laser pulse ablated/fabricated polymer film having graphitic structures with defects and pores for necessary electro-catalytic activity,
said three electrode assembly (1) including said 3 number of channels and connecting track regions connecting said channels on said strip includes said working electrode (2) linking said sample holding spot, pseudo-reference electrode (3) preferably coated with silver (or) silver/silver chloride paste or electrodeposited silver from silver salts together with counter electrode (6) which is laser pulse ablated, possess graphitic structures and is devoid of any further deposition/modification and connecting pads (5) coated with conductive silver paste or covered with copper tapes for operatively connecting said electrode assembly (1) based sensor strip to said portable potentiostat.

Another aspect of the present invention, the electrochemical non-enzymatic analyte/urea sensor device wherein said laser induced graphene-based (LIG) electrodes as 3-electrode system is fabricated of precursor polymer substrates/films that include materials selected from polyimide, polyethyleneimine, polyether ether ketone, polybenzimidazole, polyamide imide, polysulfone, polyethersulfone, polystyrene, epoxy, phenolic resin, and cellulosic content rich substrates as paper, wood, cardboard, where the material of substrate dictates the necessity for pre-treatment, which substrates/films upon laser ablation/irradiation exhibits increase in carbon content and decrease in oxygen and nitrogen content due to breakage of imide bonds, decomposition of diaryl ether groups, and formation of aliphatic hydrocarbon upon laser irradiation.

Yet another aspect of the present invention, the electrochemical non-enzymatic analyte/urea sensor device wherein said working electrode (2) of said electrode assembly (1) having laser engraved/ ablated graphitic patterns of precursor polymer films is modified with said metallic dendrites including silver dendrites based on electrodeposited silver on said laser engraved/ ablated graphitic patterns.

Still another aspect of the present invention, the electrochemical non-enzymatic analyte/urea sensor device wherein said precursor polymeric materials preferably includes polyimide film of thickness varying in the range of 5 mil (175 μm) to 10 mil (250 μm) for subjecting to continuous (or) pulsed laser source (or) laser diodes of different wavelengths of CO2 laser (1064 μm), visible laser (~450 nm), UV-excimer laser source (190 to 350 nm) providing graphitic structures with defects and pores exhibiting an carbon content of 82 to 95 % and oxygen and nitrogen content of 5 to 11% and 0.5 to 6.5 % respectively.

Another aspect of the present invention, the electrochemical non-enzymatic analyte/urea sensor device wherein said counter electrode have empirical geometrical area much larger than working electrode (4 times) to ensure the rate-limiting reactions occur at the working electrode and such that relevant analyte information pertaining to reaction of interest is gained from sensor strips.

Another aspect of the present invention, the electrochemical non-enzymatic analyte/urea sensor device wherein said working electrode (2) is responsive to provide quantification of analytes in presence of redox mediators and supporting electrolytes, including outer-sphere redox couple as ruthenium hexamine (II)/(III) (or) inner-sphere redox couple such as ferro/ferricyanide in supporting electrolyte for graphene-based electrodes, and monovalent salt solutions including potassium chloride, sodium chloride (or) divalent salt solutions such as calcium chloride, magnesium chloride (or) solutions of phosphate buffered saline with concentration varying in the range of 0.1 to 1 M respectively.

Another aspect of the present invention, the electrochemical non-enzymatic analyte/urea sensor device wherein said non-centrifuged saliva samples for drop casting on reaction spot upon dilution includes monoacidic alkaline solutions corresponding to sodium hydroxide, potassium hydroxide (or) diacidic alkaline solution corresponding to calcium hydroxide, magnesium hydroxide for said dilution to reach analyte concentration levels of 100 to 500 µM.

Yet another aspect of the present invention, the method of fabrication of electrochemical non-enzymatic analyte/urea sensor device comprising the steps of providing metallic dendrite modified porous graphene based electrode favouring metallic Nano interface including silver Nano interface as working electrode (2) for configuring as electrode assembly based electrochemical sensor strip capable of holding diluted analytes samples including non-centrifuged saliva samples as analyte for testing when drop casted on sample holding spot in said strip, enabling generation of said metal oxide/silver oxide based soluble complex with hydrolysis products of urea thereby exposing underlying metal/silver for its related stoichiometric electrochemical oxidation based peak currents corresponding to analyte/urea levels in samples providing for their quantification.

Another aspect of the present invention, the method of fabrication of electrochemical non-enzymatic analyte/urea sensor device wherein said providing metallic dendrite modified porous graphene-based electrode favouring metallic Nano interface including silver Nano interface as working electrode (2) includes the following steps:
(a) Configuring a three-electrode assembly based electrochemical sensor strip and creating sample holding spot on said strip for holding said drop casted diluted non-centrifuged saliva samples as analyte for testing in said strip,
(b) Connecting said sensor strip with a portable potentiostat for operation which in turn is operated by placing a smart phone/laptop in connection and in having said stoichiometry supporting analytics enables said real time sensing and electrochemical quantification of urea levels in saliva samples held in said sample holding spot of said strip.

Still another aspect of the present invention, the method of fabrication of electrochemical non-enzymatic analyte/urea sensor device wherein said step (a) of configuring the three electrode assembly based electrochemical sensor strip includes the sub-steps of:

(i) providing precursor polymer substrates including polyimide films and subjecting said films to laser engraving/laser ablation and creating graphitic patterns on said film for fabrication of said three electrode assembly and their connecting track,
(ii) modifying working electrode (2) of the electrode assembly by electro-deposition of the thus attained laser engraved patterns on said film with metal-based electro-catalytic material giving metallic dendrites including silver dendrites adapted for said metallic Nano interface to facilitate electro-oxidation of analytes including urea and obtaining said working electrode (2) as three electrode assembly based electrochemical sensor strip therefrom.

Another aspect of the present invention, the method of fabrication of electrochemical non-enzymatic analyte/urea sensor device wherein
in said step (i) for creating graphitic patterns and fabricating a three electrode assembly based electrochemical sensor strip is based on providing preferably polyimide film within thickness range of 5 mil (175 μm) to 10 mil (250 μm) followed by subjecting to rapid lasing pluses sourced from laser diodes including different wavelengths corresponding to CO2 laser (1064 μm), visible laser (~450 nm), UV-excimer laser source (190 to 350 nm) resulting in formation of graphitic structures with defects and pores with porosity ranging from 1 to 4 µm while maintaining vertical height of the electrodes, and connecting tracks thus laser engraved at 3 to 5 mm, raster scanning of 20 mm/s to 40 mm/s, hatching distance of 0.01 to 0.05 mm, and angle at 0o to 90o, to attain said laser induced graphene (LIG) electrodes with high porosity and electrocatalytic activity, and
in said step (ii) for modifying the thus engraved patterns and channel based electrodes on said film is subjected to electro-deposition of the working LIG electrode (2) with a salt concentration of silver corresponding to (1 to 15 mM), potential window for LSV (0 to -0.4 V - 0 to -1.5 V), scan rate of LSV (5 to 100 mV/s) with number of repetitions/cycles (1 to 5) selected for deposition of silver suiting SWV features of the sensor device to yield high peak current of 4 to 6 µA with minimum standard deviation of 0.4 to 0.6 µA quantifying urea levels.

Another aspect of the present invention, the method of fabrication of electrochemical non-enzymatic analyte/urea sensor device wherein regions (4) excluding the electrode channels and their connecting tracks on said strip is passivated with a relatively hydrophobic, non-conducting material including Kapton tape (or) cello tape ensuring the confinement of analyte/diluted saliva sample within said 3-electrode region of the sensor strip.

Another aspect of the present invention, the method of fabrication of electrochemical non-enzymatic analyte/urea sensor device wherein said electrode assembly having pseudo-reference electrode (3) is manually coated with silver (or) silver/silver chloride paste or silver salts are electro-deposited, and the connecting pads (5) are coated with conductive silver paste or covered with copper tapes for establishing connection with said potentiostat.

Another aspect of the present invention, the method of fabrication of electrochemical non-enzymatic analyte/urea sensor device wherein said sensor is evaluated on basis of square wave voltammetry (SWV) features, which includes responses in said redox mediator including outer-sphere redox couple selected as ruthenium hexamine (II)/(III) (or) inner-sphere redox couple such as ferro/ferricyanide in supporting electrolyte for said graphene-based LIG electrodes, and wherein said supporting electrolytes provided varies in the range of 0.1 to 1 M and includes monovalent salt solutions selected as potassium chloride, sodium chloride (or) divalent salt solutions selected as calcium chloride, magnesium chloride (or) solutions of phosphate buffered saline.

Another aspect of the present invention, the method of fabrication of electrochemical non-enzymatic analyte/urea sensor device wherein the thus fabricated sensor device responds to SWV features of amplitude of 10 to 100 mV, increment voltage of 2 to 10 mV and frequency of 1 to 10 Hz, to correspond to values of peak current and standard deviation.

Still another aspect of the present invention, the method of fabrication of electrochemical non-enzymatic analyte/urea sensor device wherein the thus fabricated sensor device gives idiosyncratic sharp oxidation peaks in the cathodic (or) reduction half cycle for urea analyte corresponding to the complex formation of hydrolysis products of urea with silver (I) oxide present on Nano interface that dissolves into the solution and exposes the underlying silver structures.

The advancement according to the present invention is discussed in further detail in relation to the following non-limiting exemplary illustrations as per the Figures given below wherein:
Fig. 1 shows the schematic of a sensor strip in cooperation with a portable potentiostat integrated with smart phone for salivary urea detection of the test apparatus.

Fig 2 shows the fabricated sensor strip of the invention.

Fig 3 is an exploded view of the sensor strip in Fig 2.

Fig. 4 shows the Raman (a), UV-Vis (b) elemental composition (c) of graphitic material (LIG) present in the salivary urea sensor strip of the test apparatus, which is formed via laser ablation method.

Fig. 5 is a plot of linear sweep voltammograms, illustrating the reproducible performance of the LIG electrodes associated with the (a) electro-deposition in silver nitrate solution and (b) response of silver electrodeposited electrodes in 0.1 M KOH solution.

Fig. 6 (a) shows the it curves associated with electro-deposition in silver nitrate solution for 150 (s) and (b) shows the LSV response of silver electrodeposited (via 'it' technique) electrodes in 0.1 M KOH solution.

Fig. 7 illustrates the multivariate parametric optimization of square wave voltammetry (SWV) features on LIG electrodes that is performed to maximize higher mean current and minimize standard deviation values. The subfigures include chart, main effects & interaction plot(s) for mean peak current & RSD values and composite desirability factor corresponding to optimization.

Fig. 8 shows the data representing the mean current and standard deviation values for SWV curves that is obtained from full factorial combination of amplitude, increment voltage and frequency features of SWV technique.

Fig. 9 (a) shows the peak current values obtained from SWV and DPV technique for electrodes fabricated using different combinations lasing speed and power, at constant vertical height, hatching angle and hatching distance (b) shows the RSD values of peak current values obtained from SWV and DPV technique for electrodes fabricated with different hatching angle and fixed vertical height, lasing speed, laser power, and hatching distance.

Fig. 10 illustrates the cyclic voltammograms of the sensor strip, in the absence & presence of urea in 0.1 M KOH buffer solution.

Fig. 11 illustrates the optimization of choice of electro-analytical technique performed on the sensor strip for urea detection.

Fig. 12 illustrates the SWV curves following pre-treatment with (a) chronoamperometry technique (b) LSV technique and (c) peak current changes at different scan rates of LSV technique of the sensor strip.

Fig.13 illustrates the SEM images corresponding to silver dendrites electrodeposited LIG electrodes wherein the influencing parameter was varied one at a time sequentially, holding the other at fixed values. The influencing parameters varied were (a-c) concentration of silver nitrate salt in 0.1 M potassium nitrate (e-f) potential window of LSV technique (i-k) scan rate of LSV technique (m-o) number of repetitions of LSV scans. The peak current changes for a particular urea concentration were compared and shown in d, h, i and p.

Fig. 14 shows the step-wise urea detection flow chart with the present system for salivary urea estimation.

Fig. 15 depicts the (a) batch-wise fabrication of LIG electrodes of customized designs (b) batch packaging of developed sensor strips (c) collection of saliva samples via passive drooling technique (d) customized connecting tracks to facilitate plug-&-play arrangement of sensor strip (e) dilution of saliva sample with buffer solution and (f) working demonstration of sensor device.

Fig. 16 (a) SWV curves for urea spiked in 0.1 M KOH buffer (b) calibration curve for experiment described in 16 (a), (c) recovery studies for urea spiked in diluted human saliva samples (d) comparison of sensor device with gold standard method.

Description of the Invention with reference to the accompanying figures:
As stated hereinbefore, the present invention discloses a non-enzymatic, economic, simple, and portable apparatus configured to rapidly determine the concentration of clinically relevant molecules, such as urea from human saliva samples. The concentration of urea in saliva is strongly correlated with the blood urea levels, and is differentially expressed with different stages of chronic kidney disease (CKD). Subsequently, its quantification can provide a non-invasive diagnosis strategy for the early detection/screening of CKD. Moreover, with CKD being an endemic in India, rapid diagnosis and mass screening programs shall create awareness amongst citizens and ameliorate the public health, thereby contributing to society's well-being. Such initiatives shall be augmented by rapid detection of urea from saliva samples, which can be easily collected by volunteers and unskilled personnel. In the context of such initiatives/programs, the sensor device is configured to be easily operated by individuals without expertise knowledge or prior training.

The present invention consists of a graphene-based electrochemical sensor strip to contain diluted saliva samples, which can be connected to a portable potentiostat system operated in conjunction with a smart phone/laptop, for the real time electrochemical quantification of urea levels from saliva samples. Reference in this context is invited from Fig. 1 which shows a schematic of salivary urea sensor device. As shown in Fig. 1, the present invention consists of a sensor strip connected to a portable potentiostat, smart phone arrangement for the non-enzymatic detection of urea from saliva samples. The working electrode consists of a metal (or) metal oxide to facilitate the electro-oxidation of urea. The saliva collection method involves passive drooling technique, which is a non-stimulated, non-invasive method for sample collection. Additionally, as shown in Fig. 1 the sensor strip includes a reaction spot onto which a minute amount of saliva/reagent mixture is drop casted. The reagent used for dilution preferably includes monoacidic alkaline solutions such as sodium hydroxide, potassium hydroxide (or) may be a diacidic alkaline solution such as calcium hydroxide, magnesium hydroxide and others, of a particular concentration. Urea present in the diluted saliva samples is electrochemically oxidized, and this is assessed stoichiometrically from redox reactions occurring at the nano interface.

In the present invention, polyimide film is used as the substrate & precursor material for the fabrication of graphene-based electrodes. This is due to the considerable amount of benzene/aromatic content in polyimide films, which on encountering high temperature and pressure conditions, such as on being subjected to rapid lasing pluses, will result in formation of graphitic structures with defects and pores. The hence formed structures show electro-catalytic activity and hence find applications in electro-analytical chemistry. Moreover, this technique facilitates a maskless strategy for the large-scale production of highly customizable electrodes. The polyimide films used for the intended purpose can be of thickness ranging from 5 mil (175 μm) to 10 mil (250 μm). Additionally, other polymeric substrate materials can be selected such as, polyethyleneimine, polyether ether ketone, polybenzimidazole, polyamide imide, polysulfone, polyethersulfone, polystyrene, epoxy, phenolic resin, and cellulosic content rich substrates such as paper, wood, cardboard, where the chemical composition of substrate may dictate the necessity for pre-treatment procedures. Also, the laser source used for the intended purpose can be continuous (or) pulsed laser source (or) laser diodes of different wavelengths such as CO2 laser (1064 μm), visible laser (~450 nm), UV-excimer laser source (190 to 350 nm), to name a few.

The sensor strip excluding the 3-electrode system and conducting pads is passivated with a relatively hydrophobic, non-conducting material. The hydrophobic, non-conducting coating /modification on the sensor strip ensures the diluted saliva sample is confined to the 3-electrode region of the sensor surface. Reference in this context is invited from Fig. 2, which contains a graphene-based electrode obtained via lasing strategy that includes the 3-electrode system (1 & 2) containing a pseudo-reference electrode (2). The pseudo-reference electrode can be manually coated with silver (or) silver/silver chloride paste or achieved via other means such electro-deposition of silver salts. The hydrophobic coating (3) on the fabricated sensor strip may be achieved using Kapton tape (or) cello tape, etc. The connecting pads (4) can be left unmodified or in some cases be coated with conductive silver paste or covered with copper tapes.

Furthermore, reference is invited from Fig. 3, which depicts an exploded view of sensor strip consisting of 3-electrode arrangement that comprises of a working, counter, and reference electrode, wherein the working electrode is further modified with a metal-based electro-catalytic material (2), and the reference electrode is manually coated with silver (or) silver/silver-chloride paste (3) to act as pseudo reference electrode. Additionally, the connecting tracks is passivated with a non-conducting coating, for example, insulating tape, (or) cello tape (4). Moreover, empirically, the geometrical area of counter electrode is much larger than working electrode and is designed in this manner to ensure the rate-limiting reactions occur at the working electrode, so relevant information pertaining to reaction of interest is gained from sensor strips.

Fig. 4 depicts the characterization results of fabricated laser induced graphene (LIG) electrodes as described above, wherein the structural characterization via Raman spectroscopy, shown in Fig. 4(a) indicated the LIG electrodes to possess graphitic structures with significant defects in basal plane. Further, UV-Vis spectrum, shown in Fig. 4(b), revealed an intense peak at 266 nm and a shoulder at 290 nm, corresponding to π-π* and n-π* transitions of C=C and C=O bonds respectively. Additionally, owing to the breakage of imide bonds, decomposition of diaryl ether groups, and formation of aliphatic hydrocarbon upon laser irradiation, the LIG materials exhibits an increase in carbon content and decrease in oxygen and nitrogen content. This phenomenon is confirmed from the elemental composition changes shown in Fig. 4(c).

The working electrode of the sensor strip is modified with metallic Nano interface to facilitate the electro-oxidation of analyte such as urea. This occurs in communion with redox reaction of metallic Nano interface, wherein the stoichiometric changes in latter are used to assess the urea levels. Enhancement of the electro-oxidation of urea and the associated redox reactions of silver dendrites, is achieved by optimal choice of electro-analytical technique, optimization of technique's features and pre-treatment procedures. Fig. 5 & 6, illustrates the electro-deposition curves using chronoamperometry (CA) and linear sweep voltammetry (LSV) techniques in silver nitrate solutions, and the corresponding LSV curves in 0.1 M KOH buffer solution, which revealed the LSV based electro-deposition technique exhibited greater reproducibility in comparison with CA procedure.

Reference is invited from Fig. 7 which illustrates the method associated with design of experiment based multivariate optimization factorial experiments of square wave voltammetry (SWV) features of the LIG electrode. This involves use of redox mediator, wherein the latter can be outer-sphere redox couple such as ruthenium hexamine (II)/(III) (or) inner-sphere redox couple such as ferro/ferricyanide in supporting electrolyte for graphene-based electrodes. Moreover, the supporting electrolyte used can be prepared from monovalent salt solutions such as potassium chloride, sodium chloride (or) divalent salt solutions such as calcium chloride, magnesium chloride (or) solutions of phosphate buffered saline.

The full factorial experiments performed on SWV features of amplitude, increment voltage and frequency, involved varying their values across a range and the corresponding values of peak current and standard deviation were recorded. The results are illustrated in Fig 8, wherein set 6 exhibited maximal peak current values with minimal relative standard deviation. The minimization of standard deviations improves the reproducibility characteristics of the sensor strip, while the higher peak current value increases the sensitivity of the sensor device. Hence, the combination of both shall improve the overall performance of the sensor device. Further, from Fig. 9, it is evident that LIG electrode fabricated using the parameter 20s, 30p (s - mm s-1/60 and p - %), exhibited higher current values and reproducibility traits, and hence used for the sensor device development.

SWV technique used for detection was preceded by a pre-treatment procedure involving LSV technique. The latter generated the required surface metal oxides, such as silver oxides on the electrode surface that took part in complex formation with hydrolysis products of urea, and yielded the oxidation peaks in the SWV curves, when scanned along the negative direction. This reaction agrees with the cyclic voltammograms shown in Fig. 10, where the silver modified LIG electrode shows several oxidation and reduction peaks in absence and presence of urea. Here, the peaks observed in oxidation half cycle correspond to different surface oxide states (1-4) of silver structures, and that obtained in reduction half correspond to reduction of the silver oxides to silver (5 &6) whose peaks are labelled in figure 10. However, in presence of urea, idiosyncratic sharp oxidation peaks (7 & 8 shown in figure 10 is observed in the cathodic (or) reduction half cycle. The peaks, 7 & 8 may correspond to the complex formation of hydrolysis products of urea with silver (I) oxide present on Nano interface that dissolves into the solution and exposes the underlying silver structures.
While porous electrodes, such as LIG electrodes inherently contribute to increased capacitance currents, they are generally modified with viscous, conducting polymers and other materials to diminish the capacitance currents. However, such modifications clog the pores and minimize the active surface area of the electrodes. Referring to Fig. 11, it could be seen that the summation SWV electro-analytical technique gave away higher changes in peak current values in comparison to other technique. Herein, the use of alternative resultant SWV current, which involved summation of forward and reverse currents, produces output currents that is inclusive of capacitance and faradic contributions. This not only increased the electro-oxidation current changes, but also negated further modification steps, thus devising a simple fabrication strategy.

To yield optimal results relating to electro-oxidation of urea, the parameters associated with electro-deposition of silver, such as concentration of precursor salt (1 to 15 mM), potential window for LSV (0 to -0.4 V - 0 to -1.5 V), scan rate of LSV (5 to 100 mV/s) and number of repetitions of technique (1 to 5) were sequentially optimized. The precursor salt suitable for the intended purpose, can include any of the following like silver nitrate, silver acetate and others, and the choice of voltammetry technique can be LSV or CV. Reference in this context is invited from Fig. 13, which shows SEM images that reveal formation of silver dendritic structures, which are attached to porous graphene electrode surface underneath. Also, the change in current values of the sensor in presence of urea (~ 500 µM) is shown.

Identification of peak current corresponding to peaks characteristic for electro-oxidation of urea is a crucial step to appropriately estimate the urea levels from buffer samples and bio fluids. Subsequently devising algorithms (or) strategy which can readily be automated shall aid in minimizing the manual errors and improve the reliability of the sensor device. Reference in this context is invited from flowchart shown in Fig. 14, which delineates the technique adopted for urea detection using the present invention. The peak current detection procedure involves performing a non-linear baseline correction on the obtained SWV curves, following which the peak potential is identified from commonly used peak detection algorithms. This is followed by choosing the current values corresponding to the approximated peak potentials form the data files. The non-linear baseline correction procedure may utilize asymmetric least squares smoothing method, and the associated parameters such as asymmetric, threshold, smoothing factor and number of iterations may be varied to yield the optimal results. Further, peak detection algorithms such as Gaussian fit method, derivative-based method, Wavelet transform method, Savitzky-Golay filter method, and others may be used for peak finding purposes.

Referring to Fig. 15, it is evident that the sensor strips can be batch produced, packaged, and be readily integrated with pocket potentiostat/smart phone arrangement. Furthermore, the sample collection and dilution procedure are shown in Fig 15 (c & e). Moreover, Fig. 16 (b) illustrates that the sensor device shows characteristic linear changes in peak current values of SWV curves corresponding to urea concentrations. In regression statistical analysis, R2 coefficient determines the relation between independent variable (Y) and dependent variable (X). If R2 approaches 1, the relation between the variables is very strong. In the present invention, the calibration curve exhibited R2 = 0.993 indicating a very strong correlation between urea concentrations with SWV peak current values. The sensor device exhibited a slope of 49.9 μA/mM with current values in linear response for urea concentrations of 100 μM to 500 μM. These concentrations corrected to dilution factor lie in the clinically relevant range for CKD patients. Moreover, Figures 16 (C) revealed very good recovery characteristics (> 90%) of the sensor device in urea spiked human saliva samples, indicating minimal interference or matrix effects. Further, the performance of the sensor device was compared with the standard pathological results obtained using auto analyzer, which revealed the results from the sensor device to be in correlation (r = 0.95) with that obtained via gold standard method.

EXAMPLE 1

Fabrication and characterization of laser induced graphene-based (LIG) electrodes:

A blue laser diode (2.8 W, 450 nm) compatible with multi tool 3D printer, Zmorph (Wroclaw, Poland) was utilized for laser engraving of graphitic patterns on polyimide substrate. The electrodes were fabricated using the following parameters: vertical height 5 mm, power 0.84 W (30%), work speed 20 mm s-1, travel speed 20 mm s-1, hatching angle 90o, hatching distance 0.05 mm with a one-time laser raster scan. Structural and chemical characterization using Raman, UV-Vis, and EDAX spectroscopy studies were performed on these as fabricated electrodes.

The Raman spectrum depicted in Fig 4(a) was collected using Hiroba Spectrophotometer (Kyoto, Japan), using 532 nm laser source, 10% power (100x, 100% power 5 mW), grating 1800, objective (50x), acquisition time (15 s) and accumulation time (1 s). The spectrum consisted of D, G and 2D bands that are characteristic for graphene-based structures. UV-Vis spectrum, shown in Fig 4(b) was obtained, using UV-Vis spectrophotometer, Thermo Fisher Scientific Instruments, USA. The LIG powder was scrapped and dispersed in Dimethyl formamide solution, and UV-Vis studies was performed across a scan window from 200 to 800 nm. This revealed n-π* and π-π* bands indicative of presence of oxygen/nitrogen atoms and conjugation (respectively) in obtained graphene-based structures. The elemental analysis was performed on the scanning electron microscopy, energy dispersive elemental analysis (SEM/EDAX) instrument (JSM-7601F, Joel, Japan). A thin layer conductive coating of gold was performed on LIG sample under vacuum for 120s and then used for analysis. Subsequently, gold was de-selected during the EDAX element estimation. It is evident from Fig 4(c) that LIG material exhibited an increased weight % of carbon and decreased weight % of nitrogen and oxygen owing to laser ablation and decomposition of
polyimide film.

EXAMPLE 2

Multivariate parametric optimization of SWV features:

A dual parameter optimization was carried out to obtain SWV features that maximizes peak current values and minimizes relative standard deviation (RSD). The experiments were performed on fabricated LIG electrodes using ferro/ferricyanide redox couple in a supporting electrolyte of potassium chloride. The multivariate optimization of amplitude, frequency, and increment voltage of SWV was performed, wherein a full-factorial design (FFD) experiment was developed. The most influencing parameter was varied to obtain the optimal SWV features. Fig. 7 shows the Pareto chart and main effects plot, which revealed only the frequency and amplitude to influence the SWV response. Moreover, the interaction plots shown in Fig. 7 indicated that while peak current values increased with frequency and amplitude, the RSD also increased with increasing frequency, step potential and decreasing amplitude. Furthermore, the FFD screening yielded a composite desirability of 0.95 thus indicating ideal optimization been performed.

COMPARITIVE EXAMPLE 2
Other fabrication parameters of LIG electrode are:
The fabrication parameters such as power and speed of lasing were varied, by keeping the hatching distance, hatching angle and vertical height fixed. The experimental conditions were same as performed in example 1. Fig 9(a) revealed, parameters of 20 mms-1, 30 % (0.84 W) power yielded the highest mean peak currents in SWV and DPV studies. Following this, the hatching angle was varied every 15o from 0 to 90o, wherein Fig 9(b) revealed the hatching angle of 90o to yield peak current results with minimal RSD for SWV technique, and hence used for remaining studies.

EXAMPLE 3
Cyclic voltammetry studies in presence and absence of urea on silver modified sensor strips:
Cyclic voltammetry studies were performed on silver electrodeposited LIG electrodes, in the absence and presence of 5 mM urea in 0.1 M KOH. Cyclic voltammograms shown in Fig. 10 revealed 4 anodic peaks (1-4). A small anodic peak 1, seen at ~ -0.1 V, is attributed to the electro dissolution of Ag to soluble [Ag(OH)2]-, (eq (1.1) & (1.2)). This is followed by a small peak 2 and a well-defined peak 3, which correspond to formation of compact Ag2O monolayer and porous, thick Ag2O, respectively. While the former results from precipitation of supersaturated [Ag(OH)2]- on electrode surface, the latter, porous Ag2O multilayers grows on 3D islands of the compact Ag2O monolayer and is controlled by diffusion of Ag+ ions through the primary oxide layer (eq 2). Lastly, while a well-defined peak between 0.6 to 0.8 V is attributed to electro-oxidation of Ag2O to AgO layers (eq 3.1).

--- (1.1)
--- (1.2)
--- (2)
--- (3.1)

While the oxidation half cycle was similar in the presence & absence of urea, changes could be seen in the reduction half where, in presence of urea a peculiar oxidation peak occurred, just after the reduction of AgO to Ag2O. The peculiar peak may be attributed to the continuous removal of Ag2O layers owing to complexation of Ag2O with hydrolysis products of urea (such as NH3), to yield soluble complex diamine silver (Ag(NH3)2+). The complex thus formed dissolves in the solution and exposes the underlying silver nano interface, which at the corresponding potentials of ~0.55 (peak 8) and ~0.4 V (peak 7) electrochemically oxidizes to form surface oxides (eq 5.1) and hydroxides (eq 5.2) of silver (respectively).

--- (4)
--- (5.1)
--- (5.2)


COMPARATIVE EXAMPLE 3
Cyclic voltammetry studies in presence and absence of urea on bare/non-electrodeposited sensor strips:

In case of non-modified LIG electrodes, similar experiments were performed in alkaline solution, 0.1 M KOH, and no peaks were observed. Moreover, the electro-oxidation of urea did not occur in unmodified electrodes.

EXAMPLE 4
Electro-deposition of Silver on LIG electrode using LSV technique
In contrast to the standard reference electrodes, the LIG electrodes possess pseudo-reference electrodes, and hence it is difficult to maintain a precise/stable potential on WE surface. Hence, utilizing voltammetry over amperometry techniques for electro-deposition is advantageous. Therefore, the reduction cycle of the LSV technique was used for electro-deposition of silver on LIG surface. Moreover, the as fabricated LIG electrodes exhibited excellent reproducibility, which could be seen from the LSV curves shown in Fig 5. Fig 5(a) depicts the LSV curves associated with electro-deposition of silver, with experiments performed in AgNO3 prepared in KNO3. Fig 5(b) depicts the LSV studies performed in 0.1 M KOH solution, on the silver electrodeposited electrodes. The meanings of anodic peaks are discussed in Example 3.

Furthermore, the parameters influencing electro-deposition, such as AgNO3 concentration, potential window, scan rate of LSV and number of repetitions of LSV scans, were optimized to improve the sensor performance towards urea detection. The optimization was carried out sequentially, wherein the concentration of AgNO3 was varied from 1 mM to 15 mM, the potential window was varied from 0 to -0.4V, 0 to -1.2 V, the scan rate of LSV was varied between 5 mV/s to 100 mV/s and 1 to 5 repetitions.

The corresponding SEM images shown in Fig 13 revealed the formation of dendritic structures, implying the growth mechanism followed the Diffusion limited aggregation (DLA) model, wherein potential, scanned along the negative direction propels the diffusion of [Ag+] ions, which reaches the surface via a random walk, and forms Ag aggregates to minimize the surface energy. The Ag+ ions diffusing towards the surface grow on these aggregates and forms dendritic structures. Moreover, the electro-deposition parameters conferring the optimal urea sensing performance is also revealed from Fig 13, wherein along with current changes, the standard deviations were also taken into considerations. Since, higher standard deviation is associated with higher error values, optimization parameters with lower standard deviation and competitive peak current values were chosen. Such choices shall improve the reliability of sensor device. Subsequently, the optimized parameters were used for calibration curve generation and sensor device validation purposes.

COMPARITIVE EXAMPLE 4
Electro-deposition of silver on LIG electrode using chronoamperometry (CA) technique:
Instead of LSV technique described in example 4, CA-technique was used to electrodeposit silver on prepared LIG electrodes (described, example 1). From the Fig 6(a), it could be seen that the CA-curves were not reproducible. Even though Fig 6(b) showed oxidation peaks for silver, which indicated their electro-oxidation ability for urea, the LSV curves were not reproducible, hence being less reliable for sensor device development.

EXAMPLE 5
Peak current changes corresponding to urea detection using SWV technique
As discussed in example 3, the electro-oxidation of urea is observed along the negative scan in cyclic voltammogram of silver modified electrode. Hence, voltammetric studies focusing on the reduction half cycle was performed to identify optimal technique for urea detection. In this regard, the SWV summation current, which involved adding the forward and reverse currents, gave away the highest current response associated with urea electro-oxidation. While porous electrodes inherently contribute to higher background/capacitance currents, they are generally modified with conducting polymers or other nanomaterial, to attenuate such background currents. However, utilization of SWV summation current produces output currents that is inclusive of capacitance and faradic contributions. This not only increased the electro-oxidation current changes, but also negated further modification steps, thus devising a simple fabrication strategy. The electrode performance was evaluated in blank 0.1 M KOH buffer and 500 μM urea spiked in 0.1 M KOH buffer samples and the difference in current values were compared and is shown in Figure 9.

COMPARITIVE EXAMPLE 5

Peak current changes corresponding to urea detection using other voltammetry techniques:
With similar experimental conditions to that described in example 5, the peak current changes corresponding to electro-oxidation of urea was studied using CV and LSV techniques. Figure 11 depicts that, while these techniques could also be used to detect urea, the current changes were less in comparison to that obtained via SWV summation method. However, the SWV and LSV technique may require a pre-treatment procedure, unlike the CV technique. Nevertheless, as shown in Fig. 12, the pre-treatment protocol when combined with detection technique (SWV summation), both together takes just over 2 min (~125 s) for completion. Further this method shows higher sensitivity in comparison to other techniques, and hence used in subsequent experiments.

EXAMPLE 6
Pre-treatment of silver modified LIG electrode using LSV technique:
From example 3, it is evident that the characteristic peaks for electro-oxidation of urea is obtained in the reduction half cycle. Moreover, as discussed in example 5, the SWV summation method exhibited greater peak current changes corresponding to electro-oxidation of urea. However, this requires conversion of electrodeposited silver to silver oxide. This shall initiate the subsequent chemical reactions and aid the electrochemical detection of urea. Subsequently, pre-treatment was carried and as shown in Figure 12(b), LSV based oxidation of silver modified surface revealed significant changes in summation peak currents. Moreover, the scan rate of LSV was varied between 10 to 50 mV/s and the optimal scan rate was chosen.

COMPARITIVE EXAMPLE 6

Absence of Pre-treatment procedure and pre-treatment of silver modified LIG electrode using chronoamperometry technique:
The SWV summation technique was performed on non-pre-treated, silver modified LIG electrodes that were exposed to 0.1 M KOH and urea spiked in 0.1 M KOH solutions. While these electrodes exhibited peaks corresponding to electrochemical redox reactions for silver oxide, silver hydroxide formation, and showed electro-catalytic activity for urea, the sensitivity was less than that exhibited for pre-treated electrodes. The non-pretreated electrodes exhibited a slope of 31 μA/mM, while the pre-treated electrodes exhibited slope of 49 μA/mM (discussed in example 7). The presence of surface silver oxides is necessary for the reaction mechanism to take place. While, the response is observed even in absence of pre-treatment procedure, this could be attributed to optimized parameters associated with SWV technique, wherein the potentials are scanned at low frequency from a positive value (+1 V) to 0 V. The lower frequency potential scanning provides sufficient time per potential, for conversion of silver-to-silver oxide(s), which interacts with the hydrolysis products of urea and exhibits peak currents corresponding to it. However, in this case, the formation of surface silver oxide may be much lower to that compared in case of pre-treated electrodes and may result in lower sensitivity.

Furthermore, instead of LSV technique utilized in example 6, CA method at a fixed potential of +1.25 V for 30 (s) was evaluated as a pre-treatment protocol for urea detection. From the Fig 12, it is evident that the current changes corresponding to ~0.4 V, was lesser in CA method in comparison to that of LSV-based pre-treatment method, hence LSV method was chosen for further experiments

EXAMPLE 7

Urea sensing in 0.1 M KOH buffer using sensor device:
The sensor device arrangement shown in Figure 15 was evaluated by spiking different concentrations of urea in 0.1 M KOH buffer solutions. As seen from Figure 16(a), there was a progressive increase in SWV summation peak current values with corresponding increase in urea concentrations. The peak current corresponding to peak potential ~0.4 V was plotted against concentration and the respective calibration curve was obtained. The sensor device exhibited a slope of 49.9 μA/mM, with a limit of detection of 26 μM, and linear response obtained for urea concentrations 100 μM to 500 μM. The peak current finding procedure is delineated in Fig. 14, wherein an asymmetric least squares baseline correction procedure is performed on SWV summation curves. Following this, the approximate peak potential is identified and the SWV summation current values corresponding to the identified potential is utilized for urea concentration calculations.

EXAMPLE 8

Recovery studies in urea spiked in diluted human saliva samples using sensor device at identified potential ~0.4 V

To validate the performance of the sensor device and comment on possible interferences from other molecules/analyte present in saliva samples, studies were performed on urea spiked saliva samples. The human saliva samples were diluted 40x using 0.1 M KOH. If the recovery % is greater than 90%, it means that the sensor device can reliably detect the added amounts of desired analyte, urea, without any interference from other analyte present in bio fluids. Moreover, performing such experiments directly on bio fluids without individually spiking the interfering species in buffer samples confers many advantages, such as, aid us to validate the matrix effects/combination of interfering species offered by saliva samples on the performance of sensor device. Subsequently, the peak current values corresponding to peak potential ~0.4 V was obtained according to the procedure described in Figure 14 and the recovery % calculated is shown in table 1. It could be seen from Figure 16(c) and table 1 that the recovery % was well above 90% implying minimal interference from other analyte in human saliva samples.

Table 1. Recovery studies corresponding to peak current values at ~0.4 V

Urea concentration spiked (μM) Recovery %
100 92.6 ± 10.8
200 95.8 ± 2.6
300 91.8 ± 6.4
400 95.9 ± 3.5
500 90.2 ± 5.8

COMPARITIVE EXAMPLE 8

Recovery studies in urea spiked in diluted human saliva samples using sensor device at identified potential ~0.55 V

From Figure 16(a), it is evident that peak current values corresponding to peak potential ~0.55 V also increased with increasing urea concentrations in buffer solutions. However, very poor recovery % was obtained when current values corresponding to ~0.55 V was considered (for corresponding calibration curve). This confirmed that current values and calibration curve obtained for ~0.4 V is appropriate for sensor device development.

EXAMPLE 9

Comparison of sensor device performance with gold standard auto analyzer method:

The saliva samples were collected from patients at the Vivacity multispecality hospital. The purpose of the study was explained to the patients in local language (Bengali/Hindi) and samples were collected following consent from the individuals. Saliva samples were collected via passive drooling method. The sensor device performance was compared with the gold standard auto analyzer method. Referring to Fig. 16 (d), it is evident that there was a strong correlation (r = 0.95) between the sensor device and gold standard method, thus indicating reliable performance of the sensor device, and usability for rapid, point-of-care detection applications.

Advantages:

• Most of the small metabolite sensing works utilizing porous LIG electrodes involve a polymeric modification step to minimize capacitance/background currents. However, such modification decreases the inherent surface area offered by LIG electrodes and introduces additional step in sensor fabrication process. In the present invention described, such modifications are negated, and further leverage the capacitance currents to enhance the sensor performance. Such an achievement was done by utilizing the alternative resultant summation current from the SWV response whose output is inclusive of capacitance and faradic contributions.
• Furthermore, while most of the works utilize one-parameter-at-a-time optimization methodology to improve the sensor/technique's performance, such methods fail to consider the interaction and combinatorial effects between factors. Addressing these issues, utilization of a design-of-experiment based multivariate parametric optimization strategy to optimize the SWV features of the sensor device to yield high peak current and minimal standard deviation values. Moreover, such a dual response optimization increases the sensitivity (peak currents) and reliability (reproducibility) of the sensor device.
• While most of the works carry out peak identification via a cursor-pointer manual procedure, they are prone to manual error. In the invention described, utilization of a non-linear asymmetric least squares baseline subtraction to identify the peak potential, and report the current values corresponding to the approximate peak potential (thus identified). The procedure adopted here can be easily automated via simple programming, has minimal manual involvement, and hence largely minimizes the error associated with peak current identification.
• The sensor device utilizes a non-enzymatic sensing strategy which addresses the issues of oxygen evolution reactions associated with reported works, such as with nickel-based non-enzymatic urea sensing in alkaline medium
• The sensor device enables quantification of urea from non-centrifuged, simply diluted human saliva samples
• The sensor device shows appreciable recovery characteristics (>90%) and high selectivity to urea in minimally processed complex bio fluid such as saliva
• The operation of sensor device does not require any prior training and can be carried out by unskilled individuals
• The sensor device is operated using a smart phone and enables rapid detection of urea within 3 min (~130 s)
• The electrode manufacturing process is a single-step, maskless, wet-chemical free procedure and is environmentally benign in contrast to other electrode fabrication strategies
• The sensor strips can be mass produced with each costing less than 0.1 $

Applications:

The proposed sensor device can be utilized for the point-of-care detection of urea from minimally processed saliva samples. Moreover, the sensor strip/device arrangement is highly portable, can be integrated with mobile phone/app and is highly suitable for mass screening programmes like that organized by Governmental and Non-Governmental organizations. Additionally, the utilization of SWV summation current approach negates the necessity to perform complex modification, such as that performed to address capacitance/background currents on sensor surface and aids in excellent utilization of sensor's effective surface area, such as that offered by porous electrodes.
, Claims:We Claim:
1. An electrochemical non-enzymatic analyte/urea sensor device suitable for Point-of-Care Settings comprising:
metallic dendrite modified porous graphene based electrode favouring metallic Nano interface including silver Nano interface for circumventing oxygen evolution reactions and adapted to generate metal oxide/silver oxide based soluble complex with hydrolysis products of urea to thereby expose underlying metal/silver for its related stoichiometric electrochemical oxidation based peak currents corresponding to analyte/urea levels in samples and their quantification.

2. The electrochemical non-enzymatic analyte/urea sensor device as claimed in claim 1 adapted as a portable, rapid, real time, salivary urea sensing and detection device wherein :
said electrode is configured as a three-electrode assembly based electrochemical sensor strip consisting of a working electrode (2) of silver dendrite modified graphene surface and is capable of holding diluted non-centrifuged saliva samples as analyte for testing when drop casted on sample holding spot in said strip,
with said sensor strip for desired sensing is operationally connected to a portable potentiostat operable by a smart phone/laptop in connection having said stoichiometry supporting analytics for said real time sensing and electrochemical quantification of urea levels in saliva samples held in said strip.

3.The electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 1 or 2 wherein said silver dendrite modified porous graphene based electrode favouring silver Nano interface is laser induced graphene-based (LIG) electrodes fabricated from precursor polymer substrates including polyimide films having graphitic patterns thereon based on laser engraving/ ablation, in which the working electrode region is modified with metal-based electro-catalytic material including silver adapted for said metallic Nano interface to facilitate electro-oxidation of analytes including urea.

4. The electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 1-3 wherein:
said electrode is configured as a three electrode assembly (1) consisting of working electrode (2) on said sensor strip and includes 3 number of channel as electrodes and connecting track regions connecting said channels on said strip with the remaining regions of the strip (4) passivated with hydrophobic, non-conducting material including insulating tape/cello tape to ensure confinement of the diluted saliva sample within said 3-electrode channel regions of the sensor strip, said electrode channels and connecting tracks being laser pulse ablated/fabricated polymer film having graphitic structures with defects and pores for necessary electro-catalytic activity,
said three electrode assembly (1) including said 3 number of channels and connecting track regions connecting said channels on said strip includes said working electrode (2) linking said sample holding spot, pseudo-reference electrode (3) preferably coated with silver (or) silver/silver chloride paste or electrodeposited silver from silver salts together with counter electrode (6) which is laser pulse ablated, possess graphitic structures and is devoid of any further deposition/modification and connecting pads (5) coated with conductive silver paste or covered with copper tapes for operatively connecting said electrode assembly (1) based sensor strip to said portable potentiostat.

5. The electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 1-4 wherein said laser induced graphene-based (LIG) electrodes as working electrode is fabricated of precursor polymer substrates/films that include materials selected from polyimide, polyethyleneimine, polyether ether ketone, polybenzimidazole, polyamide imide, polysulfone, polyethersulfone, polystyrene, epoxy, phenolic resin, and cellulosic content rich substrates as paper, wood, cardboard, where the material of substrate dictates the necessity for pre-treatment, which substrates/films upon laser ablation/irradiation exhibits increase in carbon content and decrease in oxygen and nitrogen content due to breakage of imide bonds, decomposition of diaryl ether groups, and formation of aliphatic hydrocarbon upon laser irradiation.

6. The electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 1-5 wherein said working electrode (2) of said electrode assembly (1) having laser engraved/ ablated graphitic patterns of precursor polymer films is modified with said metallic dendrites including silver dendrites based on electrodeposited silver on said laser engraved/ ablated graphitic patterns.

7. The electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 1-6 wherein said precursor polymeric materials preferably includes polyimide film of thickness varying in the range of 5 mil (175 μm) to 10 mil (250 μm) for subjecting to continuous (or) pulsed laser source (or) laser diodes of different wavelengths of CO2 laser (1064 μm), visible laser (~450 nm), UV-excimer laser source (190 to 350 nm) providing graphitic structures with defects and pores exhibiting an carbon content of 82 to 95 % and oxygen and nitrogen content of 5 to 11% and 0.5 to 6.5 % respectively.

8. The electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 1-7 wherein said counter electrode have empirical geometrical area much larger than working electrode (4 times) to ensure the rate-limiting reactions occur at the working electrode and such that relevant analyte information pertaining to reaction of interest is gained from sensor strips.

9. The electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 1-8 wherein said working electrode (2) is responsive to provide quantification of analytes in presence of redox mediators and supporting electrolytes, including outer-sphere redox couple as ruthenium hexamine (II)/(III) (or) inner-sphere redox couple such as ferro/ferricyanide in supporting electrolyte for graphene-based electrodes, and monovalent salt solutions including potassium chloride, sodium chloride (or) divalent salt solutions such as calcium chloride, magnesium chloride (or) solutions of phosphate buffered saline with concentration varying in the range of 0.1 to 1 M respectively.

10. The electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 1-9 wherein said non-centrifuged saliva samples for drop casting on reaction spot upon dilution includes monoacidic alkaline solutions corresponding to sodium hydroxide, potassium hydroxide (or) diacidic alkaline solution corresponding to calcium hydroxide, magnesium hydroxide for said dilution to reach analyte concentration levels of 100 to 500 µM.

11. A method of fabrication of electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 1-10 comprising the steps of
providing porous graphene based electrode favoring metallic dendrite modified, metallic Nano interface including silver Nano interface as working electrode (2) for configuring as electrode assembly based electrochemical sensor strip capable of holding diluted analytes samples including non-centrifuged saliva samples as analyte for testing when drop casted on sample holding spot in said strip, enabling generation of said metal oxide/silver oxide based soluble complex with hydrolysis products of urea thereby exposing underlying metal/silver for its related stoichiometric electrochemical oxidation based peak currents corresponding to analyte/urea levels in samples providing for their quantification.

12. The method of fabrication of electrochemical non-enzymatic analyte/urea sensor device as claimed in claim 11 wherein said porous graphene based electrode with metallic dendrite modified, favoring metallic Nano interface including silver Nano interface as working electrode (2) includes the following steps:
(a) Configuring said electrode (1) as three electrode assembly based electrochemical sensor strip and creating sample holding spot on said strip for holding said drop casted diluted non-centrifuged saliva samples as analyte for testing in said strip,
(b) Connecting said sensor strip with a portable potentiostat for operation which in turn is operated by placing a smart phone/laptop in connection and in having said stoichiometry supporting analytics enables said real time sensing and electrochemical quantification of urea levels in saliva samples held in said sample holding spot of said strip.

13. The method of fabrication of electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 11 or 12 wherein said step (a) of configuring said electrode (1) as three electrode assembly based electrochemical sensor strip includes the sub-steps of:

(i) providing precursor polymer substrates including polyimide films and subjecting said films to laser engraving/laser ablation and creating graphitic patterns on said film for fabrication of said three electrode assembly and their connecting track,
(ii) modifying working electrode (2) of the electrode assembly by electro-deposition of the thus attained laser engraved patterns on said film with metal-based electro-catalytic material giving metallic dendrites including silver dendrites adapted for said metallic Nano interface to facilitate electro-oxidation of analytes including urea and obtaining said working electrode (2) as three electrode assembly based electrochemical sensor strip therefrom.

14. The method of fabrication of electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 11-13 wherein:
in said step (i) for creating graphitic patterns and fabricating working electrode (2) as three electrode assembly based electrochemical sensor strip is based on providing preferably polyimide film within thickness range of 5 mil (175 μm) to 10 mil (250 μm) followed by subjecting to rapid lasing pluses sourced from laser diodes including different wavelengths corresponding to CO2 laser (1064 μm), visible laser (~450 nm), UV-excimer laser source (190 to 350 nm) resulting in formation of graphitic structures with defects and pores with porosity ranging from 1 to 4 µm while maintaining vertical height of the electrodes, and connecting tracks thus laser engraved at 3 to 5 mm, raster scanning of 20 mm/s to 40 mm/s, hatching distance of 0.01 to 0.05 mm, and angle at 0o to 90o, to attain said laser induced graphene (LIG) electrodes with high porosity and electrocatalytic activity, and
in said step (ii) for modifying the thus engraved patterns and channel based electrodes on said film is subjected to electro-deposition of the working LIG electrode (2) with a salt concentration of silver corresponding to (1 to 15 mM), potential window for LSV (0 to -0.4 V - 0 to -1.5 V), scan rate of LSV (5 to 100 mV/s) with number of repetitions/cycles (1 to 5) selected for deposition of silver suiting SWV features of the sensor device to yield high peak current of 4 to 6 µA with minimum standard deviation of 0.4 to 0.6 µA quantifying urea levels.

15. The method of fabrication of electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 11-14 wherein regions (4) excluding the electrode channels and their connecting tracks on said strip is passivated with a relatively hydrophobic, non-conducting material including Kapton tape (or) cello tape ensuring the confinement of analyte/diluted saliva sample within said 3-electrode region of the sensor strip.

16. The method of fabrication of electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 11-15 wherein said electrode assembly having pseudo-reference electrode (3) is manually coated with silver (or) silver/silver chloride paste or silver salts are electro-deposited, and the connecting pads (5) are coated with conductive silver paste or covered with copper tapes for establishing connection with said potentiostat.

17.The method of fabrication of electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 11-16 wherein said sensor sensor is evaluated on basis of square wave voltammetry (SWV) features, which includes responses in said redox mediator including outer-sphere redox couple selected as ruthenium hexamine (II)/(III) (or) inner-sphere redox couple such as ferro/ferricyanide in supporting electrolyte for said graphene-based LIG electrodes, and wherein said supporting electrolytes provided varies in the range of 0.1 to 1 M and includes monovalent salt solutions selected as potassium chloride, sodium chloride (or) divalent salt solutions selected as calcium chloride, magnesium chloride (or) solutions of phosphate buffered saline.

18. The method of fabrication of electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 11-17 wherein the thus fabricated sensor device responds to SWV features of amplitude of 10 to 100 mV, increment voltage of 2 to 10 mV, and frequency of 1 to 10 Hz, to correspond to values of peak current and standard deviation.

19. The method of fabrication of electrochemical non-enzymatic analyte/urea sensor device as claimed in claims 11-18 wherein the thus fabricated sensor device gives idiosyncratic sharp oxidation peaks in the cathodic (or) reduction half cycle for urea analyte corresponding to the complex formation of hydrolysis products of urea with silver (I) oxide present on Nano interface that dissolves into the solution and exposes the underlying silver structures.




Dated this the 11th November, 2024 Anjan Sen
Of Anjan Sen & Associates
(Applicants Agent & Advocate)
IN/PA-199

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