Field of the invention
The present invention is related to a sensitive method for determining blood glucose level by the collection and analysis of saliva.
Background
Diabetes is one of the most talked about metabolic diseases across the world and especially in India. The lack of awareness about the disease can well be estimated by the fact that India today has more people with type-2 diabetes (more than 50 million) than any other nation in the world. The WHO also estimates that 80 per cent of diabetes deaths occur in low and middle-income countries and projects that such deaths will double between 2016 and 2030. It has been further estimated that the global burden of type-2 diabetes is expected to increase to 438 million by 2030 from 285 million people (recorded in 2010). Similarly, for India this increase is estimated to be 58%, from 51 million people in 2010 to 87 million in 2030.

Type-1 diabetes is an insulin dependent diabetes wherein the body's immune system destroys the cell that release insulin. Type-2 diabetes is a non-insulting dependent diabetes wherein body isn't able to use insulin the right way. This is called insulin resistance. Monitoring and managing the disease can prevent complications in both the types.

People with diabetes are required to test the blood sugar level at least two times a day. Most of the glucose monitoring devices in market are invasive and involves use of needles leading to increased chances of infection. To use the device, patients have to prick their finger for a drop of blood. Pricking fingers is painful, inconvenient and requires puncturing the skin which may results in development of peripheral neuropathy. Patient usually, is not willing to puncture skin so many times per day. Further, the blood performs glucose oxidation upon pricking and therefore the measurement of sample changes.

Therefore, non-invasive glucose monitoring devices are being widely investigated and studied over the past few years for their ability to monitor glucose under highly controlled conditions. Non-invasive monitoring of glucose levels eliminates the need of painful pricking with increased risk of infection, and amount of damage caused to the finger tissue.

Various techniques that involves measurement of blood glucose levels non-invasively includes, infrared (IR) spectroscopy, Near Infrared Spectroscopy (NIRS), Mid-Infrared Spectroscopy (Mid-IRS), fluorescence spectroscopy, Raman Spectroscopy, optical polarization rotation measurement, Photo-acoustic probe (PA), Optical Coherence Tomography (OCT), surface plasma resonance and many more. Most are for laboratory use only and are not suitable for routine glucose monitoring at home due to size, cost and complexity. Results from these techniques are limited by spectral signal-noise levels and sample thickness, and have to be correlated with direct blood glucose measurements. There is always a need to develop easy, cost-effective simplified device for accurate glucose measurements.

Presently, there are few non-invasive devices are available in market for glucose estimation. These devices include ‘Flash glucose monitoring system’ that introduces sensor inside the skin. ‘GlucoTrack®’ by Integrity applications which detects blood sugar through skin (clamp placed on ear-lobe) by applying ultra-sonic, electromagnetic and thermal technology for the detection. ‘GlucoWise®’ by MediWise also detects glucose via skin with high frequency radio waves. ‘Google Smart Contact Lens’ by Google and Novartis detect glucose in tears via integrated chip in contact lens. ‘Symphony’ is another glucose detection device by ‘Echo therapeutics’ which uses transdermal micro-abrasion system for glucose detection in sweat.
All these technologies are minimal/ non-invasive used only for monitoring purpose, however, have a common disadvantage which is ‘cost’. The cost of such device is unaffordable to major Indian population.

There is a proven and reliable scientific correlation between saliva and glucose levels. In saliva, glucose concentration is low as compared to blood. It is reported that salivary glucose levels are significantly higher in diabetic patients than in people without diabetes under similar conditions. Thus, measurement of saliva glucose level can be utilized as an alternative diagnostic method for diabetics and as a health indicator of a subject who is normal or suspected of having diabetes.

U.S. Pat. No. US6102872 by Pacific Biometrics discloses a method for determining subject's blood glucose level by performing a chemical analysis of glucose level. Upon collecting filtered saliva, it is put on colorimetric glucose film containing enzymes glucose oxidase and horseradish peroxidase along with dyes and accessory reagents for producing coloured spot on it. Colour intensity on film is proportional to the glucose concentration in saliva sample. The invention however does not provide information regarding quantitative measurement of glucose level.

U.S. Pat. No. 9244034 by Northeastern University discloses a system containing glucose sensor using electrochemical method. The sensor is made of three electrodes viz working, counter and reference electrode. Working electrode is attached with glucose oxidase. When drop of saliva comes in contact with all three electrodes, the amount of glucose is measured via amperometric method. The system contains display for outputting the results.

US20080177166A1 by Provex Tech LLC discloses amperometric saliva glucose sensor for detection of concentration of glucose. The device described to comprise of support, multi-layer electrode with glucose oxidase as enzymatic catalyst, sample region and measurement region. However, the application is abandoned and it seems that development of device has been discontinued.

DiaStrips is a non-invasive method of detecting elevated glucose levels in saliva with an app-based platform available in market currently. The main disadvantage of ‘DiaStrips’ is that these are only qualitative. It detects diabetes but does not give the accurate glucose value.
Presently, there is no workable device available in market for blood glucose estimation from Saliva, which can be used by patient single-handedly, easy to operate and which provide accurate and quantitative blood glucose value.

Thus, there was a requirement of a device that will sense glucose in PBS with desired sensitivity and repeatability. The device for better and accurate detection of salivary glucose, which can be used by patients including pediatric population at home and which is affordable to every sector of the society. By providing better disease control, it will ultimately lead to decrease in morbidity, mortality, and health care expenditure.
The present invention is based on principles of nanotechnology for glucose measurements. It provides an easy and accurate detection of Diabetes by quantitative measurement of blood glucose level in untreated saliva.

Summary of Invention
The present invention provides a highly sensitive glucose sensor for detection of glucose in saliva samples using an electrochemical method. The saliva glucose sensor is suitable for use in diagnosing diabetes and monitoring glucose levels in diabetic patients using only a small amount of unmodified saliva, and with results obtained in seconds. The sensor is provided in both reusable and disposable embodiments.
In one aspect of invention, the invention is a glucose sensor which is a strip comprising a screen printed electrode;
at least one working electrode, a counter electrode, and a reference electrode, and a sample placement area on a surface of screen printed electrode for containing the saliva; wherein; the working electrode, counter electrode, and reference electrode are connected to an amperometry circuit; and; wherein; an output voltage of the amperometry circuit correlates with a glucose concentration in the saliva deposited in the placement area.
The glucose sensor is a disposable device or a reusable device.
In another aspect of invention, it elaborates a method of fabricating a glucose sensor, comprising the steps of:
(a) dropcasting one or more working electrodes, a reference electrode, and a counter electrode on the surface of a screen printed electrode, wherein each of said electrodes contacts a sample placement area on the screen printed electrode;
wherein, dropcasting of 2 µL of 40 mM Ferrocene, 10 µL of 2 mg/mL Chitosan, 7 µL of gold nanoparticles, 10 µL of 1 unit / µL Horse-Raddish Peroxidase and 7 µL of BSA-Glu- Glucose Oxidase.
In further aspect of invention, it describes method of determining a glucose concentration in a saliva sample using the glucose sensor, wherein, the method consists the steps of:
(a) providing the glucose sensor of claim 1;
(b) introducing a saliva sample into the sample placement area of the sensor; and
(c) determining the glucose concentration in the saliva sample from an electrical output of the sensor in a device like glucometer or its variants.
(d) removing the liquid sample introduced in step (b); (e) introducing a new liquid sample into the sample placement area of the sensor; and
(f) determining a new glucose concentration in the new liquid sample from an electrical output of the sensor.
The glucose sensor described in the present invention is capable of sensing and detecting glucose concentration in saliva as low as 0.18 mg/decilitre.

Brief Description of Figures
Figure 1 relates to the device in the form of ‘strip‘ for the detection of blood glucose from saliva.
Figure 2 relates to flowchart methodology for preparation of non-invasive glucose sensor for detection of blood glucose from saliva.
Figure 3 referes to combination 1. A) A chronoamperometric plot measured for SPCE/Fc/CS/HRP/CS/GOx/Nafion in PBS saline pH 7.4 at a fixed potential of -0.3V vs. Ag/AgCl. The concentration of glucose was varied from 10 µM to 80 µM . B) The corresponding calibration curves of glucose.
Figure 4 refers to combination 2. First graph relates to A chronoamperometric plot measured for SPCE/Fc/CS/GDp/HRP/CS/GOx/Nafion in PBS saline pH 7.4 at a fixed potential of -0.3V vs. Ag/AgCl. The concentration of glucose was varied from 10 µM to 200 µM . B) The corresponding linear calibration curve
Second figure relates to Cyclic voltammogram of SPCE/Fc-CS in 0.1 M PBS saline buffer of pH 7.4.
Third figure relates to A) Cyclic voltammograms of the SPCE/Fc-CS in 0.1 M PBS saline buffer of pH 7.4 at potential scan rates: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mV/ and (B) Linearity of the plot of anodic peak currents vs. potential scan rate.
Figure 5 refers to combination 3 and 4. First figure relates to cyclic voltammetry of SPCE/Fc-CS/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with and without 5 mM glucose.
Second figure relates to- A) Chronoamperometric plot ofSPCE/Fc-CS/GDp/HRP/Chitosan-Gox-BSA-Gla/ Nafion in PBS saline buffer (pH 7.4) at a fixed potential of -0.32 V. The concentration of glucose was varied from 10 µM to 2 mM. and B) The corresponding calibration plot.
Figure 6 refers to combination 3 and 4. First two figures relate to Cyclic voltammetry of A) SPE/Fc-Cs (1 mg/mL) / GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion, B) SPE/Fc-Cs(2mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion, C) SPE/ Fc-Cs(3mg/mL)/ GDp/HRP/ Chitosan-Gox-BSA-Gla/ Nafion, D) D) SPE/Fc-Cs(4 mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion, E) SPE/Fc-Cs(5 mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with and with varying concentrations of Glucose
Third figure relates to A) Chronoamperometric plot of SPCE/Fc-CS (4mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion in PBS saline buffer (pH 7.4) at a fixed potential of -0.3V. The concentration of glucose was varied from 10 µM to 5 mM . B) The corresponding calibration plot.
Figure 7 refer to combination 3 and 4. First and second figure relate to Cyclic voltammetry of A) SPE/Fc-Cs(1 mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion, B) SPE/Fc-Cs(2 mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion and C) SPE/Fc-Cs(3 mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion D) SPE/Fc-Cs(4 mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion, E) SPE/Fc-Cs(5 mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with and with varying concentrations of Glucose.
Third and fourth figure relate to Cyclic voltammetry of A) SPE/Fc-Cs(0.5 mg/ mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with varying concentrations of Glucose B) Repeated experiment C) SPE/Fc-Cs(1 mg/ mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with varying concentrations of Glucose D) Repeated experiment
Figure 8 refer to combination 5. First figure relates to Cyclic voltammetry of SPE(550)/CS-Fc/GDp/HRP in pH 7.4 PBS buffer at a scan rate of 50 mV/s with varying concentrations of H2O2
Second figure relates to Cyclic voltammetry of SPE(550)/CS-Fc/GDp/HRP/CS/BSA-Gox-Gla in pH 7.4 PBS buffer at a scan rate of 50 mV/s with varying concentrations of glucose
Third figure relates to Cyclic voltammetry of A) SPE/Cs-Fc(0.5 mg/mL)/GDp/HRP and B) SPE/Cs-Fc(1 mg/mL)/GDp/HRP in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with varying concentrations of H2O2
Figure 9 refer to combination 5. It relates to Cyclic voltammetry of 1) SPE/HRP (1unit/µL) and 2) SPE/HRP (1mg/mL) in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with varying concentrations of H2O2.
Figure 10 refer to combination 6. First figure relates to A) Cyclic voltammetry of SPE/Fc-Cs-CNT/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with varying concentrations of Glucose and B) Repeated experiment
Second figure relates to A) Cyclic voltammetry of SPE/Gox-BSA-Gla in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with varying concentrations of Glucose B) Same experiment with reverse scan window
Figure 11 refer to combination 7. First figure relates to electropolymerisation of Carbon nanotube - Aniline composite
Second figure relates to Cyclic voltammetry of SPE/CNT-PANI/GDp/Gox/Nafion in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with varying concentrations of Glucose
Third figure relates to Calibration plot showing the relation between Oxygen reduction current and Glucose concentration
Figure 12 refer to combination 7. First figure relates to Cyclic voltammetry of the electropolymerization of Prussian blue on SPCE
Second figure relates to Cyclic voltammetry of the SPCE/PB in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with varying concentrations of H2O2
Third figure relates to Cyclic voltammetry of the electropolymerization of Prussian blue on SPE (550)
Figure 13 refer to combination 7. It relates to Cyclic voltammetry of the SPE (550)/PB in pH 7.4 PBS saline buffer at a scan rate of 50 mV/s with varying concentrations of H2O2
Figure 14 refer to combination 8. First figure relates to A Cyclic voltammetry of SPE(550)/PB/CS/BSA-Gox-Gla in pH 7.4 PBS buffer at a scan rate of 50 mV/s with varying concentrations of Glucose B) Repeated experiment
Second figure relates to Cyclic voltammetry of SPE (550)/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion in pH 7.4 PBS
Third figure relates to a Chronoamperometric response of SPE (550)/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion towards glucose at an applied potential of 50 mV. B) The corresponding linear calibration plot
Figure 15 refer to combination 8. First figure relates to A Cyclic voltammetry of SPCE/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion in pH 7.4 PBS buffer at a scan rate of 50 mV/s with varying concentrations of Glucose B) Chronoamperometric response of SPCE/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion towards glucose at an applied potential of -30 mV
Second figure relates to: A, B, C cyclic voltammetry of SPCE/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion in pH 7.4 PBS buffer at a scan rate of 50 mV/s with varying concentrations of Glucose
Third figure relates to: A, B, C Chronoamperometric response of SPE (550)/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion towards glucose at an applied potential of 50 mV
Fourth figure is Linear calibration plot
Figure 16 refer to combination 8. First figure relates to A Cyclic voltammetry of A) SPE(550)/Prussian blue(50 µg/mL) in pH 7.4 PBS buffer at a scan rate of 50 mV/s with varying concentrations of Glucose B) SPCE/Prussian blue(50 µg/mL) in pH 7.4 PBS buffer at a scan rate of 50 mV/s with varying concentrations of H2O2
Second figure relates to A CV response of the SPCE/PB (1 mg/mL) towards H2O2. B) SPCE/PB (1 mg/mL)-BSA-GOx towards glucose
Figure 17 refer to combination 9. First figure relates to CV response of the SPE (550)/PB (1 mg/mL) towards H2O2
Second and third figure relates to a Amperometry on SPE (550)PB (1mg/mL)-BSA-GOx at an applied potential of -0.4 V and Linear calibration plot for glucose determination
Figure 18 refer to combination 9. A Amperometry on SPCE/Fc/CS/GDp/HRP/CS/GOx/Nafion (Electrode 1) at an applied potential of -300 mV. B) The corresponding linear calibration plot C) Amperometry on SPCE/Fc/CS/GDp/HRP/CS/GOx/Nafion (Electrode 2) at an applied potential of -300 mV. D) The corresponding linear calibration plot
Figure 19 refer to combination 10. A Amperometry on SPCE/CS/Fc/GDp/HRP/CS/GOx/Nafion (Electrode 1) at an applied potential of -400 mV. B) The corresponding linear calibration plot. C) Amperometry on SPCE/CS/Fc/GDp/HRP/CS/GOx/Nafion (Electrode 2) at an applied potential of -400 mV. D) The corresponding linear calibration plot.

Brief Description of Invention
Definition
Throughout the description the following terms, unless otherwise indicated, shall be understood to have following meanings:
The term ‘chronoamperometry’ refers to an electrochemical technique in which the potential of the working electrode is stepped and the resulting current from faradaic processes occurring at the electrode (caused by the potential step) is monitored as a function of time.
The term ‘electrochemical method’ refers to study of an analyte by measuring the potential (volts) and/or current (amperes) in an electrochemical cell containing the analyte.
The term ‘electrode’ refers to a small piece of metal or other substance or an electrical conductor used to establish contact a non-metallic part of a circuit.
The term ‘sample’ refer to the biological liquid sample such as saliva obtained from patient’s body for further measurement of glucose level.
The term ‘sensor’ refer to a strip made of sample placement area and other components described in present invention to detect glucose level in sample.
The term ‘saliva’ or ‘untreated/unaffected saliva’ refers to liquid sample of salivary secretion collected from patient and not exposed to filtration or other modification before using as sample for the present invention. The saliva primarily consists of water, essential electrolytes, glucose, amylase, glycoprotein, and antimicrobial enzymes.
The term ‘Screen-printed electrodes (SPEs)’ refers to economical electrochemical substrates for electrodes manufactured for electrochemical analysis in environmental, clinical or agri-food areas. These substrates include carbon, gold, platinum, silver or carbon nanotubes inks etc.

In the present invention, an electrochemical method was used to quantify blood glucose levels based on the amount of the glucose detected in saliva. The method consists of use of sensor which is for measuring the glucose level in untreated saliva sample by electrochemical method. The sensor particularly consists of
1. screen printed electrode with placement area
2. one or more working electrode(s) with conductive metal layer
3. counter electrode (carbon) with conductive metal layer
4. reference electrode (silver/platinum electrode) with conductive metal layer
5. protective membrane
wherein, working electrode(s), counter electrode and reference electrode are further connected to amperometry circuit. The output voltage of the same provides a measure of the glucose concentration in untreated saliva sample.

In another aspect of present invention, it discloses the method for detecting the amount of glucose in the sample of saliva, wherein, the method comprises of below steps:

1. Collect small amount of saliva from patient (as per instruction)
2. Place a drop of saliva on the sample area on glucose sensor (strip).
3. Insert the strip into the device (glucometer)
4. Determine glucose concentration in sample from electric output on sensor
5. Check device display for blood sugar level value in few seconds
6. remove the strip from device
7. insert another strip in the device for another sample

The strip detects glucose in saliva within few seconds upon insertion into a device such as glucometer which also quantify the amount of glucose in saliva.

Various experiments were performed to determine the two parameters of interest such as
(i) concentration range of linear behavior, and,
(ii) sensitivity
Inventors studied ten different combinations of screen printed electrode, working electrode, reference electrode and sensory electrode to find the most suitable combination with respect to its sensitivity and range. Inventors further fabricated the strip for detection of glucose in saliva (figure 1)
The sensitivity was optimizing by adjusting the working electrode. The sensor can serve as a stand-alone device, or it can be incorporated into another device.

Example 1
The example 1 elaborate details of combinations of screen printed electrode, working electrode, reference electrode and sensory electrode to find the most suitable combination with respect to its sensitivity and range.
These combinations of screen printed electrode, working electrode, reference electrode and sensory electrode were prepared by the drop casting method. The flowchart of methodology (figure 2) refer to the flow of drop-casting method to prepare the working glucose sensor for detection of glucose in sample of saliva.
These were further tested by Chronoamperometry, in which the potential of the working electrode is stepped and the resulting current from faradaic processes occurring at the electrode (caused by the potential step) was monitored as a function of time.
Also, Voltammetry was done to investigate electrolysis mechanism. A voltage or series of voltages are applied to the electrode and the corresponding current that flows was monitored.

Combination 1) SPCE/Fc/CS/HRP/CS/GOx/Nafion
The SPCE/Fc/CS/HRP/CS/GOx/Nafion electrode was fabricated by dropcasting 2 µL of 40 mM Ferrocene, 10 µL of 2 mg/mL Chitosan, 10 µL of 1 unit / µL HRP and 7 µL of BSA-Glu-GOx. The electrode shown reduction of H2O2 at -300 mV. The chronoamperometric results showed a linear range of 10µ-80 µM with a sensitivity of 0.09 µA/µM (Figure 3).

Combination 2) SPCE/Fc/CS/GDp/HRP/CS/GOx/Nafion
The SPCE/Fc/CS/GDp/HRP/CS/GOx/Nafion electrode was fabricated by dropcasting 2 µL of 40 mM Ferrocene, 10 µL of 2 mg/mL Chitosan, 7 µL of gold nanoparticles, 10 µL of 1 unit / µL HRP and 7 µL of BSA-Glu-GOx. The electrode shown reduction of H2O2 at -300 mV. The chronoamperometric results showed a linear range of 10 µM-200 µM with a sensitivity of 0.483 µA/µM (Figure 4 first graph).
Preparation of Ferrocene carboxaldehyde- Chitosan composite
75.0 mg of chitosan was dissolved in a 0.1 M acetic acid solution and added dropwise to a methanolic solution of ferrocene carboxaldehyde (1 mg/ mL). The mixture was stirred for 2 hours at room temperature and 80.0 mg of sodium cyanoborohydride was dropped to the reaction mixture and stirred for 24 hours. The reaction was stopped by adding 5% NaOH and the resulting yellow precipitate was exhaustively washed with water and methanol. The product was dried in air and dispersed in 0.2 M acetate buffer using sonication. Six concentrations of Ferrocenecarboxaldehyde-Chitosan composite namely 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL and 5 mg/mL.
Electrochemical studies of SPCE/Fc-CS in 0.1 M PBS saline pH 7.4
10 L of Ferrocenecarboxaldehyde-Chitosan composite of concentrations 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL and 5 mg/mL were drop-casted on six separate screen-printed electrodes and the CV response were recorded in 0.1 M PBS saline buffer of pH 7.4. As shown in Figure 4:second graph, increase in redox current is observed with increase in the loading of Fc-CS. This is consistent with the increased numbers of Fe(II) centers of ferrocene carboxaldehyde.
Scan rate studies
For scan rate studies, the SPCE/Fc-CS(5 mg/mL) electrode was used. CV was recorded at potential scan rates: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mV in a 0.01 K4[Fe(CN)6] solution mixed with 0.1 M KCl supporting electrolyte. The peak current vs scan rate plot was consistent with the surface-confined redox process (Figure 4-third graph).

Combination 3 and 4) SPE/Fc-Cs(0.5 mg/mL)/ GDp/ HRP/ Chitosan - Gox-BSA-Gla/Nafion electrode and SPCE/Fc-CS(4mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion
Fabrication of SPE/Fc-Cs/ GDp/ HRP/ Chitosan - Gox-BSA-Gla/Nafion electrode
The SPCE was first electrochemically scanned in the potential window -0.6 V to 0.6 V in 0.1 M PBS buffer of pH 7.4. The electrodes were washed with buffer and dried. 10 µL of chitosan-Ferrocene carboxaldehyde, 3 units HRP, 7 µL of the gold nanoparticles, and a composite of 0.5 U Glucose oxidase glutaraldehyde (0.25%) and BSA (3%) were used for the fabrication. The layers were washed with buffer drying overnight. Finally, a 3 µL solution of Nafion (0.1 wt%) was added to prevent the leaching out of enzymes to the buffer solution.
Electrochemical response of the fabricated sensor SPE/Fc-Cs (0.5 mg/mL)/ GDp/ HRP/ Chitosan - Gox-BSA-Gla/Nafion towards glucose was recorded. The electrode showed reduction of H2O2 (byproduct of glucose oxidation) around -320 mV (Figure 5 first graph). This potential was similar to the reduction of H2O2 observed on LBL modification of Chitosan and Ferrocene. The Amperometry response was recorded at -320 mV. The observed linear range was from10 µM to 1 mM with a sensitivity of 2.15 nA/µM (Figure 5 second graph). The experiment is repeated on other modified electrodes also and similar poor performance is observed (Figures 6-7-first graph). The electrodes were refabricated and similar response was observed (Figure 7-second graph).

Combination 5) SPE(550)/CS-Fc/GDp/HRP
The chitosan- ferrocene carboxaldehyde composites were unable to mediate the electron transfer between carbon electrode and HRP. To see the effect of electrode surface, Screen printed platinum electrodes were used to fabricate SPE(550)/CS-Fc/GDp/HRP and electrochemical response towards H2O2 was recorded. SPE(550)/CS-Fc/GDp/HRP electrode shown good response (Figure 8-first graph) for low concentrations of H2O2. Therefore, the electrode SPE(550)/CS-Fc/GDp/HRP/CS/BSA-Gox-Gla was fabricated and electrochemical response of towards Glucose was recorded. The electrode showed good response for glucose in the range of 1-20 µM after 20 µM no increase in current was observed with increase in glucose concentration (Figure 8-second graph)
To see if the Ferrocene carboxaldehyde-Chitosan composite can mediate the electron transfer from HRP, two electrodes were prepared as SPE/Cs-Fc(0.5 mg/mL)/GDp/HRP and SPE/Cs-Fc(1 mg/mL)/GDp/HRP and electrochemical response towards H2O2 were recorded. The results (Figure 8-third graph) showed that the Ferrocene carboxaldehyde-Chitosan composite was not suitable for mediating the electron transfer between screen printed electrode and HRP
Two electrodes were prepared as SPE/HRP (1unit/µL) and SPE/HRP (1mg/mL) and electrochemical response towards H2O2 is recorded the modified electrodes showed no response towards H2O2 (Figure 9). The stock solution of HRP may be denatured. Freshly prepared HRP solutions were used for the subsequent experiments.

Combination 6) SPE/Fc-Cs-CNT/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion
Preparation of Ferrocene carboxaldehyde- Chitosan – Carbon nanotube composite
75.0 mg of chitosan was dissolved in a 0.1 M acetic acid solution and added dropwise to a methanolic solution of ferrocene carboxaldehyde (1 mg/ mL). The mixture was stirred for 2 hours at room temperature and 80.0 mg of sodium cyanoborohydride was dropped to the reaction mixture and stirred for 24 hours. The reaction was stopped by adding 5% NaOH and the resulting yellow precipitate was exhaustively washed with water and methanol. The product was dried in air and dispersed in 0.2 M acetate buffer using sonication. Subsequently, 1.0 mg of SWNTs functionalized with carboxylic groups were ultrasonically dispersed in a 2.0 mL solution of 1.0 mg /mL Chitosan-Ferrocene carboxaldehyde solution to form a black mixture

Fabrication of SPE/Fc-Cs-CNT/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion
New electrode was fabricated by drop casting 10 µL of Chitosan-Ferrocene –Carboxaldehyde-Carbon nanotubes and the remaining layers were drop cast by the same procedure described before. The electrode showed response towards glucose up to 10 µM glucose. Above 10 µM concentration of glucose, only negligible increase in current is observed (Figure 10-first graph-A). The experiment was repeated and same response was observed (Figure 10-first graph-B).
To check the activity of the Glucose oxidase enzyme in the prepared stock solution of Glucose Oxidase-BSA-Glutaraldehyde, one electrode was prepared as SPE/Gox-BSA-Gla and electrochemical response towards Glucose was recorded. Oxidation (Figure 10-second graph-A) and reduction (Figure 10-second graph-B) of H2O2 was observed consistent with Glucose oxidase activity

Combination 7) SPE/CNT-PANI/GDp/Gox
Electropolymerisation of Carbon nanotubes-Aniline and fabrication of SPE/CNT-PANI/GDp/Gox
The CNT-PANI composite films were formed by electropolymerization of 0.35 M aniline dissolved in 0.5 M H2SO4 in the presence of 0.8 wt.% CNT w. r. to the weight of aniline. The Electropolymerisation was carried out by cycling in the potential range from -0.2 to -1.2V at a scan rate of 100 mV/s. The peak current of three distinctive processes increased with the successive scan number (Figure 11-first graph), suggesting successful electropolymerization. The peak A is due to the formation of a radical cation of aniline. The peak B is due to the oxidation of tail to tail dimer and the peak C is due to the conversion of emeraldine structure of polyaniline to pernigraniline structure
Electrochemical response of the prepared SPE/CNT-PANI/GDp/Gox/Nafion electrode was recorded towards glucose. As shown in Figure 11-second graph, a decrease in the Oxygen reduction current was observed with increase in concentration of Glucose. The CNT-PANI/GDp composite was more active towards O2 reduction and less active towards H2O2 reduction. The Glucose Oxidase reaction consumes Oxygen, and with increase in concentration of Glucose, the dissolved Oxygen also decreases proportionately. Calibration plot of figure 11-third graph showed the relation between Oxygen reduction current and Glucose concentration.

Electropolymerization of Prussian blue on SPCE and its electrochemical response towards H2O2
Prussian Blue layer was electrodeposited on screen-printed carbon electrode by cyclic scan within the limits of -0.2 to 1 V in a solution containing 1.5 mM K3Fe(CN)6 and 2 mM FeCl3 in 0.1 M KCl and 2 mM HCl (pH 2.0)(Figure 12-first graph). The electrode showed no response towards H2O2 (Figure 12-second graph). This may not be suitable for the fabrication of Glucose Sensor. There is a possibility that the reference and counter electrodes may be contaminated during the electropolymerization process.

Electropolymerization of Prussian blue-CNT composite on SPE (550) and its electrochemical response towards H2O2
Prussian Blue-CNT composite layer was electrodeposited on screen-printed carbon electrode by cyclic scan within the limits of -0.2 to 1 V in a solution containing 1.5 mM K3Fe(CN)6, 2 mM FeCl3 and 100 µg CNT in 0.1 M KCl and 2 mM HCl (pH 2.0)(Figure 12-third graph). The electrode showed good response towards low concentrations of H2O2 (Figure 13). This electrode is suitable for the fabrication of Glucose Sensor.

Combination 8) SPE (550)/PB/CS/BSA-Gox-Gla and SPE (550)/PB-CNT/CS/BSA-Gox-Gla
Fabrication and electrochemical response of SPE (550)/PB/CS/BSA-Gox-Gla and SPE (550)/PB-CNT/CS/BSA-Gox-Gla towards Glucose
Chitosan (10 µL of 2 mg/mL) and 3 µL of BSA-Gox-Gla were drop cast on SPE (550) electropolymerized with Prussian blue and electrochemical response towards Glucose is recorded. The electrodes showed good response for glucose at low concentrations (Figure 14-first graph-A-B). However, linear range was limited. This may be due to electrode poisoning

Fabrication of SPE (550)/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion
The electrodes prepared by layer by layer approach was often damaged by the spreading of the drop casted liquid to the reference and counter electrodes. This was especially noticed after the drop casting of enzyme solution. It was found that a small quantity of nanodiamond powder (100 µg) added during the preparation of ferrocene carboxaldehyde-chitosan composite made a porous like composite. The drop cast liquid were adsorbed into this composite and prevented spreading of the subsequent layers. The electrochemical response of the nanodiamond powder modified composite were similar to the composite without nanodiamond.

CV response of SPE (550)/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion towards Glucose
Cyclic voltammetry response of SPE (550)/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion towards glucose were recorded in pH 7.4 PBS buffer at a scan rate of 50 mV/s. The SPE (550)/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion electrode showed detection of low concentrations of Glucose at potential close to 50 mV (Figure 14-second graph). The electrode was saturated above 100 µM Glucose. Chronoamperometric response of SPE (550)/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion electrode was recorded at an applied potential of 50 mV(Figure 14-third graph-A). The linear range observed was from 500 nM to 100 µM with a sensitivity of 0.06 µA/µM (Figure 14-third graph-B). The experiment was repeated and similar kind of results were observed

CV response of SPCE/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion towards Glucose
The same experiment performed on screen-printed carbon electrodes. Four stack of electrodes were prepared. The electrodes showed poor electrochemical response towards glucose (Figure 15). Lower limit of detection was 1 µM with a linear range 1-50 µM and sensitivity 23 nA/µM

Fabrication of Prussian blue nanoparticle modified screen-printed electrodes.
Prussian blue nanoparticle modified screen-printed electrodes were fabricated as SPE (550)/Prussian blue (50 µg/mL) and ) SPCE/Prussian blue(50 µg/mL) and CV response towards H2O2 were recorded. Both the electrodes shown poor response towards H2O2. The SPCE/PB shown good oxidation current for H2O2 (Figure 16-first graph-B). This was inconsistent with the literature reports. Therefore, SPCE electrode was refabricated with higher Prussian blue loading as SPCE/PB (1 mg/mL) and SPCE/PB (1 mg/mL)-BSA-GOx and CV response was recorded. The electrodes showed good response for H2O2 (Figure 16-second graph-A) and Glucose (Figure 16-second graph-B).

Combination 9) SPE (550) PB (1mg/mL)-BSA-Gox
SPE (550) electrode was refabricated with higher Prussian blue loading as SPE (550)/PB (1 mg/mL) and SPE (550)/PB(1 mg/mL)-BSA-GOx and CV response was recorded. The electrodes showed good response for H2O2 (Figure 17-first graph) and Glucose (Figure 17-second-third graph). Lower limit of detection was 10 µM with a linear range 10 µM -2 mM and sensitivity 7 nA/µM.
The sensor was refabricated and the sensor parameters of linear range and sensitivity was reproduced. Figure 18 shows the corresponding linear calibration plot.

Combination 10) SPCE/CS/Fc/GDp/HRP/CS/GOx/Nafion
Fabrication of SPCE/CS/Fc/GDp/HRP/CS/GOx/Nafion
The electrode SPCE/Fc/CS/GDp/HRP/CS/GOx/Nafion showed good sensitivity towards glucose detection. Therefore, electrodes were fabricated by moving the ferrocene near to the HRP enzyme as SPCE/CS/Fc/GDp/HRP/CS/GOx/Nafion. Moving ferrocene near to HRP didn’t produced expected results. The reduction peak is observed around -400 mV. The SPCE/CS/Fc/GDp/HRP/CS/GOx/Nafion electrode was able to detect Glucose in the linear range of 10-150 µM with a sensitivity ˜ 74 µA/mM. Figure 19D shows the corresponding linear calibration plot.

Table 1 summarizes the results of concentration range of linear behavior and sensitivity studied for all the combinations of fabricated electrodes for detection of glucose in saliva.

SI. No. Combination Current Statua Comments
1 SPCE/Fc/CS/HRP/CS/GOx/Nafion 10µM to 80 µM linear range
Sensitivity = 0.09 µA/µM Lower sensitivity. It is possible to improve the sensitivity by using gold nanoparticles.
2 SPCE/Fc/CS/GDp/HRP/CS/GOx/Nafion 10 µM to 200 µM linear range
Sensitivity = 0.483 µA/µM Good sensitivity.
Possible to go below 10 µM.
This is suitable for the sensing of Glucose from saliva
3 SPCE/Fc-CS(0.5mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion Linear range from
10 µM to 1 mM
Low sensitivity of 2.15 nA/µM Result failed to reproduce multiple times
The ferrocene-Chitosan complex is not a suitable platform.
4 SPCE/Fc-CS(4mg/mL)/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion Linear range from10 µM to 5 mM
Low sensitivity of 1.68 nA/µM Reproducibility issues
5 SPE(550)/CS-Fc/GDp/HRP No Response
6 SPE/Fc-Cs-CNT/GDp/HRP/Chitosan-Gox-BSA-Gla/Nafion No Response
7 SPE/CNT-PANI/GDp/Gox/Nafion Decrease in Oxygen reduction current The Glucose oxidase consumes Oxygen and causes a decrease in Oxygen concentration of the solution. Here, the CNT-PANI composite is more active towards Oxygen reduction and less active towards H2O2 reduction. It is possible to detect Glucose quantitatively by measuring the decrease in O2 reduction current.
8 SPE (550)/CS-Fc-D/GDp/HRP/CS/Gox-BSA-Gla/Nafion in pH 7.4 PBS Linear range from 500 nM to 100 µM
sensitivity of 0.06 µA/µM The observed sensitivity is very less
9 SPE(500)/PB(1 mg/mL)-BSA-Gox Linear range from 100 µM to 2 Mm
Sensitivity 7.22 nA/ µM The observed sensitivity is very less
10 SPCE/CS/Fc/GDp/HRP/CS/GOx/Nafion Reduction current is observed around -400 mV Sensitivity is low.
Linear range = 10-150 µM Sensitivity ˜ 0.074 µA/mM

Table1: Summary of the results
Note: 1 µM = 18 x 10 -8 gm/ml; 1 mM = 18 x 10 -5 gm/ml

Combination 2 was finalized for preparation of glucose sensor cosisting of electrodes for detection of glucose in saliva. The glucose sensor consists of following:
1. Screen Printed Carbon Electrode (SPCE)
2. Ferrocene (Fc)
3. Chitosan (CS)
4. Glucose Dehydrogenase (GDp)
5. Horse radish Peroxidase (HRP)
6. Glucose Oxidase (GOx)

Example 2:
Inventors further studied the correlation between concentration of salivary glucose, and plasma glucose in both diabetic and healthy patients. Serum and salivary glucose estimation was done by the use of an enzymatic colorimetric test kit (Bio Vision Inc), by GOD-POD (glucose oxidase and peroxidase) method. In GOD-POD method, the proportion is 1000µL of reagent and 10µL of saliva.
Through this study, inventors tried to predict the values of average serum glucose, for a given salivary glucose by using the following regression equations in diabetic patients.
Random selection of 13 diabetic and non-diabetic patients were done. In diabetic patients both type 1 and type 2 diabetic patients were included.
Inclusion criteria
1. Any well-established cases of diabetes mellitus (either insulin dependent diabetes mellitus [IDDM] / non-insulin dependent diabetes mellitus [NIDDM] diagnosed with features of polyuria, polydipsia, polyphagia and unexplained weight loss and elevated blood glucose levels or fasting blood sugar (FBS) as per the criteria established by the Expert Committee on Diagnosis and Classification of Diabetes Mellitus in 1980.
2. Patients should not have taken any medicines or insulin prior to sampling.
3. For healthy controls, patient should be non-diabetic and blood glucose levels should be within normal limits.
4. All the patients in this study should not have any other systemic disease.
Exclusion criteria
1. Patients with any other systemic disease or diabetes related severe complications were excluded from the study.
2. Patients on medications for diabetes were excluded.
3. Patients with habits of tobacco or alcohol or smoking were excluded from the study.
4. Patients with current pregnancy were excluded from the study.
The subjects were briefed on the study being undertaken and a written consent was obtained for the procedure to be carried out to obtain the sample. A pre-structured questionnaire was prepared and relevant information of all the subjects was recorded. The statistical analysis was done by using ANOVA.

Fasting Blood Sugar (FBS)
FBS is measured after 8 h of whole night fasting. The normal range for fasting blood glucose is 70 to 100 mg/dl. Levels between 100 and 126 mg/dl are referred to as impaired fasting glucose or pre-diabetes. Diabetes is typically diagnosed when fasting blood glucose levels are 126 mg/dl or higher.

Post Prandial Blood Sugar (PPBS)
Blood sugar 2 hours after meals. Normal for person without diabetes: Less than 140 mg/dl, 140-200 mg/dl is prediabetes i.e. impaired postprandial glucose and = 200 mg/dl is diabetes.

Altered salivary composition is commonly found in patients with diabetes mellitus. It is confirmed that Salivary glucose level can be used as a monitoring tool to assess the glycemic status of diabetes mellitus patients as it offers distinct advantages over other body fluids.
,CLAIMS:We Claim:
1. A glucose sensor for determining a concentration of glucose in a saliva, wherein, the sensor comprising:
a screen printed electrode;
at least one working electrode, a counter electrode, and a reference electrode, and
a sample placement area on a surface of the screen printed electrode for containing the saliva;
wherein the working electrode, counter electrode, and reference electrode are connected to an amperometry circuit; and
wherein an output voltage of the amperometry circuit correlates with a glucose concentration in the saliva deposited in the placement area.
2. The glucose sensor of claim 1, wherein the glucose sensor is a strip.
3. The glucose sensor of claim 2, wherein the strip comprises a carbon screen printed paper electrode.
4. The glucose sensor of claim 1, wherein, the working electrode contains layer of ferrocene chitosan complex.
5. The glucose sensor of claim 4, wherein the working electrode comprises one or more materials selected from the group consisting of carbon nanotubes, graphite, gold nanoparticles, platinum nanoparticles, diamond nanoparticles, and chitosan and its derivatives made of.
6. The glucose sensor of claim 1, wherein working electrode have coating of at least one enzyme selected from the group of glucose oxidase, horse radish peroxidase, glucose isomerase, glucose peroxidase or combination thereof,
7. The glucose sensor of any of the preceding claims that is capable of detecting glucose concentration in saliva at as low as 0.18mg/decilitre.
8. The glucose sensor of any of the preceding claims is a disposable device or a reusable device.
9. A glucose analysis system comprising:
the glucose sensor of any of the preceding claims; and
a signal conditioning and/or analysis device that processes an electrical signal from the sensor.
10. A method of determining a glucose concentration in a saliva sample, the method comprising the steps of:
(a) providing the glucose sensor of claim 1;
(b) introducing a saliva sample into the sample placement area of the sensor; and
(c) determining the glucose concentration in the saliva sample from an electrical output of the sensor using device glucometer or its variants thereof.
11. The method of claim 10 further comprising the steps of:
(d) removing the liquid sample introduced in step (b); (e) introducing a new liquid sample into the sample placement area of the sensor; and
(f) determining a new glucose concentration in the new liquid sample from an electrical output of the sensor.
12. The method of claim 10 or claim 11, wherein the saliva sample is from a subject who has diabetes or is suspected of having diabetes.
13. The method of any of claims 10-11, wherein the saliva sample is from a subject who is tested as part of a health screening process.
14. A method of fabricating a glucose sensor, comprising the steps of:
(a) drop casting one or more working electrodes, a reference electrode, and a counter electrode on the surface of a screen printed electrode, wherein each of said electrodes contacts a sample placement area on the screen printed electrode;
wherein, drop casting of 2 µL of 40 mM Ferrocene, 10 µL of 2 mg/mL Chitosan, 7 µL of gold nanoparticles, 10 µL of 1 unit / µL Horse-Radish Peroxidase and 7 µL of BSA-Glu- Glucose Oxidase.