FIELD OF THE INVENTION:

The present invention relates to a biosensor for detecting multi analyte in oral fluid. More particularly, the present invention relates to a saliva-based glucose, urea and pH monitoring biosensor using a developed portable handheld optical device with a possibility of detecting other such analytes in saliva using the same device.

BACKGROUND OF THE INVENTION:

In current health scenario non-communicable diseases, such as heart disease, diabetes, or kidney disease have become more prevalent than communicable diseases and is amongst significant cause of premature death worldwide. The people who have diabetes have side effects like kidney disease and the tooth loss related problem. Besides, there are various diseases which can be monitored through saliva, albeit the concentration of these biomarkers are very low in saliva. It has been proven that diabetes, chronic kidney disease (CKD), and periodontal diseases can be monitored by analyzing salivary glucose, urea and pH respectively. Millions die each year because they do not have access to affordable treatment. Enormous economic impact of Diabetes and CKD on world GDP and its Expenses. Scale up prevention, strengthened care, and enhance surveillance of diabetes (measurement of glucose), kidney failure (Measurement of urea) is required. For monitoring of periodontal disease like gingivitis and periodontitis pH measurement is needed as this is the primary cause of tooth decay in millions of people. Regular control is essential to prevent disorders.

The standard glucose and urea monitoring machines use invasive techniques which is a painful method. Thus, in traditional tools, the patient always needs to prick his/her body to get a blood sample for glucose and urea monitoring. The handheld devices are also developed for glucose and urea monitoring, but they use blood as a sample and not cost effective. Moreover, conventional machines are not available to masses in underdeveloped and developing nations, because of their cost. Also, the strips developed for glucose, and urea monitoring is costly. The tracking of periodontitis requires several parameters to monitor in blood to reveal systematic factors that relate to the disease. Latest study showed that there is a good correlation found between blood glucose and blood urea nitrogen (BUN) to saliva glucose and saliva urea also the saliva pH to gingivitis and periodontitis. Thus, there exists a need, for a non-invasive monitoring system. Further, there exists a need for diabetes and monitoring of its complocations which include non-invasive glucose, urea, and pH monitoring system that may use saliva of the patient as a sample instead of blood and is also cost-effective.

Indian patent application no. 1587/DEL/2015 dated 1/6/2016) uses a Smartphone and android app to monitor salivary glucose concentration on a filter paper having co -immobilized glucose oxidase and pH responsive dye and the detection principle was based on RGB based color change analysis. This system has various drawbacks. The invention uses punch machine based technology for the manufacturing of the paper strips. Thus, the strips are costly and has volume variation effect and poor sensitivity.

The article with title“Inkjet-Printed Microfluidic Multianalyte Chemical Sensing Paper”, Anal. Chem. 2008, 80, 6928-6934, presents an inkjet printing method for the fabrication of entire microfluidic multi-analyte chemical sensing devices made from paper suitable for quantitative analysis, requiring only a single printing apparatus. The invention uses inkjet printing to make the hydrophobic barrier which requires hours of time to make the strips and also costly. They require one more inkjet paper to dispense the assay on the detection zone. It uses flatbed scanner using the refection based intensity measurement which is costlier and can only be used in the labs and non-portable and the response time of the invention is about ten minutes.

Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva https://www.ncbi.nlm.nih.gov/pubmed/20425l07. This document reports the use of paper- based microfluidic devices fabricated from a novel polymer blend for the monitoring of glucose, and salivary nitrite. Reagents were spotted on the detection pad of the paper device and allowed to dry prior to spotting of samples. Following the spotting of the reagents and sample solution onto the paper device and subsequent drying, color images of the paper chips were recorded using a flatbed scanner, and images were converted to a format in Adobe Photoshop where the intensity of the color change was quantified using the same software. The cited paper uses the technology which is costlier and takes more time in making strips. Also the cited paper uses flatbed scanner using the refection based intensity measurement which is costlier and can only be used in the labs and non-portable.

Paper-based analytical devices for clinical diagnosis: recent advances in the fabrication techniques and sensing mechanisms https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529l45/. The cited literature reports calorimetric detection based on paper substrates. It recites how color intensity and uniformity depend on the type of paper substrates and the volume of reagents used. The cited paper uses different technologies which are costlier and take more time to make strips and the color gradient of the strip is poor.

Thus, a cost effective biosensor and a fast responsive detective instrument is required which eliminates the drawbacks of the above mentioned prior arts.

OBJECTS OF THE INVENTION:

The main object, of the present invention is to provide a saliva-based analyte detecting biosensor.

Another object, of the present invention is to provide a portable handheld optical instrument for detecting color and intensity change of the paper of the biosensor and method for the same. SUMMARY OF THE INVENTION:

An aspect of present disclosure provides a biosensor (100) for detecting multi analyte in oral fluid, the biosensor (100) comprises: a sheet (101) of supporting material having a top; a notch

(102) provided at the top for enabling the detection of insertion of the biosensor (100); a hole A

(103) provided proximal to the top of the sheet for identifying the type of analyte which can be detected by the biosensor (100); a strip (105) of filter paper provided over the sheet with a lamination layer between them; a paper affixed over the strip (105) with a filter paper (106), characterized in that the strip (105) of filter paper comprises one wax printed hole for detecting the analyte (120) and one wax printed hole for collecting the oral fluid (110) which upon heating form a hydrophobic microchannel (130) between the holes for the flow of the oral fluid, the hole for detecting the analyte (120) is immobilized with a solution followed by a coating of poly vinyl alcohol, and the paper fixed over the strip (105) partially blocking the hydrophobic microchannel to control the rate of flow of the oral fluid. In an embodiment, the present disclosure provides that the analyte is selected from the group consisting of glucose, urea and pH.

In another embodiment, the present disclosure provides that the hole A (103) has dimensions predetermined for the type of analyte which can be detected by the biosensor (100). In yet another embodiment, the present disclosure provides that the hole A (103) has diameter is in the range from 2.5mm to 4.5mm.

In still another embodiment, the present disclosure provides that the solution comprises mixture of a base, a dye and phosphate buffer having a pH value 7.

In further embodiment, the present disclosure provides that the base is Glucose oxidase where the analyte is glucose.

In preferred embodiment, the present disclosure provides that the base is Urease where the analyte is urea.

In another embodiment, the present disclosure provides that the dye is selected from the group consisting of nitrazine yellow, methyl red, methyl orange, bromocresol purple, bromothymol blue and cresol red.

In yet another embodiment, the present disclosure provides that the solution comprises pH indicator where the biosensor is used for determining pH.

In still another embodiment, the present disclosure provides that an instrument for detecting multi analyte in oral fluid, the instrument comprising: a means for placing biosensor (100) with oral fluid; a plurality of light sources (210) for providing light on biosensor; at least a detector configured for receiving light passing through a notch (102) of the biosensor (100) for determining the insertion of the biosensor (100) and for receiving light passing through a hole A (103) of the biosensor (100) for determining the type of analyte to be detected by the biosensor (100); a plurality of colour sensors (220) for detecting the changing in colour of the hole for detecting the analyte (120); a microcontroller (250) configured for receiving and processing data from the detector and colour sensors (220); and a display (200) configured for receiving and displaying the processed data received from microcontroller (250). In further embodiment, the present disclosure provides that the instrument comprises a plurality of temperature sensors (290) for detecting the temperature of ambient sample and uses 3D calibration curve for temperature compensated readings, a power supply, a data transfer means and data storage means. In preferred embodiment, the present disclosure provides that the data transfer means is Bluetooth (270) based on USB 2.0 protocol (280).

In another embodiment, the present disclosure provides that the data storage means is multimedia memory card (260).

In still another embodiment, the present disclosure provides a method for detecting multi analyte in oral fluid by the instrument as claimed in claim 10, the method comprising: applying oral fluid on a biosensor (100) and placing the biosensor (100) in the instrument for detecting multi analyte; providing light from the plurality of light sources (210) on the biosensor (100); receiving the light passes through a notch (102) and a hole A (103) of the biosensor (100) by the detector for detecting the presence of the biosensor (100) in the instrument and for determining the type of analyte to be detected by the biosensor (100); detecting the change in color of a hole for detecting the analyte (120) after a predetermined time by the color sensors (210); receiving the processing data from the color sensors (210) by the microcontroller (250); and displaying the processed data received from the microcontroller (250) on the display (200).

In yet another embodiment, the present disclosure provides that the method the predetermined time is in the range from 22.5 seconds to 240 seconds for allowing the oral fluid to arrive the hole for detecting the analyte and change the color of the biosensor (100).

These and other features, aspects, and advantages of the present subject matter will become better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF DRAWINGS:

Figure 1A and 1B represents the design of the strip. Figures 2A, 2B, 2C, 2D, 2E, and 2F represent the front and back view of a urea, glucose and pH monitoring strips respectively, according to various embodiments.

Figure 3 Block diagram of the instrument and its components.

Figures 4A and 4B represent the internal view of the instrument. Figures 5A and 5B represents method steps for measuring analyte quantity using the instrument shown in figure 4, by way of the flowchart.

Figure 6 represents procedure used for obtaining sample readings through the instrument.

Figures 7A, 7B and 7C represent the RGB profile of the calibration curve obtained using different concentrations of synthetic glucose, synthetic urea and synthetic pH (1% in 7pH I mM phosphate buffer) spiked in saliva sample giving a response time of 60 seconds.

Figures 8 A, 8B, 8C and 8D represent the R and G intensities integration of the calibration curve for and ambient temperature and different concentrations of synthetic glucose and synthetic urea spiked in saliva sample giving a readout time of 7 and 5 seconds respectively for temperature range (l5°C- 50°C), and Fig 8E represents the B intensity change for thirty second for saliva pH variations.

Figure 9 shows the volume variation effect removing of the developed biosensor for the three analytes at specific wavelength of the source used. (620nm for glucose, 530nm for urea and 480nm for pH strips).

Figure 10A and 10B shows the front view of color change of various glucose concentration strips. 10C, 10D and 10E shows the front view of color change of various urea concentration strips. 10F and 10G shows the front view of color change of various pH change strips.

DETAILED DESCRIPTION OF DRAWINGS:

The present invention relates to non-invasive glucose, urea, and pH monitoring system. The present invention relates to saliva-based glucose, urea, and pH monitoring system comprising a glucose, urea, and pH monitoring biosensor strip and a single portable handheld optical device to detect all the three analytes. A saliva sample of a diabetic patient, chronic kidney disease patient, gingivitis, and periodontitis may be applied onto the strip, wherein the strip may change its color on application of saliva. Further, the said handheld device may be used to monitor the type of strip applied to detect analyte and the color change to recognize the glucose, urea and pH level of the patient. Also, the system provides the ambient temperature compensation for the enzymetic chemical kinetics of the strips.

For preparing the monitoring biosensor strip the design of the biosensor is illustrated in Figure 1A. The biosensor (100) comprises a sheet (101) of supporting material, wherein the supporting material may be Plastic sheet and cardboard. In particular, Polyethylene Terephthalate Sheet and white cardboard may be used as supporting material. In an embodiment, said sheet (101) may include a length of 5.0 cm to 6.0 cm, the width of 8mm to 12mm and thickness of 400 microns to 600 microns. Specifically, in an exemplary embodiment, the plastic sheet (101) may be 5.9 cm long, 12 mm wide and 400 microns thick. The sheet (101) contains a hole (104) of 2.5 mm to 4.5 mm width, placed at a distance of around 2 cm from the top of the sheet (101). In particular, the width of the hole (104) may be 3.5 mm. The sheet (101) also contains one more hole A (103) of variable length as per the strip analyte of 0 mm to 3.5 mm placed at around 10 mm from the top of the sheet (101). In particular, the length is 0 mm, 2 mm and 3 mm respectively. One notch (102) at the top of the sheet (101) having the size of 1.5 mm triangular shape to provide the detection of the biosensor (100) by the instrument. In order, the sample should not be absorbed onto the cardboard; the entire sheet may be laminated using a thin lamination film. Further, a strip (105) of grade one filter paper having a length of 3.8 cm and width of 1.1 cm is printed using solid ink printer and designed as shown in figure 1A that contains two holes: one hole (120) is for detecting the analyte is of 4.5mm diameter as detection zone and the other hole for collecting the sample (110) is of 6 mm diameter after heating of the wax. The channel (130) width for traveling the liquid can be 0.1 mm to 2 mm. The shape of the channel may be trapezoidal as shown in figure 1A or rectangular as shown in figure 1B. In particular, the 1.5 mm x 2.5 mm of width for trapezoidal microchannel or 1.5 mm width and 28.5 mm length of the rectangular microchannel has been used. The strip (105) is heated at l20°C for the wax to absorb over the paper for 120 seconds and to make the strip hydrophobic for the liquid sample to flow through it. The top layer was cut as per the design is shown in figure 1A and laminated with the filter paper sandwiched in between to fix the paper on the strip. The top layer also contains one 3mm length of the block in the path of the sample flow to control the sample flow rate to improve the color gradient of the detection zone as shown in figure 1B. This strip (105) design can be altered for other usage, such as detection of multiple analytes simultaneously. In such case, multifurcated channel can be created on strip and in each detection well one particular enzyme dye combination can be used. The meter shall have to modified in that case to have multiple source-detector pair aligned with detection zone of the strip.

For preparing the glucose strip in an exemplary embodiment for design as shown in figure 10A the detection zone may further be immobilized with a solution of Glucose oxidase and dye nitrazine yellow. In the exemplary embodiment, for immobilization of 100 such strips, 110 mΐ solution may be prepared by adding 5U of Glucose oxidase per strip and 50 mΐ of dye (0.2% solution) along with 50 mΐ of 1 mM Phosphate buffer having a pH value 7. Further, a coating of polyvinyl alcohol (PVA), with a concentration of 0.5% may be added to each strip by adding ten mΐ of PVA to stabilize the immobilized components. The Dithiothreitol (DTT) is also added to 2 mg /ml in the prepared assay to increase the shelf life of the enzymetic strips. The said immobilization procedure may comprise steps of adsorption of 1.1 mΐ solution on each strip with the help of a pipette and allowed to dry for five minutes at 25°C.

For preparing the glucose strip for design of figure 10B the detection zone may further be immobilized with a solution of Glucose oxidase and dye nitrazine yellow along with reducing agent i.e. 2-mercapto ethanol(2-ME). In an example, for immobilization of 100 such strips, 115 mΐ solution may be prepared by adding 5U of Glucose oxidase per strip and 114 mΐ of dye (0.1% solution prepared in 0.5% PVA in 7pH ImM phosphate buffer). The 1 mΐ of 2-ME is added as 13.27 mM concentration to increase the shelf life of the enzymatic strips. The said immobilization procedure may comprise steps of adsorption of 1.15 mΐ solution on each strip with the help of a pipette and allowed to dry for five minutes at 25°C. Such formulation cab be optimized even further to test any other analyte where colorometric detection is involved, such as sensing of antibiotic in water or milk, detection of pharmaceutical product or blood based analyte. In such case enzyme-dye combination have to be changed and any color change on strip may be detected using our meter. For preparing the urea strip of figure 10C, the detection zone may further be immobilized with a solution of urease and dye cresol red (CR). Whereas the dye phenol red (PR) is used for urea strips of figure 10D. In an exemplary embodiment, for immobilization of 100 such strips of figure 10C, 100 mΐ solution may be prepared by adding lOU/strip of Urease and 50 mΐ of dye (0.2% solution) along with 40 mΐ of 1 mM Phosphate buffer having a pH value 7. Further, a coating of PVA, with a concentration of 0.5% can be added to each strip by adding 10 mΐ of PVA to stabilize the immobilized components. In an exemplary embodiment, for immobilization of 100 such strips of figure 10D, 115 mΐ solution may be prepared by adding 3U/strip of Urease and 115 mΐ of PR dye (0.1% solution) in 1% PVA in 1 mM Phosphate buffer having a pH value 7. The said immobilization procedure may comprise steps of adsorption of 1.0 mΐ solution for figure 10C and 1.15 mΐ for figure 10D on each strip with the help of a pipette and allowed to dry for five minutes at 25°C.

For preparing the urea strip for design of figure 10E the detection zone may further be immobilized with a solution of urease and dye PR along with reducing agent i.e. 2-mercapto ethanol (2-ME). In an exemplary embodiment, for immobilization of 100 such strips, 125 mΐ solution may be prepared by adding 0.08U of urease per strip and 122 mΐ of PR dye (0.1% solution prepared in 1.25% PVA in 7 pH 20mM phosphate buffer). The Imΐ of 2-ME is added as 12.67 mM concentration to increase the shelf life of the enzymetic strips. The said immobilization procedure may comprise steps of adsorption of 1.25 mΐ solution on each strip with the help of a pipette and allowed to dry for five minutes at 25°C.

For preparing the pH strip of figure 10F, the detection zone may further be immobilized with a solution of the universal indicator from Merck. The 0.5% of PVA (prepared in distilled water) solution is mixed with the Universal indicator. The said immobilization procedure may comprise steps of adsorption of 1 mΐ solution on each strip two times with five minutes of drying step of the indicator with the help of a pipette and allowed to dry.

For preparing the pH strip of figure 10G, the detection zone may further be immobilized with a solution of the universal indicator from Merck. The 0.75% of PVA (prepared in distilled water) solution is mixed with the Universal indicator. The said immobilization procedure may comprise steps of adsorption of 1 mΐ solution on each strip three times with five minutes of drying step of the indicator with the help of a pipette and allowed to dry.

The PVA is introduced into the assay to improve the color gradient of the developed strips by using the entrapment method for the protein into the filter paper as the adsorption technique is not sufficient for the degradation of the color of the detection zone as saliva sample is more viscous than water and erases the color of the detection zone as it flows into. To improve color gradient, the channel is also blocked with 3mm width of lamination sheet across the flow for restricting the free flow of the sample as compared to figure 1A to figure 1B. Filter paper used in the strip also acts as the filter for food particles and other bigger particles present in the sample. The volume variation effect is also removed from the strips as the bud is used to collect the sample from the user and then squeezed to make a drop of the sample on the strip. The volume variation effect is show in the figure 9.

For preparing the instrument the block diagram of the electronic part is shown in figure 3. It consisted of five basic components: a light source (210), light detectors, a microcontroller (250) for signal processing, power source (240), temperature sensor (290), LCD (200), Bluetooth (270) and MMC (260) for data storage. Once then biosensor (100) was placed in the meter along with saliva sample, due to the presence of an analyte, the enzyme caused a change in pH of reaction medium (saliva) thereby changing the color of the paper in the detection zone. The pH of the saliva changes the pH thereby changing the color of the paper. The instrument had an RGB LED (CLX6B-FKC: 460-480nm, 520-540nm, 6l9-624nm) as a light source and an RGB color sensor (220) S 11092 from Hamamatsu which is highly sensitive among the family of the 16-bit digital color sensor. The RGB sensor (220) would capture the color change on the strip, and the data was sent to a 16-bit PIC Microcontroller (250) using Inter- Integrated Circuit (I2C) protocol. It was found after the test the NY is most sensitive to 619 nm source, CR is most sensitive to 520 nm, and UI is most sensitive to 460 nm. For detecting the biosensor (100) type inserted into the instrument the LDR and white LED (SM0805UWC) pair is used to detect the type of the analyte the instrument is going to detect. The biosensor (100) contains the hole (103) which is specific to the analyte type (1 mm for pH, 2mm for glucose and 3mm for urea) and aligned with the LDR source pair. Due to the variation in the length of the hole, the variable light reaches the LDR, and varying intensity is detected through the LDR thereby changing the analog voltage detected by the lObit ADC of the microcontroller (250). The LMT86 temperature sensor (290) is used to detect the temperature of the ambient and is saved with the response curve of the analyte using the lObit ADC of the microcontroller (250). The microcontroller (250) process the unknown analyte quantity and displayed onto inbuilt alphanumeric Liquid Crystal Display (LCD) (200). The data was also saved in a 4Gb Multimedia memory card (MMC) (260) having serial peripheral interface (SPI) protocol, along with date and time using the Real Time Clock (RTC) inbuilt into the microcontroller (250). Such details were personalized for user point of view. A BL5C, rechargeable Lithium-ion battery (240) was used to provide the power source for all the electronic load of the instrument which is cost effective and have high energy density as compared to other types of chargeable power source available. In addition, a low power buzzer (230) was incorporated to indicate if the user did not apply the saliva sample within the specified time interval and also to indicate low battery condition. There was provision to transfer data to computers/laptops/android mobiles using USB2.0 protocol (280) via Bluetooth (270). The meter design can be changed for multiplexed reaction and altered channel design as stated in 0024 and 0025a. Also, instead of RGB analysis, plain light intensity measurement can also be employed using white LED and visible range photodiode pair for other sensing applications involving colorometric change.

For preparing the chassis of the optically isolated handheld equipment was printed using a 3D printer to house the electronic circuitry. The printing material of the box was Acrylonitrile- Butadiene-Styrene, and the color was chosen as black to reduce interference from the ambient light. A double-sided PCB was used for assembling the electronic components. The electronic circuit consisted of five basic components: light source, light guides, wavelength selector device, light detectors, signal processing electronics, Bluetooth and other components for data transmission. The internals of the instrument are shown in figure 4A and 4B respectively. Switches (320), charging connector (402) and charging indicator (401) is provided in the internal electronic circuit as shown in figure 4A. Buttons for the switches (320) and cut (310) for the LCD are provided on the black body of the instrument as shown in figure 4B.

For making the instrument software the flowchart of the C program of the instrument is shown in figure 5A. Figure 5A program is used in for figure 10A, 10C, 10D and 10F biosensors. The steps indicate that the instrument first detects the strips and awake from the sleep mode by using the switch and notch pair present in the system. Then the instrument detects the type of analyte to be identified with the help of the cut present into the biosensor. After this, the instrument waits for the saliva to arrive into the detection zone and then start the calculation of the RGB profile and then calculate the analyte quantity as per present in the sample with the help of the equation stored into the instrument for that analyte. Then the data is derived from 60 seconds and displaying it on the LCD. Then the data can be saved into the MMC along with the date, time and name of the user. The data can also be sent to the computer/ android mobile for further using the data. For taking the sample to measure the analyte quantity the method of saliva collection was such that an end user could collect it by self as shown in figure 6.

EXAMPLE:

The following examples are given by way of illustration of the present disclosure and should not be construed to limit the scope of present disclosure. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the subject matter.

The sample volume that can be used is from 15 to 45 pL range and in particular 30m L is used for all the tests done. Also, the sequence of sample addition and monitoring was designed in the device so that a patient can quickly follow the instructions by placing the biosensor strip into the instrument and complete the procedure of reading the salivary glucose level on display. As per standard operating procedure (SOP), fresh saliva was collected from beneath the tongue from volunteers using a medical grade cotton bud and was applied to the test strip’s sample application zone by forming a droplet. After utterly absorbing the sufficient amount of saliva on the strip, the sample would reach detection zone, and enzymatic reaction between the enzyme and salivary analytes would initiate a pH change, resulting in a change in strip color that was recorded by using RGB detector on the handheld instrument.

Figures 7A, 7B and 7C represent the RGB profile of the calibration curve obtained using different concentrations of synthetic glucose, synthetic urea and synthetic pH (1% in 7pH ImM phosphate buffer) spiked in saliva sample giving a response time of 60 seconds. Figures 8 A, 8B, 8C and 8D represent the R and G intensities integration of the calibration curve for and ambient temperature and different concentrations of synthetic glucose and synthetic urea spiked in saliva sample giving a readout time of 7 and 5 seconds respectively for temperature range (l5°C- 50°C), and Fig 8E represents the B intensity change for thirty second for saliva pH variations. The color change of the three analytes detected by the instrument is shown in figure 10. Figure 10A and 10B shows the front view of color change of various glucose concentration strips. 10C, 10D and 10E shows the front view of color change of various urea concentration strips. 10F and 10G shows the front view of color change of various pH change strips.

Although the present invention has been described in considerable detail concerning figures and certain preferred embodiments thereof, other versions are possible. Therefore, the spirit and scope of the present invention should not be limited to the description of the favorite versions contained herein.