WO2021019552A1

WO2021019552A1 - System for estimation of plasma glucose by integrated paper-based device and method thereof

Info

Publication number: WO2021019552A1
Application number: PCT/IN2020/050615
Authority: WO
Inventors: Sujay Kumar BISWAS; Sampad LAHA; Subhamoy CHATTERJEE; Vinay Manaswi PEDIREDDI; Shikhar PATEL; Satadal Saha; Suman CHAKRABORTY
Original assignee: Indian Institute Of Technology, Kharagpur
Priority date: 2019-07-27
Filing date: 2020-07-17
Publication date: 2021-02-04

Abstract

A low-cost paper-based microfluidic device is developed for detection of blood glucose level. The major advantage of this innovation is its ability to perform plasma separation from a single drop of blood and subsequent colorimetric detection of plasma glucose in a single platform. The present-day glucometers are based on detection of whole blood glucose using electrochemical sensing or reflectance photometric detection techniques. However, in the gold standard technique measure plasma glucose which is accurate and widely acceptable. Compare to plasma glucose level, whole blood glucose shows 10-12% variations. Moreover, the invented device is almost instrument free and requires only a paper strip of invention and smartphone with the developed App to operate and diagnose plasma glucose level.

Description

TITLE: SYSTEM FOR ESTIMATION OF PLASMA GLUCOSE BY INTEGRATED PAPER-BASED DEVICE AND METHOD THEREOF.

FIELD OF INVENTION

The present invention relates to system for estimation of plasma glucose level and more specifically, to a point of care simple and user friendly device which can effectively enable estimation of plasma glucose. Importantly, the plasma glucose estimation system would also take care of factually incorrect results usually encountered in case of glucose estimation from whole blood samples. The system is workable with even only a drop of blood as a test sample for estimation and thus is highly recommended for wide scale and user friendly estimation of blood glucose levels as a reliable and cost effective manner of estimation of blood glucose level.

BACKGROUND OF THE INVENTION

As per the WHO report, high blood sugar and diabetes are two leading causes of global mortality. In 2016, an estimated 1.6 million deaths were caused directly by diabetes and another 2.2 million deaths were reported due to high blood sugar in 2012. Therefore, proper diagnosis and monitoring of blood glucose level is an utmost necessity to start on time treatment and take control over this serious health issue. It is clearly undeniable that there are several glucose monitoring kits are commercially available in the market. However, most of these devices estimate whole blood glucose instead of plasma glucose which is factually incorrect. Therefore, a sense of urgency is prevailing to develop an affordable, instrument- free and user-friendly POC device which can estimate plasma glucose only.

Since last decades, a significant progress has been made to develop various techniques and methods for point of care diagnosis of blood glucose. These techniques can be classified into two categories namely colorimetric detection and electrochemical sensing . Noiphung et al. (3 Noiphung, T Songjaroen, W Dungchai, Charles S Henry, O Chailapakul, W Laiwattanapaisal. Electrochemical detection of glucose from whole blood using paper-based microfiuidic devices, Analytica Chimica Acta. 2013 :788 :39-45) have developed an electrochemical paper-based analytical device with intermediate plasma separation for glucose detection in a dumbbell shaped paper-strip. The authors have used VF2 filter paper for plasma separation at two separation zones and Whatman filter paper for detection of glucose in the middle.

Leah et al. (Leah A. Cohn, Dudley L. McCaw, Deborah J. Tate. Assessment of five portable blood glucose meters, a point-of-care analyzer, and color test strips for measuring blood glucose. JAVMA. 2000; 216: 198-202) have assessed five different POC glucose detection devices and color test strips using blood sample from dog.

A review article on recent developments in blood glucose sensors has been reported by Wang et al. (Hui-Chen Wang, An-Rong Lee. Recent developments in blood glucose sensors. Journal of food and drug analysis. 2015;23 : 191-200). In this review the authors have discussed and analyzed about both of enzyme-based and enzyme-free glucose sensors.

In another review article, Klonoff et al (David C. Klonoff, David Ahn, Andjela Drincic. Continuous glucose monitoring : A review of technology and clinical use. Diabetes research and clinical practice . 2017; 133 : 178-192) have provided an overview of the technical and clinical features of continuous glucose monitoring devices. They have also discussed about the issues related to accuracy of detection of glycemic variability, strategies for optimal use etc.

Kim et al. (Jayoung Kim, Alan S. Campbell, Joseph Wang. Wearable non-invasive epidermal glucose sensors: A review. Taianta. 2018; 177 : 163- 170) have reviewed about the recent advancement and challenges of development of non-invasive epidermal electrochemical glucose sensing systems.

J. Liu et al. [J Liu, Z Geng, Z Fan, Jian Liu, H Chen. Point-of-care testing based on smartphone: The current state-of-the-art (2007-2018), Biosensors and Bioelectronics. 2019; 132: 17-37] have done a comprehensive review on recent development of the Smartphone-based point-of-care devices and also examined the merits and demerits of these devices. The authors also have concluded the req uirements and future prospect of the point-of-ca re devices.

A Smartphone-based non-invasive saliva glucose detection device has been developed and reported by Soni et al. (A Soni, S K Jha. Smartphone based non- invasive salivary glucose biosensor. Analytics Chimica Acta. 2017; 996: 54-63). This in-vitro sa liva g lucose detection device uses glucose oxidase enzyme with a pH responsive dye on filter paper. Once the color changes d ue to the presence of g lucose in saliva, Smartphone is used to ta ke image and analyze through RGB profiling .

In a review article reported by Chinnyadayyala et al . (S R Chlnnadayyala, J Park, H T N Le, M Santhosh, A N Kadam, S Cho. Recent advances in microfiuidic paper- based electrochemiiuminescence analytical devices for point-of-care testing applications. Biosensors and Bioelectronics. 2019; 126: 68-81) has highlighted the current developments in electrogenerated chemiluminescence (ECL) paper-based microfiuidic devices. They have further reviewed va rious ECL signal amplification labels, inexpensive a nd portable devices which have replaced traditional instruments a nd different light driven detection technolog ies used in ECL devices.

Most of the state-of-art POC devices use whole blood sample to estimate blood glucose either by electrochemical or reflectance photometric detection techniques. Recently, some of the POC devices calibrated to provide the result as a plasma equivalent. However, gold standard pathological technique is based on plasma glucose estimation 'which will provide ~ 12% higher value than the result obtain from whole blood glucose measurement. Moreover, it makes easier for a physician to take quick decision if the result of POC device is comparable to the pathology obtained result.

US patent no, 3092465 describes a paper-based glucose detection method using glucose oxidase and o-tolidine for colorimetric detection. In this method, a semi- permeable membrane was set surrounding the test pad to avoid contact of the hemoglobin with o-tolidine. A glucose detection method from whole blood and device has been provided in the US patent No. 5426032. Optical detection of whole blood glucose on a porous matrix has been described using reflectance spectroscopy.

A non-invasive salivary glucose detection process has been reported in the patent No, US0023324A1. The inventor has used electrochemical sensing for quantification of the saliva glucose. Subsequently, this saliva glucose has been mapped to the blood glucose level.

US5101814 disclosed a blood glucose monitoring device using electrochemical detection technique. In this advancement, glucose sensitive living cells are implanted in the body tissue to monitor blood glucose concentration.

US0118175A1, a paper-based microfluidic device has been disclosed for detection of analytes and biomarkers. The paper substrate is chemically patterned by the method of chemical vapor deposition for making the microfluidic device.

WO2016038529 A1 disclosed a device and method for collecting and retaining bio analytes with electrodes connected to conductive tracks arranged on a substrate. This innovation can also measure blood glucose level from small amount of sample.

A plasma glucose estimation system is disclosed in the US patent no. US 20140244181 Al. This device comprises a sensor for generation of signal based on the presence of glucose concentration in the medium.

OBJECT OF THE INVENTION

The primary objective of the present invention is to develop paper based device for simultaneous separation of plasma from whole blood drop and measure the amount of glucose level in plasma. Another objective of the present invention is to develop device where req uirement of external instruments will be very less other than smart phone.

Another object of the present invention is the integ ration of both blood plasma sepa ration module and plasma glucose detection cha mber in the same platform of the pa per strip.

Another object of the present invention is to develop paper based device for colorimetric detection of concentration of glucose present in the plasma .

Yet further objective of the present invention is to develop paper based device which would provide concentration of glucose reading compa rable with other sta ndard methods for the same.

Yet further objective of the present invention is to develop paper based device which would be chea p a nd cost effective.

Yet further objective of the present invention is to develop paper based device which would provide an adaptive time window based on reaction completion provid ing accurate result as a result of sensing the color saturation by the system intelligence and would be a pplicable uniformly without depending on geog raphical orig in or nature of ethnicity of the population .

SUMMARY OF THE INVENTION

Thus the basic aspect of the present invention is directed to provide a system for estimation of plasma glucose comprising :

integrated blood plasma sepa ration module and plasma glucose detector.

Another preferred aspect of the present invention is directed to provide a system for estimation of plasma glucose wherein said glucose detector include image processing mea ns a nd display unit. Another aspect of the present invention is directed to provide the system comprising paper based integrated platform as POC device having said blood plasma separation module and plasma glucose detector.

Yet another aspect of the present invention is directed to provide the system wherein said paper based integrated platform having said blood plasma separation module and plasma glucose detector comprises microfluidic paper strip of combination of first and second filter paper said first filter paper enabling introducing blood drop and and subsequent plasma seperation from blood and said second filter paper providing for reaction chamber for plasma glucose detection.

In a further aspect of the present invention is directed to provide the system comprising a microfludic strip of said first filter paper comprising Whatman® LF1 filter paper adapted for said introducing the blood drop and subsequent plasma separation from blood ceils and defining a source pad and channel body and of said second filter paper providing said reaction chamber comprising of whatman® cellulose filter paper grade 1 and placed at the end of the body of the channel.

Still further aspect of the present invention is directed to provide the system compriisng microfiuidic strip on a hydrophobic paper layer fixed on a cartridge type substrate.

Another aspect of the present invention is directed to provide the system wherein said cartridge type substrate comprises a

a 3D printed cartridge having an enclosure.

Yet another aspect of the present invention is directed to provide the system comprising paper strip based substrate platform wherein said first and second filter papers are provided with some overlaps to each other on a hydrophobic paper layer such that when a drop of blood is introduced onto the source pad, the said first filter paper LF1 does not allow RBC to transport through the LF1 filter paper but only plasma can easily tra nsport ena bling separation of plasma only to reach to the reaction pad,

sa id reaction pad provided with pre-embedded glucose detecting chemica ls, sa id paper strip based substrate platform disposed such a way that one need to put only a drop of blood onto the source pad then subseq uent separated plasma based reaction detection functionalities are a utomatically continued .

Still another aspect of the present invention is d irected to provide the system comprising pre-embedded glucose detecting chemicals including potassium iodide (KI) with gold standard g lucose reagent for producing brown color based on the presence of glucose into the plasma .

Further aspect of the present invention is directed to provide the system comprising image processing means ada pted for color intensity based estimation of glucose.

Yet fu rther aspect of the present invention is directed to provide the system comprising image processing mea ns including ca mera means for imaging the colour change of plasma on reaction with glucose detecting chemicals a nd colorimetric based detector for estimation of glucose.

Another aspect of the present invention is d irected to provide the system comprising a smartphone including sa id camera means for imaging colour change of plasma on reaction with glucose detecting chemicals in said plasma glucose detector of said paper based integrated platform having said blood plasma sepa ration module and plasma glucose detector and analytic tool for mapping the parameters with plasma glucose level for producing quantitative results.

Still another aspect of the present invention is directed to provide the system comprising an enclosure for supporting a ca rtridge based said integrated blood plasma separation module and plasma glucose detector and a smartphone unit with sa id camera means and a na lytic tool for estimation of g lucose. Still further aspect of the present invention is directed to provide a process for manufacture of the system comprising : providing a paper based integrated blood plasma separation module and plasma glucose detector in the form of microfluidic paper strip made of two different filter papers.

Yet another aspect of the present invention is directed to provide a method of manufacture comprising providing said microfluidic paper strip made of two different filter papers, wherein said two different filter papers used comprises a first filter paper obtained of Whatman® LF1 filter paper for introducing the blood drop and subsequent plasma separation from blood cells which form the source pad and channel body and a second fileter paper providing a reaction chamber involving whatman® cellulose filter paper grade 1 and placed at the end of the body of the channel, said microfluidic strip being placed on a hydrophobic paper layer .

In a further aspect of the present invention is directed to provide a method comprising providing the said paper strip fixed on a cartridge type substrate preferably 3D printed cartridge and insert into an enclosure.

DETAILS OF THE INVENTION

There are many techniques available for blood plasma separation such as centrifugation, sedimentation or magnetic separation techniques. These processes are costly and require expensive instruments and skilled manpower. The existing point-of-care devices for blood plasma separation reported comprise of various other methods such as agglutination of RBC, filtration using RBC specific membranes, capillary driven diffusion, RBC crenation by salt and electrochemical methods etc. A paper-based device have been developed by the present invention for estimating plasma glucose at the bedside from a single drop of blood using colorimetric detection technique.

The uniqueness of this device is the integration of both blood plasma separation module and plasma glucose detection chamber in the same platform of the paper strip.

It is to be understood that both the foregoing general description and the following detailed description are exemplary, and are intended to provide further explanation of the invention as claimed. The advancement according to the present invention is discussed in further detail in relation to the following non-limiting exemplary illustrations wherein:

Figure 1 : a) Blood plasma separation and color change due to the presence of glucose in the blood b) Smart phone based image acquisition using 3D printed enclosure.

Figure 2: Smartphone app for image processing and result display. A single touch on the app starts the mobile camera and take images automatically after a certain time duration. Multiple images are taken in a regular interval. Then this images are processed and mapped the greyscale intensity to the glucose level (mg/dl)

Figure 3: Calibration curve for colorimetric glucose detection on paper using Smartphone. Standard glucose is used for producing the calibration curve.

Figure 4: Clinical validation of POC plasma glucose estimation device. The result of POC device are plotted against gold standard clinical results for all patient samples (n = 30).

Figure 5: Top part of the enclosure to hold the Smartphone

Figure 6: Bottom part of the enclosure holding LEDs and insert cartridge Figure 7 : Upper part of the cartridge to hold the paper strip

Figure 8 : Bottom part of the cartridge to fix the paper strip.

A single drop of finger prick blood is enough to extract sufficient amount of plasma and subsequent detection of glucose level. This method is almost instrument-free plasma glucose detection technique requiring only a smart-phone consisting of an indigenously developed app for image processing and result display.

A 3D printed enclosure has been developed to hold the Smartphone and to insert the cartridge with paper strip.

To validate the efficiency of present device, more than 60 human blood samples were tested and results were compared with the results obtained from gold standard technique or conventional techniques. The accuracy of the device is well within the standard set by ISO 15197 as ± 15 mg/dl for concentration below 100 mg/dl and ± 15% of the laboratory standard for concentration above 100 mg/dl.

Example-1

The microf!uidic paper strip is made of two different filter papers. Whatman® LF1 filter paper is used for introducing the blood drop and subsequent plasma separation from blood cells. The source pad and channel body is made of LF1 filter paper. The reaction chamber is fabricated using whatman® cellulose filter paper- grade 1 and placed at the end of the body of the channel. This microfluidic strip is placed on a hydrophobic paper layer (figure 1). The paper strip is fixed on a 3D printed cartridge and insert into the enclosure.

The present device first separates plasma from whole blood and subsequently estimates plasma glucose using colorimetric detection technique. The paper strip consists of two types of filter papers -i) Whatman® LF1 filter paper and ii) Whatman® cellulose filter paper grade 1. The source pad and the channel body are made of LF1 filter paper. The reaction pad is made of Whatman® filter paper grade 1. These two papers were fixed with some overlaps to each other on a hydrophobic paper layer (Figure 1). When a drop of blood is introduced onto the source pad, RBC could not transport through the LF1 filter paper but plasma can easily transport. Once the separated plasma reach to the reaction pad, plasma glucose reacts with the pre-embedded chemicals and produce specific brown color. The design of the paper strip is done in such a way that one need to put only a drop of blood onto the source pad then other functionalities are taken care of automatically.

Example-2

The developed paper-based device for estimating plasma glucose at the bedside from a single drop of blood using colorimetric detection technique. The uniqueness of this device is the integration of both blood plasma separation module and plasma glucose detection chamber in the same platform of the paper strip. A single drop of finger prick blood is enough to extract sufficient amount of plasma and subsequent detection of glucose level. This method is almost instrument-free plasma glucose detection technique requires only a smartphone consists of an indigenously developed app for image processing and result display. A 3D printed enclosure has been developed to hold the smartphone and to insert the cartridge with paper strip. The design of the said cartridge is depicted in Figure -5 to Figure 8. Saving the design parameters into 3D printing machine and supplying material of construction - provides the cases with proper dimension. The material of construction used for 3D printing of the cartridge is PVC.

Example-3

This innovation is a paper-based microfluidic device for colorimetric detection of plasma glucose using Smartphone app. This device is developed to estimate and monitor plasma glucose for the diabetic patient. Glucose oxidase catalyses the oxidation of glucose to yield glucoronic acid and hydrogen peroxide. The concentration of hydrogen peroxide liberated is measured using a peroxidase step coupled to a colored oxygen acceptor or an electrode. These reactions form the basis of both reagent strips and bench top glucose electrode methods (Harish J, Srinivas H. A, Soumya A. "Comparative Study of Glucometer and Laboratory Glucose Oxidase Method for the Estimation of Blood Glucose Levels in Neonates". Journal of Evolution of Medical and Dental Sciences 2015; Vol.4, Issuel6, February23; Page : 2652-2663, DOI: 10.14260/jemds/2015/383)

Glucose + Oxygen --- ® gluconolactone + Hydrogen Peroxide

2 I- + H₂O₂ + 2H⁺ --- I₂ + 2H₂O

Presence of ammonium molybdate or peroxidases catalyze the second step of the reaction i.e. generation of I₂ Glucose oxidase breaks down glucose in the presence of oxygen into hydrogen peroxide and glucoronic acid. Horseradish peroxidase/ammonium molybdate catalyses the oxidation of potassium iodide by hydrogen peroxide (formed by the action of glucose oxidase) to iodine, which is brown. The intensity of this brown colour can be taken as a measure of the amount of iodine present.

Potassium iodide (KI) has been used along with the gold standard glucose reagent for producing brown color based on the presence of glucose into the plasma. The color intensity depends on the concentration of glucose. The glucose level has been quantified by taking images of the colored reaction spot and subsequently by image processing using an in-house developed algorithm.

The present device has adopted the colorimetric detection technique along with Smartphone based analysis for identification and quantification plasma glucose level from a single drop of whole blood. Gold standard glucose reagent was modified by adding potassium iodide for paper-based colorimetric detection. Plasma glucose reacts with water and consumed oxygen in presence of glucose oxidase and produce hydrogen peroxide. Hydrogen peroxide is highly unstable and the potassium iodide catalyzes production of oxygen. Either presence of peroxidase or ammonium molybdate catalyses liberation of iodine. The color of iodine is brown. The color intensity depends on the presence of the glucose concentration into the blood plasma.

Images of the brown colored reaction chamber were taken using smartphone camera and extract grayscale intensity, color saturation value, no of color pixels etc. An algorithm is developed to map these parameters with the plasma glucose level for producing quantitative results. A correlation has been developed between plasma glucose level and aforementioned properties of the image using statistical analysis tool. An android based app has been developed for image acquisition, image processing and result display on the smartphone screen.

Example-4

Calibration and Validation of the device.

For calibration of newly designed device solutions of different concentration of glucose were prepared and the responses were measured. The obtained values were compared with the actual concentration of the glucose used to make the solution and it was observed that experimental values are well within the actual range (R² = 0.9629) (Figure 3).

Until now, the calibrated paper-based POC device is tested with 60 human blood samples. So far the blood samples used for validation encompass the range of 70 mg/dl to 400 mg/dl of blood glucose level and therefore it is well covering the entire range of high blood sugar to healthy condition (Figure 4). We have subsequently estimated the glucose level from the measured intensity values, by appropriately evoking the calibration curve (Figure 3). For a particular blood sample, the experiment has been repeated 5 times to check the repeatability of the device. The POCD measured glucose level has been compared with the pathological gold-standard estimates; see Figure 4. The plasma glucose level measured with the help of the POC device exhibits a good correlation with the pathological estimates. The accuracy of invented device is well within the standard set by ISO 15197 as ± 15 mg/dl for concentration below 100 mg/dl and ± 15% of the laboratory standard for concentration above 100 mg/dl. Hence it can be inferred that this method of colorimetric detection of plasma glucose has a high degree of repeatability.

Thus by way of the present invention the following things can be attained :

An affordable, paper-based POC device for measurement of plasma glucose whereas, most of the existing POC glucose meters available in the market estimate whole blood glucose using electrochemical or reflectance photometric detection techniques in spite of the knowledge that measurement of plasma glucose is more accurate than whole blood glucose and directly comparable to the gold standard pathological results.

The uniqueness of this device is its ability to perform blood plasma separation and subsequent analysis of glucose concentration on a single paper platform. Till date, there is no POC device available in the market which can perform both of these functionalities together on a single platform.

This is almost an instrument-free plasma glucose detection technique requiring only test strip and a smartphone based app for image processing and result display.

Most of the existing POC glucose meters are validated with some ethnic groups or demographically specific population. Therefore, these devices may not work universally. The present device will work universally for all ethnic and geographically diversified population.

There is an inherent limitation with most of the existing glucose meters i.e. a fixed data reading time window. This leads to inaccuracy in varying condition where reaction kinetics may change. Here we are proposing an adaptive time window based on reaction completion providing accurate result after sensing the color saturation by the system intelligence.

It is thus strongly believed that possible end users of the present device such as diabetic patients, pathologists and health workers, doctors at the bedside of the patients, hospitals and nursing home would be immensely benefitted by the present invention.

Claims

CLAIMS:

1. System for estimation of plasma glucose comprising :

integrated blood plasma separation module and plasma glucose detector.

2. The system as claimed in claim 1 wherein said glucose detector include image processing means and display unit.

3. The system as claimed in anyone of claims 1 or 2 comprising paper based integrated platform as POC device having said blood plasma separation module and plasma glucose detector.

4. The system as claimed in anyone of claims 1 to 3 wherein said paper based integrated platform having said blood plasma separation module and plasma glucose detector comprises microfluidic paper strip of combination of first and second filter paper said first filter paper enabling introducing blood drop and and subsequent plasma seperation from blood and said second filter paper providing for reaction chamber for plasma glucose detection.

5. The system as claimed in claim 4 comprising a microfiudic strip of said first filter paper comprising Whatman© LF1 filter paper adapted for said introducing the blood drop and subsequent plasma separation from blood cells and defining a source pad and channel body a nd of said second filter paper providing said reaction chamber comprising of whatman© cellulose filter paper grade 1 and placed at the end of the body of the channel.

6. The system as claimed in anyone of claims 1 to 5 compriisng microfluidic strip on a hydrophobic paper layer fixed on a cartridge type substrate.

7. The system as claimed in claim 6 wherein said cartridge type substrate comprises a

a 3D printed cartridge having an enclosure.

8. The system as claimed in anyone of claims 1 to 7 comprising paper strip based substrate platform wherein said first and second filter papers are provided with some overlaps to each other on a hydrophobic paper layer such that when a drop of blood is introduced onto the source pad, the said first filter paper LF1 does not allow RBC to transport through the LF1 filter paper but only plasma can easily transport enabling separation of plasma only to reach to the reaction pad,

said reaction pad provided with pre-embedded glucose detecting chemicals, said paper strip based substrate platform disposed such a way that one need to put only a drop of blood onto the source pad then subsequent separated plasma based reaction detection functionalities are automatically continued.

9. The system as claimed in anyone of claims 1 to 8 comprising pre-embedded glucose detecting chemicals including potassium iodide (KI) with gold standard glucose reagent for producing brown color based on the presence of glucose into the plasma.

10. The system as claimed in anyone of claims 1 to 9 comprising image processing means adapted for color intensity based estimation of glucose.

11. The system as claimed in anyone of claims 1 to 10 comprising image processing means including camera means for imaging the colour change of plasma on reaction with glucose detecting chemicals and colorimetric based detector for estimation of glucose.

12. The system as claimed in anyone of claims 1 to 11 comprising a smartphone including said camera means for imaging colour change of plasma on reaction with glucose detecting chemicals in said plasma glucose detector of said paper based integrated platform having said blood plasma separation module and plasma glucose detector and analytic tool for mapping the parameters with plasma glucose level for producing quantitative results.

13. The system as claimed in anyone of claims 1 to 12 comprising an enclosure for supporting a cartridge based said integrated blood plasma separation module and plasma glucose detector and a smartphone unit with said camera means and analytic tool for estimation of glucose.

14. A process for manufacture of the system as claimed in anyone of claims 1 to 13 comprising :

providing a paper based integrated blood plasma separation module and plasma glucose detector in the form of microfluidic paper strip made of two different filter papers.

15. A method of manufacture as claimed in claim 14 comprising providing said microfluidic paper strip made of two different filter papers,

wherein said two different filter papers used comprises a first filter paper obtained of Whatman® LF1 filter paper for introducing the blood drop and subsequent plasma separation from blood cells which form the source pad and channel body and a second fileter paper providing a reaction chamber involving whatman® cellulose filter paper grade 1 and placed at the end of the body of the channel, said microfluidic strip being placed on a hydrophobic paper layer.

16. A method as claimed in anyone of claims 14 or 15 comprising providing said The paper strip fixed on a cartridge type substrate preferably 3D printed cartridge and insert into an enclosure.