WO2023180899A1 - Device and method for detecting diarrhea - Google Patents

Abstract

Disclosed herein are a device and method for detecting viral and bacterial based diarrhea of infants and children. More particularly, invention relates to development of point-of-care microfluidic-paper analytical device (µ-PAD) based rapid diagnostic kit and method for differentiating viral and bacterial diarrhea of infants and children.

Description

[000] CROSS-REFERENCE TO RELATED APPLICATIONS

This application which claims the benefit of Indian Provisional Application 202241015578 filed March 21, 2022, titled “DEVICE AND METHOD FOR DETECTING DIARRHEA”. The entire contents of the above-identified applications are hereby fully incorporated herein by reference

TECHNICAL FIELD OF THE INVENTION

[001] The present invention is in the technical field of device and method for detecting viral and bacterial based diarrhea of infants and children.

[002] BACKGROUND OF THE INVENTION

[003] As per the study conducted in National Family Health Survey-4, it was revealed that the death rate of children underage-group 5 in rural India is 56 per 1000 live birth out of which 13% comprises diarrheal cases [1]. In India, the average occurrence of diarrhea is three times a year (i.e., 13% prevalence rate) with death rate of approximately 9-9.5% (among -12% world-wide proportion) claiming an average of 300 lives/day mortality [2]. There are mainly three types of acute diarrheal infections among which 70 % are caused by viruses whereas 10-20% by bacteria and <10% by protozoans and helminths as depicted in figure 1 [1]. It was observed that pediatricians working with government sector prescribed antibiotics to only 23% of patient children, while private practitioners prescribed antibiotics more often to children with diarrhea [3]. Figure 1A showsclassification of acute diarrheal infections with their respective abundance rate among infants and children.

[004] Diarrhea is defined as the passage of loose or watery stools with an increased frequency of three or more times per 24 hours leading to dehydration and electrolyte imbalance (Na, K, Cl) among infants. However, change in consistency and character of the stools is a more crucial observation rather than the number of stools [4] .

[005] Based on the etiology of the disease, diarrhea can be classified as infectious and non- infectious. Bacteria and viruses are the prime sources of causing infectious diarrhea and non-infectious causes include cow milk protein allergy and inflammatory bowel disease (Crohn’s disease and ulcerative colitis) which are usually very rare in children under 5 age group [5].

[006] It was also observed that the major cause of inclination in the mortality and morbidity rate among infants and young children is diarrheal infection [6]. This diarrhea- derived illness can cause malnutrition in children under five years old. In a few cases, it is sometimes even difficult to determine whether diarrhea is caused by a virus or by a bacteria. However, doctors can determine the cause of infection by reviewing the patient’s medical history (history of blood in stools) and by physical examination (tenderness of the abdomen, etc.). The identification of diarrheal etiology in children can be carried out through stool microscopy examination (microscopic analysis of white cells in stool) by collecting a stool sample from patients [7]. In rural health care setups, it is difficult to perform culturing of a stool sample, due to non-availability, time-consumption, and cost- expensive test prices. Parents are not enough educated to provide a proper medical history (history of blood and/or mucosa in the stool) to the physician/pediatricians [6].

[007] In most cases, the infection is self-limiting in nature and does not require antibiotic therapy or any other treatment other than oral rehydration therapy. However, some cases of diarrhea, especially invasive bacterial diarrhea would require antibiotic treatments.

[008] The unavailability of a point-of-care (POC) test facility has put the clinicians in difficulty to differentiate between viral and bacterial diarrheal infections thereby, resulting in the prescription of antibiotics for most of the diarrheal infant patients due to lack of POC device to detect the type of infection and can cause misuse of antibiotics [8]. The overconsumption of antibiotics can also result in antimicrobial resistance to antibiotics, serious adverse effects in children’s body and unnecessary outflow to the government and the parents.

[009] Hence, there is an urgent need for developing a Point-of-Care (POC) diagnostic tool, which is sensitive, rapid, reliable, affordable, easy-to-use, indigenous and assist in early diagnosis and treatment.

[010] SUMMARY OF THE INVENTION

[Oi l] A microfluidic -paper analytical device for detecting a diarrheal infection, wherein the device comprises: a sample-application zone, wherein a sample obtained from a subject is applied to the sample-application zone through an applicator; two reaction zones, wherein the two reaction zones comprise immobilized antibodies that bind with protein bio markers in the sample to form a protein- antibody complex, wherein the protein biomarkers comprise calprotectin (CP) and lactoferrin (LF); and two detection zones, wherein the two detection zones comprise immobilized labeled antibodies conjugated with gold nanoparticles, wherein the protein- antibody complex binds with the immobilized labeled antibodies in the two detection zones releasing a color in the presence of an enhancer that indicates the presence of the diarrheal infection in the subject, wherein the device is configured to allow the flow of the sample that is applied to the sample-application zone through capillary action along microfluidic channels toward the two reaction zones and the two detection zones.

[012] According to an exemplary aspect of the invention, the device as described above, wherein the immobilized antibodies in the two reaction zones comprise monoclonal antibodies specific against the protein biomarkers comprising calprotectin (CP) and lactoferrin (LF).

[013] According to an exemplary aspect of the invention, the device as described above, wherein the enhancer comprises silver ions that nucleates and precipitates around the gold nanoparticles at varying concentrations to show the color change with respect to a concentration of the protein biomarkers in the sample.

[014] According to an exemplary aspect of the invention, the device as described above, wherein the enhancer is obtained as a solution comprising silver ions and applied on the two detection zones to visualize the color change.

[015] According to an exemplary aspect of the invention, the device as described above, wherein the labeled antibodies in the two detection zones comprise anti- calprotectin and anti- lactoferrin monoclonal antibodies.

[016] In yet another aspect of the invention, the device as described above, wherein the color change to dark grey or black indicates the diarrheal infection caused by bacteria comprising Salmonella, Campylobacter, and Shigella.

[017] In yet another aspect of the invention, the device as described above, wherein the lack of color change in the two detection zones indicates the diarrheal infection caused by protozoa, rotavirus, norovirus, adenovirus, and helminths.

[018] In yet another aspect of the invention, the device as described above, wherein the sample comprises stool of the subject diluted in Phosphate Buffer Solution (PBS).

[019] In yet another aspect of the invention, the device as described above, wherein an intensity of color developed in the two detection zones is determined using colorimetry. [020] In yet another aspect of the invention, the device as described above, wherein the subject is an infant or child.

[021] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.

[022] BRIEF DESCRIPTION OF THE DRAWINGS

[023] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.

[024] FIG. 1A illustrates the classification of acute diarrheal infections with their respective abundance rate among infants and children, according to the aspects of the invention.

[025] FIG. IB illustrates the diagrammatic representation of Paper analytical device (PAD), according to the aspects of the invention.

[026] FIG. 2 illustrates the process of lateral flow assay occurring in paper analytical device, according to the aspects of the invention.

[027] FIG. 3 illustrates the schematic representation of essentialities of the bacterial diarrhea detection kit, according to the aspects of the invention.

[028] FIG. 4 illustrates stepwise representation of the procedure for conduction of detection test, according to the aspects of the invention.

[029] FIG.5A illustrates the final design of disposable PoC p-PAD based test-platform for diagnosing and differentiating bacterial diarrheal infection from others, according to the aspects of the invention.

[030] FIG. SB illustrates standardization and fabrication on paper using different dye on designing photomask and standardization of microfluidic channel, flow rate and channel integrity using different dye showing color variation, according to the aspects of the invention.

[031] FIG. 6 illustrates in-vitro spike sample study on p-PAD (possible proposed solution) and determination of LOD and cut-off value, according to the aspects of the invention.

[032] FIG. 7 illustrates working LFA mechanism on p-PAD. It shows the diagrammatic representation of Paper analytical device (PAD), according to the aspects of the invention.

[033] FIG. 8A illustrates the optimization of core components CP and LF Cut-off value determining through concentration, according to the aspects of the invention.

[034] FIG. 8B illustrates the direct proportionality of Diarrheal infection severity with respect to High cut-off value. Maximum cut-off value means higher is the diarrheal infection, according to the aspects of the invention.

[035] FIG. 8C illustrates the detection of bioconjugation by color changes in presence or absence of CP & LF. Color change means bioconjugation of antigen with antibody LF & CP. If no color changes then there is no bioconjugation of CP & LF, according to the aspects of the invention.

[036] FIG. 9 illustrates Bioconjugation of gold nanoparticles (AuNPs) with antibodies (Ab.) specific to target biomarkers, according to the aspects of the invention.

[037] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[038] DETAILED DESCRIPTION OF THE INVENTION

[039] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. [040] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[041] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.

[042] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

[043] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.

[044] Example embodiments of the present invention are described with reference to the accompanying figures.

[045] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[046] DEFINITIONS

[047] The term "antigen" refers to a substance, such as a virus or bacteria, that triggers an immune response in the body. In a lateral flow assay occurring in a paper analytical device, the antigen of interest is added to the sample solution as the first step.

[048] The term "antibody" refers to a protein produced by the immune system in response to an antigen. In a lateral flow assay, a specific antibody that can bind to the antigen of interest is added to the sample solution to form an antigen-antibody complex.

[049] The term "immobilized" refers to the process of attaching or fixing a substance to a specific location or surface. In a lateral flow assay, the specific antibody is typically immobilized on a specific zone on the paper analytical device, known as the antibody capture zone.

[050] The term "bioconjugate" refers to a complex of two or more biomolecules that are chemically linked together. In a lateral flow assay, gold nanoparticles coated with antibodies are added to the second zone on the paper, and these nanoparticles that can bind to the antigen-antibody complex are referred to as the bioconjugate.

[051] The term "detection zone" refers to the specific area on the paper analytical device where the gold nanoparticles bind to the antigen-antibody complex to create a visible signal.

[052] The term "silver enhancement solution" refers to a solution containing a silver compound that reacts with the gold nanoparticles in the bioconjugate to create a more visible line on the detection zone. This solution is added to the paper as the final step in the lateral flow assay.

[053] The term "lateral flow assay" refers to a type of diagnostic test that uses a paperbased device to detect the presence of a specific substance, such as an antigen. The process involves the movement of the sample solution through the paper via capillary action, with the test result being visible on the device.

[054] The term "paper analytical device" refers to a type of diagnostic test device that uses paper as a substrate to perform chemical and biochemical assays. The paper analytical device used in a lateral flow assay typically has three distinct zones: the detection zone, antibody capture zone, and sample zone.

[055] The term "specificity" refers to the ability of an antibody to bind to a specific antigen. In a lateral flow assay, a specific antibody that has high specificity for the antigen of interest is used to ensure accurate detection.

[056] The term "complex" refers to the combination of the antigen and antibody that interact with each other in a lateral flow assay, forming a visible line on the detection zone.

[057] The term "gold nanoparticles" refers to tiny particles made of gold that are used in lateral flow assays to bind to the bioconjugate and create a visible signal on the detection zone. [058] The term "visible signal" refers to the result of a lateral flow assay, which is visible to the naked eye and indicates the presence or absence of the antigen of interest in the sample solution. The visible signal is typically in the form of a visible line on the detection zone.

[059] EMBODIMENTS OF THE INVENTION:

[060] A microfluidic -paper analytical device for detecting a diarrheal infection, wherein the device comprises: a sample-application zone, wherein a sample obtained from a subject is applied to the sample-application zone through an applicator; two reaction zones, wherein the two reaction zones comprise immobilized antibodies that bind with protein bio markers in the sample to form a protein- antibody complex, wherein the protein biomarkers comprise calprotectin (CP) and lactoferrin (LF); and two detection zones, wherein the two detection zones comprise immobilized labeled antibodies conjugated with gold nanoparticles, wherein the protein- antibody complex binds with the immobilized labeled antibodies in the two detection zones releasing a color in the presence of an enhancer that indicates the presence of the diarrheal infection in the subject, wherein the device is configured to allow the flow of the sample that is applied to the sample-application zone through capillary action along microfluidic channels toward the two reaction zones and the two detection zones.

[061] The said invention is a device and method for detecting viral and bacterial based diarrhea of infants and children.

[062] The acute diarrheal infections ( 100) are classified with their respective abundance rate among infants and children (102) as viral (104), bacterial (105) or protozoan/helminths (106) infection as shown in FIG. 1A. A paper analytical device (120) is used to detect bacterial diarrhea infection in a sample solution (stool + PBS). The device has different regions with specific functions as shown in FIG. IB:

[063] Hydrophobic region ( 110): This is a region on the paper that repels water or other aqueous solutions. It helps to contain the sample solution within the designated areas and prevent it from spreading. [064] Hydrophilic region (112): This is a region on the paper that attracts water or other aqueous solutions. It helps to transport the sample solution by capillary action from the sample zone to other regions.

[065] Detection zone for bacterial diarrhea infection (114): This is a region on the paper that contains specific reagents that can detect the presence of bacteria causing diarrhea. If the sample solution contains these bacteria, it will react with the reagents and produce a visible signal.

[066] Antibody capturing zone (118): This is a region on the paper that contains antibodies specific to the bacteria causing diarrhea. These antibodies capture the bacteria from the sample solution as it flows through the paper.

[067] Sample zone (120): This is the area where the user drops the sample solution (stool + PBS) onto the paper. It is the starting point for the sample to flow through the paper via capillary action. During diarrheal infection, a group of intracellular proteins i.e., calprotectin (CP) and lactoferrin (LF) are released from activated granulocytes, particularly neutrophils, as part of the inflammatory response, and have been considered as a potential diagnostic bio marker for inflammatory bowel disease (IBD). Among children infected with bacterial diarrhea caused by Salmonella, Campylobacter and Shigella, the concentrations of fecal calprotectin and lactoferrin are high in contrast to viral infectious diarrhea. In accordance to the diversified research carried out on bacterial diarrheal infection among infants, it is estimated that the cut off concentration values for fecal calprotectin and fecal lactoferrin concentration are very high in contrast to viral diarrhea infection. In a study performed by Chen et al., for children under age group of 3 months to 10 years, the level of fecal calprotectin was less in patients infected with rotavirus infection (2.27xlO"⁶nM/ml), norovirus infection (2.54xlO'⁶nM/ml), or adenovirus infection (2.6xlO"⁶nM/ml) [10]. According to Borkowska et al., levels of fecal lactoferrin was higher in patients with Salmonella (0.0036 nM/ml) and Campylobacter (0.0033 nM/ml) infections and lower in patients with rotavirus (0.0009 nM/ml) or norovirus (0.001 nM/ml) infections. The concentration of fecal lactoferrin was inclined significantly in patients with severe (0.0036 nM/ml) or moderate (0.0012 nM/ml) disease activity when compared with subjects with mild (0.0005 nM/ml) disease activity [11]. [068] The FIG. 2 (200) describes the process of a lateral flow assay occurring in a paper analytical device. The first step is to add the antigen (202) of interest (e.g., virus, bacteria) to the sample solution. Next, an antibody (204) specific to the antigen is added to the sample solution. The antibody is typically immobilized on a specific zone on the paper analytical device, called the antibody capture zone. The antigen and antibody interact (206) with each other, forming a complex. The complex reaches a second zone on the paper, which contains gold nanoparticles coated with antibodies (208) that can bind to the antigen-antibody complex. These nanoparticles are referred to as the bioconjugate (210). The bioconjugate binds to the antigen-antibody complex, creating a visible line on the detection zone of the paper. Finally, a silver enhancement solution (212) is added to the paper, which reacts with the gold nanoparticles to create a more visible line on the detection zone. The paper analytical device (214) used in this process has three distinct zones: the detection zone (216), antibody capture zone (218). and sample zone (220). The detection zone is where the gold nanoparticles bind to the antigen-antibody complex to create a visible signal. The antibody capture zone contains immobilized antibodies that capture the antigen-specific antibodies. The sample zone is where the sample solution is added to initiate the lateral flow assay.

[069] The anti-calprotectin and anti-lactoferrin monoclonal antibodies exhibit higher affinity towards fecal biomarkers calprotectin (CP) and lactoferrin (LF), and these antibodies can undergo bio -conjugation with gold nanoparticles that can be visualized using silver enhancement solution. The silver ions that are present in enhancement solution nucleates and precipitates around gold nanoparticle at varying concentrations which shows different colour change with respect to concentration change that can be properly visualized. This change in colour from low to high concentration can detect the level of infection, thereby depicting as mild, moderate or severe as shown in FIG. 2.

[070] EXPERIMENTAL EMBODIMENTS

[071] The main concept behind development of point-of-care device for detection of bacterial diarrheal infection has been derived from the principle of photolithography technique, a paper-based lateral flow assay encompassing of anti-calprotectin and anti- lactoferrin monoclonal antibodies as capture antibodies. [072] Initially, the paper strip is immersed adequately into the infectious stool sample to detect the type of diarrheal infection in order to give an accurate and specific passage for treatment and thereby declining the uplift of AMR to antibiotics for non-severe cases. After sample immersion of paper strip, phosphate buffer will be added to the strip to prevent contamination and to neutralize the sample for further processing.

[073] Through capillary action, the sample on the paper strip start moving and reaches to the capture zone where targeted CP and LF biomarkers if present in the sample will interact with their respective monoclonal antibodies and will be tagged further with the prepared bio-conjugate comprising of gold nanoparticles in order to check the antigen- antibody interaction. Finally, silver enhancement solution will be added on the paper strip to attain visualization results of the type of infection.

[074] If there is a colour change on the strip showing dark grey or blackish showing high concentration of biomarkers indicates bacteria-caused diarrheal infection that requires antibiotic treatment. No or low limit colour change depicts non-bacterial diarrheal infection (either virus- or parasite-derived) which can treated with ORT and nutrition therapy. FIG. 2 shows demonstration of process of lateral flow assay occurring in paper analytical device.

[075] Table 1: Analysis of existing closest known solutions commercialized in the market worldwide

[076] II) POINT OF CARE SYSTEM

[077] Detection of the presence of calprotectin and lactoferrin are conducted through ELISA and LFA procedures, which are also employed to detect inflammatory bowel disease (IBD) from irritable bowel syndrome. The existing commercialized POC test strips have a fixed cut-off detection value mentioned in table 1, which is applicable for IBD but not in case of invasive diarrhea infection. However, recent studies have shown that the cut off value for both biomarkers are constantly high among bacterial diarrheal cases in contrast to IBD cases.

[078] It has been observed that the incidence of pediatric IBD is relatively low (5.2 per 100000 per year) and moreover, the detection kits/products are not available in India yet, while India is contributing an increase in the abundance of diarrheal cases [14]. Apart from Lactoferrin + Calprotectin Rapid Test Strip device, all other products can detect only a either lactoferrin or calprotectin biomarker. The Lactoferrin+ Calprotectin rapid test strip also has some pitfalls including limited cut-off values mentioned detection for invasive bacterial diarrhea. Besides, these products are using lateral flow assay (LFA) approach for the detection and management of IBD which are rationally costly and shows limitations in the aspect of specificity and selectivity with specific cut-off ranges for bacterial diarrhea is not mentioned.

[079] The prime objective of our study is to detect invasive bacterial diarrhea from viral diarrhea among infants and children under age group of 5 years. The point-of-care device follows the established lateral flow immunoassay technique which utilizes anti- calprotectin and anti- lactoferrin monoclonal antibodies along with bioconjugation with AuNPs and silver enhancement solution for diagnosis of bacterial diarrhea. The main goal of POC test strip is to make it effortless, rapid, and exhibits desirable with high specificity and sensitivity. This POC is made up of by using simple photolithography technology in chromatography paper making it cost-effective and eco-friendly in nature. This easy-to-use device is portable that can be used in resource-limited settings and attain diversified range of application due to simple methodology and cost-effective large-scale production ability. This can play a great role for clinicians to prescribe antibiotics in case of bacterial invasive diarrhea and to avoid overuse of antibiotics in case of viral diarrhea.

[080] The major source of bacterial diarrhea infection among infants and children includes Salmonella, Campylobacter and Shigella that can increase CP and LF in stool [8, 10]. This Point-of-care test strip is widely applicable due to many benefits including simple methodology approach, cost-effectiveness, high sensitivity and specificity, greater portability, greater cut-off value detection, requires resource-limited settings and have large-scale production ability. It will be applicable for the detection of these two biomarkers altogether that will provide qualitative outcome for the conformation of bacterial diarrhea infection. Hence, it can act as an effective helping hand for clinicians, frontline health workers to prescribe antibiotics to infants and children only in case of bacterial diarrhea, thereby, avoiding antimicrobial resistance, side effects from antibiotics, and for parents in terms of saving money.

[081 ] III) STEPS OF THE SYSTEM

[082] FIG. 3 (300) shows essentialities of the bacterial diarrhea detection kit that consists of loop for collecting stool (302), Eppendorf containing buffer (304), dropper (306). silver enhancement solution (308). p-PAD (310) and FIG. 4: Stepwise representation of the procedure for conduction of detection test.

[083] Step 1: Take out the sample collecting loop/swab from the kit and collect small amount of stool/feces specimen (402) .

[084] Step 2: Open the dilution vial by from the led cap and insert the stool sample containing the loop inside the Eppendorf tube containing phosphate buffer solution (PBS) (404).

[085] Step 3: Take out the loop after inserting the sample and close the cap of tube and then shake gently and carefully for proper mixing of the sample with the PBS to attain good sample dispersion (406).

[086] Step 4: Take out the paper analytical device (PAD) from the kit for further procedure (408).

[087] Step 5: Now open the cap of the tube and fill required of the solution in the dropper (410). [088] Step 6: Dispense 2-3 drops of the solution in the sample zone as mentioned in the illustration using dropper.

[089] Step-7: Wait for the sample to reach the proximal end i.e., detection zone of the device and then add few drops of silver enhancement solution in the detection zone (412).

[090] Step-8: Once added, wait for few minutes to check whether there is change in the color or not to confirm the type of diarrheal infection (414).

[091] (Note: If color changes to greyish black, it indicates bacterial diarrhea infection and if no color change, then viral or protozoan/helminth infection) (416).

[092] FIG. 5A illustrates the final design of disposable PoC p-PAD based testplatform for diagnosing and differentiating bacterial diarrheal infection from others. By using photolithography technology, inventors have prepared the hydrophobic and hydrophilic zones in p-PAD. The PAD has different regions (500) such as Hydrophobic region (502). hydrophilic region (504). detection zone for bacterial diarrhea infection (506). antibody capturing zone (508), sample zone for dropping sample solution (Stool + PBS) (510).

[093] The standardization and fabrication of microfluidic channels on paper using different dyes on a designed photomask was performed as shown in FIG. 5B (530). The process involves testing the microfluidic channels to ensure uniformity, consistency, and integrity of the channels (522). The figure illustrates the color variation of the dyes as they flow through the channels. The different shades of color indicate the differences in the flow rate, channel size, and channel integrity. The channel integrity refers to the absence of any leakage or blockage in the channels, which can affect the accuracy of the assay. By using different dyes, it is easier to standardize the microfluidic channels to ensure that they are uniform, consistent, and have a high degree of integrity. This is important for ensuring the accuracy and reliability of the assay. The figure also shows the photomask used to design the microfluidic channels on the paper (524). The photomask is a template that defines the size, shape, and position of the microfluidic channels. By using a well-designed photomask, the researchers can fabricate microfluidic channels with high precision and accuracy.

[094] FIG. 6 (600) shows an in-vitro spike sample study conducted on the p-PAD to determine the limit of detection (LOD) and cut-off value. The inventors prepared spike samples containing target biomarkers (610), specifically Cp and LF (602). Immunoassay was conducted of the spike sample and was compared with the control (604) showing p - PAD without spike samples (606) and p -PAD with spike sample containing biomarker (608). The target biomarker interference study was performed to determine the selectivity and sensitivity of p -PAD (612). The results of the study are presented in the form of color changes, which indicate the bioconjugation of the antigen and antibody. The inventors observed that the p-PAD with spike protein showed a visible color change, whereas the p- PAD without spike protein did not show any color change. This is because there was no bioconjugation of the antigen and antibody in the absence of the spike protein. The inventors determined the LOD and cut-off value of the p-PAD based on the color changes observed in the spike samples (614). The LOD is the lowest concentration of the analyte that can be detected, whereas the cut-off value is the concentration above which a sample is considered positive.

[095] The working LFA mechanism on p-PAD as shown in FIG. 7 (700). is the diagrammatic representation of Paper analytical device (PAD), wherein, the process of a lateral flow assay occurring in a paper analytical device is explained. The first step is to add the antigen (202) of interest (e.g., virus, bacteria) to the sample solution. Next, an antibody (204) specific to the antigen is added to the sample solution. The antibody is typically immobilized on a specific zone on the paper analytical device, called the antibody capture zone. The antigen and antibody interact (206) with each other, forming a complex. The complex reaches a second zone on the paper, which contains gold nanoparticles coated with antibodies (208) that can bind to the antigen-antibody complex. These nanoparticles are referred to as the bioconjugate (210). The bioconjugate binds to the antigen-antibody complex, creating a visible line on the detection zone of the paper. Finally, a silver enhancement solution (212) is added to the paper, which reacts with the gold nanoparticles to create a more visible line on the detection zone. The paper analytical device (702) used in this process has three distinct zones: the detection zone (216), antibody capture zone (218), and sample zone (220). This mechanism having the components like loop for collecting stool sample that transferred to Eppendorf tube containing buffer.

[096] FIG. 8(A-C) (800) are related to the optimization and determination of the cutoff value of the paper analytical device for the detection of diarrheal infection. FIG. 8A (801) describes the optimization of the core components CP and LF and the determination of the cut-off value through concentration. The inventors optimized the concentration of the antibody for CP and LF at different concentrations, and the maximum color concentration obtained was taken as the standard concentration. In FIG. 8B, the inventors demonstrated the direct proportionality of diarrheal infection severity with respect to high cut-off value (802). The maximum cut-off value indicates a higher degree of diarrheal infection. FIG. 8C shows the detection of bioconjugation by color changes in the presence or absence of CP (803) and LF (804) . A color change was observed, which indicated the bioconjugation of the antigen with the antibody LF and CP. If there is no color change, it means that there is no bioconjugation of CP and LF. Together, these demonstrate the effectiveness of the approach to optimize the core components of the assay and determine the cut-off value for accurate detection of diarrheal infection. The direct proportionality of diarrheal infection severity with respect to high cut-off value highlights the clinical relevance of the paper analytical device. The detection of bioconjugation through color changes in the presence of CP and LF is a crucial step in the assay and helps to ensure the accuracy and reliability of the results.

[097] FIG. 9 (900) demonstrates the bioconjugation of gold nanoparticles (AuNPs) with antibodies specific to target biomarkers (902). The inventors incubated AuNPs with antibodies and observed that when they bind, it shows bioconjugation. This incubation was carried out for 30 minutes under vortex conditions (904), followed by 5 minutes of centrifugation (906). To confirm the bioconjugation, UV spectroscopy was performed, and the results showed the successful binding of antibodies to AuNPs (908). After the successful bioconjugation of AuNPs with antibodies, calorimetric analysis was carried out (910). The results of this analysis showed the presence of antigenic biomarkers. A p-PAD was used to detect the presence of these biomarkers.

[098] IV) USES, BENEFITS AND ADVANTAGES OF THE SYSTEM

[099] As will be appreciated by a person skilled in the art the present invention provides a variety of following advantages.

[0100] Point-of-care paper-based test strips can be considered portable, easy to use, and very effective due to its sensitivity and specificity for tier -2 and tier-3 level areas as there are comparatively very fewer diagnostic facilities available. The clinicians, ANMs, AWW are the people who will be using this strip for infants and children below 5 years old to detect bacterial diarrhea.

[0101] The device obeys WHO’s Assured criteria (Affordable, Sensitive, Specific, User-Friendly, Rapid, Equipment-Free, delivered to end user) as well as the Reassured criteria (Real-time connectivity, Ease of specimen collection, Affordable, sensitive, Specific, User-friendly, Rapid, Equipment-free, delivered to end user).

[0102] It also reduces the hassle of frequent visit to the hospitals and help in quick decision making. In the absence of laboratory diagnostics, the kit is inexpensive and practical. It can reduce the syndromic management approach. In other words, the device is simple, economical, rapid, and works without access to laboratories, thus giving results quickly.

[0103] According to a further aspect of the present invention, the best mode to practice the inventions are,

1. Device for on-filed, bedside, diagnostic labs, hospitals, clinic and community settings. Further same principle could be used to develop other biomarkers as well.

2. It could be a revolutionary substitute for conventional in the laboratory setting by development of the aforementioned assay.

[0104] V) NOVELTY OF THE SYSTEM

[0105] The said biosensors are promising due to their high sensitivity, high signal-to- noise-ratio, and relative simplicity.

[0106] Cost-effective, rapid and highly reliable and user friendly compared to the existing technologies.

[0107] Highly sensitive.

[0108] Specific diagnostic kit.

[0109] Point of care, rapid and early detection of diarrhea.

[0110] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed. [0111] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.

[0112] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0113] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.

[0114] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0115] References:

]Lakshminarayanan S, Jayalakshmy R. Diarrheal diseases among children in India: Current scenario and future perspectives. J Nat Sci Biol Med. 2015 Jan-Jun;6(l):24-8. doi: 10.4103/0976-9668.149073. PMID: 25810630; PMCID: PMC4367049.

[2] Ghosh K, Chakraborty AS, Mog M. Prevalence of diarrhoea among under five children in India and its contextual determinants: A geo-spatial analysis. Clinical Epidemiology and Global Health. 2021 Oct 1; 12: 100813

[3]Bruzzese E, Giannattasio A, Guarino A. Antibiotic treatment of acute gastroenteritis in children. FlOOORes. 2018 Feb 15;7: 193. doi: 10. 12688/flOOOresearch. 12328.1. PMID: 29511533; PMCID: PMC5814741 [4]Paulauskaite I, Eidukaite A, Orentaite R. Fecal Calprotectin as a Biomarker Distinguishing Infectious Cause in Acute Intestinal Infections, Comparing Different Age Groups of Children. Iranian Journal of Pediatrics. 2020 Apr 30;30(2).

[5] Lee HM, Lee S, Lee BI, Jekarl DW, Song JY, Choi HJ, Kang BK, Im EJ, Kim JS, Kim JI, Kim BW. Clinical significance of fecal lactoferrin and multiplex polymerase chain reaction in patients with acute diarrhea. Gut and liver. 2015 Sep;9(5):636.

[6] Bjarnason I. The use of fecal calprotectin in inflammatory bowel disease. Gastroenterology & hepatology. 2017 Jan;13(l):53.

[7] Abraham BP, Kane S. Fecal markers: calprotectin and lactoferrin. Gastroenterol Clin North Am 2012 Jun;41(2):483-95. doi: 10. 1016/j.gtc.2012.01.007. Epub 2012 Feb 16. PMID: 22500530.

[8] Bunn SK, Bisset WM, Main MJ, Gray ES, Olson S, Golden BE. Fecal calprotectin: validation as a noninvasive measure of bowel inflammation in childhood inflammatory bowel disease. Journal of pediatric gastroenterology and nutrition. 2001 Jul l;33(l):14-22.

[9] Chen CC, Huang JL, Chang CJ, Kong MS. Fecal calprotectin as a correlative marker in clinical severity of infectious diarrhea and usefulness in evaluating bacterial or viral pathogens in children. J Pediatr Gastroenterol Nutr. 2012 Nov;55(5):541-7. doi: 10.1097/MPG.0b013e318262a718. PMID: 22699836.

[10] Chen CC, Chang CJ, Lin TY, Lai MW, Chao HC, Kong MS. Usefulness of fecal lactoferrin in predicting and monitoring the clinical severity of infectious diarrhea. World J Gastroenterol. 2011 Oct 7;17(37):4218-24. doi: 10.3748/wjg.vl7.i37.4218. PMID: 22072854; PMCID: PMC3208367.

[11]Borkowska A, Liberek A, Luczak G, Jankowska A, Plata-Nazar K, Korzon M, Kaminska B. Fecal lactoferrin, a marker of intestinal inflammation in children with inflammatory bowel disease. Acta Biochim Pol. 2015;62(3):541-5. doi: 10.18388/abp.2015_982. Epub 2015 Sep 4. PMID: 26339799

[12]Reenaers, C., Bossuyt, P., Hindryckx, P., Vanpoucke, H., Cremer, A., &Baert, F. (2018). Expert opinion for use of faecal calprotectin in diagnosis and monitoring of inflammatory bowel disease in daily clinical practice. United European gastroenterology journal, 6(8), 1117-1125. [13] Sorrentino, D., Nguyen, V. Q., & Love, K. (2021). Fecal Lactoferrin Predicts Primary Nonresponse to Biologic Agents in Inflammatory Bowel Disease. Digestive Diseases.

[14]Holtman GA, Lisman-van Leeuwen Y, Reitsma JB, Berger MY. Noninvasive Tests for Inflammatory Bowel Disease: A Meta-analysis. Pediatrics. 2016 Jan;137(l). doi: 10.1542/peds.2015-2126. Epub 2015 Dec 17. PMID: 26681783

Claims

CLAIMS Uwe claim:

1. A micro fluidic-paper analytical device for detecting a diarrheal infection, wherein the device comprises: a sample-application zone, wherein a sample obtained from a subject is applied to the sample-application zone through an applicator; two reaction zones, wherein the two reaction zones comprise immobilized antibodies that bind with protein biomarkers in the sample to form a protein-antibody complex, wherein the protein biomarkers comprise calprotectin (CP) and lactoferrin (LF); and two detection zones, wherein the two detection zones comprise immobilized labeled antibodies conjugated with gold nanoparticles, wherein the protein-antibody complex binds with the immobilized labeled antibodies in the two detection zones releasing a color in the presence of an enhancer that indicates the presence of the diarrheal infection in the subject, wherein the device is configured to allow the flow of the sample that is applied to the sample-application zone through capillary action along microfluidic channels toward the two reaction zones and the two detection zones.

2. The device as claimed in claim 1, wherein the immobilized antibodies in the two reaction zones comprise monoclonal antibodies specific against the protein biomarkers comprising calprotectin (CP) and lactoferrin (LF).

3. The device as claimed in claim 1, wherein the enhancer comprises silver ions that nucleates and precipitates around the gold nanoparticles at varying concentrations to show the color change with respect to a concentration of the protein biomarkers in the sample.

4. The device as claimed in claim 3, wherein the enhancer is obtained as a solution comprising silver ions and applied on the two detection zones to visualize the color change. i ]

5. The device as claimed in claim 1, wherein the labeled antibodies in the two detection zones comprise anti-calprotectin and anti-lactoferrin monoclonal antibodies.

6. The device as claimed in claim 1 , wherein the color change to dark grey or black indicates the diarrheal infection caused by bacteria comprising Salmonella, Campylobacter, and Shigella.

7. The device as claimed in claim 1, wherein the lack of color change in the two detection zones indicates the diarrheal infection caused by protozoa, rotavirus, norovirus, adenovirus, and helminths.

8. The device as claimed in claim 1, wherein the sample comprises stool of the subject diluted in Phosphate Buffer Solution (PBS).

9. The device as claimed in claim 1, wherein an intensity of color developed in the two detection zones is determined using colorimetry.

10. The device as claimed in claim 1, wherein the subject is an infant or child. i ]