[002] This application which claims the benefit of Indian Provisional Application, 202211012046 filed March 7, 2022, titled “PAPER ANALYTICAL DEVICE (PAD) FOR DETECTION OF VAGINAL INFECTIONS The entire contents of the above-identified applications are hereby fully incorporated herein by reference.

[003] TECHNICAL FIELD OF THE INVENTION

[004] The present invention is in the technical field of paper analytical device (pad) for detection of vaginal infections. Also provided are kits and electrochemical biosensor arrays.

[005] BACKGROUND OF THE INVENTION

[006] Vaginal infection or Vaginitis, also called vulvovaginitis, is an inflammation or infection of the vagina, which can also affect the vulva, which is the external part of a woman's genitals. Vaginitis can cause itching, pain, discharge, and odor. ^1-3

[007] The vaginal walls in a woman become inflamed due to some irritant that had disturbed the balance of the vaginal area, vaginal infection occurs. Bacteria, yeast, viruses, and/or chemicals in creams or sprays, or even clothing contribute to manifestations of infections. Vaginal infections often occur from organisms that are passed between sexual partners. Some additional factors that affect the vaginal environment include the overall health of the woman, personal hygiene, medications, hormones (particularly estrogen), and the health of her sexual partner. A disturbance in any factor or combination of these factors can trigger vaginitis. Abnormal vaginal discharge is a frequent complaint of women. The three most common reason for vaginal infection includes vulvovaginal candidiasis (VVC), bacterial vaginosis (BV), and trichomoniasis (TV) which have been associated with poor pregnancy outcomes but typically unattended to. ^2-9

[008] Bacterial vaginosis is the most common cause of vaginal symptoms among women ages 15-44. BV is caused when there is the replacement of the normal hydrogen peroxide producing Lactobacillus sp. in the vagina with high concentrations of anaerobic bacteria (e.g., Prevotella sp. and Mobiluncus sp.), G. vaginalis, Ureaplasma, Mycoplasma, and numerous fastidious or uncultivated anaerobes. It causes dysuria, itching, thin white or gray vaginal discharge; a strong fish-like odor. ⁶ “ 9

[009] Bacterial vaginosis has been associated with premature rupture of membranes, preterm delivery, infection of the chorion and amnion, infection of amniotic fluid, intrauterine death. This infection has been associated with pelvic inflammatory disease (PID), and increased acquisition of human immunodeficiency virus (HIV) infection and other sexually transmitted diseases (STDs). ^10,11

[010] Trichomoniasis is a widespread sexually transmitted infection around the world. According to WHO, 156.0 million trichomoniasis cases were reported worldwide ¹³ . Trichomonas vaginitis is caused by a one-celled parasite called Trichomonas vaginalis (TV), which passes between partners during sexual intercourse ¹⁰ . It causes dysuria, dyspareunia itching, and burning; thin green-yellow vaginal discharge; foul odor. ^9,12,13

[Oi l] Trichomoniasis is associated with many adverse reproductive health outcomes such as low birth weight (LBW) and premature birth. Many infections are asymptomatic and syndromic management leads to under-detection of TV. Newborns bom to mothers infected with Trichomoniasis may contact infection during delivery. ¹⁵

[012] VVC usually is a yeast infection caused by Candida species. The symptoms are dysuria, itching, thick white cottage cheese odor-free discharge. ¹⁶ An estimated 75% of women will experience at least one symptomatic episode during their lifetime and 6-9% will experience chronic recurrent vulvovaginal candidosis (at least four episodes per year). Vulvovaginal Candidiasis (VVC) has a risk factor of developing Candida-colonization of the infant, which in turn can lead to the development of Candida- septicemia in the newborn. ¹⁷

[013] Misdiagnosed, incorrectly diagnosed, or undiagnosed vaginitis is a major health issue that calls for the development of Successful diagnosis and treatment modalities.

[014] Current methods of diagnosing vaginal infections are few and having many drawbacks. For bacterial vaginosis, diagnosis is done using Amsel criteria: adherent and homogenous vaginal discharge; vaginal pH greater than 4.5; detection on the saline wet mount of clue cells; and/or amine odor after the addition of potassium hydroxide (positive whiff test). ¹ ’ ^2,3 ’ ^6,9 Trichomoniasis is diagnosed using a wet mount with microscopic visualization of motile T. vaginalis on slide preparations from vaginal secretions. ¹ ’ ^2,3 ’ ^9,14 The diagnosis of Candidiasis requires pelvic examination. The pH of vaginal secretions is pH < 4.5, and budding yeast and pseudohyphae may be seen on a wet mount on preparation with KOH solution. ¹ ’ ^2,3 ’ ^9,16

[015] 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.

[016] SUMMARY OF THE INVENTION

[017] According to the aspect of the present invention, a paper-based device for detecting Vagina Infections in a subject, wherein the device comprises: a sampleapplication zone, wherein a sample obtained from a subject is applied to the sampleapplication zone; two reaction zones, wherein the two reaction zones comprise immobilized antibodies that bind with antigens in the sample to form an antigenantibody complex; and five detection zones, wherein the five detection zones comprise immobilized labeled antibodies with enhancers in two zones, wherein the antigenantibody complex binds with the immobilized labeled antibodies with enhancers in the respective detection zones and analytes in three zones, two control zone with immobilized anti-conjugated antibody, releasing a color that indicates the presence of the Vaginal 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 hydrophilic channels toward the two reaction zones and the five detection zones, wherein the device detects the vaginal infections caused by Bacterial vaginosis (BV), Vulvovaginal candidiasis (VVC), and Trichomoniasis.

[018] In another aspect of the present invention, the paper-based device as described above, wherein the immobilized antibodies in the two reaction zones comprise monoclonal antibodies specific against a Fungal Beta Glucan, Trichomoniasis adhesion protein AP65.

[019] In another aspect of the present invention, the paper-based device as described above, wherein the five detection zones are configured to perform Amine test, Whiff s test, pH test, Candida Antigen-Antibody test, and Trichomoniasis Antigen-Antibody test respectively.

[020] In another aspect of the present invention, the paper-based device as described above, wherein are configured to perform control test. [021] In another aspect of the present invention, the paper-based device as described above, wherein the labeled antibodies are tagged with gold nanoparticles or Alkaline Phosphatase [ALP], wherein the enhancers comprise a silver enhancer or a substrate enhancer comprising Nitrote trazolium Blue Chloride or 5-Bromo-4-Chloro-3'- Indolyphosphate P-Toluidine.

[022] In another aspect of the present invention, the paper-based device as described above, wherein the labeled antibodies comprise the Trichomonas vaginalis Monoclonal Antibody with the substrate enhancer Nitro tetrazolium Blue Chloride or 5-Bromo-4-Chloro-3'- Indolyphosphate P-Toluidine tagged with the Alkaline Phosphate to detect infection caused by the Trichomoniasis.

[023] In another aspect of the present invention, the paper-based device as described above, wherein the labeled antibodies comprise the Candidiasis Monoclonal Antibody tagged with gold nanoparticles and the silver enhancer to detect infection caused by the Vulvovaginal candidiasis (VVC).

[024] In another aspect of the present invention, the paper-based device as described above, wherein detection of the Bacterial vaginosis is performed following Amsel’s criteria, wherein the pH test, the Whiff’s test, and the amine test are performed in the respective detection zones.

[025] In another aspect of the present invention, the paper-based device as described above, wherein particle size of the gold nanoparticle tagged with the labeled antibody is 720nm with zeta potential of 2.9 mv.

[026] In another aspect of the present invention, the paper-based device as described above, wherein an intensity of color developed in the five detection zones is determined using colorimetry.

[027] In another aspect of the present invention, the paper-based device as described above, wherein the subject is a woman of reproductive age group.

[028] 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.

[029] BRIEF DESCRIPTION OF THE DRAWINGS

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

[031] FIG. 1 illustrates the contents in the diagnostic kit for detection of vaginal infections A) Vile with Buffer Solution and dropper B) sterilized cotton swab C) Paper device, according to the aspects of the invention.

[032] FIG. 2 illustrates A) Design of Photomask used for Fabrication, B) Fabricated paper device C) Structural Integrity Test, D) and E) The paper device for detection of Vaginal Infections, according to the aspects of the invention.

[033] FIG. 3 illustrates the schematic representation of conjugates in a lateral flow assay using sandwich format for A) Colorimetric detection of Candidiasis using gold nanoparticle, antibody conjugate assay with silver enhancer B) Trichomoniasis using enzyme linked antibody and NBT/BCIP substrate enhancer, according to the aspects of the invention.

[034] FIG. 4 illustrates Bioconjugation of Gold nanoparticle (AuNP) with Antibody (Ab), according to the aspects of the invention.

[035] FIG. 5 illustrates UV-Vis Spectroscopy data for conjugation of gold nanoparticle and antibody of interest, according to the aspects of the invention.

[036] FIG. 6 illustrates Dynamic light scattering study of gold nanoparticle and gold nanoparticle and antibody conjugate, according to the aspects of the invention.

[037] FIG. 7 illustrates Bioconjugation of antibody of interest with Alkaline Phosphatase (ALP), according to the aspects of the invention.

[038] FIG. 8 illustrates Colorimteric analysis and LOD for different concentration of Candidiasis marker, according to the aspects of the invention.

[039] FIG. 9 illustrates Bar graph representing the gray values for different concentration of Candidiasis marker, according to the aspects of the invention.

[040] FIG. 10 illustrates Calibration curve for gray value versus different concentration of Candidiasis biomarker, according to the aspects of the invention.

[041] FIG. 11 illustrates Sensitivity test of Candidiasis biomarker and other biomarkers, according to the aspects of the invention.

[042] FIG. 12 illustrates Colorimetric analysis for different concentrations of Trichomoniasis biomarker, according to the aspects of the invention. [043] FIG. 13 illustrates Bar graph representing the gray values for different concentration of Trichomoniasis biomarker, according to the aspects of the invention.

[044] FIG. 14 illustrates Calibration curve for gray value versus different concentration of Trichomoniasis biomarker, according to the aspects of the invention.

[045] FIG. 15 illustrates Sensitivity test of Trichomoniasis biomarker and other biomarkers, according to the aspects of the invention.

[046] FIG. 16 illustrates Optimisation of pH Test, according to the aspects of the invention.

[047] FIG. 17 illustrates Optimisation of Amine Test, according to the aspects of the invention.

[048] 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.

[049] DETAILED DESCRIPTION OF THE INVENTION

[050] 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.

[051] 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.

[052] 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.

[053] 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.

[054] 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.

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

[056] 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.

[057] DEFINITIONS

[058] The term "vaginal infections" refers to various types of infections caused by bacteria, viruses, or fungi in the vaginal area of women.

[059] The term "detection zones" refers to the specific areas on the paper device that are designated for the detection of different types of infections.

[060] The term "capillarity" refers to the process by which fluid is drawn through narrow spaces or tubes due to surface tension and adhesion.

[061] The term "Amine test" refers to a test used to detect the presence of amines, which are produced by bacteria in the vaginal area and can cause a fishy odor.

[062] The term "Whiffs test" refers to a test used to detect the presence of bacterial vaginosis, which involves adding potassium hydroxide to a sample of vaginal discharge and observing for a fishy odor.

[063] The term "pH test" refers to a test used to measure the acidity or alkalinity of a substance, such as vaginal discharge.

[064] The term "Candida Antigen-Antibody test" refers to a test used to detect the presence of Candida, a type of fungus that can cause vaginal yeast infections.

[065] The term "Trichomoniasis Antigen- Antibody test" refers to a test used to detect the presence of Trichomonas vaginalis, a type of parasite that can cause vaginal infections. [066] The term "Antibody" refers to a blood protein produced in response to and counteracting a specific antigen.

[067] The term "Enhancer" refers to a DNA sequence that increases the level of transcription of a gene that is located nearby on the same chromosome.

[068] EMBODIMENTS OF THE INVENTION:

[069] According to the aspect of the present invention, a paper-based device for detecting Vagina Infections in a subject, wherein the device comprises: a sampleapplication zone, wherein a sample obtained from a subject is applied to the sampleapplication zone; two reaction zones, wherein the two reaction zones comprise immobilized antibodies that bind with antigens in the sample to form an antigenantibody complex; and five detection zones, wherein the twodetection zones comprise immobilized labeled antibodies with enhancers, wherein the antigen- antibody complex binds with the immobilized labeled antibodies with enhancers in the respective detection zones, three detection zone comprises of analytes, two control zone comprising of the anti-conjugated antibody, releasing a color that indicates the presence of the Vaginal 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 hydrophilic channels toward the two reaction zones and the five detection zones, two control zones wherein the device detects the vaginal infections caused by Bacterial vaginosis (BV), Vulvovaginal candidiasis (VVC), and Trichomoniasis.

[070] The said invention is a paper-based point-of-care diagnostic kit for vaginal infections. A colorimetric test will be performed using the concept of lateral flow technique. The invention is capable of detecting three causative agents’ mainly Bacterial vaginosis, Candidiasis and Trichomasiasis.

[071] The device for detecting vaginal infections in women comprises five detection zones, two reaction zones, two control zones and one sample zone. The paper device is capable of allowing the flow of fluid applied to the sample zone through capillarity within the device, in the following order,

[072] a sample-application zone, [073] a reaction zone, and

[074] a detection zone

[075] a control zone

[076] The following test will be performed in the detection zones:

[077] Amine test,

[078] Whiffs test,

[079] pH test,

[080] Candida Antigen-Antibody test and

[081] Trichomoniasis Antigen- Antibody test

[082] I) EXPERIMENTAL EMBODIMENTS

[083] The sample from the subject is added to a sample zone at a center of a paper device, whereby the sample moves by capillary action along the hydrophilic channels toward detection zones. The sample migrates towards the reaction zone or detection zone wherein the antigen/amines will bind to the analytes immobilized in their respective zones giving colour.

[084] For immunoassay, the sample migrates towards the reaction zone wherein the antigen from the sample will be capable of binding to the capture antibodies on the membrane. The antigen-bound antibody migrates through hydrophilic channels and reaches the detection zone which contains labelled antibodies (gold nanoparticle or Alkaline Phosphatase [ALP]) along with signal enhancers-silver enhancer and NBT/BCIP. The sample, together with the labelled antibody bound to the target antigen, releasing a color that shows the presence of infection in women. The detection of Trichomoniasis and Candidiasis will be done using an immune assay.

[085] The sample applied to the sample zone travels through a reaction zone were non-immobiliazed captured monoclonal antibody specific against a Fungal Beta Glucan. The target antibodies are Beta D Glucan which is found in abundance in the cell wall of fungi.

[086] The antigen in the sample reacts with capture antibody in the reaction zone to form a mobile antibody complex and migrates toward detection zone. In the detection zone, a labeled antibody with gold nanoparticle will be immobilized. This step includes detecting the presence or absence of antigen antibody complex in the detection zone. The labeled antibody forms a sandwich immunoassay with the antigen captured antibody and releases color in presence of silver enhancer determine the presence of infection in the subject.

[087] The same principle will be used to detect antigens from Trichomoniasis. The antibody specific to Trichomonas which in this case is an adhesion protein AP65 will be immobilized in the reaction zone. The antigen from the sample reacts with the capture anibody forming a complex and then migrating to the detection zone. In the detection zone, an APL labeled antibody will make an immune complex and color is released in presence of BCIP/NBT substrate enhancer.

[088] Similar reaction happens at the control line, the only difference is that the antibodies fixed at the control line are to the test antibody itself, and no bridging occurs. The gold colloid stuck to the test antibodies will become visible confirming that the test works.

[089] The detection of Bacterial vaginosis will be done following the Amsel’s criteria wherein pH test, Whiff s test, and amine test will be performed. A simple colorimetric test will be performed telling about the presence of the infection. The normal pH of vaginal fluid is in the range of 3.8-4.2, after application of the sample in the sample zone migrates towards the detection zone.

[090] The change in color, indicates that the pH is above 4.7, depicting a positive result. The next test is Whiff s test which is used to detect odor. A foul or fishy odor is associated with BV. The infected sample when reaches the detection zones reacts with potassium hydroxide (KOH) releasing a pungent smell. The amine test is the confirmatory test for Whiffs test to measure the presence of amines in the sample. The amines present in the sample react with ninhydrin showing deep blue color determining the presence of Bacterial vaginosis.

[091 ] II) POINT OF CARE SYSTEM

[092] The point of care kit for detection of vaginal infections will consist of a lysis buffer solution to dilute the sample, a dropper for adding sample to the sample zone on the paper device, and a sterilized cotton swab. The health care worker will collect the sample from the vaginal walls of awomen . Once the sample is collected, it is added to the vile and mixed the swab with the buffer solution and later keeping the swab inside the solution for some minutes. Using a dropper, add the solution to the sample zone and wait for some minutes for the result to arrive. Change in color in any of the detection zone suggest that the woman is suffering from either Trichomoniasis, Candidiasis and Bacterial vaginosis. If there is a change in color in all the detection change it suggests the presence of all the causative agents causing Vaginal infections. The interpretation of the test result will be as follows:

[093] Colour development in both control and test zone for Vulvovaginal Candidiasis

- Positive case

[094] Colour development in both control and test zone for Trichomoniasis - Positive case

[095] Colour development for pH (as per the interpretation table), amine test and development of odour- Positive Bacterial vaginosis case

[096] Development of colour in all the five zones, presence of all three pathogens.

[097] FIG. 11 : Illustration of the contents in the diagnostic kit for detection of vaginal infections A) Vile with Buffer Solution and dropper B) sterilized cotton swab C) Paper device.

[098] III) PAPER DEVICE

[099] FIG. 2A-D describes the Paper Device. Details of the paper device for detection of Vaginal Infections.

[0100] pH test

[0101] Whiffs test

[0102] Amine test

[0103] Trichomonas vaginalis Monoclonal Capture Antibody

[0104] Alkaline Phosphate tagged Trichomonas vaginalis Monoclonal Antibody with

NBT/BCIP substrate.

[0105] Fungal beta-glucan Captured Monoclonal Antibody

[0106] Fungal beta-glucan Antibody tagged with Gold Nanoparticle and Silver enhancer.

[0107] Sample zone

[0108] Hydrophilic zone

[0109] Hydrophobic zone

[0110] Designing and Fabrication of the Paper Analytical Device

[0111] Design of the photomask and fabrication of the Paper Analytical Device

[0112] Different size and dimension of the channel were designed. In-house paper fabrication technique was done using Photolithographic technique. A structural integrity test was conducted using coloured dyes to study the channel dimensions and flow rate. After the result from integrity test the photomask was designed again so as to increase the flow rate of the dye. (FIG 2A-E) The label of Fig. 2D and 2E suggest the following

[0113] 1- pH test

[0114] 2 -Whiff s test

[0115] 3 -Amine test

[0116] 4-Trichomonas vaginalis Monoclonal Capture Antibody

[0117] 5-Alkaline Phosphate tagged Trichomonas vaginalis Monoclonal Antibody with NBT/BCIP substrate

[0118] 6-Control Zone

[0119] 7-Fungal beta-glucan Captured Monoclonal Antibody

[0120] 8- Fungal beta-glucan Antibody tagged with Gold Nanoparticle and Silver enhancer

[0121] 9- Control Zone

[0122] 10- Sample zone

[0123] 11 - Hydrophilic zone

[0124] 12- Hydrophobic zone

[0125] III) LATERAL FLOW ASSAY:

[0126] FIG. 3 : Schematic representation of conjugates in a lateral flow assay using sandwich format [300] for A) Colorimetric detection [316] of Candidiasis using Gold nanoparticle [326], antibody conjugate assay [302], [306], [320], [332] with silver enhancer [324] B) Trichomoniasis using enzyme linked antibody and NBT/BCIP [312] substrate enhancer [308].

[0127] Antigen [304], [318] & [330]

[0128] Ab: Antibody

[0129] ALP: Alkaline Phosphatase [310] &[314]

[0130] AuNP: Gold Nanoparticle

[0131] NBT/BCIP: Nitro tetrazolium Blue Chloride/ 5-Bromo-4-Chloro-3'-

Indolyphosphate P-Toluidine.

[0132] Sample collection: A small amount of the patient's vaginal discharge is collected.

[0133] Sample application: The patient's sample is applied to the sample pad and migrates through the device via capillary action. [0134] Conjugate addition: A gold nanoparticle-antibody conjugate [328] specific to the target analyte (e.g., Candidiasis or Trichomoniasis) is added to the conjugate pad. For Trichomoniasis, an enzyme-linked antibody is used instead.

[0135] Sandwich formation: If the target analyte is present in the patient's sample, it will bind to both the conjugate and the test line antibodies, forming a sandwich complex.

[0136] Signal amplification/signal generation [322]: For Candidiasis, a silver enhancer is added to amplify the signal, making it visible to the naked eye. The enhancer reacts with the gold nanoparticle, producing a silver precipitate that accumulates at the test line, resulting in a visible colored line. If the target analyte is not present, no colored line will appear at the test line. For Trichomoniasis, an NBT/BCIP substrate enhancer is added, which reacts with the enzyme on the conjugate, producing a colored signal at the test line. The intensity of the color is proportional to the amount of the target analyte present in the patient's sample.

[0137] Optimisation of Nano-bioconjugate

[0138] Bioconjugation of Candidiasis antibody with gold nanoparticles

[0139] The conjugation of gold nanoparticle (AuNP) [402] and antibody of interest [404] was done following the protocol from Nature’s [416] journal. First, in the gold nanoparticle antibody of interest was added and incubated for half-hour [408] at four- degree temperature [410], Later the conjugate [406] was centrifuged for 5 minutes [412], and the supernatant was removed, and buffer solution was added to dilute the conjugate [414]. The conjugate was studied in UV vis spectroscopy [418] to check the conjugation between antibody of interest with gold nanoparticle (FIG. 4).

[0140] Characterization of the prepared Gold nanoparticle and Antibody (AuNPs- Ab) bioconjugate with respect to the naked AuNPs were done using UV-vis absorption spectroscopy. As shown in FIG. 6, the naked AuNPs depicted a common surface plasmon resonance peak (SPR) at 520.0 nm. After the bioconjugation of AuNPs with Antibody of interest, the SPR band shifted to 523.0 nm (redshift) as indicated in FIG. 5. This spectral shift is due to the change in AuNPs size or due to the aggregation resulted from the physical adsorption of antibody of interest onto AuNPs, during the bioconjugation process.

[0141] Dynamic light scattering (DLS) study was done to measure nanoparticles size, and also zeta-potential to characterize nanoparticles surface charge, obtaining information about their stability and surface interaction with other molecules as shown in FIG. 6. The particle size of gold nanoparticle is 420 nm, this is due to the clumping of the particle together and the zeta potential is -4.8 mv (Negative polarity). On the other hand, particle size of gold nanoparticle and antibody conjugate is 720nm with zeta potential of 2.9 mv (Positive polarity).

[0142] Bioconjugation of Trichomoniasis Antibody with Alkaline Phosphatase (ALP)

[0143] A protocol from Sigma-Aldrich was followed for the conjugation of antibody of interest with ALP. The mentioned steps were followed for the conjugation process.

[0144] Combine 10 mg of antibody and 5 mg of high activity alkaline phosphatase (Cat.

No. P0114) in 1 mL PBS (20 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl).

[0145] Dialyze against 0.1 M sodium phosphate, pH 6.8, overnight to remove any free amine-containing compounds.

[0146] While gently stirring in a fume hood, add 0.05 mL of a 1% glutaraldehyde solution. Stir for 5 minutes.

[0147] Incubate for 3 hours at room temperature, and then add 0.1 mL of 1 M ethanolamine pH 7.0.

[0148] Incubate for an additional 2 hours at room temperature.

[0149] Dialyze overnight at 4 °C against three changes of PBS.

[0150] Centrifuge at 40,000 g for 20 minutes to remove unwanted precipitate.

[0151] Store clarified supernatant at 4 °C in the presence of 50% glycerol, 1 mM ZnC12, 1 mM MgC12, and 0.02% sodium azide. (FIG. 7)

[0152] Optimization detection analytes for the recognition of BV, Trichomoniasis, and VVC

[0153] Optimiza tion of Candidiasis biomarker

[0154] Optimization of Candidiasis antigen

[0155] Testing was performed of the AuNPs-based colorimetric detection of antibody of interest as shown in Figure 8. When adding different concentrations of the antigen for Candidiasis to different detection zones (10% 10% 10 ¹² 10 ¹⁵ g/ml and control respectively), the immunocomplex showed different colours. The colour change was attributed to different amounts of AuNP bio-probes bound to different concentrations of antigen during the immune recognition (FIG. 8). The absence of antigen exhibited no colour change on the detection zones (control), while the presence of 10' ⁶ , 10' ⁹ , 10 1 ² 10’ ¹³ g/mL showed dark grey colour (FIG. 9). [0156] Establishing limit of detection for Candidiasis antigen

[0157] The grey values of digitally photographed detection zones were measured of different concentrations of antigen using ImageJ software. The grey intensity values increased gradually as the concentrations of antigen varied from low (10 ¹⁵ g/mL) to high (10“ ⁶ g/mL) (FIG. 10). The established limit of detection for Candidiasis biomarker is 2.218 fg/ml.

[0158] Sensitivity test of Candidiasis biomarker

[0159] A sensitivity test of Candidiasis biomarker was done to check the ability to detect infection in patients in whom the disease is truly present. Various biomarker of different infections/diseases was immobilised on the paper and tests were done to see the sensitivity of the biomarker (FIG. 11).

[0160] Optimization of Trichomoniasis biomarker

[0161] Optimization of Trichomoniasis antigen

[0162] Usually, antigen of varied concentrations was prepared, and optimisation was done while maintaining a constant incubation time and temperature in order to optimize staining. The development of enzyme-based colorimetric immunoassays was done using enzyme labels as the signal amplification strategies, alkaline phosphatase (ALP) was used due to their high catalytic activity, high specificity, mild reaction conditions, and easy conjugation to antibodies. FIG. 12 and 13 shows the colorimetric analysis of Trichomoniasis biomarker.

[0163] Establishing limit of detection for Trichomoniasis antigen

[0164] The limit of detection was calculated using the similar method as mentioned above. The grey value intensity was measured using ImageJ software and later the limit of detection was established (FIG. 14). The limit of detection of Trichomoniasis biomarker is 1.7017 fg/ml.

[0165] Sensitivity test of Trichomoniasis biomarker

[0166] Biomarkers of various other infections were used along with the biomarker of Trichomoniasis to see the sensitivity of the paper device as shown in FIG. 15.

[0167] Optimisation of pH

[0168] The optimisation of the pH was done between the range of 2-13. This pH paper changes colour in different pH solutions because of a pigment present in red cabbage. This is soluble in water and changes colour in the presence of various types of solutions. In the presence of an acidic solution, it turns red. In the presence of a basic solution, it turns greenish. In the presence of a neutral solution, it turns purple (FIG. 16).

[0169] Optimisation of Amine

[0170] The ninhydrin test was optimised to check whether a given analyte contains amines or a-amino acids. In this test, ninhydrin is added to a test solution of the analyte. The development of a deep blue colour indicates the presence of ammonia, primary/secondary amines, or amino acids in the analyte (FIG. 17).

[0171] IV) NOVELTY OF THE SYS EM

[0172] The present technique which is used for detection is the PAP smear and observational diagnosis based on discharge. PAP smear method takes around 1-2 days to give result, so the decision gets related and the patients have to come to and fro to the hospital. It also needs a laboratory facility to perform the tests. The diagnosis based on the discharge type is based on the assumption of the clinicians. The other available products which are available in the market are either

[0173] Expensive

[0174] Uses sophisticated instruments

[0175] Not user friendly

[0176] Unavailable for the Indian market

[0177] Time-consuming

[0178] Target a certain pathogen causing other ignoring the other possible cause of infection

[0179] The current invention is focusing on all the pathogens and is based on a simple colorimetric method that gives results within few minutes. The technology is

[0180] Easily to use

[0181] Faster turn-around-times (TAT),

[0182] The device does not require any special equipment

[0183] The tests will be performed in 2-3 steps

[0184] Affordable and cost-effective diagnostics that can be used in resource-limited environments.

[0185] Rapid medical decisions [0186] Portable

[0187] V) USES, BENEFITS AND ADVANTAGES OF THE SYSTEM

[0188] As will be appreciated by a person skilled in the art the present invention provides a variety of following advantages.

[0189] The Point of Care testing kit can be used as a screening tool for the detection of vaginal infections. It can be used in communities and primary healthcare centers and have a tremendous potential in reducing delays in diagnosing and initiating treatment for Bacterial vaginosis (BV), Vulvovaginal candidiasis (VVC), and Trichomoniasis.

[0190] 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).

[0191] 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 not only 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.

[0192] During a field visit, in antenatal checkup, a woman came in with the complaint of itching and discomfort, the woman was in her fourth month of pregnancy and the doctor suggested her to get the vaginitis test done.

[0193] There was a case of miscarriage and premature rupture of membrane reported by Gynaecogist because of this infection. Some of the doctors also mentioned that a vaginal infections test is recommended in the antenatal period but it is not performed mostly. Pregnant women are also not aware of this condition and get overlooked by them especially in the rural areas. Observations books, as well as lab notebooks, are maintained of all the observations and the experiments conducted.

[0194] 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. [0195] Following are the novel features of the invention,

[0196] The said biosensors are promising due to their high sensitivity, high signal-to- noise-ratio, and relative simplicity.

[0197] Cost-effective, rapid and highly reliable and user friendly compared to the existing technologies.

[0198] Highly sensitive.

[0199] Specific diagnostic kit.

[0200] Point of care, rapid and early detection of vaginal infection in a patient sample.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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. [0206] References:

[0207] L. Paladine, H. and A. Desai, U., 2018. Vaginitis: Diagnosis and Treatment. [ebook] American Family Physician, pp.322-329. Available at:

<https://www.aafp.org/afp/2018/0301/afp20180301p321.pd f> [Accessed 20 May 2021],

[0208] WHO, Sexually transmitted and other reproductive tract infections: a guide to essential practice. 2005.

[0209] CDC. Vaginal Discharge. https://www.cdc.gov/std/tg2015/vaginal- discharge.htm (Last Accessed on 20 ^th February, 2021)

[0210] Hainer BL, Gibson MV. Vaginitis. Am Fam Physician. 2011 Apr l;83(7):807- 15.

[0211] Quan M. Vaginitis: diagnosis and management. Postgrad Med. 2010 Nov; 122(6): 117-27. doi: 10.3810/pgm.2010

[0212] CDC. Bacterial vaginosis.https://www. cdc.gov/std/tg2015/bv.htm (Last Accessed on 20 ^th February, 2021)

[0213] Coudray MS, Madhivanan P. Bacterial vaginosis — A brief synopsis of the literature. European Journal of Obstetrics & Gynecology and Reproductive Biology. 2020 Feb 1;245: 143-8.

[0214] Sobel J, Mitchell C. Bacterial vaginosis: Clinical manifestations and diagnosis. UpToDate, Waltham, MA, (Accessed on April 25, 2017): Wolters Kluwer. 2017

[0215] Sherrod J, Wilson J, Donders G, Mendling W, Jensen JS. 2018 European (IUSTI/WHO) International Union against sexually transmitted infections (IUSTI) World Health Organisation (WHO) guideline on the management of vaginal discharge. Int J STD AIDS. 2018;29(13): 1258-1272.

[0216] Krauss-Silva L, Almada-Horta A, Alves MB, Camacho KG, Moreira ME, Braga A. Basic vaginal pH, bacterial vaginosis and aerobic vaginitis: prevalence in early pregnancy and risk of spontaneous preterm delivery, a prospective study in a low socioeconomic and multiethnic South American population. BMC pregnancy and childbirth. 2014 Dec;14(l):l-0.

[0217] Leitich H, Kiss H. Asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome. Best Pract Res Clin Obstet Gynaecol. 2007;21(3):375-90. [0218] Van Schalkwyk J, Yudin MH, Allen V, Bouchard C, Boucher M, Boucoiran I, Caddy S, Castillo E, Kennedy VL, Money DM, Murphy K. Vulvovaginitis: screening for and management of trichomoniasis, vulvovaginal candidiasis, and bacterial vaginosis. Journal of Obstetrics and Gynaecology Canada. 2015 Mar l;37(3):266-74.

[0219] https://www.who. int/bulletin/volumes/97/8/18-228486.pdf.

[0220] CDC. Trichomoniasis, https://www.cdc.gov/std/tg2015/trichomoniasis.htm

(Last Accessed on 20 ^th February, 2021)

[0221] Gaydos CA, Klausner JD, Pai NP, Kelly H, Coltart C, Peeling RW. Rapid and point-of-care tests for the diagnosis of Trichomonas vaginalis in women and men. Sexually transmitted infections. 2017 Dec l;93(S4):S31-5.

[0222] CDC. Vulvovaginal

Candidiasis.https://www.cdc.gov/std/tg2015/candidiasis.ht m (Last Accessed on 20 ^th February, 2021)

[0223] Chokoeva A, Kouzmanov A, Ivanova Z, Zisova L, Amalie G, Petleshkova P, Miteva-Katrandzhieva T, Krasteva M, Uchikova E. [Investigation on antifungal susceptibility of Candida yeasts in pregnant patients with confirmed vulvovaginal candidiasis and their newborns.]. Akush Ginekol (Sofiia). 2016;55(4):20-29.

[0224] Other References

[0225] Lisi, P. J., Dondero, R. S., Kwiatkoski, D. I. A. N. E., Spence, M. R., Rein, M. F., & Alderete, J. F. (1988). Monoclonal-antibody-based enzyme-linked immunosorbent assay for Trichomonas vaginalis. Journal of clinical microbiology, 26(9), 1684-1686.

[0226] Petrin, D., Delgaty, K., Bhatt, R., & Garber, G. (1998). Clinical and microbiological aspects of Trichomonas vaginalis. Clinical microbiology reviews, 11(2), 300-317.

[0227] Garber, G. E. (2005). The laboratory diagnosis of Trichomonas vaginalis. Canadian Journal of Infectious Diseases and Medical Microbiology, 16(1), 35-38.

[0228] Garcia, A. F., & Alderete, J. F. (2007). Characterization of the Trichomonas vaginalis surface-associated AP65 and binding domain interacting with trichomonads and host cells. BMC microbiology, 7(1), 1-10.

[0229] Lee, H. Y., Hyung, S., Lee, J. W., Kim, J., Shin, M. H., Ryu, J. S., & Park, S. J. (2011). Identification of antigenic proteins in Trichomonas vaginalis. The Korean journal of parasitology, 49(1), 79.

[0230] Espiritu, C. A. L., Justo, C. A. C., Rubio, M. J., Svobodova, M., Bashammakh, A. S., Alyoubi, A. O., ... & O’Sullivan, C. K. (2018). Aptamer selection against a Trichomonas vaginalis adhesion protein for diagnostic applications. ACS infectious diseases, 4(9), 1306-1315.

[0231] Zhang, Z., Song, X., Zhang, Z., Li, H., Duan, Y., Zhang, H., ... & Wang, M. (2021). The molecular characterization and immune protection of adhesion protein 65 (AP65) of Trichomonas vaginalis. Microbial Pathogenesis, 152, 104750.

[0232] Hoenigl, M., Prattes, J., Spiess, B., Wagner, J., Prueller, F., Raggam, R. B., ... & Buchheidt, D. (2014). Performance of galactomannan, beta-d-glucan, Aspergillus lateral-flow device, conventional culture, and PCR tests with bronchoalveolar lavage fluid for diagnosis of invasive pulmonary aspergillosis. Journal of clinical microbiology, 52(6), 2039-2045.

[0233] Bullarbo, M., Andersch, B., Samuelson, E., Lindgren, A., & Kondori, N. (2017). Self-diagnosis of vulvovaginal candidiasis is poor — a comparison of diagnostic methods introducing P-glucan as a complement. Reprod Syst Sex Disord, 6(202), 2.

[0234] Huppler, A. R., Fisher, B. T., Lehrnbecher, T., Walsh, T. J., & Steinbach, W. J. (2017). Role of molecular biomarkers in the diagnosis of invasive fungal diseases in children. Journal of the Pediatric Infectious Diseases Society, 6(suppl_l), S32-S44.

[0235] Hammarstrom, H., Stjarne Aspelund, A., Christensson, B., HeuBel, C. P., Isaksson, J., Kondori, N., ... & Friman, V. (2018). Prospective evaluation of a combination of fungal biomarkers for the diagnosis of invasive fungal disease in high- risk haematology patients. Mycoses, 61(9), 623-632.

[0236] Singh, S., Kaur, H., Choudhary, H., Sethi, S., Malhotra, P., Gupta, K. L., ... & Chakrabarti, A. (2019). Evaluation of biomarkers: Galactomannan and 1, 3-beta-D- glucan assay for the diagnosis of invasive fungal infections in immunocompromised patients from a tertiary care centre. Indian journal of medical microbiology, 36(4), 557-563.

[0237] Pacora, P., Romero, R., Erez, O., Maymon, E., Panaitescu, B., Kusanovic, J. P., ... & Hassan, S. S. (2019). The diagnostic performance of the beta-glucan assay in the detection of intra-amniotic infection with Candida species. The Journal of Maternal-Fetal & Neonatal Medicine, 32(10), 1703-1720. [0238] K Hussain, K., Malavia, D., M Johnson, E., Littlechild, J., Winlove, C. P., Vollmer, F., & Gow, N. A. (2020). Biosensors and Diagnostics for Fungal Detection. Journal of Fungi, 6(4), 349.