DESC:F O R M 2

THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]

1. TITLE OF THE INVENTION: NON-INVASIVE DIAGNOSTIC KIT FOR DETECTION OF URINARY TRACT INFECTION (UTI)

2. APPLICANT (A) NAME: SURAJIT BHATTACHARJEE

(B) ADDRESS: Department of Molecular Biology & Bioinformatics, Tripura University, Suryamaninagar-799022, Tripura, India.

3. NATIONALITY (C) NATIONALITY: INDIA

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
TECHNICAL FIELD OF THE INVENTION

[001] The present invention relates to a pathogen detection device and method related to a portable instrument required for point-of-care analysis, wherein the portable instrument may include functionalized micro bead particles, a sample collection device and/or sample reservoir, reagent and fluid delivery systems, one or more glass slides, and/or waste reservoirs. More particularly, the said kit can be used for rapid and non-invasive diagnosis kit for detection of Urinary Tract Infection (UTI) through agglutination method. Detection of UTI causative bacterial markers from non-invasive sample like urine can be utilized for diagnosis of type and stage of infection.

[002] BACKGROUND OF THE INVENTION

[003] Urinary tract infections (UTI) has been regarded as major cause of infection suffered by women and about 50% of all females get a UTI during their lifetime. Major bacteria species which are involved in UTI infection includes Escherichia coli, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella sp., Enterobacter and Proteus species etc. [1, 2].
[004] The pathogens traditionally associated with UTI are changing many of their features, particularly because of antimicrobial resistance through development of biofilm. The etiology of UTI is also affected by underlying host factors that complicate UTI, such as age, diabetes, spinal cord injury, or catheterization.
[005] Because of biofilm formation and increasing antimicrobial resistance, undiagnosed and untreated UTI can be fatal as well. Diagnosis of UTI from urine samples or clinical isolates is a mandatory step.
[006] At present, conventional methods of diagnosis of UTI involve urine routine examination (RE), urine culture and antibiotic sensitivity test, ultrasonography, pap smear (Papanicolaou smear test) examination along with measurement of ESR from blood sample (erythrocyte sedimentation rate) and CRP (C-Reactive Protein) from serum sample of patient [1, 2, 3, 4].
[007] However, urine RE is a less sensitive assay whereas antibiotic sensitivity assay is costly as well as very tedious process and may take 2-3 days for getting results.
[008] Furthermore, during examination time organism can grow very rapidly in planktonic form whereas biofilm can become very firm to adopt higher antibiotic resistance.
[009] In addition, it is noteworthy to mention that organisms during their growth in urinary tract both in planktonic and biofilm form release a variety of virulence factor that can be potential marker molecule of respective bacteria.
[010] Therefore, there is an urgent need for affordable rapid diagnosis method required for identification and detection of multiple uropathogen.
[011] In addition, there is a need for detection of these UTI causative bacterial markers from non-invasive sample like urine that can be utilised for diagnosis of type and stage of infection.
[012] Furthermore, there is a need for diagnosis procedure that will be less costly, less tedious and very effective procedure that can be performed in complete absence of any laboratory instruments.
[013] Therefore, there is an urgent need for affordable low-cost, quick and easy to operate diagnostic point-of-care solution that requires limited training and is accessible to remote areas at affordable price.
[014] In addition, a point-of-care technology platform would help to identify the bacterial agent causing diseases. It will help in delivering appropriate and prompt treatment to improve clinical outcomes.
[015] Therefore, it is desirable to develop a point-of-care technology platform that can be available at rural healthcare centres in India and other developing countries and to detect and treat and/or other bacterial infected patients at an earlier stage of their therapeutic itinerary.
[016] In Summary, there is an urgent need in the art to develop new methods and systems capable of rapid and non-invasive diagnostic kit for detection of Urinary Tract Infection (UTI). Furthermore, it is desirable that the methods and systems facilitate rapid screening of microbes for further confirmatory testing.

[017] SUMMARY OF THE INVENTION

[018] According to an exemplary aspect, the present invention relates to pathogen detection device and method related to a portable instrument required for point-of-care analysis. In particular, the present invention relates to a pathogen detection device and method related to a portable instrument required for point-of-care analysis. The portable instrument may include functionalized micro bead particles, a sample collection device and/or sample reservoir, reagent and fluid delivery systems, one or more glass slides, and/or waste reservoirs. More particularly, the said kit can be used for rapid and non-invasive diagnosis kit for detection of Urinary Tract Infection (UTI) through agglutination method. Detection of UTI causative bacterial markers from non-invasive sample like urine can be utilised for diagnosis of type and stage of infection. Furthermore, it is desirable that the methods and systems facilitate rapid screening of microbes for further confirmatory testing. Furthermore, the functionalized diagnostic kit may be used in conjunction with membrane- and/or particle-based analysis through agglutination technique. Furthermore, different types of cells and/or microbes and/or pathogens can be detected through the present invention.
[019] Thus the diagnostic kit can be denoted as rapid and non-invasive diagnosis kit for detection of Urinary Tract Infection (UTI) through agglutination method.
[020] Yet another exemplary aspect of the present invention, the present kit can also detect suspected UTI patients under developing condition. To develop the kit, antibody coated sepharose beads were used for agglutination with bacterial proteins from patient urine. Clean and dry slides were used for agglutination of sepharose beads with patient samples. Furthermore, titre value of each sample was determined for characterizing the amount of beads needs to be used during the detection through agglutination.
[021] Thus, in one particular embodiment, detection of UTI patients using the kit is based on the principle of agglutination. The test urine samples are mixed with above mentioned reagents and allowed to react/ agglutination. If bacterial proteins are present in the samples then a clear visible agglutination is observed. If bacterial proteins are not present in the sample agglutination would not be observed.
[022] The said method is a quantitative method wherein the titre value can be determined through serial dilution of urine sample with fixed quantity of antibody immobilised bead.
[023] In yet another embodiment, the agglutination is observed by naked eye and if required may be confirmed through microscopy.
[024] In yet another embodiment, the possible uses of this invention include: point-of-care technology platform that can be made available to remote and low resource rural areas in India for detecting bacterial pathogens in clinical samples. It can be fabricated into a portable device for wider usage.
[025] This point-of-care technology platform of bacterial pathogen detection from clinical samples and/or pathogen contaminated food or water sources would help in alleviating the socio-economic loss that bacterial diseases inflict on low-income communities. In urban areas, point-of-care technology platform would enable faster bacterial pathogen identification than is currently possible with culture-dependent techniques.
[026] As would be appreciated by the person skilled in the art, invention provides following advantages,
[027] A device for diagnosis and/or monitoring of the presence of a urinary tract infection (UTI) in a sample/ in a subject comprising:
• disposable cartridge of integrated electrodes,
• functionalized particles,
• an optical detector, a sample collection device and/or sample reservoir,
• reagent and fluid delivery systems,
• one or more channels, and
• waste reservoirs.
[028] The device of [027], wherein microgel particles is functionalized with antibody/tailspike protein, wherein antibody/tailspike proteins are fluorescently labelled.
[029] The device of [027], wherein the device can be used for rapid and non-invasive diagnosis kit for detection of Urinary Tract Infection (UTI) through agglutination method, wherein the detection of UTI causative bacterial markers from non-invasive sample can be utilized for diagnosis of type and stage of infection.
[030] An in vitro method for diagnosis and/or monitoring of the presence of a urinary tract infection (UTI) in a subject/ sample comprising:
• interdigitated electrodes;
• functionalized microgel particles, wherein the functionalized microgel particles conjugated with antibody and/or phage tailspike, wherein at least one of the particles is configured to entrap microbes under an electric field trapping method; and
• one or more optical detection systems, wherein at least one of the detection systems is configured to interact with at least a portion of the sample to allow detection of the analyte under an optical detector.
[031] The method of [030], wherein one or more detection systems comprise one or more particle-based detection systems, wherein at least one of the particle-based detection systems is coupled to electric field based interdigitated electrodes.
[032] The method of [030], wherein the detection system comprises culture independent diagnosis of analyte in a sample.
[033] The method of [030], wherein at least one of the microgel particles comprises a receptor molecule coupled to a fluorescent polymeric microgel beads, and wherein the particle further comprises a first indicator (fluorescence), the first indicators being coupled to the receptor, and wherein interaction of the receptor with the analyte causes the first indicator to produce the fluorescent signal.
[034] The method of [030], wherein the functionalized fluorescent microgel particles conjugated with antibody/tailspike protein used for targeted bacterial attachment, wherein microgel-targeted bacterial particles are pulled-down using an external electric field.
[035] The method of [030], wherein the one or more sample droplets comprise a substance selected from the group consisting of whole blood, lymphatic fluids, serum, plasma, seminal fluids, vaginal excretions, serous fluids, synovial fluids, pericardial fluids, sweat, tear, saliva, sputum, cerebrospinal fluids, amniotic fluids, peritoneal fluids, pleural fluids, transudates, exudates, cystic fluids, bile, urine, gastric fluids, intestinal fluids, fecal samples, fluidized tissues, fluidized organisms, biological swabs and biological washes, wherein the method is used to identify different types of cells and/or microbes and/or pathogens.
[036] A method for assessing the status of a disease, condition, pathology, or cell state, wherein the disease, condition, pathology, or cell state is associated with the presence or level of a cellular component, the method comprising the steps of detecting and/or isolating or removing the cellular component associated with the disease, condition, pathology, or cell state by a method according to any of [027-030], and correlating the presence or level of the cellular component with disease, condition, pathology, or cell state.
[037] 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.

[038] BRIEF DESCRIPTION OF THE DRAWINGS

[039] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.
[040] FIG. 1(A, B & C) illustrates the agglutination of Staphylococcal a-toxin, E. coli alkaline phosphatase (Bap) and both antibody with water (Negative control). This was observed that both antibody alone and in combination with water did not show any visible agglutination. 1A, Staphylococcal a-toxin antibody with water. 1B, E. coli alkaline phosphatase (Bap) antibody with water. 1C, Staphylococcal a-toxin + E. coli alkaline phosphatase (Bap) antibody with water.
[041] FIG. 2 (A, B & C) illustrates the agglutination of Staphylococcal a-toxin [A], E. coli alkaline phosphatase (Bap) [B] and both antibody with S. aureus and E. coli culture supernatant (positive control). Staphylococcal a-toxin antibody and BAP antibody did not show any agglutination with E. coli and S. aureus culture supernatant respectively. Combination of both antibodies [C] against bacterial culture supernatant gives maximum agglutination. 2A (i), Staphylococcal a-toxin antibody with S. aureus supernatant. 2A (ii) Staphylococcal a-toxin antibody with E. coli supernatant. 2B (i) Bap antibody with S. aureus supernatant 2B (ii) Bap antibody with E. coli supernatant. 2C (i) Staphylococcal a-toxin + E. coli alkaline phosphatase (Bap) antibody with S. aureus supernatant. 2C (ii) Staphylococcal a-toxin + E. coli alkaline phosphatase (Bap) antibody with supernatant.
[042] FIG. 3 (A & B) illustrates the agglutination of Staphylococcal a-toxin antibody with urine sample 1 [A] and 2 [B]. This was observed that sample 1 shows better agglutination than that of sample 2 with fixed volume of Staphylococcal a-toxin antibody. The minimum volume of Staphylococcal a-toxin antibody required for positive agglutination reaction was determined through titre value of respective reaction. 3A, Agglutination with sample 1. 3B, Agglutination with sample 1.
[043] FIG. 4 (A & B) depicts the agglutination of E. coli alkaline phosphatase (Bap) with urine sample 1 [A] and 2 [B]. This was observed that sample 2 shows better agglutination than that of sample 1 with fixed volume of BAP antibody. The minimum volume of BAP antibody required for positive agglutination reaction was determined through titre value of respective reaction. 4A, Agglutination with sample 1. 4B, Agglutination with sample 1.
[044] FIG. 5 (A & B) depicts the agglutination of Staphylococcal a-toxin and E. coli alkaline phosphatase (Bap) with urine sample 1 [A] and 2 [B]. This was observed that both sample 1 and 2 shows dense agglutination because of presence of both antibodies. The minimum volume of combination of both antibodies required for positive agglutination reaction was determined through titre value of respective reaction. 5A, Agglutination with sample 1. 5B, Agglutination with sample 1.
[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] DETAILED DESCRIPTION OF THE INVENTION

[047] 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.
[048] 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 item. 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.
[049] 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.
[050] 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.
[051] 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.
[052] Example embodiments of the present invention are described with reference to the accompanying figures.
[053] 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.
[054] Definitions
[055] "Diagnostic" means identifying a pathologic condition.
[056] The terms "detection", "detecting" and the like, may be used in the context of detecting markers or biomarkers.
[057] A "test amount" of a marker refers to an amount of a marker present in a sample being tested. A test amount can be either in absolute amount (e.g., µg/ml) or a relative amount (e.g., relative intensity of signals).
[058] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. "Polypeptide," "peptide" and "protein” can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
[059] "Detectable moiety" or a "label" refers to spectroscopic, photochemical, biochemical, immunochemical, or chemical means of detection of a composition. For example, labels may include enzyme labelled antibody molecules. The detectable moiety generates a measurable signal that can quantify the amount of bound detectable moiety in a sample. In the present kit quantitation of the signal is done by determination of titre of the agglutination reaction.
[060] "Antibody” refers to a polypeptide ligand encoded by an immunoglobulin gene(s), which specifically binds and recognizes an epitope.
[061] The terms "subject", "patient" or "individual" generally refer to a human or mammals. "Sample" refers to a polynucleotide, antibodies fragments, polypeptides, peptides, genomic DNA, RNA, or cDNA, polypeptides, a cell, a tissue, and derivatives thereof may comprise a bodily fluid or a soluble cell preparation, or culture media, a chromosome, an organelle, or membrane isolated or extracted from a cell.
[062] “Agglutination” refers to the clumping of particles. Agglutination is the process that occurs if an antigen is mixed with its corresponding antibody.
[063] The present kit can also detect suspected UTI patients under developing condition. To develop the kit antibody coated sepharose beads were used for agglutination with bacterial proteins from patient urine. Clean and dry slides were used for agglutination of sepharose beads with patient samples. Furthermore, titre value of each sample was determined for characterizing the amount of beads needs to be used during the detection through agglutination.
[064] REAGENTS USED
a. Tube 1: Contain suspension of Staphylococcal a-toxin antibody immobilised bead.
b. Tube 2: Contain suspension of bacterial (E. coli) alkaline phosphatase (BAP) antibody immobilised bead.
c. Tube 3: Contain suspension of Staphylococcal a-toxin and BAP antibody immobilised bead.

[065] PRINCIPLE
[066] Detection of UTI patients using the kit is based on the principle of agglutination. The test urine samples are mixed with above mentioned reagents and allowed to react/ agglutination. If bacterial proteins are present in the samples then a clear visible agglutination is observed. If bacterial proteins are not present in the sample agglutination would not be observed.
[067] SAMPLE COLLECTION AND PREPARATION
[068] Early morning urine samples should be collected from UTI patients in sterile vial under aseptic condition. After collection sample should be checked within 2-3 hrs. If necessary samples can be stored at 40C for upto 48 hrs.
[069] MATERIAL REQUIRED
[070] Glass slide, Mixing stick, Pipette and Tip.
[071] TEST PROCEDURE
[072] Bring reagent and samples to room temperature before use.
[073] Qualitative Method
a. Take Clean and dry slide, label as 1, 2 & 3 on three different spots on the slide.
b. Mix properly the contents in tube labelled as 1, 2 and 3.
c. Pipette one drop (15-20 µl) of bead (from each of the above mentioned tubes) on the labelled spot over the glass slide.
d. Add one drop (15-20 µl) of sample (in 1:1 ratio) to each drop of bead.
e. Using a mixing stick, mix each bead with the sample uniformly over the slide and wait for 5 minutes.
f. Titre value can be determined through serial dilution of urine sample with fixed quantity of antibody immobilised bead.
[074] Observe the agglutination by naked eye and if required may be confirmed through microscopy.
[075] CONDITION FOR STORAGE AND STABILITY
a. Store reagent tubes (labelled 1, 2 and 3) at 2-8 0C. DO NOT FREEZE.
b. The shelf life of the reagent is as per the expiry date mentioned on the reagent vial label. Do not use reagents after the expiry date.
c. Reagents are light sensitive. Avoid direct contact with light.
[076] SHELF LIFE
[077] The product is stable upto 24 months if stored as recommended.
[078] INTERPRETATION OF RESULTS
[079] Qualitative Method
[080] Observation of clear and uniform agglutination is a positive result and indicates the presence of bacterial proteins i.e. UTI causative bacterial virulent proteins in the test urine sample.
[081] Observation of non-uniform/no agglutination is a negative result indicates the absence of bacterial proteins of UTI causative agents in the test sample.
[082] EXPERIMENTAL PROCEDURE:
a. Bacterial CFU count from UTI infected patient urine sample:
[083] Early morning urine samples were collected from UTI patients, samples were cultured in separate media and CFU count was measured to confirm the presence of UTI in the patient. Results obtained from patient sample are tabulated below.
[084] TABLE 1: Results obtained from patient sample
No. of patient CFU/ml
Sample 1 4,50,000
Sample 2 3,31,00

[085] After collecting urine sample from UTI patient, samples were cultured over LB agar. From the urine culture a single colony was isolated and sub-cultured. Presence of type of bacteria in the sub-cultures were identified through gram staining. From gram staining it was observed that urine sub culture contain both gram positive and gram negative cocci.
[086] 2. Confirmation of presence of specific bacteria in urine of UTI patient:
[087] Inventors purchased antibody and immobilised with CNBr activated sepharose micro bead surface. Further, these Ab-immobilised beads was used for agglutination assay and determination of titre value for detection of UTI. For determination of titre value, fixed quantity of Ab-immobilised bead was placed over a glass slide and to that patient sample or bacterial culture supernatant was added in serial dilution. The least quantity of patient sample or bacterial culture supernatant with which agglutination is positive was recorded as the titre value. More the value higher will be the stage of infection in UT. Based on titre value respective type of responsible bacteria were determined using respective Ab-immobilised CNBr micro bead (a-toxin of S. aureus, Bacterial alkaline phosphatase of E. coli).
[088] The study was compared with the culture supernatant of bacteria i.e. E. coli (MTCC 40) and S. aureus (MTCC 96) as a control. Agglutination with a-toxin antibody was higher in case of S. aureus compared to E. coli. Whereas agglutination with alkaline phosphatase antibody was higher in case of E. coli compared to S. aureus. No agglutination was observed in case of negative control (autoclaved Milli-Q water). FIG 1 (A, B and C): Agglutination of Staphylococcal a-toxin, E. coli alkaline phosphatase (Bap) and both antibody with water (Negative control). This was observed that both antibody alone and in combination with water did not show any visible agglutination.
[089] FIG 2 depicts the agglutination of Staphylococcal a-toxin [A], E. coli alkaline phosphatase (Bap) [B] and both antibody with S. aureus and E. coli culture supernatant (positive control). Staphylococcal a-toxin antibody and BAP antibody did not show any agglutination with E. coli and S. aureus culture supernatant respectively. Combination of both antibodies [C] against bacterial culture supernatant gives maximum agglutination.
[090] FIG 3 depicts the agglutination of Staphylococcal a-toxin antibody with urine sample 1 [A] and 2 [B]. This was observed that sample 1 shows better agglutination than that of sample 2 with fixed volume of Staphylococcal a-toxin antibody. The minimum volume of Staphylococcal a-toxin antibody required for positive agglutination reaction was determined through titre value of respective reaction.
[091] FIG 4 depicts the agglutination of E. coli alkaline phosphatase (Bap) with urine sample 1 [A] and 2 [B]. This was observed that sample 2 shows better agglutination than that of sample 1 with fixed volume of BAP antibody. The minimum volume of BAP antibody required for positive agglutination reaction was determined through titre value of respective reaction.
[092] FIG 5 depicts the agglutination of Staphylococcal a-toxin and E. coli alkaline phosphatase (Bap) with urine sample 1 [A] and 2 [B]. This was observed that both sample 1 and 2 shows dense agglutination because of presence of both antibodies. The minimum volume of combination of both antibodies required for positive agglutination reaction was determined through titre value of respective reaction.
[093] Till this phase, antibody was immobilised against bacterial proteins over micro bead surface. The agglutination protocol was also standardised for identification of UTI from urine sample. Inventors have also standardised the protocol for titre value
[094] TABLE 2: Standardized the protocol for titre value
Report of the UTI patients diagnosed using present agglutination kit.
[095]
Sl. No. No. of patient Source of sample Bead-A Bead-B Bead-A+B Total positive Total negative
1 350 Aastha Diagnostic & Research Centre. Bhubaneswar. 72 234 55 274 44
2 150 Vivekananda Daignostics, Unit-6, Ekrma Marg, Near Capital Hospital, Bhubaneswar. 92 52 29 144 6
2 5 Tripura Medical College, Agartala. 3 2 5 5 0
3 8 Agartala Govt. Medical College 5 2 7 7 1
4 2 ILS, Agartala 2 0 2 2 0
determination for detection of UTI. At this phase large scale screening of detection of UTI from patient urine sample is going on. Successful completion of the screening helped in commercialization of the kit.
[096] TABLE 3: The available detection kits in the market vs our indigenously designed diagnostic kit

[097] COST OF UTI DETECTION KIT
[098] Total cost involvement for 100 patients around rupees 5226.32/-
[099] Cost per patient sample is less than 100 rupees.

[0100] Benefits and uses of the invention:

[0101] Detection of UTI patients using the kit is based on the principle of agglutination. The test urine samples are mixed with above mentioned reagents and allowed to react/ agglutination. If bacterial proteins are present in the samples then a clear visible agglutination is observed.

[0102] Advantages of the UTI kit
[0103] Low amount is required for diagnosis
[0104] Detects the presence and absence of pathogen
[0105] Identifies the specific strain of pathogens involve in infection
[0106] Cost effective
[0107] Time saving and promotes early detection and helps in quick diagnosis
[0108] can be made easily available
[0109] As would be appreciated by the person skilled in the art, invention provides following advantages,
[0110] A device for diagnosis and/or monitoring of the presence of a urinary tract infection (UTI) in a sample/ in a subject comprising:
• disposable cartridge of integrated electrodes,
• functionalized particles,
• an optical detector, a sample collection device and/or sample reservoir,
• reagent and fluid delivery systems,
• one or more channels, and
• waste reservoirs.
[0111] The device of [0109], wherein microgel particles is functionalized with antibody/tailspike protein, wherein antibody/tailspike proteins are fluorescently labelled.
[0112] The device of [0109], wherein the device can be used for rapid and non-invasive diagnosis kit for detection of Urinary Tract Infection (UTI) through agglutination method, wherein the detection of UTI causative bacterial markers from non-invasive sample can be utilized for diagnosis of type and stage of infection.
[0113] An in vitro method for diagnosis and/or monitoring of the presence of a urinary tract infection (UTI) in a subject/ sample comprising:
• interdigitated electrodes;
• functionalized microgel particles, wherein the functionalized microgel particles conjugated with antibody and/or phage tailspike, wherein at least one of the particles is configured to entrap microbes under an electric field trapping method; and
• one or more optical detection systems, wherein at least one of the detection systems is configured to interact with at least a portion of the sample to allow detection of the analyte under an optical detector.
[0114] The method of [0112], wherein one or more detection systems comprise one or more particle-based detection systems, wherein at least one of the particle-based detection systems is coupled to electric field based interdigitated electrodes.
[0115] The method of [0112], wherein the detection system comprises culture independent diagnosis of analyte in a sample.
[0116] The method of [0112], wherein at least one of the microgel particles comprises a receptor molecule coupled to a fluorescent polymeric microgel beads, and wherein the particle further comprises a first indicator (fluorescence), the first indicators being coupled to the receptor, and wherein interaction of the receptor with the analyte causes the first indicator to produce the fluorescent signal.
[0117] The method of [0112], wherein the functionalized fluorescent microgel particles conjugated with antibody/tailspike protein used for targeted bacterial attachment, wherein microgel-targeted bacterial particles are pulled-down using an external electric field.
[0118] The method of [0112], wherein the one or more sample droplets comprise a substance selected from the group consisting of whole blood, lymphatic fluids, serum, plasma, seminal fluids, vaginal excretions, serous fluids, synovial fluids, pericardial fluids, sweat, tear, saliva, sputum, cerebrospinal fluids, amniotic fluids, peritoneal fluids, pleural fluids, transudates, exudates, cystic fluids, bile, urine, gastric fluids, intestinal fluids, fecal samples, fluidized tissues, fluidized organisms, biological swabs and biological washes, wherein the method is used to identify different types of cells and/or microbes and/or pathogens.
[0119] A method for assessing the status of a disease, condition, pathology, or cell state, wherein the disease, condition, pathology, or cell state is associated with the presence or level of a cellular component, the method comprising the steps of detecting and/or isolating or removing the cellular component associated with the disease, condition, pathology, or cell state by a method according to any of [0109-0112], and correlating the presence or level of the cellular component with disease, condition, pathology, or cell state.
[0120] 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.
[0121] According to a non-limiting exemplary aspect of the present invention, the markers can be used for the development of kits that enable sample collection, processing and pathogen detection. These diagnostic kits developed can then be utilized by hospitals/private clinics/dental doctors or the public as such to screen/diagnose different pathogens.
[0122] 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.
[0123] 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.
[0124] 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.
[000] References:
1. Minardi, D., d'Anzeo, G., Cantoro, D., Conti, A., & Muzzonigro, G. (2011). Urinary tract infections in women: etiology and treatment options. International journal of general medicine, 4, 333-43.
2. Al-Badr, A., & Al-Shaikh, G. (2013). Recurrent Urinary Tract Infections Management in Women: A review. Sultan Qaboos University medical journal, 13(3), 359-67.
3. B. Yildiz, H. Poyraz, N. Cetin, N. Kural, O. Colak. High sensitive C-reactive protein: a new marker for urinary tract infection, VUR and renal scar. Eur Rev Med Pharmacol Sci 2013; 17 - N. 19; 2598-2604.
4. Ehsanipour F, Noorbakhsh S, Zarabi V, Movahedi Z, Rahimzadeh N. Comparison the Serum STREM1 Levels Between Children with Upper and Lower UTI. Curr Pediatr Rev. 2017;13(2):152-156.
,CLAIMS:Claims
I/We Claim,
1. A device for diagnosis and/or monitoring of the presence of a urinary tract infection (UTI) in a sample/ in a subject comprising:
(a) disposable cartridge of integrated electrodes,
(b) functionalized particles,
(c) an optical detector, a sample collection device and/or sample reservoir,
(d) reagent and fluid delivery systems,
(e) one or more channels, and
(f) waste reservoirs.
2. The device of claim 1, wherein microgel particles is functionalized with antibody/tailspike protein, wherein antibody/tailspike proteins are fluorescently labelled.
3. The device of claim 1, wherein the device can be used for rapid and non-invasive diagnosis kit for detection of Urinary Tract Infection (UTI) through agglutination method, wherein the detection of UTI causative bacterial markers from non-invasive sample can be utilized for diagnosis of type and stage of infection.
4. An in vitro method for diagnosis and/or monitoring of the presence of a urinary tract infection (UTI) in a subject/ sample comprising:
a) interdigitated electrodes;
b) functionalized microgel particles, wherein the functionalized microgel particles conjugated with antibody and/or phage tailspike, wherein at least one of the particles is configured to entrap microbes under an electric field trapping method; and
c) one or more optical detection systems, wherein at least one of the detection systems is configured to interact with at least a portion of the sample to allow detection of the analyte under an optical detector.
5) The method of claim 4, wherein one or more detection systems comprise one or more particle-based detection systems, wherein at least one of the particle-based detection systems is coupled to electric field based interdigitated electrodes.
6) The method of claim 4, wherein the detection system comprises culture independent diagnosis of analyte in a sample.
7) The method of claim 4, wherein at least one of the microgel particles comprises a receptor molecule coupled to a fluorescent polymeric microgel beads, and wherein the particle further comprises a first indicator (fluorescence), the first indicators being coupled to the receptor, and wherein interaction of the receptor with the analyte causes the first indicator to produce the fluorescent signal.
8) The method of claim 4, wherein the functionalized fluorescent microgel particles conjugated with antibody/tailspike protein used for targeted bacterial attachment, wherein microgel-targeted bacterial particles are pulled-down using an external electric field.
9) The method of claim 4, wherein the one or more sample droplets comprise a substance selected from the group consisting of whole blood, lymphatic fluids, serum, plasma, seminal fluids, vaginal excretions, serous fluids, synovial fluids, pericardial fluids, sweat, tear, saliva, sputum, cerebrospinal fluids, amniotic fluids, peritoneal fluids, pleural fluids, transudates, exudates, cystic fluids, bile, urine, gastric fluids, intestinal fluids, fecal samples, fluidized tissues, fluidized organisms, biological swabs and biological washes, wherein the method is used to identify different types of cells and/or microbes and/or pathogens.
10) A method for assessing the status of a disease, condition, pathology, or cell state, wherein the disease, condition, pathology, or cell state is associated with the presence or level of a cellular component, the method comprising the steps of detecting and/or isolating or removing the cellular component associated with the disease, condition, pathology, or cell state by a method according to any of claims 4 to 9, and correlating the presence or level of the cellular component with disease, condition, pathology, or cell state.