The present disclosure pertains to a biosensor device. In particular the present disclosure relates to a biosensor device and a kit for identifying microorganisms in a given sample for DNA-based detection of infection and antimicrobial resistance in a shorter time duration within 2 hours.

BACKGROUND
[0002] Infection is a critical factor in many patients suffering from other ailments. Infection needs to be accurately identified and treated when the infection occurs especially in immune compromised patients. However, infection being a major challenge in managing patients suffering from other diseases, physicians do straightaway start prophylactic antibiotics in most of the cases due to lack of time because all the existing modalities are very tedious, and time consuming. Current detection techniques are time consuming like microbial culture, polymerase chain reaction (PCR), sequencing, next generation sequencing (NGS), matrix-assisted laser desorption/ionization (MALDI), are very complex in terms of experimental and instrumental aspects, and are human labour and expenditure intensive. Hence, not only the test results for detection of infection or antimicrobial resistance are not available soon, but also costs of such testing are high for a patient. Further, such tools and techniques mostly can provide either qualitative or quantitative results.
[0003] In view of the above, it is evident that there is an urgent requirement of providing a means for detection of infection detection and antibiotic resistance which can overcome one or more shortcomings of the existing modalities, in terms of giving qualitative and quantitative test results, in simple, cost-effective and instantaneous manner.

OBJECTS OF THE PRESENT DISCLOSURE
[0004] Some of the objects of the present disclosure, which at least one embodiment herein satisfies are as listed herein below.
[0005] It is an object of the present disclosure to provide a biosensor device for identifying microorganism(s) in a biological sample.
[0006] It is an object of the present disclosure to provide a biosensor device for identifying microorganism(s) in a given sample for detecting infection and antimicrobial resistance.
[0007] It is another object of the present disclosure to provide a portable device for identifying microorganism(s) in a given sample in DNA/plasmid DNA -based detection of an infection and antimicrobial resistance simultaneously in a shorter duration (less than 2 hour).
[0008] It is an object of the present disclosure to provide a biosensor device with high sensitivity to presence of microorganism that cause infection and/or antibiotic resistant bacterial cells.
[0009] It is an object of the present disclosure to provide a biosensor device capable of identifying infection causing and/or antibiotic resistant bacterial cells in a given sample in qualitative and quantitative manner.
[0010] It is an object of the present disclosure to provide a biosensor device for identifying infection causing and/or antibiotic resistant bacterial cells in a given sample with specificity for DNA based detection of infection, and their antibiotic resistance.
[0011] It is an object of the present disclosure to provide DNA biosensor device with a simpler set-up for identifying infection causing and/or antibiotic resistant bacterial cells in a given sample in cost effective manner.
[0012] It is an object of the present disclosure to provide a biosensor device that can be used as a point of care device for fast detection of infection or antibiotic resistance to enable physician to arrive at an immediate and appropriate antibiotic treatment regimen.

SUMMARY
[0013] The present disclosure relates to a portable bio-sensor device for identifying a microorganism in a sample for DNA-based detection of an infection and antimicrobial resistance. The device can include housing for enclosing one or more components of the device and a sensor cabinet, wherein said sensor cabinet can include a receptacle portion for receiving a sensor including one or more working electrodes capable of oxidation-reduction process. In an embodiment, the sensor can be configured to receive the biological sample and provide a surface for immobilization of a set of hybridization probes that form a three-dimensional complex upon independent hybridization with a target nucleic acid sequence of a gene corresponding to the microorganism in the biological sample to be identified, wherein the sensor upon being placed in the sensor cabinet enables detection of the three-dimensional complex by the oxidation-reduction process that enables generation of an electrical signal measurable by the device that may facilitate the identification of the microorganism in the biological sample in DNA-based detection of any or a combination of an infection and an antimicrobial resistance caused by the microorganism.
[0014] In an embodiment, the set of hybridization probes can include a capture probe and a detector probe, wherein the capture probe can include a first oligonucleotide sequence that may be a single-stranded oligonucleotide tagged with a conjugating agent including biotin. The detector probe can include a second oligonucleotide sequence that may be a single-stranded oligonucleotide tagged with at least one fluorescent compound. In an embodiment, each of the first oligonucleotide sequence and the second oligonucleotide sequence can be single-stranded oligonucleotide complimentary to each strand of a pair of strands in the target nucleic acid sequence. In an embodiment, each of the first oligonucleotide sequence and the second oligonucleotide sequence may be capable of independently hybridizing with each strand of the pair of strands of target nucleic acid sequence to form the three-dimensional complex, wherein the target nucleic acid sequence may be obtained after cell lysis of the microorganism
[0015] In an embodiment, the Biosensor can further include a potentiostat arrangement that can include at least one counter electrode and at least one reference electrode that are coupled to the working electrodes for measurement of the electrical signal in form of dataset including measurement of voltage and current associated with the oxidation-reduction process. In an embodiment, the housing can be cuboid-shaped and the device can include a display screen to indicate the generation data. In an embodiment, the device can include a thermal printer for printing the generated data.
[0016] In an embodiment, the first oligonucleotide sequence may be a single-stranded oligonucleotide tagged with a conjugating agent at 5-’ or 3’- end of the oligonucleotide. In an exemplary embodiment, the conjugating agent may be biotin.
[0017] In an embodiment, the one or more working electrodes may be carbon-based electrodes printed on a cellulosic substrate. In an exemplary embodiment, the cellulosic substrate may be paper.
[0018] In an embodiment, the one or more electrodes may be functionalized by coating with at least one protein compound selected from streptavidin and avidin, wherein the capture probe can be localized on the one or more electrodes by conjugation interaction between the protein and the conjugating agent.
[0019] In an embodiment, the second oligonucleotide sequence can be a single-stranded oligonucleotide tagged with at least one fluorescent compound at 5-’ or 3’- end of the oligonucleotide. In an exemplary embodiment, the fluorescent compound can be fluorescein.
[0020] In an embodiment, the device can detect the microorganism that may include microbial strain selected from wild type strain, a pathogenic strain, an antibiotic resistant bacterial strain, a multidrug resistant bacterial strain, an extreme-drug-resistant strain, , and pan drug-resistant strain.
[0021] In an exemplary embodiment, the detection of the sample for identifying the microorganism can be done in a time duration in the range of 10 mins to 120 mins, wherein the sample may be biological fluid can be selected from blood, urine and other biological fluids of a body.
[0022] In an embodiment, the detection of the formation of the three-dimensional complex can be done by using one or more reagents selected from any or a combination of anti-fluorescein monoclonal Fab fragment, horseradish peroxidase, a buffer, reagents for cell lysis and detection reagents.
[0023] In an embodiment, the formation of the three-dimensional complex can be carried out inside an acrylamide cassette, wherein the sensor is placed inside the cassette to enable hybridization between the probes and the target nucleic acid sequence on the surface of the sensor.
[0024] In another aspect, the present disclosure provides an acrylamide cassette for accommodating one or more sensors as described hereinabove, for enabling hybridization experiment.
[0025] In another aspect, the present disclosure provides a kit for identifying a microorganism in a sample for DNA-based detection. The kit can include a set of hybridization probes and one or more reagents selected from any or a combination of streptavidin, biotin, anti-fluorescein monoclonal Fab fragment, horseradish peroxidase, a buffer, reagents for cell lysis and detection reagents. The set of hybridization probes can include capture probes and detector probes complementary to and capable of hybridizing to a target nucleic acid sequences of a gene for identification of infection causing and drug resistant strain(s) of the microorganism.
[0026] The device, cassette and kit of the present disclosure provide a convenient, rapid and cost-effective way of identifying a microorganism in a sample for DNA-based detection of any or a combination of the infection and the antimicrobial resistance.
[0027] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF DRAWINGS
[0028] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
[0029] The diagrams are for illustration only, which thus is not a limitation of the present disclosure, and wherein:
[0030] FIG. 1 illustrates an exemplary biosensor device 100 in accordance with an embodiment of the present disclosure.
[0031] FIG. 2 illustrates an exemplary cassette of a kit, in accordance with an embodiment of the present disclosure.
[0032] FIG. 3 illustrates a voltammetry data using biosensor device, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION
[0033] The following is a detailed description of embodiments of the present disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0034] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0035] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0036] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0037] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
[0038] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[0039] All methods described herein can be performed in suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0040] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0041] Various terms are used herein. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0042] The oxidation-reduction potential means the standard potential of an atom or ion that undergoes oxidation at the anode or reduction at the cathode in an electrochemical cell as compared to the redox potential of a standard carbon-based printed electrodes
[0043] The present disclosure relates to a bio-sensor device for identifying a microorganism in a sample for DNA or plasmid DNA-based detection of any or a combination of an infection and an antimicrobial resistance. The device can be designed and customized for detecting a wide range of microorganisms causing the infection (gram positive, TB, sepsis, fungal, viral, hospital acquired). In an embodiment, the device can detect a microbial strain selected from wild type strain, a pathogenic strain, an antibiotic resistant bacterial strain, a multidrug resistant bacterial strain, an extreme-drug-resistant strain, and pan drug-resistant strain. The device of the present disclosure can be an effective alternative for most of the existing modalities in term of time, experimental-expenditure, human labor and cost for infection detection and antibiotic resistance to obtain the results in a qualitative and quantitative manner.
[0044] As per an embodiment of the present disclosure, the bio-sensor device can include a housing for enclosing one or more components of the device and a sensor cabinet. The sensor cabinet can include a receptacle portion for receiving a sensor including one or more working electrodes capable of oxidation-reduction process. In an embodiment, the sensor can be configured to receive the biological sample and provide a surface for immobilization of a set of hybridization probes that form a three-dimensional complex upon independent hybridization with a target nucleic acid sequence of a gene corresponding to the microorganism in the biological sample to be identified, wherein the sensor upon being placed in the sensor cabinet may enable detection of the three-dimensional complex by the oxidation-reduction process that enables generation of an electrical signal measurable by the device that may facilitate the identification of the microorganism in the biological sample in DNA-based detection of any or a combination of an infection and an antimicrobial resistance caused by the microorganism.
[0045] In an embodiment, the set of hybridization probes can include a capture probe and a detector probe. The capture probe can include a first oligonucleotide sequence that may be a single-stranded oligonucleotide. The first oligonucleotide sequence may be tagged with a conjugating agent so that the capture probe can be localised on the one or more electrodes. The conjugating agent may be any agent capable of conjugating with a protein. In an exemplary embodiment, the conjugating agent can be biotin that may be tagged at 5-’ or 3’- end of the first oligonucleotide.
[0046] In an embodiment, the detector probe can include a second oligonucleotide sequence that may be a single-stranded oligonucleotide. The second oligonucleotide sequence may be tagged with at least one fluorescent compound or marker. In an exemplary embodiment, the fluorescent compound can be fluorescein that may be tagged at 5-’ or 3’- end of the second oligonucleotide.
[0047] In an embodiment, the one or more working electrodes may be functionalized by coating with at least one protein compound. The functionalized protein on the electrodes may provide a platform for localization of the capture probe on the electrodes. In an embodiment, the electrodes can be a carbon-based electrode printed on a cellulosic substrate. In an exemplary embodiment, the cellulosic substrate can be paper. In an exemplary embodiment, the functionalization may be done with proteins selected from streptavidin and avidin. These protein compounds have extraordinarily high affinity for conjugating compounds like biotin and hence can provide a good localization of the capture probes on the electrodes.
[0048] In an embodiment, the sensor can further include a potentiostat arrangement that can include at least, one working electrode, one counter electrode and at least one reference electrode. The counter and reference electrodes can be coupled to the working electrodes for measurement of the electrical signal in form of dataset including measurement of voltage and current associated with the oxidation-reduction process. In an exemplary embodiment, the housing can be cuboid-shaped which enables space for the one or more components of the device.
[0049] In an embodiment, the device can include a display screen to indicate the generated data. The display screen can be any one selected from LCD screen or LED screen. The housing can further enclose a printed circuit board for various electrical connections between the one or more components. In an embodiment, the device can include a thermal printer for printing the generated data. FIG. 1 illustrates an exemplary biosensor device 100 in accordance with an embodiment of the present disclosure. The portable device 100 of FIG. 1 can include a housing 112 having an upper surface 112a. The housing 112 can be cuboid shaped as shown in FIG. 1. The device 100 can include a sensor cabinet 114 including a receptacle portion 102 to receive one or more sensors including working electrodes capable of oxidation-reduction process. The sensor can be configured to receive the biological sample and provide a surface for immobilization of a set of hybridization probes that form a three-dimensional complex upon independent hybridization with a target nucleic acid sequence of a gene corresponding to the microorganism in the biological sample to be identified, wherein the sensor upon being placed in the sensor cabinet 114 enables detection of the three-dimensional complex by measurement of an electrical signal due to the oxidation-reduction process at the working electrodes, wherein the electrical signal may be measurable by the device that may facilitate the identification of the microorganism in the biological sample in DNA-based detection of any or a combination of an infection and an antimicrobial resistance caused by the microorganism capture probe and a detector probe. The cabinet can enable to secure the sensor within the housing for detection purpose, wherein the sensor cabinet can also avoid spillage of any sample/reagents during experiment and enables to keep the surface steady for obtaining good precision. The sensor can include a potentiostat arrangement including at least one counter electrode and at least one reference electrode that are coupled to the working electrodes for measurement of the electrical signal in form of dataset including measurement of voltage and current associated with the oxidation-reduction process. The device 100 can include a LED display screen 104 to view the generated data and a thermal printer 106 to print the generated date to give immediate results.
[0050] Based on the requirements, the display screen can be used to display one or more aspects of the analysis i.e. for result description, for displaying the graph and excel data of species specific infection and drug resistance as well as other details of the analysis. In one embodiment, multiple onboard LED may be present to enable the indication/display of results. The thermal printers 106 may enable to obtain quick printed form of data/results to the physician and patient, especially in remote rural areas wherein getting access to such facilities may be challenging and practically difficult. The data can also be transferred to a laptop or computer via a USB port 108. The device 100 can also have a power button 110 for charging/connecting to a power source. In an embodiment, the device 100 can also be powered with a rechargeable battery. In addition to the components shown, the device 100 can also have a control panel which can provide control to one or more menu buttons or switch on/off option.
[0051] The device can include a printed circuit board (PCB) wherein the PCB can be placed within the housing. The PCB can include LCD headers, potentiostat connections and for counter electrode (CE), reference electrode and working electrode respectively. In an exemplary embodiment, the PCB dimensions can be 100 x 60 mm whereas the complete device dimension can be 150 x 100 x 20 mm, although the embodiments of the present invention are not limited by the mentioned size/dimensions. The PCB can also include connection points for power connectivity and USB port respectively. The PCB can include mounting holes for mounting the PCB within housing of the device. Thus the device is a complete and robust solution which can detect microbial infection and drug resistance and provide immediate result onscreen, which can also be physically available in terms of paper using thermal printer. Further, the biosensor DNA based technology can enable detection of infection and antibiotic resistance in less than 2 hours. The portable device can be used for infection drug resistance, which can be used to detect any infection i.e. hospital acquired infection, Viral infection, TB infection, sepsis and fungal infection and the like. Using specific capture and detector probe for wild strains, pathogenic strain, microbial infection in blood sample, urine sample and other biological fluid of body, it is possible to obtain fast, accurate and cost-effective detection of the microorganism. Using specific gene of accessory genome from genomic DNA or plasmid, antimicrobial resistance in blood and urine sample and other biological fluid of body can be known easily and effectively. Thus, the device is unique and a practically viable solution for detecting infection and antimicrobial resistance based on paper based microfabrication technology, which create can enable high sensitivity due to complex formed by double hybridization using specific probes.
[0052] In an embodiment, each of the first oligonucleotide and the second oligonucleotide can be single-stranded oligonucleotide complimentary to each strand of a pair of strands in a target nucleic acid sequence of a gene corresponding to the microorganism to be identified. Upon cell lysis of the microorganism, the target nucleic acid sequence may be accessed for independent hybridization with the detector probe and the capture probe. In one embodiment, upon cell lysis, the detector probe may be hybridized with one strand of the pair of strands of the target nucleic acid sequence to form a target nucleic acid-detector probe complex, followed by hybridization of the capture probe with another strand of the pair of strands of the target nucleic acid sequence to form a target nucleic acid-capture probe-detector probe complex, which may be a three-dimensional complex between the target nucleic acid, the capture probe and the detector probe.
[0053] In an embodiment, formation of three-dimensional complex can be detected by the one or more electrodes by the oxidation-reduction process that enables generation of an electrical signal for identifying the microorganism in the sample in DNA-based detection of the infection and antimicrobial resistance. In an embodiment, the detection of the formation of the three-dimensional complex can be done by using one or more reagents selected from any or a combination of anti-fluorescein monoclonal Fab fragment, horseradish peroxidase, a buffer, reagent for cell lysis and detection reagents. The reagent anti-fluorescein monoclonal Fab fragment may be used for detection of fluorescein-labelled compounds whereas reagent horseradish peroxidase is a metalloenzyme that can catalyse the oxidation of various organic substrates. This reagent may be added after formation of the three dimensional complex, wherein the anti-fluorescein monoclonal Fab fragment may interact with the fluorescein of the detector probe and enable detection of the three-dimensional complex. The reagent horseradish peroxidase may enable to promote the oxidation and reduction process to generate the electrical signal that may be measured using one or known techniques. Thus, these reagents may enable cell-lysis and/or measurement of one or more attributes related to qualitative and/or quantitative analysis of the three-dimensional complex for effective identification of the type of the microorganism and their quantitative analysis.
[0054] In certain embodiments the present disclosure provides a capture probe and a detector probe specific to a target nucleic acid sequence of a specific gene(s) for identifying a pathogenic bacteria type bacteria; a capture probe and a detector probe specific to virulent nucleic acid sequence of a specific gene, for identifying an antibiotic resistant bacterial strain, a multidrug resistant bacterial strain, an extensively drug-resistant strain; or pan drug-resistant strain, a capture probe and a detector probe specific to drug resistant nucleic acid of a specific gene was used. The set of capture probe and detector probe non-specific to any nucleic acid is used as a positive control (universal probe positive for gram negative and gram-positive bacteria)). Based on the type of oligonucleotide being used in the capture probe and the detector probe, the type of microorganism may be identified. The formation of the three-dimensional complex may be confirmed by detection using one or more methods that can enable measurement of electrical signal due to formation of the complex, wherein the three-dimensional complex formation may depend on the nature of the target nucleic acid sequence and thus provide a qualitative data on the type of microorganism being detected.
[0055] Based on the measurement, the current output can provide an estimate of hybridization between capture probe, target nucleic acid sequence and the detector probe, wherein the result can indicate number of bacterial cells in the sample that may be directly proportional to current output. In an embodiment, the current output may be measured by using voltammetry. In an exemplary embodiment, a potential of ±2.5V may be applied for a time duration in the range of 30 seconds to 1.5 minutes with scan rate of 0.1 second to obtain the current output that may provide a quantitative idea regarding the number of bacterial cells in the sample.
[0056] In an embodiment, the detection of the sample for identifying the microorganism may be done in a time duration in the range of 1 min to 120 mins. In an exemplary embodiment, the detection may be done in a time period of less than 2 hours. In another exemplary embodiment, the sample may be detected for identification of the microorganism in the range of 30 seconds to 1.5 minutes. The sample may be a biological fluid selected from blood, urine and other biological fluids of a body. The device of the present disclosure may enable a detection limit in the range of 101 to 1010 CFU/ml in a sample such as blood and urine samples. The device may be very specific due to its gene specific probes hybridization to the target sequence.
[0057] In another aspect, the present disclosure provides a kit for identifying a microorganism in a sample for DNA based detection of an infection and antimicrobial resistance. The kit can include a set of hybridization probes and one or more reagents selected from any or a combination of streptavidin, biotin, anti-fluorescein monoclonal Fab fragment, horseradish peroxidase, a buffer, reagents for cell lysis and detection reagents. The set of hybridization probes can include capture probes and detector probes complementary to and capable of hybridizing to a target nucleic acid sequences of a gene for identification of infection causing and drug resistant strain(s) of the microorganism selected from a wild type strain, an infection causing strain and an antibiotic resistant microorganisms.
[0058] In an embodiment, the formation of the three-dimensional complex may be carried out inside an acrylamide cassette, wherein the sensor is placed inside the cassette to enable hybridization between the probes and the target nucleic acid sequence on the surface of the sensor. FIG. 2 illustrates an exemplary cassette, in accordance with an embodiment of the present disclosure. . In an embodiment, the length of the cassette can be in the range of 15 cm to 20 cm and the breadth can be in the range of 5 cm to 10 cm. In an exemplary embodiment, the length and width of the cassette can be 18 cm and 7 cm, respectively. The bottom of cassette can be made up of opaque white colour acrylamide material and the cover of the cassette can be made up of transparent acrylamide material for better visibility and transparency. The cassette can have a panel having plurality of columns in each panel, wherein the space between two adjacent columns can be in the range of 0.1 cm to 0.6 cm, so that solution of each sensor will not spill out on each other. The length, width and depth of each column can be in the range of 1cm to 5 cm. In an exemplary embodiment, the space between two adjacent column can be 0.5 cm, and the width, length and depth of each can be 1.5 cm, 5 cm and 1 cm respectively as shown in fig 2. is the cassette can have a good heat resistance, temperature tolerance >650 C such that hybridization can take place in the cassette within an incubator. The screen-printed electrode can tend to be very thin and slippery on surface, and hence the surface can be made comparatively rough for ease of retrieval after the completion of experiment.
[0059] In an embodiment the hybridization between the fluorescein tagged single stranded oligonucleotide detector probe with the target nucleic acid sequence may be done at 65 0 C in incubator. The method in accordance with the present disclosure may be carried out by adopting cyclic voltammetry principle for measuring the reduction potential of a microbial species in the reaction solution. In an embodiment, the suitable equipment to measure the oxidation-reduction response may be portable potentiometer, which being, a low cost equipment can add to the cost-effectiveness of the testing.
[0060] In an embodiment the hybridization may be carried out by applying current to the electrodes. The potential applied can be of ±2.5V for about 30 seconds to 1.5 minutes, preferably from about 1 minute to about 1.5 minutes with a scan rate of 0.1 second to about 0.5 seconds. The current output may impart threshold of hybridization between capture probe-target nucleic acid sequence-detector probe. The result may be generated in format of number of bacterial cells in the sample directly proportional to current output using voltammetry.
[0061] The localization of probes, and double hybridization between capture probe, target nucleic acid sequence and detector probe provide three-dimensional structure on screen printed electrode acting as a DNA biosensor, and thereby result in profound sensitivity in detecting infection causing and antibiotic resistant microorganisms and thereby enables detection of infection and antibiotic or multidrug resistance. The method in accordance with the present invention is capable of detecting infection causing and antibiotic resistant microorganisms within 1 minute to 120 minutes.
[0062] The device and kit in accordance with the present disclosure is capable of detecting microbial cells from 101 to 10 10 CFU/ml from a given sample, the sample preferably being blood and urine samples or any other biological body fluid. The device and kit of the present disclosure may be capable of providing results in terms of number of bacterial cells causing infection and antibiotic resistant in the sample, are accordingly more sensitive up-to >98% as compared to the conventional culture tools up-to 26% and method used to detect infection. Further, due to ability to identify specific bacterial cells due to specificity to target nucleic acid, the device, kit and the method of the present disclosure are contemplated to be superior to the existing PCR and sequencing tools and techniques. The device, and kit provided in accordance with the present disclosure can be used by a physician to prescribe specific antibiotic course to patients who are suffering from infection based on the output received. The device and kit of the present disclosure can also be used by a physician to identify infection in each and every disease where it can be used as monitoring tool to treat infection, thus providing faster and cost-effective analysis that can overcome the disadvantages of the conventional systems.
[0063] The present disclosure is further explained in the form of following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
Example-1 - Biosensor device (experimental details of sample preparation and measurement)
We developed 6 step quick technology based biosensor to detect infection and antimicrobial resistance using paper-based portable device to detect infection and antibiotic resistance in form of reduction current during oxidation reduction reaction. it will complete within 2 hour and the steps are as follows.
Experiment was done inside the cassette, as shown in FIG.2. First of all, screen-printed electrodes (SPE) was placed inside the acrylamide cassette and then the following steps were followed:
Step-1: functionalization of screen-printed electrodes (SPE) or sensors was done with streptavidin
Step-2: Immobilization of capture probe was done on the SPE
Step-3: A biological sample containing human urine (or blood sample) was taken from an acute leukaemia patient. The urine sample was used in undiluted form whereas a blood sample was diluted with water in 1:1 ratio before usage. Initially, the sample was centrifuged at 14000 rpm for 15 minutes at room temperature to obtain a sedimented portion/pellet of DNA/plasmid DNA which was used for further analysis whereas the supernatant was discarded. Further, a 350µL cocktail mixture was prepared for adding to the pellet obtained from the urine sample for carrying out cell lysis of the gram negative bacterium to be detected. The cocktail mixture included 1 molar sodium hydroxide solution, 10% Tween+ 20mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl), 1 mM EDTA, Lysozyme (10mg/ml) in 10mM tris HCL, wherein the volume ratio of all the five ingredients was 1:1:1:0.5:0.25. Upon adding the cocktail mixture to the pellet obtained by the centrifugation of the urine sample, the urine sample released target nucleic acid sequence after cell lysis. The resultant mixture was centrifuged at 14000 rpm for 15 minutes and the resultant supernatant solution was taken and allowed for first hybridization between released DNA or Plasmid DNA and detector probe takes place at 65 °C in incubator for 10 minutes.
Step-4: Second hybridization between capture probe, DNA/Plasmid and detector probe was done inside a cool 4 °C box takes place at 65 °C in incubator for 15 minutes to form a three dimensional complex.
Step-5: Addition of anti-fluorescein monoclonal Fab fragment was done
Step -6: The sensor (with the 3-D complex) was taken out from cassette and placed into the sensor cabinet present within the housing (cuboid box) of the device (FIG.1), where electrical connection for electrodes (Working, reference and counter) was already available. This was followed by addition of detection reagents on the sensor and reading of the sample on test instrument/device.
Using specific capture and detector probe for wild strains, pathogenic strain, microbial infection and antimicrobial resistance was reported in blood and urine sample and other biological fluid of body.
The sensitivity of the sensor was the result of location of probes hybridization. The current out-put gave an indication of the threshold of hybridization between capture probe-target sequence -and detector probe. The result was obtained in format of no. of bacterial cells in blood/urine directly proportional to current output. Potential of ±2.5 was applied for 30 sec to 1.5 minutes with scan rate of 0.1 second. Detection limit of our sensor was in the range from 101 to 10 10 CFU/ml from blood and urine samples. This paper based sensor was very specific due to its gene specific probes hybridization to the target sequence.
The sensor device was used to detect a biological sample such as urine (undiluted form) or blood (dilution with water in 1:1 ratio). Using the present device, better sensitivity in data was obtained. As showed in table-1, existing test methods have limitations, i.e. optical density (OD) measurement can predict growth only in a range up to 101 to 103 and hence for further downline experiments, samples need to be diluted for culture purpose. While culture can be sensitive method, but still if when we observe it closely, culture results can be read only between 103 to 106CFU (colony forming unit), and hence it is difficult to measure beyond CFU value of 107 and moreover it take almost 2-3 days’ time, to finish the whole experiment using conventional methods. So overall sensitivity of existing gold standard method culture was found to be 24-26% whereas the present disclosures >98% sensitive. The device of the present disclosure can give results up to 101-10 CFU within 2 hours so it quite sensitive and fast in compare to existing modalities. Moreover, the device does not require high manpower or skill-set for usage. you can directly take blood or urine, process it on our sensor, using our technology, you will get gene specific bacterial infection and antibiotic resistance results in 90 minutes.

Table-1: Comparison of conventional techniques with the present device

[0064]
[0065]

Using the device, detection result was obtained in format of no. of bacterial cells in blood/urine that were directly proportional to current output using portable tailored potentiostat for POC device for sensor technology. Potential of ±2.5V was applied for 30 seconds to 1.5 minutes with scan rate of 0.1 second to obtain the voltammetry data as shown FIG. 3, wherein the presence of reduction current indicated detection/presence of microorganism whereas the type of probes for which the results tested positive gave confirmation on the type of microorganism thereby enabling identification of the microorganism, which in case of FIG. 3 related experiment was E. Coli. The detection limit of the sensor in the device was found to be 101 to 10 10CFU/ml from blood and urine samples. The paper based sensor was very specific due to its gene specific probes hybridization to the target sequence.
The workable range of the present device is as illustrated in Table 2:

Table 2: Workable range of the present device
Output Range
Applied DC potential range ±2.5V
Current range 1-700µA
DC Potential resolution 0.8mV
DC Potential accuracy 0.08% of the scale
Result analysis time 30 sec-90 sec

[0064] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.

ADVANTAGES OF THE PRESENT DISCLOSURE
[0065] The present disclosure provides a biosensor device that can identify infection causing and antibiotic resistant bacterial strain simultaneously in a given sample with >98% sensitivity.
[0066] The present disclosure provides a biosensor device capable of DNA-based detection of infection and antibiotic resistance in shorter duration for example less than 2 hours.
[0067] The present disclosure provides a device that can detect infection causing and antibiotic resistant bacterial strain with high sensitivity, specificity and in cost effective manner.
[0068] The present disclosure provides a kit comprising cassette structure for holding multiple screen printed electrodes for wide range application
[0069] The present disclosure provides a biosensor device with a simpler set-up can identification of infection causing and antibiotic resistant bacterial strain in bulk samples detection of infection, or antibiotic resistance, or both.
[0070] The present disclosure provides a biosensor device that can provide the result in qualitative and quantitative terms.
[0071] The present disclosure provides a biosensor device and kit that can be used as a point of care device for fast detection of infection and/or antibiotic resistance to enable an immediate and appropriate treatment regimen.
[0072] The present disclosure provides a biosensor device an kit that can be used by a physician to identify any infection where patient is suffering only from that particular infection or infection in a patient suffering from other diseases.
[0073] The present disclosure provides a biosensor device and kit that can be used by a physician as a monitoring tool to treat infection and antibiotic resistance.

We Claim:
1. A portable bio-sensor device for identifying a microorganism in a biological sample for DNA-based detection, said device comprising:
a housing for enclosing one or more components of the device; and
a sensor cabinet provided within the housing, wherein said sensor cabinet comprises a receptacle portion for receiving a sensor comprising one or more working electrodes capable of oxidation-reduction process;
wherein the sensor is configured to receive the biological sample and provide a surface for immobilization of a set of hybridization probes that form a three-dimensional complex upon independent hybridization with a target nucleic acid sequence of a gene corresponding to the microorganism in the biological sample to be identified, and
wherein the sensor upon being placed in the sensor cabinet enables detection of the three-dimensional complex by the oxidation-reduction process that generates an electrical signal measurable by said device that facilitates the identification of the microorganism in the biological sample in DNA-based detection of any or a combination of an infection and an antimicrobial resistance caused by the microorganism.
2. The device as claimed in claim 1, wherein the set of hybridization probes comprise a capture probe and a detector probe, wherein said capture probe comprises a first oligonucleotide sequence that is a single-stranded oligonucleotide tagged with a conjugating agent, wherein the detector probe comprises a second oligonucleotide sequence that is a single-stranded oligonucleotide tagged with at least one fluorescent compound,
wherein each of the first oligonucleotide sequence and the second oligonucleotide sequence are single-stranded oligonucleotides that are complimentary to each strand of a pair of strands in said target nucleic acid sequence,
wherein each of the first oligonucleotide sequence and the second oligonucleotide sequence are capable of independently hybridizing with each strand of said pair of strands of target nucleic acid sequence to form said three-dimensional complex, and wherein said target nucleic acid sequence is obtained after cell lysis of the microorganism.
3. The device as claimed in claim 1, wherein the sensor further comprises a potentiostat arrangement that comprises at least one counter electrode and at least one reference electrode that are coupled to the working electrodes for measurement of the electrical signal in form of dataset including measurement of voltage and current associated with the oxidation-reduction process.
4. The device as claimed in claim 1, wherein the housing is cuboid-shaped, wherein the device comprises a display screen to indicate the generation data, wherein the device comprises a thermal printer for printing the generated data, and wherein the one or more working electrodes are carbon-based electrodes printed on a cellulosic substrate, wherein the cellulosic substrate is paper..
5. The device as claimed in claim 1, wherein said first probe is a single-stranded oligonucleotide tagged with a conjugating agent at 5-’ or 3’- end of the oligonucleotide, and wherein said conjugating agent is biotin, wherein said second probe is a single-stranded oligonucleotide tagged with at least one fluorescent compound at 5-’ or 3’- end of the oligonucleotide, and wherein said fluorescent compound is fluorescein.
6. The device as claimed in claim 1, wherein the one or more working electrodes are functionalized by coating with at least one protein compound selected from streptavidin and avidin, and wherein said capture probe is localized on said one or more electrodes by conjugation interaction between said protein and the conjugating agent, wherein the detection of the sample for identifying the microorganism is done in a time duration in the range of 1 minute to 120 minutes, wherein the biological fluid is selected from blood, urine and other biological fluids of a body.
7. The device as claimed in claim 1, wherein the device detects the microorganism that is a microbial strain selected from wild type strain, a pathogenic strain, an antibiotic resistant bacterial strain, a multidrug resistant bacterial strain, a extreme drug-resistant strain, and pan drug-resistant strain.
8. The device as claimed in claim 1, wherein the detection of the formation of the three-dimensional complex is done by using one or more reagents selected from any or a combination of anti-fluorescein monoclonal Fab fragment, horseradish peroxidase, a buffer, reagents for cell lysis and detection reagents,
wherein the formation of the three-dimensional complex is carried out inside an acrylamide cassette, wherein the sensor is placed inside the cassette to enable hybridization between the probes and the target nucleic acid sequence on the surface of the sensor.
9. An acrylamide based cassette for accommodating one or more sensors as claimed in claim 1, for enabling hybridization experiment.
10. A kit for identifying a microorganism in a sample for DNA-based detection of an infection and antimicrobial resistance, said kit comprising.
a set of hybridization probes comprising one or more capture probes and one or more detector probes complementary to and capable of hybridizing to a target nucleic acid sequences of a gene for identification of infection causing and drug resistant strain(s) of the microorganism; and
one or more reagents selected from any or a combination of streptavidin, biotin, anti-fluorescein monoclonal Fab fragment, horseradish peroxidase, a buffer, reagents for cell lysis and detection reagents.