Description:FIELD OF THE INVENTION:
The present invention relates to a portable point-of-care testing device, process thereof and a method for to rapid detection of microorganisms. More particularly, present invention relates to an affordable, handheld and portable point-of-care testing device using highly sensitive and selective electrochemical impedimetric biosensors to rapidly detect bacteria such as E. coli in clinical settings and real samples such as tap water, urine and other biological fluids.

BACKGROUND OF THE INVENTION:
Infections caused by anti-infective resistant microorganisms or infectious agents are a significant problem for healthcare professionals in hospitals, nursing homes, and other healthcare environments. Rapid detection of the susceptibility of such infectious agents to antibiotics or other anti-infectives is crucial in order to prevent the spread of their resistance profiles.

Patient samples, such as blood samples, urine sample, are the primary biological starting point, for assessing the etiology of a patient's disease and determining the appropriate therapy course for treating that disease. Initiating the proper therapeutic treatment of a critically ill patient at the appropriate dosage regimen as soon as possible is the priority and can be achieved only after analysis of patient samples at the earliest. The historically weak link in this process is sufficient cultivation of a microbial population to enable identification of pathogen(s) present and to determine which antimicrobial compounds the pathogen(s) will respond to in therapy. Reducing the assay time required to and assess their drug sensitivity is crucial to improving patient survival odds.
In many instances, patient samples contain multiple types of microorganisms, such as mixtures of bacteria from differing genera, species, and even strains and known as "polymicrobial" samples.

Existing methods and instruments for identification of microorganisms from clinical specimens typically require overnight subculturing to isolate individual species prior to biochemical assay-based identification, followed by growing isolated organisms in the presence of various antimicrobials to determine susceptibilities. Therefore, existing methods to prepare patient samples are time-intensive (e.g., up to 24-48 hours), and requires trained technicians and sophisticated instruments for analysis. However, those methods often require manual interpretation by skilled personnel and are prone to technical or clinician error. In addition, certain biological samples harboring infectious agents, such as samples containing animal or human blood, are often difficult to assess using prevailing optical techniques given the samples' opacity. Moreover, such optical techniques often require expensive equipment.

Therefore, more rapid, sensitive and cost-effective methods are highly desired. The use of rapid detection systems, electrochemical biosensors have proven to be a promising way to detect bacteria due to their merits of simple fabrication, rapid detection and low cost. Considering the crucial effects of a sensing surface on the performance of a biosensor, many researchers have focused on the development of bacterial biosensors.

US application no. US20060228738A1 titled as “DNA-polypyrrole based biosensors for rapid detection of microorganisms” discloses a DNA-polypyrrole based biosensor and methods of using the biosensor for the rapid detection of dangerous microorganisms for monitoring water quality of a sample from a drinking water or food source.

Another US patent application no. 62/268,340 titled as “Instrument and system for rapid microorganism identification and antimicrobial agent susceptibility testing” discloses automated, microscopy-based method that can perform bacterial/yeast identification in about one (1) hour and antimicrobial susceptibility testing in about five (5) or fewer hours directly from clinical specimens which is also quite time consuming.

As a result of the above limitations, there is a need for rapid, sensitive and cost-effective device for bacterial identification in a polymicrobial samples and quantification of bacterial cells in order to increase productivity and to reduce human error.

Accordingly, the present invention discloses a rapid, accurate, cost effective, handheld, and portable point-of-care testing device, process thereof and a method to detect Gram-negative bacteria such as E. coli in a polymicrobial samples using highly sensitive and selective electrochemical impedimetric biosensors and detection of pathogenic bacteria in real samples such as tap water, urine and other biological fluids in less than 30 minutes.

OBJECT OF THE INVENTION:
The main object of the present invention is to provide a portable point-of-care testing device,process thereof and a method for rapid detection of microorganisms.

Another object of the present invention is to provide a portable point-of-care testing device, process thereof and a method for rapid detection of pathogenic bacteria in a polymicrobial samples using highly sensitive and selective electrochemical impedimetric biosensors and in real samples such as tap water, urine and other biological fluids.

Yet another object of the invention is to provide a process to obtain a portable point-of-care testing device to detect gram-negative bacteria such as E. coli.

Yet another object of the invention is to provide a process to obtain a novel colistin passivated carbon dots (CCD) which is highly selective for Gram negative bacteria E. coli.

Yet another object of the present invention is to provide a handheld, portable, rapid, accurate, cost-effective, and automated bacterial detection device for quantification of bacterial cells.

Yet another object of the present invention is to provide a handheld and portable point-of-care testing device that would require less than 30 minutes in order to increase productivity and to reduce human error.

SUMMARY OF THE INVENTION:
The present invention provides a portable point-of-care testing device, process thereof and method for rapid detection of microorganisms. More particularly, present invention provides a portable point-of-care testing device using highly sensitive and selective electrochemical impedimetric biosensors to detect pathogenic bacteria such as E. coli in in a polymicrobial samples and in real samples such as tap water, urine and other biological fluids.

The conventional method for bacterial detection involves techniques like cell culturing, which takes about 48 hrs. It also requires trained technicians and sophisticated instruments for analysis.

The handheld and portable bacterial device of the present invention requires only less than 30 minutes for quantification of bacterial cells, identification of gram-negative bacteria such as E. coli cells in a polymicrobial samples and detection of pathogenic bacteria in real samples such as tap water, urine and other biological fluids.

The present invention further caters the need of the medical sciences by providing a cost-effective, accurate bacterial detection device in order to increase productivity and to reduce human error, therefore enables the medical professionals to initiate the proper therapeutic treatment of a critically ill patient at the earliest based on the rapid identification of the disease-causing micro-organisms.

The present invention addresses the problems of the requirement of skilled personnel for diagnostic testing, automation of conventional bacterial detection and quantification of bacterial cells and therefore provides a handheld, portable, rapid, accurate, cost-effective, and automated bacterial detection device.

Thus, the present invention provides for a portable point-of-care testing device is comprising of a non-disposable electronic module comprising an electronic meter and a disposable module comprising of a microfluidic chip. The microfluidic chip comprises micromixers, microvalves, sample well, washing buffer well, reagent well, impedimetric biosensor and redox mediator. The impedimetric biosensor is obtained by synthesizing colistin derived carbon dots coated with agarose polymer immobilized on disposable screen-printed carbon electrodes which is capable of identification of bacterial cells in polymicrobial samples, quantification of bacterial cells and detection of pathogenic bacteria in real samples thereby eliminating the need for extensive analysis by an expert.

BRIEF DESCRIPTION OF THE DRAWINGS:
Fig. 1 depicts the device for detection of Gram -negative bacteria E.Coli using Colistin Carbon Dots (CCD) immobilized disposable impedimetric sensors

Fig. 2 depicts the process for the ultrasensitive detection of E. coli using Colistin Carbon Dots (CCD) immobilized disposable impedimetric sensors

Fig. 3A depicts UV-Vis absorption spectrum of CCD with corresponding photoluminescence (PL) emission at 456 nm (inset a: photographs of CCD under ambient light (transparent) and under UV- 360 nm lamp (bright blue) inset b: A significant Stoke’s shift between UV absorption peak at 350 nm and corresponding PL emission peak at 456 nm

Fig. 3B depicts PL emission spectra of CCD with different excitation wavelength

Fig. 3C depicts Fourier transform infrared (FTIR) spectra of colistin precursor, and CCD and Fig. 3D depicts XPS wide scan spectrum of CCD

Fig. 4A, 4B and 4C depicts the de-convoluted XPS spectrum of C 1s, N 1s, and O 1s respectively to observe the presence of several functional groups on the surface of CCD

Fig. 5A depicts Transmission Electron Microscopy (TEM) micrograph of monodispersed spherical CCD particles (Inset: fringes pattern exhibiting measured interlayer distance of 0.2 nm),

Fig. 5B depicts Histogram plot for measuring average size of forty CCD particles

Fig. 5C depicts Selected Area Electron Diffraction (SAED) pattern of CCD showing diffuse rings corresponding to the amorphous nature

Fig. 6 depicts PL emission spectrum of CCD tagged E. coli and S. aureus cells at 350 nm excitation wavelength

Fig. 7A and 7B depicts the Fluorescence microscopic images of CCD tagged E. coli and S. aureus cells respectively at different excitation filters (λex ~ 365 and 470 nm) with overlay images

Fig. 8 depicts the FTIR characterization of Agr/CCD@SPCE to verify the presence of CCD within the agarose polymer scaffold by analyzing the presence of specific functional groups

Fig. 9A depicts the Bright light (top) and UV lamp (bottom) images of agarose only and Fig. 9B depicts the Transparent agarose intercalated CCD under bright light (top) and fluorescing under UV lamp (bottom)
Fig. 10A and 10B depicts the Electrochemical response obtained at the different stages of sensor fabrication with and without the presence of E. coli cyclic voltammogram and Nyquist plot respectively

Fig. 11A, 11D depicts systematic morphological characterization using Field Emission Scanning Electron Microscopy (FESEM) image acquisition of Bare electrode in absence and presence of E. coli, Fig. 11B, 11E depicts Agr@SPCE in absence and presence of E. coli and Fig. 11C, 11F depicts Agr/CCD@SPCE in absence and presence of E. coli respectively.

Fig. 12A, 12B and 12C depicts elemental mapping of bare electrode, Agr@SPCE, and Agr/CCD@SPCE respectively for confirmation of immobilization of CCD within agarose scaffold, Fig. 12D, 12E and 12F depicts Atomic force microscopy (AFM) images of bare electrode, Agr@SPCE and Agr/CCD@SPCE respectively

Fig. 13A and 13B depicts scanning electron microscope (SEM) images of Agr/CCD@SPCE with E. coli and S. aureus respectively

Fig. 14 depicts Nyquist plot of Agr/CCD@SPCE in the presence of various E. coli concentrations ranges from 102 – 108 CFU mL-1, (inset) linear plot based on the triplicate measurement and equivalent circuit model used for the data analysis

Fig. 15A and 15B depicts the selectivity of Agr/CCD@SPCE - Electrochemical Impedance Spectroscopy (EIS) response of different micro-organisms at 108 CFU mL-1 bacterial concentration (inset: histogram of Agr/CCD@SPCE response for different bacteria) and polymicrobial mixture of all the four micro-organisms with only the concentration of E. coli varying from zero to 107 CFU mL-1 (inset: histogram of Agr/CCD@SPCE response for different concentration of E. coli in polymicrobial solution) respectively

Fig. 16 depicts stability assessment of Agr/CCD@SPCE by measuring the response of the sensor from different modified electrodes stored for a different time period.

DETAILED DESCRIPTION OF THE INVENTION:
The present invention provides a portable point-of-care testing device, process thereof and a method for rapid detection of microorganisms. More particularly, present invention provides a portable point-of-care testing device using highly sensitive and selective electrochemical impedimetric biosensors to detect Gram-negative bacteria such as E. coli in a polymicrobial samples, and detection of pathogenic bacteria in real samples such as tap water, urine and other biological fluids.

Pathogens are infectious agents that cause disease. Some of the most common pathogens include viruses, fungi, protozoans and bacteria, such as E. coli and S. aureus.

Rapid and sensitive detection of pathogens such as bacteria is crucial in order to prevent the spread of their resistance profiles and has received considerable critical attention. In many instances, patient samples contain multiple types of microorganisms, such as mixtures of bacteria from differing genera, species, and even strains and known as "polymicrobial" samples.

Initiating the proper therapeutic treatment of a critically ill patient at the appropriate dosage regimen as soon as possible is the priority and can be achieved only after analysis of patient samples at the earliest. Colony counting and the polymerase chain reaction have proven promising methods for the highly sensitive and reliable determination of bacteria. However, to culture and count a colony always needs 1-2 days, which is time consuming. The conventional method for bacterial detection involves techniques like cell culturing, which takes about 48 hrs. It also requires trained technicians and sophisticated instruments for analysis. Therefore, more rapid, sensitive and cost-effective methods are highly desired.

Biosensors have the advantages of simplicity, specificity, low detection limit, simple operation, being inexpensive, easy to use, providing real-time measurement, capability of multitarget testing and automation, portability, miniaturization, and rapid detection.

The use of electrochemical biosensor has been disclosed in the present invention to meet the demand for an accurate and efficient means of detection of microorganism. Electrochemical biosensor of the present invention provides sensitive detection, rapid data acquisition, and relatively simple analytical procedures at an economical cost for effective detection of microorganisms.

Accordingly, the present invention discloses a novel, facile, label-free, and a cost-effective impedimetric sensor for the detection of E. coli using colistin carbon dots (CCD) immobilized agarose dried films on screen-printed carbon electrodes (Agr/CCD@SPCE).

The present invention is supported by non-limiting experimental data as detailed below. Various experiments have been conducted and result have been obtained showing the interaction of E.coli with the modified electrode using cyclic voltammetry, electrochemical impedance spectroscopy, and scanning electron microscopy.

Thus, the present invention provides for a portable point-of-care testing device is comprising of a nondisposable electronic module comprising an electronic meter and a disposable module comprising of a microfluidic chip. The microfluidic chip comprises micromixers (M), microvalves (V1, V2… Vn), sample well (W1), dilution buffer well (W2) containing buffer including but not limited to phosphate buffer saline PBS, Citrate buffer, Phosphate buffer and washing buffer well (W3) containing buffer including but not limited to phosphate buffer saline PBS, Citrate buffer, Phosphate buffer, deionized water), a redox reagent well (W4) well containing a solution including but not limited to potassium chloride and potassium ferricyanide, hexamine Ruthenium(II)chloride), and impedimetric biosensor (Sensor). The impedimetric biosensor is obtained by synthesizing colistin derived carbon dots coated with agarose polymer immobilized on disposable screen-printed carbon electrodes which is capable of identification of bacterial cells in polymicrobial samples, quantification of bacterial cells and detection of pathogenic bacteria in real samples thereby eliminating the need for extensive analysis by an expert. Specificity of the detection of the Gram-negative bacteria in the present invention is achieved by colistin capping on the carbon dots.

The device of the present invention for detection of Gram-negative bacteria such as E. coli using Colistin Carbon Dots (CCD) immobilized disposable impedimetric sensors is shown in Fig. 1.
The sample and the buffer flow through the micromixer, when a negative pressure is applied and reaches the sensor well, which contains the electrode. A potential is applied for the preconcentration of bacterial cells onto the sensor electrode. This is followed by the sequential delivery of the washing buffer and the final redox mediator for the electrochemical analysis. The controlled delivery of fluids is achieved by using microvalves. An indigenously developed electronics meter module perform both voltammetry for preconcentration and impedance bacterial detection. The microfluidic system integrated with the Agr/CCD@SPCE and the electronic meter display the quantified bacterial cell count, thereby eliminating the need for extensive analysis by an expert. The process for the ultrasensitive detection of E. coli using Colistin Carbon Dots (CCD) immobilized disposable impedimetric sensors of the present invention is shown in Fig. 2.

The complete automated analysis of the present invention is based on the microcontroller programme. Hence no user intervention other than the introduction of the sample. The fluid flow is controlled using the microvalves (piezoelectric and solenoid type) operate based on the programmed microcontroller. This achieves definite time for mixing, incubation, preconcentration etc. Micromixers are the meander microchannels with alternately varying diameters for enhanced mixing.

A process to obtain the novel device of the present invention that amalgamates electrochemical impedimetric spectroscopy, microfluidics, and microelectronics to detect Gram-negative bacteria comprising of:
• synthesizing a novel colistin passivated carbon dots (CCD) by a one-step microwave-assisted method using colistin, citric acid and ethylenediamine as chemical precursors,
• entrapping synthesized said novel colistin passivated carbon dots (CCD) within an agarose polymer matrix and drop-casted over disposable screen-printed carbon electrodes (Agr/CCD@SPCE) to obtain a impedimetric biosensor,
• The developed impedimetric biosensors then incorporated in a microfluidic chip for the automated point-of-care testing of the present invention.

The clean and dried films of Agr/CCD@SPCE have capability of quantification of bacterial cells, identification of Gram-negative bacteria such as E. coli cells in a polymicrobial samples and detection of pathogenic bacteria in real samples such as tap water, urine and other biological fluids.

Hydrothermal synthesis of CCD
Colistin-based carbon dots of the present invention synthesized by using simple one-step pyrolysis of citric acid (50 mg), ethylenediamine (EDA, 20 µL), and colistin (10 mg mL-1) at 180 ºC for 1 hour. The citric acid acts as an efficient carbon source and EDA provides nitrogen (N) atoms as a dopant to enhance fluorescence emissions. Colistin, also known as Polymixin E, is a polypeptide antibiotic and works efficiently against Gram-negative bacteria such as E. coli, Psedumonas, Klebsiella, and many more. The pyrolyzed CCD exhibited brown colour charred mass and diluted in 10 mL MilliQ water. The CCD solution then centrifuged at 12100 rpm for 60 minutes to filter out the large particles present in the solution. This is the first step of purification. The supernatant then collected carefully and further purified with a syringe filter (0.22 micron). The CCD further purified by dialysis using a 1 kDa Molecular weight cut-off (MWCO) membrane to filter the ionic impurities. The pale yellow colour purified solution then obtained and used for the analysis.
Fabrication of screen-printed electrodes
Indigenously fabricated, screen-printed electrodes with three-electrode configurations as working electrode, carbon, and Ag/AgCl as counter and reference electrode respectively used for the electrochemical analysis. The PET sheets cleaned with acetone before screen printing and pre-heated at 90 ºC for 30 minutes. A base layer of carbon ink and Ag/AgCl reference electrode was printed sequentially using an automated screen printer. The carbon ink was dried at 80 ºC for 30 minutes, and the Ag/AgCl layer was dried at 80 ºC for 15 minutes. The screen-printed carbon electrodes (SPCE) thus fabricated has the area of the working electrode as 3.14 mm2 (r = 1 mm).

Preparation of Agr/CCD dried film and the fabrication of Agr/CCD@SPCE biosensor
The agarose solution (1%) prepared in Milli Q water and standardized for drop-casting over the surface of SPCE. The agarose solution heated carefully to dissolve the agarose precipitate in water. The agarose solution uniformly mixed with CCD in a 1:1 volume ratio by quick and vigorous vortexing. The homogenous solution (5 µL) of Agr/CCD drop-casted over the clean working electrode surface. The Agr/CCD mixture was allowed to dry at room temperature for 4-5 hours. The dried film firmly adhering to the surface of the electrodes and found to be stable enough even after dipping into the electrolyte solution. Various electrochemical and morphological characterizations confirmed the uniformity of the developed electrode (Agr/CCD@SPCE).

Microscopic characterization of Agr/CCD@SPCE
The microscopic validation of each modification step in the present invention was performed using an Atomic Force Microscope (AFM, XE-70) and Scanning Electron Microscope (SEM, Carl Zeiss). The images of bare SPCE, Agarose drop-casted SPCE, and Agr/CCD@SPCE acquired at different magnifications and scan size with better resolution. AFM images acquired in non-contact mode within 6 µm x 6 µm scan area. Similarly, to acquire images at higher magnification and better resolution, SEM images were snapped at different magnifications. All the images were taken at 30.00 K magnification for each step of the modifications.

EIS experimental parameters
All the EIS experiments of the present invention were carried out on electrochemical analyser (608D, CH instruments) using Agr/CCD modified SPCE. E. coli cells were pre-concentrated at the Agr/CCD@SPCE in 0.1 M PBS buffer (pH 7.4) at 0.5 V for 200 seconds. After pre-concentration, the cyclic voltammetry (CV) and EIS measurements performed using 5 mM K4[Fe(CN)6] and 1.5 M KCl solution as a supporting electrolyte. The impedance experiments were carried out in a frequency range from 1×105 to 0.01 Hz, with AC amplitude of 5 mV. The equivalent circuit and values of charge transfer resistance (Rct) were deciphered by using simulation software.

E. coli detection and quantification using Agr/CCD@SPCE
The real-time monitoring and detection of E. coli of the present invention obtained by using Agr/CCD@SPCE for different concentrations ranging from 102 to 108 CFU mL-1. New electrodes were used for all the concentrations, and Rct values were obtained directly from corresponding Nyquist plots, and equivalent circuits were modeled using simulation software. The impedimetric detection assay duration was 20 mins. The detection and quantification data obtained from Nyquist plots for different bacteria concentrations statistically analyzed and represented accordingly.

Selectivity assay of Agr/CCD@SPCE in polymicrobial samples
Different bacterial strains electrochemically tested, and the acquired values analyzed to validate the selectivity of the Agr/CCD@SPCE. The bacterial strains used are E. coli, S. aureus, P. vulgaris, and M. smegmatis at the concentration of 108 CFU mL-1. Individual Agr/CCD@SPCE used to measure the impedimetric response for all the bacterial strains to confirm the selectivity of the sensor in a polymicrobial solution. The Rct values for the other bacterial strains were significantly different from the E. coli response. The polymicrobial solution used in the present invention with the known concentration of 105 CFU mL-1 bacteria viz; S. aureus, P. vulgaris, and M. smegmatis and varying concentrations of E. coli. The specificity of the modified electrodes tested with the variations in the impedimetric response.

The application of the modified Agr/CCD@SPCE of the present invention also shows commercial viability by performing the experiments using tap water and urine sample spiked with different concentrations of E. coli to screen the efficiency of the modified electrodes for the real samples. Fresh tap water and urine samples were procured and spiked with different concentrations of E. coli cells (103, 105, and 107 CFU mL-1). There was no pre-processing required for both samples. The impedimetric responses for the different E. coli concentrations measured along with the control (un-spiked).

Spectroscopic characterization of CCD
The synthesized CCD of the present invention by the hydrothermal method characterized thoroughly using various spectroscopic techniques such as UV-Vis absorption, photoluminescence (PL), IR, and XPS spectroscopy. The homogenous solution of CCD was transparent and pale yellow in daylight but bright blue under UV lamp, as shown in Fig. 3A (inset a).
The UV-Vis absorption spectrum exhibited two peaks (black curve in Fig. 3A). The feeble peak at 240 nm was attributed to the π → π* transition due to the presence of sp2 aromatic carbon. The intense second peak at 350 nm is assigned to n → π* transition and indicates the bulk availability of lone pair of electrons on nitrogen and C=O functional groups at the surface of CCD. The PL emission spectrum exhibits the highest emission peak (λem) at 456 nm corresponding to 350 nm excitation (λex). The significant Stoke's shift between maximum excitation and emission peak was measured as 106 nm (Fig. 3A, inset b). The PL emission scan, as shown in Fig. 3B, was carried out from 300 to 500 nm excitation wavelength to obtain an insight into the luminescence behaviour of CCD. A redshift was observed while scanning from lower to higher excitation wavelength with a reduction in PL emission intensity.

A weak emission was noticed towards the longer end, due to which the intensity of multicolour emission was continuously decreased from blue to red. The subdued green and red emission could be due to the dominating blue emissive fluorophores known as (1, 2, 3, 5- tetrahydro-5-oxo-imidazo [1, 2-a] pyridine-7-carboxylic acid, IPCA.

These fluorophores are formed due to condensation and dehydration reactions between citric acid and amine at low temperatures (140-150 οC) to form highly reactive amide bonds. As the temperature increases beyond 150 οC, carbon dots formation with carbogenic core takes place with high PL emission intensity.

Chemical composition analysis of CCD
The chemical composition of CCD of the present invention was investigated and confirmed by using FTIR and XPS. The FTIR spectrum of the precursors and CCD were analyzed to confirm the presence of colistin moiety on the surface of synthesized carbon dots. The FTIR spectrum of CCD exhibits peaks at 1689, 1639 and 1551 cm-1 as shown in Fig. 3C. The small peak at 1689 cm-1 corresponding to the stretching vibration showed by C=O functional group. This explains the amide bond formation on the surface of the carbon dot. The small peak at 1639 cm-1 attributed to the stretching vibrations of C=C. The peak at 1551 cm-1 corresponds to N-H bending vibrations. Therefore, the formation of an amide bond confirms the passivation of colistin over the surface of CCD. The wide scan XPS analysis results exhibit three prominent peaks correspond to the presence of carbon, nitrogen, and oxygen at 284, 399, and 530 eV, respectively, on the surface of CCD as shown in Fig. 3D. The atomic percentage is 71.46% for C 1 s (maximum), 18.39% for O 1 s, and 10.15% for N 1 s, respectively. The de-convoluted XPS spectrum for carbon, nitrogen, and oxygen shows peaks at 285.11, 397.45, 398.82, and 530.01 eV and confirms the presence of -C-O, -C=O, -C=N, and -NH2 functional groups. The XPS spectrum of C 1s (Fig. 4A) was de-convoluted to three peaks with corresponding binding energies at 282.07 eV for sp2 carbon and 283.21 eV for C-N 285.11 eV for C-H & C-O functional groups. The high-resolution spectrum of N 1s (Fig. 4B) was de-convoluted to three peaks at binding energy 396.66, 397.45, and 398.82 eV and ascribed to N-H, C=N and NH2 functional groups. The de-convoluted high-resolution spectrum for O 1s (Fig. 4C) shows two peaks at 528.51 and 530.01 eV which was attributed to physically adsorbed oxygen and organic C=O functional group. The XPS data revealed the surface functionalities of CCD which was also confirmed with the FTIR data.

Morphological analysis of CCD
The morphological analysis of CCD of the present invention was carried out by using High Resolution Transmission Electron Microscopy (HR-TEM). The micrograph displayed several near-spherical and mono-dispersed CCD with size ranges from 5-6 nm as shown in Fig. 5A. The inset of Fig. 5A shows the definite fringe patterns with measured d-spacing as 0.20 nm, corresponding to the (1 0 0) lattice plane of graphite. The average diameter of forty CCD particles was 5.41 ± 0.22 nm as shown in Fig. 5B. This attributed to highly disordered carbon atoms and confirms the availability of graphitic carbon atoms arranged in disordered planes.

The selected area electron diffraction (SAED) pattern as shown in Fig. 5C attained from TEM projects the diffused rings or halo devoid of any spots. This suggests the amorphous characteristic of the synthesized CCD.

Fluorescence spectrophotometric analysis of CCD tagged E. coli and S. aureus cells
The fluorescence spectroscopy results of CCD-tagged E. coli and S. aureus cells are also analyzed and exhibited in Fig. 6. The specific nature of CCD observed by labeling with Gram-positive (S. aureus) and Gram-negative bacteria (E. coli). The PL emission was observed at 458 nm, but the fluorescence intensity was highest for CCD-tagged E. coli cells than S. aureus cells. This justifies the specific nature of the synthesized fluorescent CCD towards E. coli because of the specific nature of polycationic colistin moiety of CCD towards Gram-negative cells.

Fluorescence microscopic analysis of CCD tagged E. coli and S. aureus cells
The fluorescence microscopic investigation of CCD-tagged E. coli and S. aureus bacterial cells is shown in Fig. 7A & 7B. The tagging was accomplished by mixing CCD solution with bacterial cells followed by incubation for 90 minutes at 37 ºC. Post incubation, the solution was washed twice with MilliQ water, and the sample was fixed on a glass slide for image acquisition. The CCD tagged E. coli and S. aureus cells were imaged separately under different excitation filters. The images show blue and green emission when excited at 365 and 470 nm, but there was no red emission observed at 545 nm excitation.

FTIR analysis of Agr/CCD@SPCE
The involvement of various functional groups on each step of electrode fabrication was characterized using IR spectroscopy (Fig. 8). The blue curve represents the IR spectra of Agr@SPCE; it shows the presence of different functional groups such as O-H, C-H, and C-O. Apart from the different peaks observed in Agr@SPCE, the incorporation of colistin into the agarose film introduced a small peak of N-H bending vibrations at 1556 cm-1 and a dual peak at 3375, and 3259 cm-1 corresponds to N-H and O-H stretching vibrations. This shows the successful immobilization of CCD within dried agarose scaffold and its availability for sensing application. The daylight and UV lamp images of CCD immobilized/embedded within the agarose scaffold have been shown in Fig. 9A & 9B.

Electrochemical characterization of Agr/CCD@SPCE
The systematic modification of the Agr/CCD@SPCE sensor characterized by EIS and cyclic voltammetry (CV) to confirm the sensor construction. The CV has been used to infer the redox behaviour as a function of input voltage, and EIS was deployed as a sensitive tool to study the charge transfer across the electrode-electrolyte interface.

The CV and EIS responses show the systematic modification of electrodes with and without the presence of bacteria. The overnight grown bacterial culture was washed twice and prepared in fresh 0.1 M PBS (pH 7.4). The bare and modified electrodes were pre-concentrated with a definite volume of bacterial samples. Since the bacterial cells are negatively charged, the pre-concentration parameters were defined accordingly. The applied voltage was set to +0.5 V for 200 seconds. The positive potential resulted in attracting negatively charged bacteria towards the surface of electrodes. Immediately after pre-concentration, the electrodes were subjected to CV and EIS in ferricyanide solution. The CV was carried out at a potential window of +0.9 V to -0.2 V at a scan rate of 0.05 Vs-1.

The CV response for different modifications is as follows;
a. bare electrode exhibited a cyclic voltammogram with definite oxidation and reduction peaks. The pre-concentration of E. coli on the bare electrode exhibits a drop in redox peak current due to the insulating nature of bacterial cells.
b. After drop-casting agarose over the working electrode surface (Agr@SPCE), the magnitude of oxidation and reduction peaks decreased further. This showed the diffusion of the redox mediator was thwarted towards the electrode surface. The bacterial pre-concentration results in further dip in the peak current values.
c. The final modification includes the drop-casting of CCD and agarose mix over the surface of the working electrode. The notable decline in oxidation and reduction of peak current values was evident. This could be due to the electrostatic repulsion experienced between the negatively charged CCD and [Fe(CN)6]3− and hence block the electron transfer on the electrode surface (Fig. 10A).

Furthermore, the pre-concentrations with a bacterial solution exhibited the lowest peak current values. This is due to the plenty of bacterial cells attached to Agr/CCD@SPCE. The exposed polycationic colistin moiety on the carbon dots selectively binds with a large number of E. coli cells resulting in negligible redox peak current.

EIS was also performed to study the electron transfer behaviour of the modified electrodes at each step, and the results are presented as Nyquist plots (Fig. 10B). A significant change in Rct values was observed at every modification step. a). The Rct value of the bare electrode was low (21700 Ω) due to the facile electron transfer across the electrode-electrolyte interface. The pre-concentration of bare SPCE with E. coli cells resulted in a subdued electron transfer. Hence, the Rct value increased, b) the Agr@SPCE electrode offered higher resistance in electron transfer (Rct ~ 26100), due to which the faradic current reduced. Rct value increased and c) on immobilizing CCD using agarose matrix of agarose polymer due to which the electron transfer was obstructed and hence, the Rct value increases further (as per the mechanism explained in CV). A remarkable increase in charge transfer resistance from 29500 to 1,32,000 Ω was observed after the pre-concentration of bacterial cells on the Agr/CCD@SPCE. This is attributed to the binding of a substantial number of E. coli cells with the polycationic colistin moiety of CCD intercalated in an agarose scaffold.

Morphological characterization of Agr/CCD@SPCE
The modified SPCE characterized by FESEM and non-contact mode AFM to analyze the morphological changes. FESEM imaging was carried out at 30 K magnifications to achieved sharp images with a clear resolution of the bare electrode and agarose coated electrodes (Agr@SPCE) and Agr/CCD@SPCE as shown in Fig. 11A, 11B & 11C.
The SEM images of bare SPCE electrode, Agr@SPCE and Agr/CCD@SPCE in the presence of E. coli is demonstrated in Fig. 11D, 11E & 11F. The SEM image of bare electrode did not show any affinity towards E. coli, and hence none of the cells were attached on the surface as shown in Fig. 11D. The Agr@SPCE shown a few E. coli cells embedded into the agarose scaffold/ matrix. The property of agarose dried films to swell in the presence of liquid solvent may allow a smaller number of E. coli cells to hold on the surface of the electrode, as shown in Fig. 11E. The Agr/CCD@SPCE has shown a greater affinity towards E. coli cells due to the presence of colistin-modified CD. The higher number of E. coli cells are attached on the modified electrode surface as shown in Fig. 11F, and therefore, the higher impedance response was obtained during electrochemical characterization of Agr/CCD@SPCE.

The elemental mapping showed the presence of carbon only as shown in Fig. 12A. The Agr@SPCE was imaged at a similar magnification, and a uniform coating was observed having a smooth surface with no irregularities. The elemental mapping projects the presence of carbon and oxygen only with different colours, as displayed in Fig. 12B. The final modification i.e., Agr/CCD@SPCE, was imaged under similar image acquisition parameters. The presence of CCD within the agarose matrix was verified by elemental mapping. This was achieved by projecting elements such as carbon, oxygen and nitrogen with different colours, as shown in Fig. 12C.

The morphological characterization of Agr/CCD@SPCE was also carried out by AFM to analyze the surface topography and roughness. The AFM images of the bare electrode, agarose coated SPCE, and Agr/CCD@SPCE were snapped at a scan rate of 0.5 Hz with the scan area of 6µ X 6µ as shown in Fig. 12D, 12E & 12F. Post imaging, the processed images were analyzed using AFM software to realize the variation in roughness quotient in each modification. It has been observed that the roughness quotient was decreasing from the bare electrode to agarose coated SPCE to Agr/CCD@SPCE. The substantial dip in roughness value from the bare electrode to agarose coated SPCE is because of the polymer coating. The vacant spaces of the agarose matrix are filled by CCD after introducing carbon dots within the polymer scaffold. This perhaps resulted in further reduction of roughness value and confirmed the presence of CCD within agarose dried films.

The SEM images of E. coli and S. aureus binding to the surface of Agr/CCD@SPCE has shown in Fig. 13A & 13B. The images clearly demonstrate the higher affinity of the modified electrodes towards E. coli as compare to S. aureus.

Quantitative detection of E. coli using Agr/CCD@SPCE sensor
The analytical detection of E. coli was performed using Agr/CCD@SPCE in a standardized experimental set-up. The samples with different E. coli concentrations (102 - 108 CFU mL-1) were prepared by serial dilution of the stock culture using 0.1 M PBS and pre-concentrated on the modified Agr/CCD@SPCE electrode. After pre-concentration, the Rct values were measured using EIS. Fig. 14 represents the linear increase in the impedance with the bacterial concentration ranges from 102 - 108 CFU mL-1. The linear increase in impedance can be attributed to the hindrance in electron transfer across the electrode-electrolyte interface offered by the bacteria attached to the electrode surface. The bacteria cells were quantified using Agr/CCD@SPCE in triplicates to ensure the repeatability of the optimized system. The inset in Fig. 14 shows the Rct values versus logarithmic concentration of E. coli cells (102 - 108 CFU mL-1) and exhibited a linear relationship. The equation of linear regression is as follows: Rct = 14085 CFU mL-1 +12951.26 and R-square value = 0.98. The detection limit (S/N = 3) of Agr/CCD@SPCE was calculated as 1.57 CFU mL-1, and the quantification limit was 102 CFU mL-1 with a pre-concentration step.

Selectivity of Agr/CCD@SPCE towards E. coli in a polymicrobial solution
The selectivity of the Agr/CCD@SPCE of the present invention observed in two phases: i) The EIS response of the sensor electrode to different micro-organisms (Escherichia coli, Staphylococcus aureus, Proteus vulgaris and Mycobacterium smegmatis) were measured separately in triplicates as shown in Fig. 15A. The bacterial concentration for all four microbes was fixed at 108 CFU mL-1. The measured EIS exhibits a higher response for E. coli in comparison to other microbes. As disclosed above, the higher Rct values for E. coli were attributed to the large number of bacteria attached to the electrode surface. The low Rct values for S. aureus and M. smegmatis can be explained based on the subdued interaction between colistin moiety of CCD and Gram-positive bacteria due to the absence of LPS membrane. Surprisingly, the Rct values for P. vulgaris were much lower than a typical response from Gram-negative bacteria as in E. coli) This is because the P. vulgaris exhibited natural resistance against colistin and hence, the lower interaction displayed between colistin and P. vulgaris. The impedance measurements were carried out in a polymicrobial mixture of all four micro-organisms. The polymicrobial mixture was prepared by using a varying concentration of E. coli from zero to 107 CFU mL-1 while keeping the concentration of all the other microbes as 105 CFU mL-1. The Rct of the Agr/CCD@SPCE was changing according to the change in E. coli concentrations as displayed in Fig. 15B. This is due to the selective nature of the developed sensor towards E. coli provided by the colistin moiety of CCD.
Storage stability of Agr/CCD@SPCE
The shelf life of Agr/CCD@SPCE sensors of the present invention monitored over a period of 32 days, as shown in Fig. 16. The electrodes were fabricated under similar conditions and stored at room temperature, and impedance measurements were carried out at a regular interval of 8 days. The experiment was performed to measure the repeatable impedimetric response of Agr/CCD@SPCE. The impedimetric response of the developed electrode was estimated using freshly prepared E. coli cells (105 CFU mL-1). The measured response was found to be precise and stable when tested on days 1, 8, 16, 24 & 32. This data indicates the potential long-term stability of Agr/CCD@SPCE without any special requirement for the storage of the electrodes.

Table 1 shows the percentage recovery of different concentrations of E. coli spiked tap water and urine samples obtained using Agr/CCD@SPCE. Various ions and minerals present in tap water and urine did not influence the quantification of E. coli. This indicates the sensitivity, specificity, and efficiency of the fabricated sensor for detecting E. coli in various real samples.

Sample Spiked concentration (CFU mL-1) Measured concentration (CFU mL-1) Recovery (%)
Tap water 8 7.84 98.0
5 4.62 92.4
3 2.75 91.6
Urine 8 6.73 84.1
5 4.83 96.6
3 2.72 90.6

Table 1 Percentage recovery estimation of E. coli cells in tap water and urine

Accordingly, the present invention discloses colistin functionalized carbon dots immobilized agarose dried films on SPCE (Agr/CCD@SPCE) to fabricate impedimetric sensors for the ultrasensitive detection of E. coli bacteria. The electrochemical and microscopic characterization of Agr/CCD@SPCE affirms the immobilization of CCD on SPCE. The impedimetric response of Agr/CCD@SPCE in the presence and absence of E. coli suggest the sensitive approach of the developed sensor. The morphological studies were carried out using SEM, and the results complemented the electrochemical characterization of Agr/CCD@SPCE.

The quantification assay shows a broad range of bacterial concentrations (102-108 CFU mL-1) having linear regression, R2 = 0.98, and the detection limit obtained as low as 1.57 CFU mL-1. The EIS response of Agr/CCD@SPCE was observed in a polymicrobial solution and shows highly selective for E. coli in the presence of other microbes such as S. aureus, P. vulgaris, and M. smegmatis. The selective nature of Agr/CCD@SPCE is due to colistin-modified carbon dots and cross-validated by the fluorescence microscopic studies using CCD-tagged bacterial cells. The sensor performance also observed with real samples such as tap water, human urine and other biological fluids with a sensitive response towards different concentrations of E. coli. The shelf life of Agr/CCD@SPCE also observed for 32 days, and the response of the sensor was constant throughout the storage span for a particular concentration of E. coli cells. This shows the sensor stability of the present invention is excellent.

The handheld and portable point-of-care testing device of the present invention using highly sensitive and selective electrochemical impedimetric biosensors requires only less than 30 minutes for quantification of bacterial cells, identification of gram-negative bacteria such as E. coli cells in a polymicrobial samples and detection of pathogenic bacteria in real samples such as tap water, urine and other biological fluids.

The present invention further caters the need of the medical sciences by providing a cost-effective, accurate bacterial detection device in order to increase productivity and to reduce human error, therefore enables the medical professionals to initiate the proper therapeutic treatment of a critically ill patient at the earliest based on the rapid identification of the disease-causing micro-organisms.

The device of present invention has potential applications in hospitals and clinical laboratories, enabling doctors to identify the disease-causing bacteria quickly. The biosensors of the present invention have capability to detect other bacteria such as Gram-positive bacteria also by adopting the specific antibiotic in place of colistin.

The present invention addresses the problems of the requirement of skilled personnel for diagnostic testing, automation of conventional bacterial detection and quantification of bacterial cells and provides a handheld, portable, rapid, accurate, cost-effective, and automated bacterial detection device.
, Claims:1. A portable point-of-care testing device is comprising of:
• a nondisposable electronic module comprising an electronic meter and
• a disposable module comprising of a microfluidic chip
wherein said microfluidic chip comprises at least one micromixer (M), at least one microvalve (V1, V2…. Vn), at least one sample well (W1), at least one dilution buffer well (W2), at least one washing buffer well (W3), at least one redox reagent well (W4), at least one impedimetric biosensor (Sensor),
wherein said impedimetric biosensor (Sensor) is obtained by synthesizing colistin derived carbon dots (CCD) coated with agarose polymer immobilized on disposable screen-printed carbon electrodes,
wherein said impedimetric biosensor (Sensor) is capable of identification of bacterial cells in polymicrobial samples, quantification of bacterial cells and detection of pathogenic bacteria in real samples such as tap water, urine and other biological fluids thereby eliminating the need for extensive analysis by an expert.

2. The portable point-of-care testing device as claimed in claim 1, wherein said CCD synthesis comprising the steps of:
• pyrolysis of citric acid, ethylenediamine, and colistin at 180 ºC for 1 hour to obtain a pyrolyzed CCD,
• diluting said pyrolyzed CCD in MilliQ water and centrifuging at 12100 rpm for 60 minutes,
• collecting supernatant and purify with a syringe filter
• dialysis of CCD using a 1 kDa Molecular weight cut-off (MWCO) membrane to filter the ionic impurities to obtain purified CCD.

3. The portable point-of-care testing device as claimed in claim 1, wherein said bacteria is selected from group of Gram-negative bacteria, Gram-positive bacteria.

4. The portable point-of-care testing device as claimed in claim 3, wherein said Gram-negative bacteria is Escherichia coli.

5. The portable point-of-care testing device as claimed in claim 1, wherein said device is rapid, accurate, cost-effective, and automated bacterial detection.

6. The portable point-of-care testing device as claimed in claim 1, wherein said device detects or identify said bacteria within 30 minutes.

7. The portable point-of-care testing device as claimed in claim 1, wherein said microvalves are V1, V2 and V3, said W2 well contains dilution buffer including but not limited to phosphate buffer saline PBS, Citrate buffer, Phosphate buffer and said W3 well contains washing buffer including but not limited to phosphate buffer saline PBS, Citrate buffer, Phosphate buffer, deionized water and said W4 redox reagent well contain a solution including but not limited of potassium chloride and potassium ferricyanide, hexamine Ruthenium(II)chloride).

8. The portable point-of-care testing device as claimed in claim 1, wherein said device uses highly sensitive and selective electrochemical impedimetric biosensors to detect Gram-negative bacteria such as E. coli in clinical settings.

9. The portable point-of-care testing device as claimed in claim 1, wherein said device used for the quantification assay with a broad range of bacterial concentrations (102-108 CFU mL-1) having linear regression, R2 = 0.98, and the detection limit low as 1.57 CFU mL-1.

10. The portable point-of-care testing device as claimed in claim 1, wherein said device uses for automation of sample handling, preparation using microfluidics, the bacterial cell count, and bacterial identification depending on the electrode response by analysing the electrochemical impedance spectroscopy.

11. A process to obtain the portable point-of-care testing device as claimed in claim 1 comprising the steps of:
• synthesizing said novel colistin passivated carbon dots (CCD) by a one-step microwave-assisted method using colistin, citric acid and ethylenediamine as chemical precursors,
• entrapping synthesized said novel colistin passivated carbon dots (CCD) within an agarose polymer matrix and drop-casted over disposable screen-printed carbon electrodes to obtain said impedimetric biosensor,
• incorporating said impedimetric biosensor in said microfluidic chip.

12. A method to detect microorganism using the portable point-of-care testing device as claimed in claim 1 comprising the steps of:
• applying a negative pressure, said sample, and said buffer flow through said micromixer and reaches the sensor well, which contains said sensor electrode,
• applying a potential for the preconcentration of bacterial cells onto said sensor electrode,
• delivering said washing buffer and said redox mediator for the electrochemical analysis,
• analysing the bacterial detection or identification by developed electronics meter module to display the quantified bacterial cell count,
wherein said microvalve controls delivery of fluids.