AU2021100312A4

AU2021100312A4 - A method for pathogenic escherichia coli (e.coli) bacteria detection through tuned nanoparticle enhancement

Info

Publication number: AU2021100312A4
Application number: AU2021100312A
Authority: AU
Inventors: Manikandan Elayaperumal; Kavitha Gnanasekaran; Gargibala Satpathy
Original assignee: Elayaperumal Manikandan Dr
Current assignee: Elayaperumal Manikandan Dr
Priority date: 2021-01-19
Filing date: 2021-01-19
Publication date: 2021-04-15
Anticipated expiration: 2029-01-19

Abstract

The present disclosure relates to a method for pathogenic Escherichia coli (E.coli) bacteria detection through tuned nanoparticle enhancement. Two nano materials (ZnO-NPs and Au NPs) are used to detect pathogenic Escherichia coli (E. Coli) with their interactions with nanomaterials. Surface plasmon resonance (SPR) nanoparticles are useful contrast agents which incorporated a 514 nm Ar+ laser used as excitation source to detect the signal intensity due to the interaction of gold nanoparticles (Au-NPs) and zinc oxide nanoparticles (ZnO NPs) with the pathogenic Escherichia coli (E. Coli) cells. The changes are further correlated to the change in the surface charge and stiffness of the cell surface with the help of the fluorescence microscopic assay. In addition, with the help of Raman microscopy and element analysis, significant changes are observed in the interaction of the two strains with Au-NPs compared to ZnO-NPs due to protein, lipid and DNA/RNA induced conformational changes. 23 LAx 3J 0M r- aE' 0) C2 0U u0 r_ 00 . 0) u a0 ma aj a0 u 0) Ei C u0 CL 0 NY 0~ EL a) (J -i a- o a' c-cU Ei-3, W'-~ -30 -F >0) CU o aiC bo = 0 u0 - + ai b uii 0 00~ r, 0 In. r, n uL ou > u E E 0 a 0 0i m0 05 4-.a ai 0 00 (r_ CL r 0 r~ 00

Description

LAx

3J 0M

aE' r- 0)

C2 0U u0 r_ 00 . 0) u a0 ma a0 aj u

0) Ei C CL 0 u0

NY 0~ EL a) -i (J a-

o a' c-cU Ei-3, W'-~ -30 -F

>0) CU = o aiC bo

u0 - + ai b 0

uii 0 00~ r, 0 In. r, nuL

ou >

u E E

0 a 0i m0 0

05 4-.a

ai 0

00 (r_

CL r 0 r~

A METHOD FOR PATHOGENIC ESCHERICHIA COLI(E.COL) BACTERIA DETECTION THROUGH TUNED NANOPARTICLE ENHANCEMENT

FIELD OF THE INVENTION The present disclosure relates to a method for pathogenic Escherichia coli (E.coli) bacteria detection through tuned nanoparticle enhancement. In more details, the present disclosure relates to a method to detect pathogenic Escherichia coli (E. Coli) bacteria using mainly non-destructive fluorescence microscopy and micro-Raman spectroscopy.

BACKGROUND OF THE INVENTION Pathogenic bacterial cells are usually detected with the help of antibody-antigen reactions on the cell wall or based on specific target strands of DNAs extracted from the cell. Charge transfer process on cell wall can be useful information but it is not clear how much specific information regarding a particular pathogenic cell can be obtained from charge induced changes. Plasmonic and fluorescence signaling based methods have been developed to analyze such effects recently. There are the strains of E.coli such as Shiga toxin-producing E. coli (STEC), Enterotoxigenic E. coli (ETEC); Diarrheagenic E. Coli can cause diarrhea, urinary tract infections, respiratory illness and pneumonia, and other illnesses. Hence, the identification of specific bacterial strain of a particular species is the most complex task to solve. There are several conventional methods found in the literature for the detection of pathogens, including polymerase chain reaction (PCR). PCR is the most sensitive method, which can detect bacterial sample with the specific complementary primers for the specific sequence specific gene. PCR detection process involves use of complex chemical for DNA extraction and detection, which is the major drawback in this process. Antigen-antibody based method, where the antibody is produced against a single antigenic determinant which can use for detection of the particular molecule as the cause for pathogenicity and for differentiation. In antigen-antibody based method, the main limitations are of the cost of the antibody and also the complicated preservation way Loop-mediated isothermal amplification (LAMP) assay another useful and powerful tool for rapid detection of pathogenic MRS strains. As compared to the molecular methods, LAMP assay in the present study offers advantages on easiness in operation and time consumption. The LAMP assay method also involves extraction of genomic DNA, to overcome this step whole cell detection is implicated in the developed method.

Later stage fluorescence plays an important role in the various field of biological research like bio-sensing, bio-imaging, DNA sequencing, genomics and diagnostics. The plasmon-induced modification has been used to study the interactions involving different molecules and plasmonic nanostructures. A very popular plasmon induced phenomenon is surface enhanced Raman scattering (SERS). The enhanced light scattering seen in SERS is due to the electromagnetic and chemical enhancement. Together, these mechanisms of enhancement increase the Raman intensity to a point where SERS can be used for applications which require greater molecular sensitivity and specificity. The method is rapid and straightforward and does not require invasive experimental from microorganisms is a result of the combination of the spectra of all molecules that form the bacterial cell (carbohydrates, proteins, fatty acids, nucleic acids, small molecules). Together, these spectra show similar discriminatory abilities of various phenotypical characterizations to other phenotypic tests like lipid analysis, whole-cell protein electrophoresis, and metabolic tests.

Another plasmon induced phenomenon is commonly known as plasmon enhanced fluorescence (PEF) and here also the signal enhancement is due to two mechanisms: excitation enhancement and emission enhancement. Metal oxide and gold (Au) nanoparticles (NPs) represent a new class of important materials that are increasingly being developed for use in research and health-related activities.

However, there are various methods available for detecting pathogenic Escherichia coli (E. Coli) bacteria, but these methods are not so effective. In view of the foregoing discussion, there exists a need to have a method for pathogenic Escherichia coli (E.coli) bacteria detection through tuned nanoparticle enhancement.

SUMMARY OF THE INVENTION The present disclosure seeks to design a method to detect pathogenic Escherichia coli (E. Coli) bacteria using mainly non-destructive fluorescence microscopy and micro-Raman spectroscopy. In an embodiment, a method for pathogenic Escherichia coli (E.coli) bacteria detection through tuned nanoparticle enhancement is provided. The method comprises: obtaining fresh colonies of E. coli pathogenic genetic of MTCC723 and MTCC443; preparing nutrient broth medium by dissolving 28 gm of nutrient agar in 1000 ml of milli-Q-water into nutrient broth containing flasks, wherein the nutrient broth solution is autoclaved subsequently at 121degreee Celsius, 15 lbs for 30 minutes; incubating the flask in a shaking incubator at 37degree Celsius at 110 rpm for 24 hours and thereby transferring 0.4 ml of the overnight inoculum to a 100 ml Erlenmeyer flask containing 25ml nutrient broth and incubating in a shaking incubator for 3-4 hours at 37degree Celsius and 100 rpm; diluting one ml of overnight inoculum serially3 fold in nutrient broth to obtain an approximate target concentration of between 1 x 105 colony forming units/ ml (CFU.ml-1); estimating number of bacteria in 4hours culture by measuring the optical density of the culture at 660nm, wherein an optical density of between 0.1 and 0.3 is roughly equal to a concentration of between 108 CFU ml-1.

In an embodiment, the method further comprises plurality of chemicals such as Zinc acetate [Zn(CH3COOH)2.2H20], Zinc nitrate [Zn (N03)2.6H20] and Hexamethylenetetramine (HMTA) [C6H12N4], Polydimethylsiloxane (PDMS), Curing agent (Sylgard 184 Silicon Elastomer Kit, Dow Corning, Midland, MI), wherein all the chemicals used are double distilled chemicals used throughout the synthesis procedure. In an embodiment, ZnO nanorods (NRs) are synthesized on PDMS substrate by sol gel based hydrothermal technique. In an embodiment, a process for synthesizing ZnO nanorods comprises: preparing 10:1 solution of PDMS and curing agent and stirring continuously until a homogenous mixture is obtained and desiccated to remove any air bubbles; performing spin coating on a clear polycarbonate sheet at 3000 rpm and curing in a hot air oven at 80degree Celsius for 48 hours; dissolving 0.2195 g of Zinc acetate dihydrate in 100 ml ethanol to form a 0.01 M solution, wherein seed solution is dropped over the PDMS bed, which is then placed in an oven at 900degree Celsius for 1 h, wherein this leads to the formation of a seed layer with homogeneous and uniform interstitial sites; adding analytic grade of 1.4875 g of Zinc nitrate hexahydrate to a small amount of DI (deionized water) in a beaker and mixed and further adding DI water to make the solution up to 200 ml and thus obtain a 0.025 M solution; adding 0.7 g of HMTA to the prepared solution and stirring the solution of Zinc nitrate and HMTA using a magnetic stirrer for 20 minutes; and pouring 50 ml of the prepared solution into Teflon vessels and placing the seed-coated beds at the bottom thereby sealing and heating the solution in a muffle furnace for 3 hours at 900degree Celsius.

In an embodiment, the seed-free growth of highly ordered ZnO NRs is very difficult on many substrates, because of the lattice mismatch, wherein a seed layer is therefore essential in order to achieve well-aligned NRs on the substrate.

In an embodiment, the morphology of ZnO-NPs and Au-NPs are determined by scanning electron microscope (SEM), wherein the surface morphology of the uncoated and coated beads is studied using scanning electron microscope (SEM, FEI ESEM Quanta 200) and its elemental analysis are done by energy dispersive X-ray analysis (EDX, JEOL-3010 electron microscope).

In an embodiment, a process for calculating fluorescence intensity of the E. coli comprises: washing the cultured E. coli strains MTCC723 and E. coli MTCC443 (concentration108 CFU.ml-1) and re-suspending in phosphate buffer (pH-7.4) for experimental study; using a SYTO 9 probe as 24uM/m concentration for the fluorescence imaging study, wherein fluorescence image of each cell with ZnO-NPs and with Au-NPs are well compared; capturing fluorescence image of each set using fluorescence microscope (Carl Zeiss Axio Observer D1) equipped with a CCD camera (AxioCam) with the excitation wavelength of 488 nm and emission wavelength of 500-532 nm; and calculating fluorescence intensity of each set of the captured image using "Image j user interface" and thereby plasmonic effect is analyses. In an embodiment, a process for sample preparation for Raman measurements comprises: allowing E. coli cells to grow in Luria Bertani broth medium Until OD (Optical density) reached to 0.17; centrifuging the cells at 12000 rpm for 30 minutes to remove the LB media prior to addition of ZnO-NPs and Au-NPs; suspending cells in 1 ml of deionized water and adding the 10 mmol-1 concentration of ZnO-NPs and 10 mmol-1 of AuNPs concentration to the bacterial cells; and drop-casting an aliquot of 2.5 pL of the mixture onto a Raman compatible CaF2 slide and air drying in ambient condition for further Raman spectroscopic measurements after vertexing.

In an embodiment, the Raman spectra are recorded at room temperature using a commercial Raman micro-spectrometer (Renishaw, InVia system) equipped with a 633 nm excitation laser and 1200 lines/mm grating.

In an embodiment, the system is calibrated to the 520.5 cm-1 line using silicon reference before acquiring the Raman spectra, wherein the excitation laser beam is focused on the samples using a 100x (NA=0.85) objective and the backscattered Raman signals are collected by the same objective. Raman spectra are recorded on a thermoelectrically cooled charge-coupled device (CCD) detector. All spectra are acquired for 60 seconds and for each spectrum 2 scans are accumulated and co-added at room temperature (RT).

An object of the present disclosure is to detect pathogenic Escherichia coli (E. Coli) bacteria using mainly non-destructive fluorescence microscopy and micro-Raman spectroscopy.

Another object of the present disclosure is to detect the signal intensity due to the interaction of gold nanoparticles (Au-NPs) and zinc oxide nanoparticles (ZnO-NPs) with the pathogenic Escherichiacoli (E. Coli) cells.

Another object of the present disclosure is to observe significant changes in the interaction of the two strains with Au-NPs compared to ZnO-NPs due to protein, lipid and DNA/RNA induced conformational changes using Raman microscopy and element analysis. Another object of the present disclosure is to discusses the interaction of ZnO-NPs and Au-NPs with pathogenic E. coli bacterial cells.

Yet another object of the present invention is to deliver an expeditious and cost effective method for pathogenic Escherichia coli (E.coli) bacteria detection through tuned nanoparticle enhancement.

To further clarify advantages and features of the present disclosure, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings.

BRIEF DESCRIPTION OF FIGURES These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

Figure 1 illustrates a flow chart of a method to detect pathogenic Escherichiacoli (E. Coli) bacteria using mainly non-destructive fluorescence microscopy and micro-Raman spectroscopy in accordance with an embodiment of the present disclosure; Figures 2A, 2B, and 2C illustrate a scanning electron microscope (SEM) image of Zinc Oxide (ZnO) nanoparticle, energy-dispersive X-ray spectroscopy (EDX) of ZnO nano rods, and table showing elements and their corresponding composition percentages SEM image of zinc Oxide nanoparticle in accordance with an embodiment of the present disclosure; Figures 3A, 3B, and 3C illustrate a scanning electron microscope (SEM) image of gold nanoparticles (Au-NPs), energy-dispersive X-ray spectroscopy (EDX) of AuNP, and table showing elements and their corresponding composition percentages SEM image of AuNP nanoparticle in accordance with an embodiment of the present disclosure; Figures 4A, 4B, and 4C illustrate a fluorescence image of E.coli bacteria strains of a MTCC723, MTCC723 in presence of ZnONPs (10mmol-1) and MTCC723 in presence of AuNPs(1Ommol-1) in accordance with an embodiment of the present disclosure; Figures 5A, 5B, and 5C illustrate a fluorescence image of E.coli bacteria strains of a MTCC443, MTCC443 in presence of ZnONPs (10mmol-1), and MTCC443 in presence of AuNPs(1Ommol-1) in accordance with an embodiment of the present disclosure; Figure 6 illustrates a Raman spectrum of E.coli Strain MTCC443 treated with mmol-1ZnONP and 10mmol-1AuNP in accordance with an embodiment of the present disclosure;

Figure 7 illustrates a Raman spectrum of E.coli Strain MTCC723 treated with mmol-1ZnONP and 10mmol-1AuNP in accordance with an embodiment of the present disclosure; Figure 8 illustrates a table of assignment of Raman modes for E. coli in accordance with an embodiment of the present disclosure; Figure 9 illustrates a table of observed spectral changes in intensity in accordance with an embodiment of the present disclosure; Figure 10 illustrates a plurality of FESEM images in accordance with an embodiment of the present disclosure; and Figures 11A and 11B illustrates a plurality of SEM images of ZnO nano-rods grown on (Polycarbonate + PDMS) substrate in accordance with an embodiment of the present disclosure. Further, skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and may not have been necessarily been drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present disclosure. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

DETAILED DESCRIPTION For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

Reference throughout this specification to "an aspect", "another aspect" or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises...a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.

Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.

Referring to Figure 1, a flow chart of a method to detect pathogenic Escherichiacoli (E. Coli) bacteria using mainly non-destructive fluorescence microscopy and micro-Raman spectroscopy is illustrated in accordance with an embodiment of the present disclosure. At step 102, the method 100 includes obtaining fresh colonies of E. coli pathogenic genetic of MTCC723 and MTCC443. Fresh colonies of E. coli pathogenic are obtained from MTCC (National Centre for Cell Science). These are the pathogenic genetic stock, MTCC723 (H 10407) and MTCC443 (25922).

At step 104, the method 100 includes preparing nutrient broth medium by dissolving 28 gm of nutrient agar in 1000 ml of milli-Q-water into nutrient broth containing flasks, wherein the nutrient broth solution is autoclaved subsequently at 121degreee Celsius, 15 lbs for 30 minutes.

At step 106, the method 100 includes incubating the flask in a shaking incubator at 37degree Celsius at 110 rpm for 24 hours and thereby transferring 0.4 ml of the overnight inoculum to a 100 ml Erlenmeyer flask containing 25ml nutrient broth and incubating in a shaking incubator for 3-4 hours at 37degree Celsius and 100 rpm.

At step 108, the method 100 includes diluting one ml of overnight inoculum serially3 fold in nutrient broth to obtain an approximate target concentration of between 1 x 105 colony forming units/ ml (CFU.ml-1); .

At step 110, the method 100 includes estimating number of bacteria in 4hours culture by measuring the optical density of the culture at 660nm, wherein an optical density of between 0.1 and 0.3 is roughly equal to a concentration of between 108 CFU ml-1.

In an embodiment, the method further comprises plurality of chemicals such as Zinc acetate [Zn(CH3COOH)2.2H20], Zinc nitrate [Zn (N03)2.6H20] and Hexamethylenetetramine (HMTA) [C6H12N4], Polydimethylsiloxane (PDMS), Curing agent (Sylgard 184 Silicon Elastomer Kit, Dow Corning, Midland, MI), wherein all the chemicals used are double distilled chemicals used throughout the synthesis procedure. In an embodiment, ZnO nanorods (NRs) are synthesized on PDMS substrate by sol gel based hydrothermal technique.

In an embodiment, a process for synthesizing ZnO nanorods includes preparing 10:1 solution of PDMS and curing agent and stirring continuously until a homogenous mixture is obtained and desiccated to remove any air bubbles. The process includes performing spin coating on a clear polycarbonate sheet at 3000 rpm and curing in a hot air oven at 80degree Celsius for 48 hours. The process includes dissolving 0.2195 g of Zinc acetate dihydrate in 100 ml ethanol to form a 0.01 M solution, wherein seed solution is dropped over the PDMS bed, which is then placed in an oven at 900degree Celsius for 1 h, wherein this leads to the formation of a seed layer with homogeneous and uniform interstitial sites. The process includes adding analytic grade of 1.4875 g of Zinc nitrate hexahydrate to a small amount of DI (deionized water) in a beaker and mixed and further adding DI water to make the solution up to 200 ml and thus obtain a 0.025 M solution. The process includes adding 0.7 g of HMTA to the prepared solution and stirring the solution of Zinc nitrate and HMTA using a magnetic stirrer for 20 minutes. The process further includes pouring 50 ml of the prepared solution into Teflon vessels and placing the seed-coated beds at the bottom thereby sealing and heating the solution in a muffle furnace for 3 hours at 900degree Celsius.

In an embodiment, a process for calculating fluorescence intensity of the E. coli includes washing the cultured E. coli strains MTCC723 and E. coli MTCC443 (concentration108 CFU.ml-1) and re-suspending in phosphate buffer (pH-7.4) for experimental study. The process includes using a SYTO 9 probe as 24uM/m concentration for the fluorescence imaging study, wherein fluorescence image of each cell with ZnO-NPs and with Au-NPs are well compared. The process includes capturing fluorescence image of each set using fluorescence microscope (Carl Zeiss Axio Observer D1) equipped with a CCD camera (AxioCam) with the excitation wavelength of 488 nm and emission wavelength of 500-532 nm. The process further includes calculating fluorescence intensity of each set of the captured image using "Image j user interface" and thereby plasmonic effect is analyzed.

In an embodiment, a process for sample preparation for Raman measurements includes allowing E. coli cells to grow in Luria Bertani broth medium Until OD (Optical density) reached to 0.17. The process includes centrifuging the cells at 12000 rpm for 30 minutes to remove the LB media prior to addition of ZnO-NPs and Au-NPs. The process includes suspending cells in 1 ml of deionized water and adding the 10 mmol-1 concentration of ZnO-NPs and 10 mmol-1 of AuNPs concentration to the bacterial cells. The process further includes drop-casting an aliquot of 2.5 pL of the mixture onto a Raman compatible

CaF2 slide and air drying in ambient condition for further Raman spectroscopic measurements after vertexing.

In an embodiment, the system is calibrated to the 520.5 cm-i line using silicon reference before acquiring the Raman spectra, wherein the excitation laser beam is focused on the samples using a 100x (NA=0.85) objective and the backscattered Raman signals are collected by the same objective. Raman spectra are recorded on a thermoelectrically cooled charge-coupled device (CCD) detector. All spectra are acquired for 60 seconds and for each spectrum 2 scans are accumulated and co-added at room temperature (RT).

Figures 2A, 2B, and 2C illustrate a scanning electron microscope (SEM) image of Zinc Oxide (ZnO) nanoparticle, energy-dispersive X-ray spectroscopy (EDX) of ZnO nano rods, and table showing elements and their corresponding composition percentages SEM image of zinc Oxide nanoparticle in accordance with an embodiment of the present disclosure. The purpose of the nondestructive and rapid method is to analyze effect of ZnO NPs and Au-NPs on pathogenic E. coli bacterial strains. ZnO-NPs, synthesized by sol-gel based hydrothermal process, are characterized by SEM as shown in (Figure 2A), it is clear that the size of ZnO-NPs is the approximately 200 nm and they have either spherical or rod like structure.

Figures 3A, 3B, and 3C illustrate a scanning electron microscope (SEM) image of gold nanoparticles (Au-NPs), energy-dispersive X-ray spectroscopy (EDX) of AuNP, and table showing elements and their corresponding composition percentages SEM image of AuNP nanoparticle in accordance with an embodiment of the present disclosure. Au-NPs is obtained from Reinste nano Ventures Pvt. Ltd. The size and structure are characterized by SEM (Figure 3A), the size of Au-NPs is approximately 300 nm, and the shape is the round and hexagonal shape.

Figures 4A, 4B, and 4C illustrate a fluorescence image ofE.coli bacteria strains of a MTCC723, MTCC723 in presence of ZnONPs (10mmol-1) and MTCC723 in presence of AuNPs(10mmol-1) in accordance with an embodiment of the present disclosure. To assess the effect of ZnO-NPs on E. coli cells MTCC 723 these cultures of concentration 108 CFU.ml-1 are inoculated in nutrient broth media each containing 10 mmol-1 ZnO-NPs. These sets are incubated overnight inside the incubator at 37degree Celsius. The effect of ZnO-NPs is clearly observed with respect to E. coli cells without any nanoparticle's treatment. The changes in the cellular membrane integrity and viability assay is confirmed from the fluorescence micrographs. When the bacterial cells are exposed to the fluorescence probe, cell membrane permeates nucleic-acid-binding to the fluorescent probe. The fluorescence intensities of two cells are compared between in presence and absence of ZnO-NPs as shown in Figure 4. The images are processed using Zeiss image analysis user interface, "Image j user interface". The effect of ZnO-NPs on the viability of MTCC723 strains are studied by fluorescence microscopy in the presence of SYTO 9 probe staining. SYTO 9 probe stains live cells which appear as green in color.

Figure 4 shows the fluorescence images of (a) MTCC723, (b) MTCC723 in presence of ZnO-NPs (10mmol-1) and (c) MTCC723 in presence of Au-NPs (10mmol-1), respectively. Results from staining suggest that (Figure 4) ZnO-NPs of 10 mmol-1 can significantly inhibit the growth of MTCC723 (Figure 4B). The less in cell viability is probably due to the altered cell membrane permeability and intracellular metabolic system in the bacterial cell caused by ZnO-NPs. There is the damage of bacterial cell membrane due to the attached of ZnO-NPs and increasing Zn2+ ions in the bacteria. Potential neutralization and colony forming unit studies together proved the adverse effect of the resultant nano bacterial interfacial potential on bacterial viability. ZnO-NPs with positive surface potential upon interaction with the negative surface potential of bacterial membrane enhances production of the reactive oxygen species and exerts mechanical stress on the membrane, resulting in the membrane depolarization. Our results show that the antimicrobial propensity of metal oxide nanoparticle mainly depends upon the interfacial potential, the potential resulting upon interaction of nanoparticle surface with the bacterial membrane.

Figures 5A, 5B, and 5C illustrate a fluorescence image ofE.coli bacteria strains of a MTCC443, MTCC443 in presence of ZnONPs (10mmol-1), and MTCC443 in presence of AuNPs(10mmol-1) in accordance with an embodiment of the present disclosure. To assess the effect of Au-NPs on E. coli cells MTCC 443, these cultures of concentration 108 CFU.ml-1 are inoculated in nutrient broth media each containing 10 mmol-1 Au-NPs. These sets are incubated overnight inside the incubator at 37degree Celsius. The effect of Au-NPs is clearly observed with respect to E. coli cells without any nanoparticle's treatment. The changes in the cellular membrane integrity and viability assay is confirmed from the fluorescence micrographs. When the bacterial cells are exposed to the fluorescence probe, cell membrane permeates nucleic-acid-binding to the fluorescent probe. The fluorescence intensities of two cells are compared between in presence and absence of Au-NPs as shown in Figure 5, respectively. The images are processed using Zeiss image analysis user interface, "Image j user interface". The effect of Au-NPs on the viability of MTCC443 strains are studied by fluorescence microscopy in the presence of SYTO 9 probe staining. SYTO 9 probe stains live cells which appear as green in color.

Figure 5 shows the fluorescence image of (a) MTCC443, (b) MTCC443 in presence of ZnO-NPs (1Ommol-1) and (c) MTCC443 in presence of AuNPs (Ommol-1), respectively. Results from staining suggest that (Figure 4) ZnO-NPs of 10 mmol-1 can significantly inhibit the growth of MTCC723 (Figure 4B) and MTCC443 (Fig. 5B) cells. The less in cell viability is probably due to the altered cell membrane permeability and intracellular metabolic system in the bacterial cell caused by ZnO-NPs. There is the damage of bacterial cell membrane due to the attached of ZnO-NPs and increasing Zn2+ ions in the bacteria.

In case of Au-NPs, there is an enhancement in fluorescence intensity for both MTCC723 (Figure 4C) and MTCC443 (Figure 5C). The nature of SPR possessed by Au-NPs resulted in enhanced fluorescence of both pathogens. This is because of the well-known SPR (the quantized oscillation of free electrons on the metal surface due to the electromagnetic field of the light) phenomenon of metallic Au-NPs. The reasons behind the enhancement of fluorescent intensity are the following (1) Au-NPs polarizes with opposite charges at the core and the surface which creates a dipole oscillation among several other modes of plasmon resonances at other wavelengths and (2) excitation of Au-NPs at particular wavelengths leads to surface charge exchange with the fluorophore (FL). The mechanism behind the enhancement is due to the opposite charge dipole moment oscillation. Au-NPs has the positive charge, and the E. coli cells have the negative charge on its outer membrane. Even ZnO-NPs is having good binding capabilities, but the amount of free electron density is very less compared to Au-NPs. As a result, the plasmon excitation for ZnO-NPs is not in the range of FL emission wavelength. Hence the radiative transfer of energy between ZnO-NPs and FL is not significant for fluorescence imaging.

Figure 6 illustrates a Raman spectrum of E.coli Strain MTCC443 treated with mmol-1ZnONP and 10mmol-1AuNP in accordance with an embodiment of the present disclosure. In order to probe and get more detailed insight into the interaction between bacterial cells and nanoparticles, Raman micro-spectroscopic fingerprint measurements are performed on the above-mentioned control and treated samples. Figure 6 shows the normalized average Raman spectra of all the processed spectra acquired for the Strain MTCC443 of control and treated, under study in the spectral range from 500-1720 cm-1.

Figure 7 illustrates a Raman spectrum of E.coli Strain MTCC723 treated with mmol-1ZnONP and 10mmol-1AuNP in accordance with an embodiment of the present disclosure. Figure 7 shows the normalized average Raman spectra of all the processed spectra acquired for the Strain MTCC723 of control and treated, under study in the spectral range from 500-1720 cm-1. Raman spectra of two strains as shown in Figure 6 and Figure 7. Consisted of features representing the main cellular components, like, proteins, lipids, carbohydrates, and nucleic acids. For example, the Raman peaks located at 547, 1000 and 1657 cm-i are assigned previously to proteins, the peak at 1454 cm-i assigned to lipids, and the peaks at 665, 778 and 1073 cm- assigned to nucleic acids (DNA/RNA). The Raman peaks located at 1046 and 1134 cm-i have been mainly to carbohydrates, but there are contributions from both proteins and lipids also. There are many ambiguities in literature concerning assignment of vibrational spectral bands of bacteria. It is not possible to assign all the Raman modes precisely due to the complexity of bacteria as a molecular system. The Raman spectral features do not specify pure molecular normal vibrations and resulting overlap of the modes arising from various vibrational modes of molecules constituting bacterial cells. The features observed in the Raman spectra in both the E. coli strains are found to be consistent with those published previously. Differences in the biochemical composition and concentration of cellular constituents, translate into characteristic differences in the Raman spectra, especially in the fingerprint region from 500 to 1800 cm-1.

All major bands are marked in the Figure 6 and Figure 7 by black lines (solid and dashed) and a tentative assignment to the vibrational modes of different cellular constituents are presented in Table 1 for the two control samples of E. coli. Here, it is observed that both the two controls of E. coli strains possess similar Raman spectra, but the subtle differences between these two are due to their differences in the cellular components present in the cell. These molecular differences can be quantified by comparing the intensities of specific Raman modes assigned to molecular vibrations associated with particular component. However, when these two are treated with Au-NPs and ZnO-NPs exhibit distinct differences as compared to their control part which are clearly visible in the Figure 6 and Figure 7. This stark difference could arise as a result of the different cellular components present in these cells and also due to their different interaction mechanisms with the Au-NPs and ZnO-NPs.

The most noticeable changes in the Raman spectra are observed for both the E. coli strains treated with Au-NPs as compared to those treated with ZnO-NPs. Significant spectral changes are also observed for the two strains treated with ZnO-NPs, but these are not as prominent as compared to treated with Au-NPs. The changes can be visualized in intensities of several peaks, including 1657, 1615, 1577, 1454, 1330, 1134 and 547 cm-1 which are Raman markers mainly associate with proteins, lipids and DNA/RNA. But the most drastic changes are noticed in case of Raman marker bands of proteins (1657, 1615 and 547 cm-1), lipids (1454 cm-1) and DNA/RNA (1577 cm-1) and are marked by black dashed lines in the Figure 6 and Figure 7. The Raman difference spectra are generated by subtracting the average control spectra of each from the average treated spectra to make sure that the observed changes (mainly, change in intensity) in the cells are due to interaction with the nanostructures as shown in Figure 6 and Figure 7. Several peak (increase in the Raman intensity) and trough (decrease in the Raman intensity) features provide valuable information related to spectral changes caused by the interaction. Differences in the structure and shape of spectra are essential for better understanding of the interactions between cells and nanostructures. Cells treated with Au-NPs resulted in major changes as compared to those treated with ZnO-NPs, to the structure of the Raman difference spectra in both the strains, shown by the deviation from the control line in Figure 6 and Figure 7.

For change in Proteins, proteins are very important constituents of the cells. The Raman spectra of both E. coli strains are mostly dominated by the vibrational modes associated with proteins. The broad Raman mode centered at 1657 cm-1, assigned to the amide I vibration composed of different secondary structural units (like, a-helix, p-sheets, random coils, turn elements, etc.) of proteins. A drastic reduction in intensity of 1657 cm-1 indicates plausible conformational changes, such as the reduction of a-helix, in the secondary structures of proteins, as shown in the Figure 6 and Figure 7. Another broad band near 547 cm-1, assigned to vibration modes associated with disulfide bonds which are very important in determining the tertiary structure of proteins. Reduction in intensity of this mode can be attributed to impairing or even breaking of disulfide bonds. On the contrary to these two modes, the Raman feature centered at 1615 cm-1, assigned to ring vibration of aromatic amino acids (like, tryptophan, tyrosine, and phenylalanine which is very much sensitive to the microenvironment, shows an increase in the intensity. This could be due to structural variations of proteins which affects the micro-environment presents around this amino acid residues and leads to hyperchromicity resulting in the increasing intensity of this mode. All these changes indicate structural degradation, which may also be a cause for the change in intensity of the peaks discussed above and affect cell physiological activities.

For change in Lipids, lipids are the major component of all cell wall which provides structural integrity to the cell. A very intense Raman marker band for lipids at 1454 cm-1, prominent in the Raman spectra of both the E. coli strains is assigned to the CH deformation modes of lipids. A dramatic decrease in intensity supposedly indicates conformational changes to the lipid bilayer, ultimately leading to damage in the lipid structures. Furthermore, such damage induces membrane disruption and eventually, leads to membrane rupture and cell leaking.

For change in DNA/RNA, the most essential part of the cells are DNA and RNA, comprised of nucleic acids residues. However, since the relative proportion of these constituents are much lower than proteins and lipids, therefore the spectral features for DNA and RNA in the Raman spectrum are quite low intense and usually masked by the vibrational modes of proteins and lipids. A Raman peak centered at 1577 cm-1, assigned to purine ring modes (stretching vibration) of guanine and adenine is considered as one of the marker band for DNA and RNA which is very susceptible to the local environment. A notable increase in intensity of this mode is clearly observed from the Figure 6 and Figure 7 the intensity increase of this marker band could be attributed to the partial denaturation of DNA and RNA via unstocking of nucleobases and by conformational transition of dRib in the corresponding nucleosides. Furthermore, these denaturation and conformational transition lead to hyperchromicity, which may also add to the intensity increment of this mode. This indicates structural breakdown of conformation of DNA and RNA molecules and leads to cell death.

Figure 8 illustrates a table of assignment of Raman modes for E. coli in accordance with an embodiment of the present disclosure. All major bands are marked in the Figure 6 and Figure 7 by black lines (solid and dashed) and a tentative assignment to the vibrational modes of different cellular constituents are presented in Table 1 for the two control samples of E. coli.

Figure 9 illustrates a table of observed spectral changes in intensity in accordance with an embodiment of the present disclosure. All the prominent spectral changes due to the interaction are categorized and summarized in Table-2 and discussed below in detail.

Figure 10 illustrates a plurality of FESEM images in accordance with an embodiment of the present disclosure. In relevance to our study, the Raman marker bands of proteins (1657, 1615 and 547 cm-1), lipids (1454 cm-1) and DNA/RNA (1577 cm-1) in our analysis is selected because these peaks show noticeable changes in intensity. The major changes observed in both the strains caused by interaction with Au-NPs as compared to that with ZnO-NPs which is clearly visible form the Raman difference spectra, highlighted by the deviation from the zero line. This enhanced interaction mechanism can be attributed due to surface plasmon resonance property of Au-NPs which is well established in the literature. On the other hand, ZnO-NPs does not possess SPR property due to absence of free electron density within it. The micro-Raman spectroscopic fingerprint results indicate significant modifications to the cellular components, like, proteins, lipids and DNA/RNA due to interaction with the small nanostructures. Our observations clearly suggest the conformational changes induced in proteins, lipids and DNA/RNA due to the interactions with the nanostructures which lead to protein synthesis inhibition, membrane rupture and cell proliferation inhibition, eventually, leading to possible cell death.

Figures 11A and 11B illustrates a plurality of SEM images of ZnO nano-rods grown on (Polycarbonate + PDMS) substrate in accordance with an embodiment of the present disclosure. In this experimental study, an attempt has been made for the latest advances, application and future trends in pathogen detection using non-destructive fluorescence and Raman micro spectroscopic techniques. This report mainly discusses the interaction of ZnO NPs and Au-NPs with pathogenic E. coli bacterial cells. The enhancement of fluorescence intensity in presence of Au-NPs happens due to the polarized dipole-dipole interaction of surface free electron or electron cloud. ZnO-NPs does not contribute toward the radiative transfer because of the lack of free electron density. Raman marker bands of proteins and lipids show drastic change as compared to DNA/RNA bands which is clearly visible from the Raman difference spectra. Also, the Raman difference spectra indicate that major changes observed in both the strains caused by interaction with Au-NPs as compared to that with ZnO-NPs. The growth of bacteria is inhibited by ZnO-NPs due to changes in cell membrane permeability and intracellular metabolic system under fluorescence microscopy. However, the nature of SPR possessed by Au-NPs resulted in enhanced fluorescence of both pathogens. In addition, with the help of Raman microscopy and element analysis, significant changes are observed in the interaction of the two strains with Au-NPs compared to ZnO-NPs due to protein, lipid and DNA/RNA induced conformational changes.

In the developed method, an attempt is made to detect pathogenic Escherichiacoli (E. Coli) bacteria using mainly non-destructive fluorescence microscopy and micro-Raman spectroscopy. Raman vibrational spectroscopy provides additional information regarding biochemical changes at the cellular level. Two nano materials (ZnO-NPs and Au-NPs) are used to detect pathogenic Escherichia coli (E. Coli) with their interactions with nanomaterials. Surface plasmon resonance (SPR) nanoparticles are useful contrast agents which incorporated a 514 nm Ar+ laser used as excitation source to detect the signal intensity due to the interaction of gold nanoparticles (Au-NPs) and zinc oxide nanoparticles (ZnO NPs) with the pathogenic Escherichiacoli (E. Coli) cells. The changes are further correlated to the change in the surface charge and stiffness of the cell surface with the help of the fluorescence microscopic assay. From these results, it is found that when two Escherichia coli (E. Coli) (MTCC723 and MTCC443) and nanoparticles are respectively mixed overnight, the growth of bacteria are inhibited by ZnO-NPs due to changes in cell membrane permeability and intracellular metabolic system under fluorescence microscopy. However, the nature of SPR possessed by Au-NPs resulted in enhanced fluorescence of both pathogens. In addition, with the help of Raman microscopy and element analysis, significant changes are observed in the interaction of the two strains with Au-NPs compared to ZnO-NPs due to protein, lipid and DNA/RNA induced conformational changes. The scanning electron microscope (SEM) with energy dispersive X-ray (EDAX) spectroscopy exhibit surface morphology and the elemental composition of the synthesized NPs.

The drawings and the forgoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims.

Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any component(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all the claims.

Claims

WE CLAIM

1. A method for pathogenic Escherichia coli (E.coli) bacteria detection through tuned nanoparticle enhancement, the method comprises:

obtaining fresh colonies of E. coli pathogenic genetic of MTCC723 and MTCC443; preparing nutrient broth medium by dissolving 28 gm of nutrient agar in 1000 ml of milli-Q-water into nutrient broth containing flasks, wherein the nutrient broth solution is autoclaved subsequently at 121degreee Celsius, 15 lbs for 30 minutes; incubating the flask in a shaking incubator at 37degree Celsius at 110 rpm for 24 hours and thereby transferring 0.4 ml of the overnight inoculum to a 100 ml Erlenmeyer flask containing 25ml nutrient broth and incubating in a shaking incubator for 3-4 hours at 37degree Celsius and 100 rpm; diluting one ml of overnight inoculum serially3 fold in nutrient broth to obtain an approximate target concentration of between 1 x 105 colony forming units/ ml (CFU.ml-1); estimating number of bacteria in 4hours culture by measuring the optical density of the culture at 660nm, wherein an optical density of between 0.1 and 0.3 is roughly equal to a concentration of between 108 CFU ml-1.

2. The method as claimed in claim 1, comprises plurality of chemicals such as Zinc acetate [Zn(CH3COOH)2.2H20], Zinc nitrate [Zn (N03)2.6H20] and Hexamethylenetetramine (HMTA) [C6H12N4], Polydimethylsiloxane (PDMS), Curing agent (Sylgard 184 Silicon Elastomer Kit, Dow Coming, Midland, MI), wherein all the chemicals used are double distilled chemicals used throughout the synthesis procedure.

3. The method as claimed in claim 1, wherein ZnO nanorods (NRs) are synthesized on PDMS substrate by sol-gel based hydrothermal technique.

4. The method as claimed in claim 3, wherein a process for synthesizing ZnO nanorods comprises: preparing 10:1 solution of PDMS and curing agent and stirring continuously until a homogenous mixture is obtained and desiccated to remove any air bubbles; performing spin coating on a clear polycarbonate sheet at 3000 rpm and curing in a hot air oven at 80degree Celsius for 48 hours; dissolving 0.2195 g of Zinc acetate dihydrate in 100 ml ethanol to form a 0.01 M solution, wherein seed solution is dropped over the PDMS bed, which is then placed in an oven at 900degree Celsius for 1 h, wherein this leads to the formation of a seed layer with homogeneous and uniform interstitial sites; adding analytic grade of 1.4875 g of Zinc nitrate hexahydrate to a small amount of DI (deionized water) in a beaker and mixed and furher adding DI water to make the solution up to 200 ml and thus obtain a 0.025 M solution; adding 0.7 g of HMTA to the prepared solution and stirring the solution of Zinc nitrate and HMTA using a magnetic stirrer for 20 minutes; and pouring 50 ml of the prepared solution into Teflon vessels and placing the seed-coated beds at the bottom thereby sealing and heating the solution in a muffle furnace for 3 hours at 900degree Celsius.

5. The method as claimed in claim 4, wherein the seed-free growth of highly ordered ZnO NRs is very difficult on many substrates, because of the lattice mismatch, wherein a seed layer is therefore essential in order to achieve well-aligned NRs on the substrate.

6. The method as claimed in claim 1, wherein the morphology of ZnO-NPs and Au-NPs are determined by scanning electron microscope (SEM), wherein the surface morphology of the uncoated and coated beads is studied using scanning electron microscope (SEM, FEI ESEM Quanta 200) and its elemental analysis are done by energy dispersive X-ray analysis (EDX, JEOL-3010 electron microscope).

7. The method as claimed in claim 1, wherein a process for calculating fluorescence intensity of the E. coli comprises:

washing the cultured E. coli strains MTCC723 and E. coli MTCC443 (concentration108 CFU.ml-1) and re-suspending in phosphate buffer (pH-7.4) for experimental study; using a SYTO 9 probe as 24uM/ml concentration for the fluorescence imaging study, wherein fluorescence image of each cell with ZnO-NPs and with Au-NPs are well compared; capturing fluorescence image of each set using fluorescence microscope (Carl Zeiss Axio Observer D1) equipped with a CCD camera (AxioCam) with the excitation wavelength of 488 nm and emission wavelength of 500-532 nm; and calculating fluorescence intensity of each set of the captured image using "Image j user interface" and thereby plasmonic effect is analysed.

8. The method as claimed in claim 1, wherein a process for sample preparation for Raman measurements comprises:

allowing E. coli cells to grow in Luria Bertani broth medium Until OD (Optical density) reached to 0.17; centrifuging the cells at 12000 rpm for 30 minutes to remove the LB media prior to addition of ZnO-NPs and Au-NPs; suspending cells in 1 ml of deionized water and adding the 10 mmol-1 concentration of ZnO-NPs and 10 mmol-1 of AuNPs concentration to the bacterial cells; and drop-casting an aliquot of 2.5 pL of the mixture onto a Raman compatible CaF2 slide and air drying in ambient condition for further Raman spectroscopic measurements after vertexing.

9. The method as claimed in claim 1, wherein the Raman spectra are recorded at room temperature using a commercial Raman micro-spectrometer (Renishaw, InVia system) equipped with a 633 nm excitation laser and 1200 lines/mm grating.

10. The method as claimed in claim 1, wherein the system is calibrated to the 520.5 cm-i line using silicon reference before acquiring the Raman spectra, wherein the excitation laser beam is focused on the samples using a 100x (NA=0.85) objective and the backscattered Raman signals are collected by the same objective. Raman spectra are recorded on a thermoelectrically cooled charge-coupled device (CCD) detector. All spectra are acquired for 60 seconds and for each spectrum 2 scans are accumulated and co-added at room temperature (RT)

0

1 102 obtaining fresh colonies of E. coli pathogenic genetic of MTCC723 and MTCC443

104 preparing nutrient broth medium by dissolving 28 gm of nutrient ent agar ag in 1000 ml of milli-Q-water into nutrient broth containing flasks, wherein the nutrient broth solution is autoclaved subsequently at 121degreee Celsius, 15 lbs for 30 minutes

1 106 incubating the flask in a shaking incubator at 37degree Celsius us at 1 110 rpm for 24 hours and thereby transferring 0.4 ml of the overnight inoculum to a 100 ml Erlenmeyer flask containing 25ml nutrient broth and incubating in a shaking incubator for 3-4 hours at 37degree Celsius and 100 rpm

108 diluting one ml of overnight inoculum serially3 fold in nutrient broth to obtain an approximate target concentration of between 1 x 105 colony forming units/ ml (CFU.ml-1)

estimating number of bacteria in 4hours culture by measuring ng the optical density of the culture at 660nm, wherein an optical 110 density of between 0.1 and 0.3 is roughly equal to a concentration of between 108 CFU ml-1

Figure 1

Figure 2 Figure 3

Figure 4 Figure 5

Figure 6 Figure 7

Figure 9

Figure 8

Figure 10

Figure 11A

Figure 11B