Description:OPTICAL BIOSENSOR AND A METHOD OF PREPARATION AND ITS APPLICATION THEREOF FOR DETECTION OF Salmonella typhi

FIELD OF THE INVENTION
The present invention is directed to the field of biosensors, more particularly the invention relates to a soluble optical biosensor and a method of preparation of said biosensors and its application thereof towards detection of food borne pathogen, in particular Salmonella typi through fluorescence spectrometry.
BACKGROUND OF THE INVENTION
Food pathogens frequently cause food borne diseases. A pre-enrichment culture or a direct culture on agar plate are standard microbiological methods. Biosensor-based methods rely on the recognition of antigen targets or receptors by antibodies, aptamers or high-affinity ligands. The captured antigens may be then directly or indirectly detected through an antibody or high-affinity and high-specificity recognition molecule. Nowadays, the developed immunoassays are based upon bacterial species-specific antibodies, aptamers [1, 2] and immuno-recognition of bacterial antigens (such as bacteriophage tailspike protein) [3]. These methods require standard conditions for the optimum binding of proteins or other highly-specific affinity compounds on: beads (Luminex, Austin, TX, USA), glass slides, gold surfaces, microplates and membranes suitable for chromatographic separation of antigen-antibody complexes, combined with dipsticks, microfluidic channels on paper (µPADs) [4] or lateral flow immuno-assays (LFIA), in which the capture antibody is conjugated with a detection molecule exploiting colorimetric methods, chemiluminescence or gold nanoparticles [5]. The sensitivity of this method applied in Salmonella spp. detection was approximately 105 CFU/mL, thus making it unsuitable for detection in pre-enrichment broth at early stages of growth (18–24 h).
A biosensor is a sensor for measuring the amount of desired material by immobilizing biological material that can react to specific substances with specific and selective combination (e.g., enzyme, antigen-antibody, ligand, or deoxyribonucleic acid (DNA) in a specific medium which can be detected by means of light, electric chemical, fluorescence, surface plasmon resonance (SPR). The optical biosensor is a kind of the biosensor, and can detect the state or the amount of biomaterial through radiation of specific wavelength.
An ideal biosensor should detect target molecules directly without the use of labelled ligands or multiple washing steps. There are several bottlenecks to be solved in order to perform an efficient analysis. The first is the use of proper surfaces. The second is to efficiently and uniformly print the capture probe (antibody, binding proteins, aptamers) and to have a constant probe density in order to obtain the detection of targets with high reproducibility. The third is to obtain a high sensibility even at a low concentration of the targets.
A number of methods are used for the rapid detection of biomolecules in solution. These include immunoassays, chromatographic methods, magnetic and biological biosensor methods (using screen-printed biosensors with immobilized enzymes), DNA biosensors and antibody-based detection methods. At the molecular level, sensitivity is huge and, in one study, a nanobiosensor able to detect even single hybridization events was demonstrated by using arrays of nanojunctions and oligonucleotides conjugated to gold nanoparticles [6]. A fiber optic portable biosensor utilizing the principle of fluorescence resonance energy transfer (FRET) has been developed for fast detection of Salmonella typhimurium in ground pork samples with limit of detection of 105 CFU/g [7]. A chip based SPR biosensor for the detection of Staphylococcal enterotoxin B (SEB) in milk has been developed for detecting SEB in buffer as low as 5 ng/ml [8]. An evanescent wave fiber-optic assay was developed to detect Salmonella and the limit of detection was found to be 103 cfu/ml in pure culture and 104 cfu/ml with egg and chicken breast samples [9].
S. Typhi is one of the serovars of Salmonella enterica. S. Typhi is the causative agent of typhoid fever. The symptoms of typhoid fever include nausea, vomiting, fever and death. S. Typhi can only infect humans, and no other host has been identified. The main source of S. Typhi infection is from swallowing infected water. Food may also be contaminated with S. Typhi, if it is washed or irrigated with contaminated water.
Salmonella LPS (6341) is a mouse monoclonal antibody raised against inactivated whole cell preparation of several Salmonella serogroups. Salmonella LPS (6341) is recommended for detection of Reactive with the A, B, and D group specific antigen (O-12) of Salmonella LPS of Salmonella anatum, Salmonella selandia and Salmonella typhi origin by solid phase ELISA. Salmonella bacteria are the most frequently reported cause of food borne illness. Salmonella typhi is a genus composed of rod-shaped, highly mobile Gram negative bacterium. This non spore-forming genus includes over 2,000 serotypes of Salmonella bacteria, organized into five different serogroups, Salmonella A, B, C, D and E. LPS is a major component of the cell membrane of all gram-negative bacteria, and it contributes greatly to the structural integrity of the bacteria, protecting the membrane from certain types of chemical attacks. LPS is an endotoxin composed of an endotoxic inner Lipid A, an O polysaccharide and an R core. All Salmonella species retain a LPS endotoxin representative of most Gram-negative bacteria.
There are several drawbacks associated with the above methods and principles for detection of bacterial pathogens by synthesis of a biosensor. Some of them are lack of proper surfaces, non uniformity in capturing the probe resulting in detection of targets with low reproducibility. Current state-of-the-art biosensors for detection of pathogens have a number of limitations such as poor detection limit and long assay time.
Despite the aforementioned disadvantages, the present inventors have made a great effort to solve the technical problems of conventional arts, and developed a nano-scale fluoro-biosensor followed by a method of preparation of said nano-scale fluoro-biosensor, wherein said method is applied in the detection of various variants of Salmonella typhi. Said method has a higher detection limit and short assay time and also uniformity in capturing the probes, along with other operational benefits such as portability, multiplexing and ease of use.
OBJECTIVE OF THE INVENTION
An objective of the present invention is to provide a nano-scale fluoro-biosensor and a method of preparation thereof for the detection of Salmonella typhi from water bodies.
Another objective of the present invention is to provide a nano-scale fluoro-biosensor that comprises of nanoparticles and a biomolecule capable of recognizing the target, wherein said target is conjugated to said nanoparticles to form nanoparticle –biomolecule conjugate.
Another objective of the present invention is to provide a nano-scale fluoro-biosensor which is soluble in nature and has a size of around 15 nm approximately and can be detected by phosphorescence at specific wavelength of 484 nm.
Another objective of the present invention is to provide a nano-scale fluoro-biosensor, wherein the nanoparticle – biomolecule conjugate obtained can interact specifically with the surface receptor of Salmonella typhi to generate nanoparticle-biomolecule -Salmonella typhi conjugate which shows effective fluorescence towards its detection.
Yet another objective of the present invention is to provide a method of preparation of said nano-scale fluoro-biosensor, wherein said biosensor is prepared by combining AgNO3, trisodium citrate and TbNO3 solution to obtain terbium ion doped silver nanoparticles that can be characterized by UV-visible spectra.
Yet another objective of the present invention is to provide such a method which detects Salmonella typhi and its various variants in a few hours or less, possibly within minutes.
Yet another objective of the present invention, the nanoparticles -biomolecule -Salmonella typhi conjugate or complex can not only be detected but also be quantified by fluorescence spectra.
These and other objectives of the present invention will be apparent from the drawings and descriptions herein. Every object of the invention is attained by at least one embodiment of the invention. However, no embodiment necessarily meets every object set forth herein.
SUMMARY OF THE INVENTION
The present invention envisions a nano-scale fluoro-biosensor and a novel method for preparation of said biosensor for detection of food borne pathogens from water bodies, particularly Salmonella typhi.
According to an embodiment of the present invention, the nano-scale fluoro-biosensor and its method of preparation is performed in two steps, first is the development of the biosensor and second is the method or process for conjugation of the biosensor (nanoparticles) with Salmonella typhi.
According to a preferred embodiment of the present invention, the nano-scale fluoro-biosensor comprises of; silver nanoparticles with terbium ion; a recognition molecule (biomolecule), preferably Salmonella LPS monoclonal antibody, wherein said biomolecule is conjugated to terbium ion doped silver nanoparticles resulting in the formation of a terbium ion doped silver nanoparticles – Salmonella LPS monoclonal antibody conjugate.
According to another preferred embodiment of the present invention the method of preparation of the nano-scale fluoro-biosensor or the synthesis of terbium ion doped silver nanoparticles, comprises the steps of:
Step 1: preparing a sample of Solution 1 (AgNO3); sample of Solution 2 (TbNO3) and a sample of Solution 3 (Trisodium citrate) solution of a defined weight separately by mixing a predetermined amount of water in each said solution(s);
Step 2: pouring a predetermined amount of water in a flask and further adding Solution 1, followed by addition of Solution 3 and continuously stirring the same to obtain a reaction mixture;
Step 3 : heating said mixture obtained in Step 2 for a fraction of seconds, thereby providing yellow colour, reflecting the formation and appearance of silver nanoparticles by UV Spectra;
Step 4: adding Solution 2 to the mixture of Step 3 and heating the same in a microwave for a fraction of seconds; and
Step 5 : generating terbium ion doped silver nanoparticles (biosensor)with the help of UV-visible spectrum showing appearance of more intense peak with Tb ion specific absorption peaks.
According to a another embodiment of the present invention, the mixture of Step 2 is heated for 90 seconds, whereas the mixture of Step 3 is heated for 30 seconds, both the help of microwave.
According to a another embodiment of the present invention, the particle size of terbium (Tb+++) doped silver nanoparticles is 15.15 nm as determined by particle size analyzer.
According to a preferred embodiment of the present invention, the method of conjugation of the nanoparticle – biomolecule conjugate (bionsensor) with Salmonella typhi, comprises the steps of:
Step 1 : pouring a predefined amount of terbium ion doped silver nanoparticles (biosensor) in tubes and further adding phosphate buffered saline (PBS);
Step 2 : mixing the solution obtained in Step 1 by tube inversion;
Step 3 : adding Salmonella LPS (6341) monoclonal antibody to said solution of Step 1 followed by continuous stirring;
Step 4 : incubating the accomplished solution overnight for conjugation in a refrigerator for a predefined time of temperature to form a nanoparticle- biomolecule Salmonella typhi conjugate or complex, wherein the particle size of said conjugated mixture is determined by particle size analyzer.
In an another embodiment of the present invention, the particle size of the conjugated mixture obtained is, 59.7 nm as determined by the particle size analyzer and is more than the particle size of the biosensor.
In an another embodiment of the present invention, the temperature of the refrigerator for incubation is between 6-8 degree Celsius.
In an another embodiment of the present invention, the conjugation of Salmonella typhi (S. typhi) bacteria to nanoparticle – biomolecule conjugate (biosensor) was studied by fluorescence spectrometer at an excitation wavelength of 240 nm and emission wavelength ranging between 460-550 nm.
In an another embodiment of the present invention, the sensitivity of the soluble optical (phosphorescent) biosensor has been found to detect at least 50 Salmonella typhi cells specifically from water body within 10 minutes.
The above and other features and advantages of the invention will become more readily apparent from the following detailed description taken with the following drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings where:
Figure 1a represents a schematic representation of the formation of the biosensor when integrated with the recognition molecule (biomolecule)
Figure 1b represents a schematic representation of the process of conjugation of the biosensor with Salomonella typhi.
Figure 2 illustrates a graphical representation of UV-visible spectra of silver nanoparticles solution and terbium doped silver nanoparticles.
Figure 3 illustrates a graphical representation of the particles size distribution of terbium doped silver nanoparticles determined by the particle size analyzer.
Figure 4 illustrates a graphical representation of the particle size distribution of the conjugated mixture determined by the particle size analyzer.
Figure 5 illustrates a graphical representation of the Fluorescence spectra of various dilutions of Salmonella typhi (S. Typhi) and one dilution of Bacillus cereus.
Figure 6 represents a correlation curve between fluorescence intensity and number of S. Typhi.
DETAILED DESCRIPTION OF THE INVENTION
The following description includes the preferred best mode of one embodiment of the present invention. It will be clear from this description of the invention that the invention is not limited to these illustrated embodiments but that the invention also includes a variety of modifications and embodiments thereto. Therefore the present description should be seen as illustrative and not limiting. While the invention is susceptible to various modifications and alternative constructions, it should be understood, that there is no intention to limit the invention to the specific form disclosed, but, on the contrary, the invention is to cover all modifications, alternative constructions, and equivalents falling within the spirit and scope of the invention as defined in the claims.
In any embodiment described herein, the open-ended terms "comprising," "comprises,” and the like (which are synonymous with "including," "having, ”and "characterized by") may be replaced by the respective partially closed phrases "consisting essentially of," consists essentially of," and the like or the respective closed phrases "consisting of," "consists of, the like.
As used herein, the singular forms “a,” “an,” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.
As used herein the term “sample” is used in a broad sense and refers to a material or medium which include, or is suspected to include, a biomolecule of interest. Exemplary samples include bodily fluids, tissues, gases, air, soil and other material. In some embodiments, the sample comprises an environmental air sample.
As used herein the term “pathogen” refers to an infectious agent that causes disease to its host. Exemplary non-limiting pathogens include bacteria, virus, molds, fungus, eukaryotic and prokaryotic microorganisms and parasites, etc.
“Serovar” or “Serotype” is the short form of referring to the serological variants of Salmonella bacteria, and is a way to distinguish between distinct types of Salmonella bacteria. The particular serovar of a Salmonella strain refers to the individual classification of that bacteria within the genus, as based upon cell membrane antigens. Serotyping often plays an essential role in determining species and subspecies. The Salmonella genus of bacteria, for example, has been determined to have over 4400 serotypes, including Salmonella enterica serovar Typhimurium, S. enterica serovar Typhi, and S. enterica serovar Dublin.
The terms “biosensor” and “biochip” as used herein refer to microelectronic-inspired construction of devices that are used for processing (delivery, analysis or detection) of biological molecules and cellular species. Thus, a biosensor or biochip as described herein is a microfluidic system with integrated electronics, inlet-outlet ports and interface schemes, high sensitivity detection of pathogen specificity, and processing of biological materials at semiconductor interfaces.
“CFU” is an abbreviation of colony-forming units, which are a form of measurement of live bacterial growth.
In microbiology, the measure of colony-forming unit (CFU or cfu) expresses the quantum of viable bacterial or fungal numbers. Unlike direct microscopic counts (where all dead and living cells are counted) CFU measures viable cells. Results are given either as CFU/mL (colony-forming units per milliliter) for liquids, or CFU/g (colony-forming units per gram) for solids. Because this invention utilizes a liquid broth to provide an incubation medium, the measurement of CFU/mL is utilized for admeasurement of Salmonella enterica density.
Plasmon as used herein refers to a quantum of plasma oscillation. The plasmon is a quasiparticle resulting from the quantization of plasma oscillations just as photons and phonons are quantizations of light and sound waves, respectively.
The present invention pertains to an optical biosensor and a method of preparation of said biosensors and its application thereof towards detection of food borne pathogen, in particular Salmonella typi through fluorescence spectrometry.
The nano-scale fluoro-biosensor in Figure 1 comprises of ;
- nanoparticles;
- and a recognition molecule (biomolecule) capable of recognizing the target, wherein the target is conjugated to said nanoparticles resulting in the formation of nanoparticle – biomolecule conjugate (biosensor).
The nano particles are selected from the list consisting of silver nanoparticles or terbium doped nanoparticles.
The recognition molecule or capture biomolecule is understood as any molecule capable of specifically recognizing a specific target through any type of chemical or biological interaction. The molecules used as recognition elements in the biosensors of the present invention must have a sufficiently selective affinity for recognizing a specific target in the presence of other compounds, in addition to being stable over time and preserving their structure as well as their biological activity once immobilized on the support and on the surface of the nanoparticles.
Antibodies, peptides, enzymes, proteins, polysaccharides, nucleic acids (DNAs), aptamers or peptide nucleic acids (PNAs) can be used as recognition molecules in the developed system.
The method of preparation of the metallic nano-scale fluoro-biosensor or the synthesis of terbium ion doped silver nanoparticles, comprises the steps of:
- preparing a 10mM AgNO3 solution by dissolving 84.9 g AgNO3 in 50 ml of Milli Q water ; followed by preparing a TbNO3 solution by dissolving 3.4 mg TbNO3 in 1 ml of Milli Q water and preparing 1% trisodium citrate solution by dissolving 0.1 g trisodium citrate in 10 ml of Milli Q water;
- pouring 25 ml Milli Q water in 150 ml conical flask and further adding 2 ml of 10 mM AgNO3 solution into it under stirring conditions;
- adding 2 ml of 1% trisodium citrate solution into the above solution under stirring condition and heating it in a microwave for 90 seconds, thereby allowing transition of colour of the solution to pale yellow showing appearance of plasmon peak (Ag np) reflecting formation of silver nanoparticles by UV Spectra; and
- summing the reaction mixture with 100 microlitre TbNO3 solution and heating the same in a microwave for 30 seconds, thereby showing appearance of more intense plasmon peak with Tb ion specific absorption peaks reflecting the formation of terbium ion doped silver nanoparticles with the help of UV visible spectrum.
The method of conjugation of the nanoparticle – biomolecule conjugate (bionsensor) with Salmonella typhi, comprises the steps of:
- pouring a predefined amount of terbium ion doped silver nanoparticles (biosensor) in tubes and further adding phosphate buffered saline (PBS);
- mixing the solution obtained in Step 1 by tube inversion;
- adding Salmonella LPS (6341) monoclonal antibody to said solution of Step 1 followed by mixing by tube inversion;
- incubating the accomplished solution overnight for conjugation in a refrigerator for a predefined amount of temperature to form a nanoparticle- biomolecule Salmonella LPS monoclonal antibody conjugate or complex, wherein the particle size of said conjugated mixture is determined by particle size analyzer.
The particle size of the conjugated mixture obtained is, 59.7 nm as determined by the particle size analyzer and is more than the particle size of the biosensor.
Figure 1 explains of the process of formation of nanoparticle – biomolecule – Salomonella LPS monoclonal antibody conjugate, wherein nanoparticles combine with the biomolecule to form nanoparticle –biomolecule conjugate which is the biosensor. Further, Salmonella typhi i.e. the bacterial pathogen combines with the nano particle biomolecule conjugate to form nanoparticle –biomolecule S. typhi conjugate or complex.
In reference to Figure 2 it illustrates a graphical representation of UV-visible spectra of silver nanoparticles solution and terbium doped silver nanoparticles at different wavelengths. It depicts a graph plot between absorbance and wavelength(nm), wherein the appearance of plasmon peak (Ag np) is depicted reflecting the formation of silver nano particles and appearance of a more intense plasmon peak formation of terbium ion doped silver nano particles. In fact, the fabrication of terbium (Tb+++) doped silver nanoparticles (phosphorescent biosensor) was monitored and confirmed by characteristic plasmon peak (414 nm).
In Figure 3 a graphical representation of the particles size distribution of terbium doped silver nanoparticles is determined by the particle size analyzer, wherein the average particle size terbium doped silver nanoparticles is 15.15 nm.
Further, Figure 4 explains a graphical representation of the particle size distribution of the conjugated mixture determined by the particle size analyzer. It mentions the particle size distribution of Salmonella LPS (6341) mouse monoclonal antibody linked terbium doped silver nanoparticles (bionsensor) wherein the average particle size of the conjugated mixture obtained is 59.7 nm. The particle size of the conjugated mixture is more as compared to the biosensor.
Referring to Figure 5 that illustrates a graphical representation of the Fluorescence spectra of Salmonella typhi (S. typhi) conjugated terbium doped silver nanoparticles. It depicts a graph plot between Fluorescence intensity and wavelength (nm) showing six different peaks of various intensities or signals with 1000 cells S. typhi having the highest peak and 50 cells S.typhi having the lowest peak. It is a Fluorescence spectra Salmonella typhi (S. typhi) conjugated terbium doped silver nanoparticles (a: 50 cells; b: 250 cells; c: 500 cells; d: 750 cells; e: 1000 cells), phosphate buffered saline (PBS) and 1000 Bacillus cereus (B.cereus) cells as control having the excitation wavelength as 240 nm, and Emission wavelength ranging between 460-550 nm.
Referring to Figure 6 represents a correlation curve between fluorescence intensity and various Salmonella typhi cell concentration. It depicts a linear correlation between terbium ion (Tb+++) specific emission at 484 nm upon excitation at 240 nm with respect to different cell concentrations of S. typhi conjugated to terbium (Tb+++) doped silver nanoparticles (phosphorescent biosensor). The data show that with increasing Salmonella cell concentration the fluorescent signal begins to rise above the baseline and reaches a maximum and finally levels off.
EXAMPLE
Hereinafter, the present invention will be described in further detail by examples. It will however be obvious to a person skilled in the art that these examples are provided for illustrative purpose only and are not construed to limit the scope of the present invention.
Example 1 : Detection of Salmonella typhi
Pure cultures of Salmonella typhi were grown in Luria broth medium for 14 hrs and diluted cultures at 10-8, 10-6 and 10-4 to control the cell numbers by colony forming unit. Pure culture of Bacillus cereus was used as negative control. The detection of Salmonella typhimurium was determined by assaying a series of samples with increasing Salmonella cell concentrations. (Table 1 ).
The process comprises of several steps ; firstly, pouring 50 microlitre of the prepared Tb+++/Ag nanoparticles in 5 ml eppendorf tubes and further adding 100 microlitre phosphate buffered saline (PBS) into it; secondly mixing the solution properly by tube inversion followed by addition of 50 microlitre of the different cell concentration of Salmonella typhi (50 cells; 250 cells; 500 cells; 750 cells; 1000 cells); repeated mixing by tube inversion and incubating the same for 5 minutes, followed by washing with 200 microlitre of PBS for removal of unbound bacteria nanoparticle biosensor; thirdly suspend the conjugated mixture in 1 ml PBS and analyze samples after 3 fold dilution in Milli Q water.
Therefore the specific binding of Salmonella typhi (S. typhi) bacteria to nanosized phosphorescent biosensor was confirmed by fluorescence (excitation wavelength 240 nm and emission wavelength 460-550 nm) of Tb+++ doped silver nanoparticles using fluorescence spectrometer (Perkin Elmer), wherein the 1000 cell S.typhi showed the highest peak or signal.
Table 1
No. of bacteria
in 50 microlitre PBS (microlitre) Antibody conjugated nanoparticles (microlitre) Mix it properly and incubate for 5 minutes
Followed by washing with 200 microlitre PBS Suspend in 1 ml PBS and analyze samples after 3 fold dilution in Milli Q water
50 S. typhi 100 50 -do- -do-
250 S. typhi 100 50 -do- -do-
500 S. typhi 100 50 -do- -do-
750 S. typhi 100 50 -do- -do-
1000 S. typhi 100 50 -do- -do-
1000 B.cereus 100 50 -do- -do-
While the present invention has been described above according to its preferred embodiments, it can be modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the present invention using the general principles disclosed herein. Further, the application is intended to cover such departures from the present disclosure as come within the known or customary practice in the art to which this present invention pertains and which fall within the limits of the following claims.

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Claims:We Claim:
1. An optical biosensor, comprising:
a. silver nanoparticles with terbium ion;
b. a recognition molecule (biomolecule), preferably Salmonella LPS monoclonal antibody, wherein said biomolecule is conjugated to terbium ion doped silver nanoparticles resulting in the formation of a terbium ion doped silver nanoparticles – Salmonella LPS monoclonal antibody conjugate.
-said biosensor is characterized in the detection of food borne pathogen, particularly Salmonella typhi by fluorescence spectrometry at an excitation wavelength of 240 nm and emission wavelength ranging between 460-550 nm.
2. A method for preparation of the biosensor as claimed in Claims 1, comprising the steps of:
a. preparing a sample of a first solution, a sample of a second solution and a sample of a third solution of a predefined weight separately by mixing a predetermined amount of water in each said solution(s);
b. pouring a predetermined amount of water to said first solution followed by addition of said third solution to form a reaction mixture;
c. heating said reaction mixture obtained in Step b for a predefined amount of time, wherein said heating results in the formation of said silver nanoparticles;
d. adding said second solution to the mixture of Step c and heating said mixture again for a predefined amount of time; and
e. generating said terbium ionic doped silver nanoparticles, wherein said particles reflect an intense peak. .
3. A method for preparation of the biosensor as claimed in Claim 2, wherein said mixture of Step b is heated for 90 seconds and said mixture of Step c is heated for 30 seconds, both the help of microwave treatment.
4. A method for preparation of the biosensor as claimed in Claim 2, wherein said first solution is AgNO3 solution, said second solution is a TbNO3 solution, and said third solution is trisodium citrate solution .
5. A method for preparation of the biosensor as claimed in Claim 2, wherein a plasmon peak (Ag np) is depicted reflecting the formation of said silver nano particles by UV visible spectra.
6. A method for preparation of the biosensor as claimed in Claims 2 or 5, wherein Step e of Claim 2 or 5 generates appearance of terbium ion doped silver nanoparticles with the help of UV visible spectrum.
7. A method for preparation of the biosensor as claimed in Claims 2 or 5 or 6, wherein the particle size of said terbium (Tb+++) doped silver nanoparticles is 15.15 nm as determined by the particle size analyzer.
8. A method of conjugation of the biosensor as claimed in Claim 1-7 with Salmonella typhi, comprises the steps of :
a. pouring a predefined amount of said terbium ion doped silver nanoparticles (biosensor) in tubes and further adding phosphate buffered saline (PBS);
b. mixing the solution obtained in Step a by tube inversion;
c. adding Salmonella LPS (6341) monoclonal antibody to said solution of Step a followed by mixing by tube inversion;
d. incubating the accomplished solution overnight for conjugation in a refrigerator for a predefined amount of temperature to form a nanoparticle- biomolecule Salmonella typhi conjugate or complex, wherein the particle size of said conjugated mixture is determined by particle size analyzer.
9. A method of conjugation of the biosensor as claimed in Claim 8 with Salmonella typhi, wherein said incubation is performed in a refrigerator with temperature ranging between 6-8 degree Celsius.
10. A method of conjugation of the biosensor as claimed in Claim 8 with Salmonella typhi, wherein said particle size of said conjugated mixture or complex is 59.7nm as determined by said particle size analyzer and is more than said terbium (Tb+++) doped silver nanoparticles.