DESC:FIBER BRAGG GRATING BASED BIOSENSOR
FIELD OF INVENTION
The invention generally relates to the field of optical fiber sensing for immunodiagnostics and particularly to a fiber bragg grating based biosensor.

BACKGROUND
The detection of biomolecules is essential in various analytical, medical, biochemical and pharmaceutical application fields. The term “biomolecule” is defined as a chemical substance generated by a living organism and includes but is not limited to nucleic acids, peptides/proteins and their building blocks.
The techniques for detection of biomolecules are broadly divided into labeled techniques and label-free techniques.
Labeled technique requires an identifying molecule, often of a fluorescent nature, to be attached to the biomolecule. Label-free techniques identify the biomolecule without the need to attach an identifying molecule.
Predominantly used labeled techniques include but are not limited to florescence, colorimetry, radioactivity, phosphorescence, bioluminescence and chemiluminescence. Fluorescent tagging, or labelling is most predominantly used technique and uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule. Fluorescent tagging is widely used method because of its high convenience and sensitivity. One significant disadvantage is maintaining the conformational and functional integrity of biomolecules.
ELISA involves detection of analytes in a sample using antibodies specific to the analytes, mostly the analytes molecules are proteins of the target cell or biomarkers. ELISA has been the gold standard of immunodiagnostics, and has high sensitivity, specificity, precision and throughput. The main drawback is that it is time consuming and cannot be used for real-time identification.
Predominantly used label-free techniques include but are not limited to surface plasmon resonance (SPR), interferometry, diffraction, quartz crystal microbalance and optical elipsometry . Surface Plasmon resonance is an optical technique which measures the thickness and density of the materials on a flat solid substrate. The application of an SPR technique requires a transparent substrate that is coated with a metal film, typically gold, of a few tens of nanometers in thickness. One significant disadvantage of the SPR technique is requirement of high quality gold coating on transparent substrate which can be costly.
Thus there is a need for a system that does not rely on any type of labelling, attachment or dyes and is not time consuming.

BRIEF DESCRIPTION OF DRAWINGS
So that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
FIG.1 shows a schematic representation of the biosensor after antibody immobilization and E.coli binding, according to an embodiment of the invention.
FIG.2 shows a schematic of the experimental setup indicating the incident and reflected light from the biosensor, according to an embodiment of the invention.
FIG. 3 shows the wavelength shift with time observed on addition of water, anti-E.coli antibody, over biosensor, according to an embodiment of the invention.
FIG. 4 shows the wavelength shift with time observed on addition of water, anti-E.coli antibody, over biosensor at the end of each stage of experiment, according to an embodiment of the invention.
FIG. 5 shows the plot of wavelength shift vs. time for antibody response, according to an embodiment of the invention.
FIG. 6 shows a SEM image of cells bound to the biosensor surface, according to an embodiment of the invention.

SUMMARY OF THE INVENTION
One aspect of the invention provides a method for preparing a bare Fiber Bragg Grating (bFBG) biosensor. The method includes selecting an optical fiber, forming a plane grating of a pre-defined period in the optical fiber, depositing at least one layer of a reactive functional group on the surface of the optical fiber having the grating and immobilising a biomolecule desirable for sensing with the functional group deposited to obtain the fiber bragg grating biosensor.
Another aspect of the invention provides a method for detection of a target biomolecule. The method includes, selecting an optical fiber, forming a plane grating of a pre-defined period in the optical fiber, depositing at least one layer of a reactive functional group on the surface of the optical fiber having the grating, immobilising the biomolecule, desirable for sensing with the functional group deposited, to obtain the fiber Bragg grating biosensor, binding the target biomolecule on the surface of biosensor and measuring the shift in bragg wavelength due to the binding of the pathogen on the biosensor.
Yet another aspect of the invention provides a bare fiber bragg biosensor. The biosensor comprises of an optical fiber, a plane grating of a pre-defined period formed in the optical fiber, a reactive functional group deposited on the surface of the optical fiber grating, and an antibody specific to the target molecule immobilised for sensing with the deposited functional group

DETAILED DESCRIPTION OF THE INVENTION
Various embodiments of the invention provide a method for preparing a fiber bragg grating biosensor. The method includes selecting an optical fiber, forming a plane grating of a pre-defined period in the optical fiber, depositing at least one layer of a reactive functional group on the surface of the optical fiber having the grating and immobilising an antibody desirable for sensing with the functional group deposited to obtain the fiber Bragg grating biosensor.
FBG is a refractive index modulation of the core introduced along the axis of a single mode photosensitive fiber by exposure to an intensity modulated UV light. When a broad band light is launched into it, the FBG reflects back a narrow band of wavelength that satisfies the Bragg's resonance condition and transmits the remaining components in the spectrum. As the cladding completely isolates the core from its surroundings, FBG is inherently insensitive to the surrounding refractive index variation.
The method described briefly herein above shall be described in detail. The technique adopted in the invention uses binding reaction between a sensing biomolecule and a target analyte for detection using a bare Fiber Bragg Grating, hereinafter referred to as bFBG. In one example of the invention, the target analyte is E.coli and the sensing biomolecule is anti-E.coli antibody. The binding of E.coli to the antibody immobilized on the surface of the functionalised bFBG is indicated and measured as shift in the reflected Bragg wavelength. The unique shift patterns in the reflected bragg wavelength indicate the capability of the biosensor to respond to the strain induced on the surface due to the binding of the pathogen to the antibody. The wavelength shift as a result of strain induced on a FBG is given by,
??_b= ?_b {1-(n_eff^2)/2 (P_12-?[P_11-P_12 ] )}*?
where ? is the Poissons ratio, and ? is the axial strain change, P11 and P12 are coefficients of the stress-optic tensor.
The invention provides a method for fabricating a real time bFBG biosensor. The method for preparing bFBG based biosensor for detection of target molecules includes selecting an optical fiber. The optical fiber includes but is not limited to a Germania doped silica fiber, aluminium doped silica fiber, Germania doped acrylic fiber. A plane grating of a pre-defined period is formed on the optical fiber. The bFBGs in the study have been fabricated using the phase mask technique. bFBG sensor of gauge length 3 mm was fabricated in photo-sensitive silica fiber (SM1500) of cladding diameter of 80 µm using a 248 nm UV Excimer Laser having pulse energy of 6 mJ at 100 Hz repetition rate. The period of the grating formed is the range of about 500nm to about 550 nm. In one embodiment of the invention, a Germania doped silica fiber with gratings in the range of 460nm to 600nm is chosen. A layer of a reactive functional group is deposited on the surface of the optical fiber having the grating. Depositing the reactive functional group is achieved by washing sequentially with 1:1 v/v methanol-hydrochloric acid solution, concentrated sulfuric acid, hot sodium hydroxide solution and boiling water, for 5 minutes each. The silica fiber surface is washed with deionized water between each wash step. The silica fiber surface is then silanylated by incubating with 5% aminopropyltriethoxysilane, herein after referred to as APTES, solution for 15 minutes. The functionalized fiber is then washed with ethanol and dried, prior to baking at 80?C for 30 minutes.
Amine functionalized fiber surface is then incubated in 1 % glutaraldehyde solution (in DI water at pH 9.2) for 15 minutes to obtain sensor surface functionalized with aldehyde group. Specific antibodies are then immobilized on the active sites of the functionalized fiber surfaces. For immobilization of antibodies, the fictionalized fiber surface is first rinsed with 0.1M carbonate-bicarbonate buffer at a pH of 9.2. In one embodiment of the invention, Horse radish peroxidase enzyme conjugated anti-E.coli antibody, hereinafter referred to as HRP enzyme conjugated anti-E.coli antibody, is used for binding to the active sites on the fiber surface. HRP enzyme conjugated anti-E.coli solution diluted in 0.1 M carbonate buffer is added over the functionalized fiber and incubated for 15 minutes. The antibody immobilized functionalized fiber is then washed with phosphate buffered saline solution, hereinafter referred to as PBS solution.
The invention also provides a method for detecting a target molecule. The method for detection of target molecules includes selecting an optical fiber. The optical fiber includes but is not limited to a Germania doped silica fiber, aluminium doped silica fiber, Germania doped acrylic fiber. A plane grating of a pre-defined period is formed on the optical fiber. bFBG sensor of gauge length 3 mm was fabricated in photo-sensitive silica fiber (SM1500) of cladding diameter of 80 µm using a 248 nm UV Excimer Laser having pulse energy of 6 mJ at 100 Hz repetition rate. The period of the grating formed is the range of about 500nm to about 550 nm. In one embodiment of the invention, a Germania doped silica fiber with gratings in the range of 460nm to 600nm is chosen. A layer of a reactive functional group is deposited on the surface of the optical fiber having the grating. Depositing the reactive functional group is achieved by washing sequentially with 1:1 v/v methanol-hydrochloric acid solution, concentrated sulfuric acid, hot sodium hydroxide solution and boiling water, for 5 minutes each. The silica fiber surface is washed with deionized water between each wash step. The silica fiber surface is then silanylated by incubating with 5% aminopropyltriethoxysilane solution, hereinafter referred to as APTES, for 15 minutes. The functionalized fiber is then washed with ethanol and dried, prior to baking at 80?C for 30 minutes.
Amine functionalized fiber surface is then incubated in 1 % glutaraldehyde solution (in DI water at pH 9.2) for 15 minutes to obtain sensor surface functionalized with aldehyde group. Specific antibodies are then immobilized on the active sites of the functionalized fiber surfaces. For immobilization of antibodies, the fictionalized fiber surface is first rinsed with 0.1M carbonate-bicarbonate buffer at a pH of 9.2. In one embodiment of the invention, HRP enzyme conjugated anti-E.coli antibody is used for binding to the active sites on the fiber surface. HRP enzyme conjugated anti-E.coli solution diluted in 0.1 M carbonate buffer is added over the functionalized fiber and incubated for 15 minutes. The antibody immobilized functionalized fiber is then washed with PBS solution.
The invention further provides a bare fiber bragg biosensor for sensing a target molecule. The bare fiber bragg biosensor comprises of an optical fiber. The optical fiber includes but is not limited to a Germania doped silica fiber, aluminium doped silica fiber, Germania doped acrylic fiber. A plane grating of a pre-defined period is formed on the optical fiber. bFBG sensor of gauge length 3 mm was fabricated in photo-sensitive silica fiber (SM1500) of cladding diameter of 80 µm using a 248 nm UV Excimer Laser having pulse energy of 6 mJ at 100 Hz repetition rate. The period of the grating formed is the range of about 500nm to about 550 nm. In one embodiment of the invention, a Germania doped silica fiber with gratings in the range of 460nm to 600nm is chosen. A layer of a reactive functional group is deposited on the surface of the optical fiber having the grating.
The reactive functional group is selected from a list including but not limited to an aldehyde group, a carbonyl group, or an amine group. In one example of the invention , the reactive functional group deposited on the surface of the optical fiber is an amine group. Antibodies specific to the target analytes are then immobilized on the active sites of the functionalized fiber surfaces. FIG.1 shows a schematic representation of the fiber bragg grating region after antibody immobilization and E.coli binding, according to an embodiment of the invention. The E.coli 101 bind to the anti-E.coli antibody 103 immobilized on the functionalized fiber bragg gratings 105.
FIG.2 shows a schematic of the experimental setup indicating the incident and reflected light from the sensor, according to an embodiment of the invention. The fiber bragg grating sensor 201 of gauge length 3 mm is pre-stretched and both sides of the fiber are taped to a clean glass slide 203. The fiber 201 is then connected to an interrogator 205. The interrogator 205 records the reflected Bragg wavelength from the fiber 201 at any given point of time. A predetermined volume approximately 300 µL of water, anti-E.coli antibody solution and sample containing E.coli bacteria are sequentially pipetted onto the fiber surface after functionalization and the resultant variation in the reflected Bragg wavelength are recorded in each case. Data is logged in before the respective samples are added and continuously recorded for another 10 minutes.
FIG. 3 shows the wavelength shift with time observed on addition of water, anti-E.coli antibody, E.coli over the biosensor, according to an embodiment of the invention. All three response curves exhibit positive shift just after addition of the respective samples over the fiber surface. Data recorded subsequently, indicate a drop in wavelength shift. As water spreads out evenly on the fiber surface, the reflected Bragg wavelength approach the reference value. However, in the other two cases, exponential drop in the response curves is observed followed by subsequent negative shifts resulting from the binding of antibody and E.coli bacteria on the fiber surface. The fiber sensor responses approached an equilibrium condition in phase three. The wavelength shift with water got normalized. The wavelength shift with antibody and E.coli solutions saturated at -10 pm and -25 pm respectively. The shifts in the wavelength is the result of the respective strain experienced by the fiber sensor due to antibody and E.coli cells bound to the surface. The dimension and the net mass of E.coli is more than that of the antibody, which is evident from the higher wavelength shift observed with E.coli binding as compared to antibody attachment.
FIG. 4 shows the wavelength shift with time observed on addition of water, anti-E.coli antibody, E.coli over biosensor at the end of each stage of experiment, according to an embodiment of the invention. The figure shows that continuous observation of reflected Bragg wavelength can be used for monitoring the interaction of different samples with the bFBG sensor. Minimal amounts of shifts are observed during
wash steps.
FIG. 5 shows the plot of wavelength shift vs. time for antibody response. The graph shows that a positive shift due to sample delivery is eliminated and a wavelength shift of 10picometer is seen.
FIG. 6 shows a SEM image of E.coli cells bound to the biosensor surface, according to an embodiment of the invention. The SEM image shows binding of E.coli on the functionalized fiber surface.
The invention as described herein provides a method for real- time detection of target molecules. One significant advantage of the invention is the sensitivity of detection which is dependent on the shift in the wavelength due to the change in the mass of the bFBG. The detection of analytes is sensitive, specific and less time consuming. The bFBG provides ease of use and is portable. The bFBG biosensor can be used in a wide range of application including but not limited to disease diagnosis, quality monitoring of water, protein binding reactions, binding kinetics of Biomolecules, biomolecular Interactions.
The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.
,CLAIMS:We Claim:
1. A method for preparing a bare fiber bragg grating biosensor, the method comprising:
selecting an optical fiber;
forming a plane grating of a pre-defined period in the optical fiber;
depositing at least one layer of a reactive functional group on the surface of the optical fiber having the grating; and
immobilising an antibody desirable for sensing with the functional group deposited to obtain the fiber Bragg grating biosensor.
2. The method of claim 1, wherein the period of the grating formed is the range of about 460nm nm to about 600nm
3. The method of claim 1, wherein the reactive functional group is an aldehyde group.
4. The method of claim 1, wherein the process of depositing the aldehyde group on the surface of the optical fiber comprises of:
a first washing of the surface sequentially with each of a 1:1 v/v solution methanol-hydrochloric acid, a concentrated sulfuric acid, a hot sodium hydroxide solution and a boiling water for about 5 minutes;
silanylating the surface subsequent to the washing by incubating the surface with an aminopropyltriethoxysilane solution wherein the concentration of the aminopropyltriethoxysilane solution is in the range of about 2% to about 7% at a pre-determined pH for a pre-defined time duration;
a second washing of the silanized surface with ethanol;
baking the ethanol washed silanyated surface at a temperature range 50-900C for a time ranging from about 15minutes to about 60minutes; and
incubating the amine functionalized sensor surface in glutaraldehyde solution wherein the concentration of the glutaraldehyde soluition is in the range of about 0.5% to about 3% at a pH of about 9 for time ranging from 5minutes to 60minutes to obtain sensor surface functionalized with aldehyde group.
5. The process of claim 4, wherein pH is in the range of about 2.0 to about 6.0.
6. The process of claim 4, wherein the time duration is in the range from about 5 minutes to about 30 minutes.
7. The method of claim 1, wherein the step of immobilisation comprises of:
rinsing the aldehyde functionalized surface with carbonate-bicarbonate buffer in a concentration range of about 0.01M to about 0.5M at pH ranging from about 8 to about 10;
incubating the rinsed functionalized surface with a diluted carbonate buffer antibody solution in a concentration range of about 0.01M to about 0.5 M for a time ranging from about 10minutes to about 90minutes;
blocking non-specific sites of the antibody incubated surface with 0.2% bovine serum albumin to 3% bovine serum albumin for a time ranging from about 5minutes to about 45minutes; and
washing the antibody incubated surface with phosphate buffered saline solution to obtain immobilized and/or antibody immobilized sensor.
8. The antibody of claim 7, wherein the antibody is specific to a target molecule sensed.
9. The antibody of claim 7, wherein the target molecule is selected from the group comprising of microorganisms, polypeptides, proteins, and any biomolecule that is capable of binding to an antibody specific to the biomolecule.
10. A method for real-time detection of a target molecule, the method comprising:
selecting an optical fiber;
forming a plane grating of a pre-defined period in the optical fiber;
depositing at least one layer of a reactive functional group on the surface of the optical fiber having the grating;
immobilising an target molecule desirable for sensing with the functional group deposited to obtain the fiber Bragg grating sensor.
binding the target molecule on the surface of biosensor; and
measuring the shift in bragg wavelength due to the binding of the target molecule on the biosensor.
11. The method of claim 10, wherein the period of the grating formed is the range of about 460nm to about 600nm.
12. The method of claim 10, wherein the reactive functional group is selected from a list comprising of an aldehyde group, a carboxyl group and an amine group.
13. The method of claim 10, wherein the target binding molecule is an enzyme, a protein, a conjugated protein-enzyme complex or a protein conjugated indicator.
14. The method of claim 10, wherein the process of depositing the aldehyde group on the surface of the optical fiber comprises of:
a first washing of the surface sequentially with each of a 1:1 v/v solution methanol hydrochloric acid, a concentrated sulfuric acid, a hot sodium hydroxide solution and a boiling water for about 5 minutes;
silanylating the surface subsequent to the washing by incubating the surface with an aminopropyltriethoxysilane solution wherein the concentration of the aminopropyltriethoxysilane solution is in the range of about 2% to about 7% at a pre-determined pH for a pre-defined time duration;
a second washing of the silanized surface with ethanol;
baking the ethanol washed silanyated surface at a temperature range 50-900C for a time ranging from about 15minutes to about 60minutes; and
incubating the amine functionalized sensor surface in glutaraldehyde solution wherein the concentration of the glutaraldehyde soluition is in the range of about 0.5% to about 3% at a pH of about 9 for time ranging from 5minutes to 60minutes to obtain sensor surface functionalized with aldehyde group.
15. The process of claim 14, wherein pH is in the range of about 2.0 to about 6.0.
16. The process of claim 14, wherein the time duration is in the range from about 5minutes to about 30 minutes.
17. The method of claim 10, wherein the step of immobilisation comprises of:
rinsing the aldehyde functionalized surface with 0.1M carbonate-bicarbonate buffer at pH of 9.2;
incubating the rinsed functionalized surface with an 0.1 M diluted carbonate buffer antibody solution for 15 minutes;
blocking non-specific sites of the antibody incubated surface with a 1% bovine serum albumin for 10 minutes; and
washing the antibody incubated surface with phosphate buffered saline solution to obtain immobilisation.
18. The antibody of claim 17, wherein the antibody is specific to a target molecule sensed.
19. The target molecule sensed in claim 10, wherein the target molecule is selected from the group comprising of microorganisms, polypeptides, proteins, and any biomolecule that is capable of binding to an antibody specific to the biomolecule
20. The method of claim 10, wherein the shift in the bragg wavelength occurs due to change in mass.
21. The method of claim 10, wherein the change in mass is due to the binding of the target molecule on the functionalised surface.
22. A bare fiber bragg biosensor for sensing a target molecule, wherein the biosensor comprises of
an optical fiber;
a plane grating of a pre-defined period formed in the optical fiber;
a reactive functional group deposited on the surface of the optical fiber having the grating; and
an antibody specific to the target molecule immobilised for sensing with the deposited functional group.
23. The biosensor of claim 22, wherein the period of the grating formed is the range of about 460nm nm to about 600nm.
24. The biosensor of claim 22, wherein the reactive functional group is selected from a list comprising of an aldehyde group, a carbonyl group and an amine group
25. The biosensor of claim 22, wherein the target molecule is selected from the group comprising of microorganisms, polypeptides, proteins, and any biomolecule that is capable of binding to an antibody specific to the biomolecule.
26. The biosensor of claim 22, wherein the biosensor is capable of performing the sensing at a point of need wherein the point of need is at least one location selected from a list comprising of disease diagnosis, quality monitoring of water, protein binding reactions, binding kinetics of Biomolecules, biomolecular Interactions.