The present invention generally relates to the field of biosensors for analyte detection. More specifically, the present invention relates to the field of liquid crystal (LC) based biosensors for histamine detection.
BACKGROUND OF THE INVENTION
Histamine is a chemical that is produced when mast cells get activated. Histamine is released into the bloodstream through white blood cells when the immune system is fighting against an allergic reaction which is caused due to pollen, mold or any certain food item. It is involved in the local immune response as well as the inflammatory response by playing a major role as a mediator of itching. Histamine imposes various effects inside the body of an organism such as contraction of smooth muscles, dilation of blood vessels that increases the permeability and lowers blood pressure and secretion of gastric acid in the stomach, etc. According to the research studies, the ability of histamine receptor antagonists to inhibit the mast cells has been proved to be developed as a group of mast cell stabilizers for the treatment of diseases caused by it.
The diagnosis of a disease or detection of chemical secretion from a body is done by various available methods. While studying or performing an experiment about the diagnosis or detection purpose, the researchers sometimes monitor key small molecules in the physiological structure of an organism in vivo to easily understand the physiology of that organism. This monitoring of physiological structure of an organism in vivo enables the clinicians to diagnose a disease easily. The in vivo monitoring of molecules includes monitoring of histamine concentration, which indicates the initiation of immune response, disease progression or response of an organism to the experiment. However, this monitoring process includes repeated blood drawing from an individual's body which may cause weakness and various other problems. Currently, the in vivo monitoring methods which are being used for the detection or diagnosis of a disease are limited by invasive implantation procedures and biofouling, thereby limiting the utility of these monitoring tools or

devices for obtaining the physiological data of an organism. For the detection of histamine, different electrochemical biosensors have been developed that are based on the sensitive amperometric response towards histamine through different surface immobilization.
To overcome these problems, the present invention provides a liquid crystal based material that detects the level of histamine in the body of an organism which liberates at particular situations and mediates cellular responses such as inflammatory responses, limited neurotransmission action in brain, allergic reaction, gastric acid secretion based on their receptors, etc.
US9816147B2 discloses about the liquid crystal based analyte detection which is used for the detection of virus, cells, bacteria, lipid-membrane containing organisms, proteins, nucleic acids, carbohydrates, etc. More specifically, the virus binding in a detection region is identified by the changes in liquid crystal orientation that is caused by the virus binding independent orientation caused by any topography associated with the detection region.
US20180052111A1 provides improved substrates and methods of using liquid crystal assays for quantitating the amount of an analyte in a sample. The present invention also provides materials and methods for detecting non-specific binding of an analyte to a substrate by using a liquid crystal assays format.
Shuzhen Liao et al. (2015) discloses about the label-free liquid crystal biosensor with high sensitivity for L-histidine-mediated formation of DNA duplex by cleaving DNAzyme using L-histidine, resulting in a remarkable optical signal. The results showed that the DNAzyme-based LC biosensor is highly sensitive to L-histidine with a detection limit of 50 nM. Compared with previously reported multi-step amplified methods, this newly designed assay system for L-histidine has no amplified procedures with comparable sensitivity.
Xiaokang Ding et al. (2012) discloses about the liquid crystal based optical sensor for detection of vaporous butylamine in air. This LC sensor doped with lauric aldehyde (LA) shows fast and distinct bright-to-dark optical response to

butylamine vapor. This LC sensor also exhibits reversibility after the sensor is exposed to open air. In addition to primary amines (such as butylamine and octylamine), this LC-based sensor responds to secondary amines (such as diisopropylamine), but the detection limit is 200 ppm, which is much higher than butylamine.
Ana Barrera et al. (2021) discloses about the dielectric properties of recycled liquid crystals (LCs) (non-purified, purified, and doped with diamond nanoparticles at 0.05, 0.1, and 0.2 wt%) were investigated. The studied LC mixtures were obtained from industrial recycling of end-of-life LC displays presenting mainly nematic phases. Results show that the dielectric anisotropy of all purified samples presents positive values and decreases after the addition of diamond nanoparticles to the LC mixtures. DC conductivity values were obtained by applying the universal law of dielectric response. In addition, conductivity of the doped LC mixtures is lower than that of the undoped and non-purified LC.
In all these inventions, there is requirement of the liquid crystal based sensor for the detection of analyte. However, none of the cited prior art till date does not discloses about the detection of histamine using liquid crystal materials through electro-optical and dielectric measurements. Moreover, the dielectric measurement performed in the instant invention helps in quantitative determination of histamine. It is different from other biosensing techniques in the sense that other biosensors presently available whether conventional or liquid crystal based did not use dielectric measurements. The process is reproducible, motivating and offers an easy way to design portable biosensors based on liquid crystal, which are highly sensitive to the amount of histamine. The main advantage of this novel biosensor is that it is highly selective, easily probe, simple and cost effective. The main advantage of this biosensing mechanism is histamine-induced LC alignment conversion from homeotropic to homogeneous that builds the foundation for this development.
OBJECT OF THE INVENTION

The main object of the present invention is to provide a liquid crystal-based biosensor for histamine detection through dielectric and optical techniques.
Another object of the present invention is to provide a biosensor to detect histamine level present in most body cells that liberate at particular situations and mediates cellular responses based on their receptors.
Yet another object of the present invention is to provide an easy portable liquid crystal-based biosensors, which is highly sensitive to the amount of histamine.
Still another object of the present invention is to provide a biosensor that is highly selective, easily probe, simple, cost effective and is based upon simple observations /measurements.
SUMMARY OF THE INVENTION
The present invention relates to provide a liquid crystal-based biosensor for histamine detection through dielectric and optical techniques.
In an embodiment, the present invention provides a method of fabrication of liquid crystal-based biosensor for histamine detection involving decorating Dimethyloctadecyl [3-(trimethoxysilyl)-propyl] ammonium chloride, forming monolayer of said DMOAP/APTMS (3-Aminopropyl)-trimethoxysilane, immobilizing diamine oxidase, binding event between histamine and diamine oxidase and preparing Liquid crystal cells. The method employs detection of histamine through dielectric and optical techniques.
In another embodiment, the present invention provides the method of decorating Dimethyloctadecyl [3-(trimethoxysilyi)-propyl] ammonium chloride (DMOAP) which comprises cleaning and separating the organic contaminants from the glass slides with ethanol followed by drying and heating in an oven for some time and then dipping them in an aqueous solution containing 0.5% (v/v) DMAOP and then rinsing the glass slides with ultrapure water.

In yet another embodiment, the present invention provides the method of monolayer formation of DMOAP/APTMS which comprises immersing the clean glass slides in 1% (v/v) DMOAP and 3% (v/v) APTMS, cleaning the glass slides and dipping them in PBS containing GA solution and again rinsing the glass slides with PBS and ultrapure water to store clean slides at 4°C for futuristic use. Furthermore, the immobilization of diamine oxidase (DAO) comprises of preparing enzymatic solution of DAO in PBS and making a layer of DAO over GA layer followed by incubation at 30°C for proper immobilization of DAO on cell surface coated with DMAOP/APTMS film using GA as a cross linker.
In yet another embodiment, the present invention provides the specific binding events between histamine and DAO which comprises preparing different concentrations of histamine by dissolving various amount of histamine in Triton X-100 and adding that prepared histamine solutions dropwise on the glass slide surface immobilized with DAO followed by rinsing the glass slides on completion of reaction. Furthermore, the liquid crystal cells are prepared by fabricating the liquid crystal cells by joining two glass plates with 6 \im thin strip of Mylar spacer and preparing three different cell types for performing whole process, keeping the cells at 40°C and filling the LC material 8CB in each cell through capillary action followed by cooling the cells slowly to room temperature.
The above objects and advantages of the present invention will become apparent from the hereinafter set forth brief description of the drawings, detailed description of the invention, and claims appended herewith.
BRIEF DESCRIPTION OF THE DRAWINGS
A complete understanding of the system and method of the present invention may be obtained by reference to the following drawing:
Figure 1 is the illustration of detection process of LC biosensor (a) no transmission of light in homeotropically aligned cell; (b) a very few transmissions in when DAO

enzyme is present; (c) more intense light transmission in the presence of histamine according to the present invention.
Figure 2 is polarizing optical images of the LC test cells filled with 8CB in (a) a homoeotropically aligned reference cell coated with DMOAP/APTMS; (b) an enzyme cell; (c)-(e) biosensing cell at various histamine concentrations as 20 mg/L, 100 mg/L and 500 mg/L respectively according to the present invention.
Figures 3(a)-3(c) are graphs showing behavior of (a) dielectric permittivity; (b) dielectric absorption and (c) dielectric loss factor, (the permittivity from 20 Hz - 2 MHz frequency is shown in the insets); as a function of frequency for different LC cells as shown in the inset with thickness 6 um filled with 8CB NLC at 1 kHz - 2 MHz frequency according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described hereinafter with reference to the accompanying drawings in which a preferred embodiment of the invention is shown. This invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art.
The present invention relates to the detection device for detecting the histamine level in the body. The detection device relates to the liquid crystal-based biosensor which detects the histamine level through dielectric and electro-optical techniques. Using these techniques, the histamine present in most body cells that liberates at particular situations and mediates cellular responses such as inflammatory responses, limited neurotransmission action in brain, allergic reaction, gastric acid secretion based on their receptors are detected. Moreover, the liquid crystal-based detection is reproducible, motivating and offers an easy way to design portable biosensors based on liquid crystal, which are highly sensitive to the amount of

histamine. The liquid crystal-based biosensor is highly selective, easily probe, simple and low cost as well as based upon the simple observations/measurements.
Thus, the most preferred embodiment of the present invention comprises a method of fabrication of liquid crystal biosensor for histamine detection comprising the steps of: (a) decorating Dimethyloctadecyl{3-(trimethoxysilyl)-propyl] ammonium chloride; (b) forming monolayer of DMOAP/APTMS ((3-Aminopropyl)-trimethoxysilane) as obtained in step (a); (c) immobilizing diamine oxidase; (d) binding between histamine and diamine oxidase; and (e) preparing liquid crystal cells; wherein the detection of histamine utilizes dieletric and optical techniques; the Liquid crystal (LC) biosensor in presence of histamine results in an extreme change at the interface which leads to a modified optical signal with precise birefringence; and the dielectric permittivity decreases as concentration of histamine increases, wherein the detection of histamine utilizes dielectric and optical techniques; the LC biosensor in presence of histamine results in an extreme change at the interface which leads to a modified optical signal with precise birefringence; and the dielectric permittivity decreases as concentration of said histamine increases.
In another preferred embodiment of the present invention comprises the decoration of Dimethyloctadecyl [3-(trimethoxysilyl)-propyl] ammonium chloride (DMOAP) comprising the steps of: (i) cleaning and separating all organic contaminants from the glass slides with ethanol; (ii) drying and heating the glass slides in an oven for some time followed by dipping them in an aqueous solution containing 0.5% (v/v) DMOAP for 30 min; and (iii) rinsing with ultrapure water to obtain clean glass slides.
In yet another preferred embodiment of the present invention comprises the monolayer formation of DMAOP/APTMS comprising the steps of: (i) immersing the clean glass slides in 1% (v/v) DMAOP and 3% (v/v) APTMS; (ii) washing the glass slides with ethanol and ultrapure water; (iii) dipping the slides in phosphate buffer saline containing 2% glutaraldehyde solution; and (iv) rinsing the glass

slides with phosphate buffer saline and ultrapure water to store clean slides at 4°C for futuristic use.
In yet another preferred embodiment of the present invention comprises the immobilization of diamine oxidase comprising the steps of: (i) preparing enzymatic solution of diamine oxidase in phosphate buffer saline; (ii) making a layer of diamine oxidase over the glutaraldehyde layer; and (iii) incubating at 30°C for 3 hours for proper immobilization of diamine oxidase on cell surface coated with DMOAP/APTMS film using glutaraldehyde as a cross linker.
In yet another preferred embodiment of the present invention comprises the specific binding event between histamine and diamine oxidase comprising the steps of: (i) preparing different concentrations of histamine such as 20 mg/L, 100 mg/L and 500 mg/L by dissolving various amount of histamine in Triton X-100; (ii) adding prepared histamine solutions dropwise on the glass slide surface immobilized with diamine oxidase; and (iii) rinsing the glass slides with phosphate buffer saline on completion of reaction wherein, different concentrations of histamine comprises of 20 mg/L, 100 mg/L and 500 mg/L.
In still another preferred embodiment of the present invention comprises the preparation of liquid crystal cells comprising the steps of: (i) fabricating the liquid crystal cells by joining two glass plates with a 6 urn thin strip fo Mylar spacer; (ii) preparing three types of cells for performing whole process, the first type of cell is made by combining two DMOAP/APTMS coated glass slides (i.e., one on the top side and the other on the bottom side), the second type of cell is coated with diamine oxide over DMOAP/APTMS Self Assembled Monolayer (SAM) and held together by maintaining a fix gap (~6 |im) and the third type of cell is prepared by loading free histamine at different concentration levels varying from 20 mg/L-500 mg/L on the diamine oxide enzymatic cell surface; (iii) keeping the cells at 40°C for 5 minutes and filling the liquid crystal material 8CB in each cells through capillary action; and (iv) cooling the cells slowly to room temperature, wherein the dielectric permittivity for 20 mg/L, 100 mg/L, 500 mg/L histamine concentration is 2.03819, 2.101718 and 2.50565 respectively.

Histamine is an organic nitrogenous compound that is involved in local immune responses, as well as regulating physiological function in the gut and acting as a neurotransmitter for the brain, spinal cord, and uterus. It is responsible for performing some of the major functions such as communicating messages to the brain, helps in releasing stomach acid for digestion and acts as an immune response which is released after injury or allergic reaction. When histamine level is too high or when it can't break down properly, it can affect the normal bodily functions. Histamine is associated with common allergic responses and symptoms as well as some of the most common reactions which include headache or migraine, nasal congestion or sinus issues, fatigue, hives, digestive issues, irregular menstrual cycle, nausea, etc. and in severe cases of histamine intolerance, the reactions include abdominal cramping, tissue swelling, high blood pressure, irregular heart rate, anxiety, etc.
Biosensors are devices that are used to detect the presence or concentration of a biological analyte, such as a biomolecule, a biological structure or a microorganism. Biosensors works on the principle of signal transduction. The transducer measures the input signal and gives the output signal which is directly proportional to the concentration of the analyte. The signal is then amplified and processed by the electronic system.
Liquid crystal biosensors are based on changes in the orientation of liquid crystal molecules induced by various events of biomolecules. These biosensors play a major role in the detection of various biomolecules, bio molecular activity, and even small key molecules which are present in our body as these biosensors are inexpensive, simple, sensitive, effective and portable to use. The liquid crystals depend on the temperature changes such as melting point and clearing point temperatures based on which these liquid crystals changes their multiple intermediate phases that are distinguished by their molecular orientations. These changes in temperature as well as in chemical structures affects the orientation sequences of liquid crystals which results in the orientation transitions. Therefore, liquid crystals are the materials which responds to the presence of foreign species or occurrence of binding events of their vicinity.

The 8CB nematic liquid crystals comprises of Smectic A (Sm A) phase. Moreover, the 8CB nematic liquid crystal has higher nemtaic-isotropic transition temperature (i.e., 41°C). Most of the analytes function at higher temperature and by using this liquid crystal material one can easily detect any type of analytes.
In a preferred embodiment of the present invention, the purpose of the invention is to detect histamine which is present in most body cells that liberate at particular situations and mediates cellular responses such as inflammatory responses, limited neurotransmission action in brain, allergic reaction, gastric acid secretion based on their receptors. The application based on the enzymatic reaction on the surface of the glass substrate and realignment of liquid crystal material.
EXAMPLE 1 Fabrication of liquid crystal-based biosensor for the detection of histamine
DMOAP Decoration
To start the experiment, firstly the glass slides are cleaned with piranha solution at 80°C for 4-5 minutes to separate all organic contaminants and then washed it properly with ethanol and copious amount of ultrapure water for several times (i.e. 5-6 times), then dried properly and heated in the oven for at least 3 hours at a temperature of 110°C. The cleaned glass slides are then dipped in an aqueous solution containing 0.5% (v/v) DMOAP at room temperature for 30 minutes and then rinsed with the overflow of ultrapure water and heated in the oven for 3 hours.
Monolayer formation of DMOAP/APTMS
The cleansed glass slides are immersed in ethanol solution containing 1% (v/v) DMOAP and 3% (v/v) APTMS at 80°C for 2 hours, washed with ethanol and ultrapure water, then heated for 1 hour at 110°C after drying properly. Then, the slides are dipped in phosphate buffer saline (PBS) containing 2% glutaraldyhyde (GA) solution for 1 hour, rinsed with PBS and ultrapure water for several times, dried properly and stored at 4°C for futuristic use.

Immobilization of Diamine Oxidase
The enzyme'atic solution of diamine oxidase (DAO) is prepared in PBS over the GA layer then incubated at 30°C for 3 hours for the proper immobilization of the enzymes. Then the glass slides are cleaned with PBS solution to was out loosely bound enzymes. The enzyme DAO is immobilized on the surface of the cell coated with DMOAP/APTMS film with the help of GA as a crosslinker.
Specific binding event between Histamine and DAO
Different concentrations of histamine such as 20 mg/L, 100 mg/L and 500 mg/L are prepared by dissolving various amount of histamine in Triton X-100. The series of prepared histamine solutions are added dropwise on the glass slide surface-immobilized with DAO at 30°C for 2 hours. After the completion of reaction, the glass slides are rinsed with PBS and large amount of ultrapure water to eliminate unbinding agents and then dried properly.
Preparation of LC cells
The LC cells are fabricated by joining two glass slides with a 6 um thin strip of Mylar spacer and then fixed it with the help of small binder clips. The active area of the cells is 16 x 16 mm2. Three types of cells are prepared to perform the whole process. Out of them, the first type cell is made by combining two DMOAP/APTMS coated two glass slides (i.e., one on the top side and the other on the bottom side). The other cell is coated with DAO over the DMOAP/APTMS Self Assembled Monolayer (SAM) and held together by maintaining a fix gap (~6 um). Finally, the third type of the cell is prepared by loading free histamine at different concentration levels varying from 20 mg/L-500 mg/L on the DAO enzymatic cell surface. All the cells are kept at 40°C for 5 minutes and then the LC material 8CB is filled in each cell through capillary action and then cooled the cells slowly up to nearly room temperature.
EXAMPLE 2 Sensing Mechanism

The sensing mechanism in the present invention is based on the following principle:
The sensing mechanism is based on optical and dielectric measurements. Owing to the long-range order inherent of LCs, the transition of the LCs will result transformation from vertical alignment to distorted orientational profile, making birefringent texture in the the optical images and the same is reflected in dielectric measurements.
Figure 1 shows the design and mechanism of LC based biosensor for histamine detection describing the whole mechanism of novel enzymatic LC based histamine biosensor. The sensor response is observed through the alignment change in LC molecules mainly due to surface effects. In part (a) of Figure 1, the glass slides are chemically operated with self-assembling DMOAP/APTMS. The homeotropic alignment in 8CB is observed and no transmitted light is detected. The DAO is then immobilized on the glass surface over the DMOAP/APTMS layer (part (b) of Figure 1). Some disruptions from homeotropic alignment begin to start and a very low transmitted light is noticed. Finally, the histamine is assembled over a pretreated glass surface and a non-zero transmitted light with high intensity is observed which is demonstrated from part (c) of Figure 1.
EXAMPLE 3 Electro-Optical textures of Different sample cells
Figure 2 illustrates the textures of polarizing optical microscope of the LC test cells filled with 8CB in a homeotropically aligned reference cell coated with DMOAP/APTMS, an enzyme cell and the biosensing cells at various histamine concentrations as 20 mg/L, 100 mg/L and 500 mg/L respectively.
The filled 8CB molecules aligned perpendicularly on the SAM of DMOAP/APTMS and a dark uniform region is viewed (part (a) of Figure 2). After incorporating DAO on the SAM surface, it has been detected that the image starts changing slowly towards brighter due to orientation change of molecules

from homeotropic alignment (part (b) of Figure 2). Different concentrations of histamine are then loaded which disrupted the inclination of LC molecules. The presence of histamine results an extreme change at the interface which leads to a modified optical signal with precise birefringence (part (c) of Figure 2). It is found that the deposition of histamine on the enzymatic SEM surface significantly benefited to alter the surface topology of sensing conjugations that leads to the effective birefringence in the LC material. At 100 mg/L, the brighter texture with improved contrast is recorded as compared to the previous concentration (part (d) of Figure 2). Finally, at the highest concentration (i.e., 500 mg/L), the maximum birefringence is observed showing the nematic liquid crystal molecules begin to start towards homogenous alignment (part (e) of Figure 2).
EXAMPLE 4 Dielectric Studies
Figures 3(a)-3(c) demonstrates the behavior of dielectric parameters such as dielectric pemittivity, dielectric absorption and dielectric loss factor as a function of frequency for different LC cells.
The profile of the permittivity along with the frequency is shown in Figure 3(a) for different LC biosensing cells. It should be noted that the dielectric permittivity of 8CB NLC in SAM of DMOAP/APTMS cell, the value of dielectric permittivity is close to 5 at 1 kHz which become lowered when enzymes were loaded on the surface. Actually, the dielectric permittivity in homeotropic alignment is more (as the molecules will be perpendicular to the substrate and electric field applied is parallel to the molecular alignment. In this configuration, the parallel component of the dielectric permittivity is found. But the values of dielectric permittivity begin to decrease when histamine is deposited with increasing concentrations. When histamine comes in contact with enzymes, the reaction takes place between them as the DAO enzyme help to excrete the extra histamine from our body. The dielectric permittivity in homogeneous alignment is less (as the molecules are parallel to the substrate and electric field applied is perpendicular to the molecular

alignment. In this configuration, the perpendicular component of the dielectric permittivity is found). The 8CB material is positive dielectric anisotropic material. The dielectric anisotropy is defined as A£ = £y-£_,_. The positive dielectric anisotropy means the parallel component is larger than perpendicular component. Higher histamine concentrations results (more homogeneous alignment) in the decreased dielectric permittivity. So, with help of these dielectric observations, different histamine concentrations are easy to detect. The response of dielectric loss is shown which is given by the imaginary part of complex dielectric permittivity (Figure 3(b), Figure 3(c)) demonstrates the dielectric loss factor which is the ratio of energy dissipated in the energy stored. It is clear from the graph that in the case of higher concentrations of histamine on the enzymatic SAM surface, the loss of 8CB molecules is slowly decreased. Also, the value of dielectric permittivity is different for different biosensing LC sample cells which are highlighted in Table 1.
Table 1 Dielectric permittivity values for different bio-sensing LC sample cells

Type of Reference Enzyme 20 wt% 100 wt% 500 wt%
LC sample cell cell histamine histamine histamine
Dielectric
permittivity 4.560826 1.354324 2.03819 2.101718 2.50565
(at around
1kHz)
From the above table, the value of dielectric permittivity (which is actually the dielectric constant) for the reference cell is 4.56 as in this case the 8CB molecules are homeotropically aligned in the presence of DMOAP and APTMS. When enzymes are incorporated in the DMOAP/APTMS coated layer, molecules tend to aligned homogeneously and the value is decreased. When Histamine is incorporated with different concentrations on the enzyme coated SAM surface, molecules still remain in the homogeneous state and there is only slight variation in their dielectric constant values. These value of the dielectric permittivity for

different biosensing LC sample cells provide the quantitative detection of Histamine using dielectric measurements.
Many modifications and other embodiments of the invention set forth herein will readily occur to one skilled in the art to which the invention pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

CLAIMS
WE claim:

1. A method of fabrication of liquid crystal-based biosensor for histamine
detection comprising the steps of:
a) decorating Dimethyloctadecyl [3-(trimethoxysilyl)-propyl] ammonium chloride;
b) forming monolayer of said DMOAP/APTMS ((3-Aminopropyl)-trimethoxysilane) as obtained in step (a);
c) immobilizing diamine oxidase;
d) binding between histamine and diamine oxidase; and
e) preparing liquid crystal cells;
wherein,
said detection of histamine utilizes dielectric and optical techniques;
said liquid crystal (LC) biosensor upon detection of said histamine results in an optical signal with birefringence in liquid crystal cells; and
the liquid crystal (LC) biosensor exhibits decrease in dielectric permittivity as concentration of said histamine increases.
2. The method as claimed in claim 1, wherein said decorating
Dimethyloctadecyl [3-(trimethoxysilyi)-propyl] ammonium chloride
(DMOAP) comprises the steps of:
i) cleaning and separating all organic contaminants from the glass slides with ethanol;

ii) drying and heating said glass slides in an oven for some time followed by dipping them in an aqueous solution containing 0.5% (v/v) said DMOAP for 30 min; and
iii) rinsing with ultrapure water to obtain clean glass slides.
3. The method as claimed in claims 1 and 2, wherein the monolayer formation
of DMOAP/APTMS comprises the steps of:
i) immersing said clean glass slides in 1% (v/v) said DMOAP and 3% (v/v) said APTMS;
ii) washing said glass slides with ethanol and ultrapure water;
iii) dipping said slides in phosphate buffer saline containing 2% glutaraldehyde solution; and
iv) rinsing said glass slides with said phosphate buffer saline and ultrapure water to store clean slides at 4°C for futuristic use.
4. The method as claimed in claim 1, wherein said immobilizing of diamine
oxidase comprises the steps of:
i) preparing enzymatic solution of said diamine oxidase in phosphate buffer saline;
ii) making a layer of diamine oxidase over said glutaraldehyde layer;
iii) incubating at 30°C for 3 hours for proper immobilization of diamine oxidase on cell surface coated with said DMOAP/APTMS film using said glutaraldehyde as a crosslinker.
5. The method as claimed in claim 1, wherein said binding between histamine and diamine oxidase comprises the steps of:
i) preparing different concentrations of said histamine by dissolving various amount of said histamine in Triton X-100;

ii) adding said prepared histamine solutions dropwise on said glass slide surface immobilized with said diamine oxidase; and
iii) rinsing said glass slides with said phosphate buffer saline on completion of reaction;
wherein,
said different concentrations of histamine comprises of 20 mg/L, 100 mg/L
and 500 mg/L.
6. The method as claimed in claim 1, wherein said preparation of liquid crystal
cells comprises of:
i) fabricating said liquid crystal cells by joining two glass plates with a 6 urn thin strip of Mylar spacer;
ii) preparing three types of cells for performing whole process;
iii) keeping said cells at 40°C for 5 minutes and filling said liquid crystal material 8CB in each cell through capillary action; and
iv) cooling said cells slowly to room temperature.
7. The method as claimed in claim 6, wherein said three types of cells
comprising of:
a) a first type of cell made by combining said two DMOAP/APTMS coated glass slides (i.e., one on the top side and the other on the bottom side);
b) a second type of cell coated with diamine oxide over said DMOAP/APTMS Self Assembled Monolayer (SAM) and held together by maintaining a fix gap (~6 |im); and

c) a third type of cell prepared by loading free histamine at different concentration levels varying from 20 mg/L, 100 mg/L, 500 mg/L on said diamine oxide enzymatic cell surface;
wherein,
the dielectric permittivity for 20 mg/L, 100 mg/L, 500 mg/L histamine
concentration is 2.03819, 2.101718 and 2.50565 respectively.