DESC:COMPLETE SPECIFICATION
Title of Invention-
Surface Plasmon Resonance (SPR) based Biosensor.
Field of the invention
Biotechnology
Technical field of Invention:
The present invention relates to an easy to operate quantification device for the identification of species responsible for the envenomation and to measure the venom present in the victim's body is described herein. The device involves the SPR based biosensor platform that enables label-free, real-time monitoring, high sensitivity and specificity and can be used in remote areas, where it may not be possible to find a hospital nearby. The present innovative device replaces the conventional non-specific polyvalent technology to specific technology and thereby significantly reduces the allergen (generally horse globulins in the polyvalent ASV sera) load on the patient and associated side effects. Further, the present invention resolves longstanding problem and much needed solution with far reaching benefit which reaches out for the benefit of civilians' and also armed forces including in remote location.
Background and prior art:
Ophitoxaemia is the exotic term that characterizes the clinical spectrum of snake bite envenomation. Snakebite envenoming is a potentially life threatening disease caused by toxins in the bite of a venomous snake. Of the 2500-3000 species of snakes distributed world-wide, about 500 are venomous. Based on their morphological characteristics including arrangement of scales, dentition, osteology, myology, sensory organs etc., snakes are categorized into families. The major families of venomous snakes are Atractaspididae, Elapidae, Hydrophidae and Viperidae. The main families in the Indian subcontinent are: Elapidae which includes Spectacled cobra, King cobra, Common Krait, etc., Viperidae which includes Russell's viper, Pit viper, Saw-scaled viper, etc., and Hydrophidae (the sea snakes). Of the 52 venomous species in India, majority of bites and consequent mortality is attributable to "Big four" species viz. Naja naja (Spectacled cobra), Daboia russelii (Russell's viper), Bungarus caeruleus (Common Krait) and Echis carinatus (Saw-scaled viper). Snakebite envenomation remains a public health concern in many countries especially India. Populations in these regions experience high morbidity and mortality because of poor access to health services, which are often suboptimal, and, in some instances, a scarcity of polyvalent antivenom, which is the only specific treatment. A large number of victims survive with permanent physical sequelae due to local tissue necrosis and, no doubt, psychological sequelae. Because most snakebite victims are young, the economic impact of their disability is considerable. Worldwide more than 5 million bites are reported per year, with 2% leading to severe sequelae.
India had the largest number of reported venomous bites and deaths. India has the highest number of deaths due to snake bites in the world, around 1.2 million snakebite deaths (representing an average of 58,000 per year) from 2000 to 2019 with nearly half of the victims aged 30-39 and over a quarter being children under 15. The states with largest number of snakebite cases include Bihar, Jharkhand, Madhya Pradesh, Odisha, Uttar Pradesh, Rajasthan, Gujarat and Telangana. In 2017, snakebite was recognized by World Health Organization as a neglected tropical disease. Snake venom is the most complex of all poisons, is a mixture of enzymatic and non-enzymatic compounds as well as other non-toxic proteins including carbohydrates and metals.
Antivenom (or antivenin or antivenene) is a biological product used in the treatment of venomous bites or stings. Antivenom is created by milking venom from the desired snake, spider or insect. The venom is then detoxified and injected into a horse, sheep, goat or cat. The subject animal will undergo an immune response to the venom, producing antibodies against the venom's active molecule which can then be harvested from the animal's blood and used to treat envenomation. Antivenom can be classified into monovalent (when they are effective against a given species' venom) or polyvalent (when they are effective against a range of species, or several different species at the same time). Conventional clinical practice is to administer polyvalent anti-snake venom (ASV), usually of horse origin at the time of hospitalization of the victim against Big Four venomous species. This often causes severe anaphylaxis reaction in the victim, (seen in up to 30% of the recipients worldwide) demanding secondary treatment. Acute pulmonary edemas, cerebellar ataxia and uveitis (an immunological complication) are some of the complications following polyvalent anti-snake venom (ASV) serum.
One of the major problems of the snake envenomation is the lack of specific snake venom detection platforms. Currently, we don’t have any quantitative detection diagnostics, we are still depending on symptomatic strategy and non-specific biochemical tests. It may cause delay in giving polyvalent ASV to victims. Because of non-specific diagnostics, we are still following the polyvalent treatment strategy to treat the snakebite patients.
Biosensors are a type of sensor that have gained prominence in recent years due to their advantages over traditional sensing methods, which are expensive and time-consuming. A generic biosensor has three main elements: target, recognition, and the transducing element. The target is the analyte molecule, which is detected when it is captured by the recognition element through some specific interactions. After binding with the target molecule, the recognition element of the sensor undergoes a change to one of its physical or chemical properties, like conductivity, refractive index (RI), pH value, etc. This change is translated to a readable signal with the help of a transducer.
An optical biosensor based on Surface Plasmon Resonance (SPR) achieves high sensitivity, is label-free and its multilayer construction allows an increase in the selectivity of the target analyte. As the choice of layers in SPR-based biosensors and the analysis of the obtained multilayer configuration is very difficult and expensive, our innovation is a SPR-based biosensor for the quantification of Snake venom, where the specific antigen and antibody interact resulting in an optical phenomenon, which will be detected by our own innovative SPR platform. When a biomolecular interaction (e.g. specific binding of analytes) takes place, the refractive index near the surface is altered. This modification of refractive index can then be detected by the SPR sensor platform. In addition to that the detection and an innovative simulation tool, developed through mathematical modeling, with an easy-to-use interface and several design options for calculating and analyzing the reflectance and angle of incidence of this type of device, which is very essential for the user-friendly approach.
In accordance with the above the present invention provides a system and method for detecting quantitative extent of envenomation useful to determine dose of monovalent ASV required to be administered to the victims bitten by poisonous snake and to monitor the venom clearance from the body using species-specific diagnostic SPR biosensor. The present inventors have designed the novel sensor detector portion and biochips, which is essentially important specific snake venom detection platform.
Figures detailed
Fig. 1. SPR Biosensor Platform scientific explanation
A – Specific Antibody/peptides/biological markers
B – Sample Analyte (venom protein)
C – Activated Surface (Surface Modification)
D – Metal oxide coating
E – Silicon wafers/Glass substrate
F – Prism
G – Polarized Light
H – Reflected Light
I – Detector
J – Light Source
K – Flow channel
Fig. 2. Design for the Portable Snake Venom Detection Biosensor

Fig. 3. SPR Biosensor Model (external view)

Fig. 4. SPR Biosensor Model (Internal view)

Fig. 5. Sensor Chip Cassette

DETAILED DESCRIPTION OF THE INVENTION:
The present invention relates to a SPR based Biosensor device for determining the quantitative load of venom toxin in the victim’s blood sample which is essentially important for venomous and non-venomous bites differentiation and detection. And further, the venom toxins under the venomous families and species-specific detection can also be specifically incorporated into the biosensor kit for effective treatment strategy. This will reduce the error and also effective quantitative methodology can lead to a specific treatment management strategy in bite cases. The quantitative detection biosensor platform of the present invention works on an optical biosensor-based Surface Plasmon Resonance (SPR) that achieves high sensitivity, is label-free and its multilayer construction allows an increase in the selectivity of the target analyte. The sensor platform (fig 1) in the device have several advantages compared to conventional ones, such as (i) real-time monitoring to uncover the binding dynamics for observing various biological interactions between biomolecules, (ii) label-free detection, (iii) short response time, (iv) simple sample treatments, (v) easy result interpretation and (vi) user friendly along with the use of minimal electrical components.
The venom from different species will be collected. The specimens collected from the wild condition will be placed in the recognized enclosures, and acclimatized for 6 months. After acclimatization, the specimens will be fasted for one week and venom will be milked from the fasted specimen with minimal stress. The venom milked in a specifically designed polypropylene container maintained at a low temperature (0oC – 20oC) for protein stability. The milked venom will be then lyophilized and used for purification steps. The crude venom of each species will be used for isolation and purification of specific protein separation. The specific proteins/antigens from each species and families will be isolated and purified using chromatographic techniques.
For the detection of venomous and non-venomous species, the major proteins of each species will be pooled together and used for antibody raising. For example Phospholipase A2, Metalloproteases, Serine proteases, L-amino acid oxidase, Three Finger toxins, Acetylcholine Esterase and Cobra Venom Factor, mainly seen in Viperidae and Elapidae families, will be isolated and purified for next antibody raising steps.
Species-specific proteins like Phospholipase A2, Metalloprotease, Serine protease and L-amino acid oxidase under the family Viperidae and species-specific Phospholipase A2, Three Finger toxins, Acetylcholine Esterase and Cobra Venom Factor from the Elapidae family will be isolated and purified using controlled conditions. For Family specific detection, especially in the present invention, Viperidae and Elapidae, responsible for major deaths in Indian subcontinent, the platform will use family specific proteins. Further, the antigens from other families or species can also be used for detection.
The species-specific proteins in each species will be isolated and purified for antibody raising steps. The isolated proteins/antigens will be used specifically against the detection platform development strategy. The species-specific proteins isolated will be used for species-specific detection.
The isolated and purified proteins (antigens) will be used for raising of the antibodies in sheep. Further, the primary antibodies can also be raised in horse, goat, shark, camel and avian models. The raised antibodies will be purified and specific non-cross reactive antibodies will be separated using family and species-specific venom column based isolation chromatographic models.
According to the present invention, the antibodies are species-specific primary antibody specific to venom. For example, identifying Elapidae in the sample, antibodies that are raised against major Elapidae venomous species in animal models and called as anti-Elapidae antibodies as these snakes are known to have neurotoxins in their venom. Similarly, primary antibodies specific to Viperidae are developed as they are known for have hemotoxins in their venom. Thus, the primary antibodies are developed in animal models against the two major venomous families. Further, for raising these antibodies whole venom or different isolated antigenic proteins can be used. Also, species-specific antibodies against each species will be used for species-wise detection from the envenomed samples.
Surface Plasmon Resonance based Biosensor Device for Venom Detection:
The present invention describes a system and method for detecting quantitative extent of envenomation useful to determine dose of monovalent ASV required to be administered to the victims bitten by poisonous species and to monitor the venom clearance from the body using species specific diagnostic biosensors. In an embodiment the present invention is based on combinatorial technology for the development of venom detection diagnostic biosensors. In practice this provides an efficient, fast, accurate and specific method doable at clinical/field environment.
Immunoassay based diagnostic sensors are identified as the best option, and is used to quantify the extent of envenomation (to determine dose of monovalent ASV required) and to monitor the venom clearance from the body. While the conventional approaches in bite kit development is to use affinity purified antibodies raised from whole venom/fractions, and there exists significant overlapping of specificity and sensitivity between the species which would pose problems in cross-reaction and false positive/negatives in the practical application, also due to diversity in the species across the country, the present invent is based on search for unique proteins in venom could give species-specific proteins, and the sensor based on it overcomes many limitations in the conventional approach like cross reactivity, sensitivity and quantification etc.
Plasmonic phenomenon originates from the collective oscillations of free charges in a material due to an applied electromagnetic field. Hence plasmonic devices require metallic components having abundant free electrons. These free electrons provide the negative real permittivity that is essential property for any plasmonic material. The Surface Plasmon Polariton (SPP), which propagates along the boundary between a metal and dielectric is extremely sensitive to changes in refractive index of the dielectric. This property is the basic principle of SPR based biosensors explained in Fig.1. In this case, biomolecules on the metal surface (Fig 1 D) recognize and capture analyte (Fig 1 B) present in a sample, and this key and lock mechanism produce local increase in the refractive index (Fig.1 H) at the metal surface. The refractive index increase can be accurately measured by optical means with the help of electronics.
Biosensor chips are at the heart of this technology because molecules can be immobilized and interact at their surface. In SPR biosensors, probe molecules are firstly immobilized on to the sensor surface. When the solution of target molecules (fig 1 B) is flown through the flow channel (Fig 1 K) into contact with the surface, a probe-target binding via affinity interaction occurs, which consequently induces an increase in the refractive index at the SPR sensor surface. This novel SPR sensor detector works on an optical technique used to monitor the Refractive Index change of a sensing layer after target molecule binding. It generates the electromagnetic (EM) resonance of the collective oscillations of free electrons associated with a plasmonic metal –dielectric semi-infinite interface. This resonance creates a coupled propagating surface EM field along the metal–dielectric interface. This field is highly sensitive to the RI change of the dielectric layer, meaning it can be used as a sensing layer to realize SPR-based sensors. SPR excitation requires a coupling medium to provide the required photon momentum along the interface. This can be achieved using a novel prism (Fig 1. F) design and detected by a novel detection platform (Fig 1. I). Transducers are a part of the biosensor that can have varying components and have the function of transducing the energy obtained in the receptor/detection surface (Fig 1.I) into a valuable analytical signal, by converting interactions from an electrical signal. There is a signal processing unit that contains an amplifier (fig 2.7) and a processor Fig2.8), which are necessary to condition and process the obtained signal.
The biosensor chip is a combination of glass chip, where the ligands/antibodies (fig1.A) are immobilized, coupled with a high–reflective index glass prism (Fig 2.3). The novel SPR based Biosensor design (Fig.1) and incorporation of the coating materials (Fig1.D) are critically important in detection sensitivity. The metal-oxide coating deposited on the silicon wafers (Fig 1.E) is the novelty of this chip component. The wafers will be diced to specific design and it is pre-treated before surface modification. The biochip samples will be prepared by depositing 400nm-600nm of metal oxides and novel surface modification will be done for ligands/antibodies immobilization. Gold and silver have been conventionally used in SPR based the plasmonic films (chips), metal oxides-based films developed against the glass substrate is one of our innovations. The microscopic glass (borosilicate or soda lime) would be used for the process and it was cleaned with various agents including distilled water and Iso-propyl alcohol. After the cleaning steps the surface was coated with metal oxides with radio frequency based sputtering units. It was then annealed with reducing gas and the surface was then modified with 5-6% epoxy silanes and placed in a nitrogen environment for 12 hours and then it was rinsed in ethanol for 10 minutes.
The epoxysilanes coated plasmonic films were immediately coated with antibodies or peptides or biological markers, which is very essential for sample molecule identification. The biomolecules to be coated was prepared in buffered solutions which is incubated for 4 hrs in a reducing environment. In the coated substrate, the surface plasmons can be excited by near infrared waves. For this, a prism geometry, adopted to excite surface plasmons. A monochromatic light of wavelength > 1400nm will be passed through a BK7 prism at a specific angle, and at this angle, an evanescent wave will be generated at the prism base penetrating the coated film. When the surface plasmon is excited, there will be a transfer of light wave energy into the surface plasmons, hence no light will be reflected, and this process is characterized by a dip in the angular spectrum of the reflected light at the angle of reference. During the binding reactions, the detection is accomplished by noting the change in dip position of the reflected light. Suitable photodetector (fig 1.I) having sensitivity can be used to capture the shift in the dip of the reflected light . The coated chip will function as a disposable chip in this novel innovative sensors.
The antibodies will be attached (fig 1.A) on the modified surface (Fig 1.C) covalently and thereafter it will be coupled with glass prism (FIG 1.F) for plasmonic excitation. After placing the biosensor chip in the detection platform, the samples will be run using innovative microfluidics technology( Fig 1.K). The presence of analytes in the samples will be detected by measuring the changes in the reflected light obtained on a novel detector (Fig1.I), which contain a user interface. The amount of surface concentration can be quantified by monitoring the reflected light intensity or tracking the resonance angle shifts. The innovative device has a detection limit on the order of 10 pg/ml. In addition to that the detection and an innovative simulation tool, developed through mathematical modeling, with an easy-to-use interface and several design options for calculating and analyzing the reflectance and angle of incidence of this type of device, which is very essential for the user-friendly approach.
The venom detection based innovative devise, SPR Biosensor, will detect venomous and non-venomous species from the patient’s sample in a real time mode. The real time detection is critically important for the treatment strategy. The delay in the detection, mainly symptomatic model, is one of the major reason for the envenomation complications. The biosensor chip (Fig 2.5) will be immobilized with antibodies raised against proteins of major venomous species, which will bind/interact with the analytes in the sample. Because of that interaction, the platform will differentiate whether it is a venomous or non-venomous bite. If it is a venomous bite, the system can quantify the venom load from the victim’s body. In addition to that the biosensor platform can detect and quantify the family-based and species-specific detection of species. The external view is detailed in Fig.3. and its internal view and detailing is done in Fig.4. The SPR chip will be immobilized with family-specific and species-specific antibodies against respective venom proteins, will be placed in a cassette as explained in Fig.5, which is a sensor chip (fig 5.1) fitted in a polymer support matix (Fig5.2) . The SPR chip will be immobilized with family-specific and species-specific antibodies against respective venom proteins. With this we can detect the species responsible for the bite and quantify the venom load, thereby we can initiate the monovalent antivenom at the earliest. This technology can reduce the deaths that usually caused by non-specific and delayed treatment strategy. These types of chip-based sensors have several advantages, such as (i) label-free detection, which simplifies the sensing device by eliminating the functionalization of multiple antibodies, like ELISA, (ii) dynamic measurement of binding–unbinding kinetics to observe the reaction mechanism occurring over the sensing surface, and (iii) high sensitivity.
The monochromatic light from the source (Fig 2.1) is fine tuned with a polarizer (Fig 2.2) moved towards the glass prism (Fig 2.3) . The sample flow is controlled using micro fluidic technology in a flow Cell (Fig 2.4) and the analyte present in the sample interacts with the antibodies/ ligands coated on the disposable immobilised chip (fig 2.5) The detection is accomplished by noting the change in dip position of the reflected light, using a photodetector (Fig.2.6 Fig1.I) having sensitivity can be used to capture the shift in the dip of the reflected light. The consumed light energy which will be converted into electric current, amplified by an amplifier (Fig.2.7). The dip of the reflected light against antigen-antibody (ligand-substrate) binding compared to the dip of standard unbound coated ligand, detected and analysed by a microcontroller embedded system (Fig.2.8) and which will be displayed in an LCD/LED panel (Fig.2.9). This innovative device contains an automated SPR dip adjuster (Fig 2.10) which is for dip positioning of reflected light. The detection units are enclosed in a panel box (fig 2.11)
The external view of innovative device is explained by Fig 3, it contains a sample loading site (fig 3.5) where the samples (urine, blood, serum etc.) will be added and using microfluidic technology it will move towards the censor chip inserted in the inserting site (Fig 3. 3). After detection the result will be displayed in the digital display (Fig3.4) and after detection the residue biological sample will be washed out through the waste out (Fig 3.1) The whole device in enclosed in an outer cover (Fig.3.2) .
The internal view of innovative device is pictured by Fig 4 covered under 1-7.
Dated this the 11th day of February 2024

,CLAIMS:CLAIMS
I Claim:-
1. This novel innovation is a portable chip based device for the detection and differentiation of venomous and non-venomous bites and also to quantify the venom present in the victim's body which is essential for venom bite diagnosis.
2. As per claim 1. This innovative device is applicable for all venomous species detection and also applicable for disease diagnosis where specific antigenic markers against the specific diseases can be identified against the corresponding antibodies or peptides or proteins as substrates.
3. As per claim 1. This innovative device (Fig.3) has a removable chip attached which has a glass substrate platform mainly borosilicate or soda lime as its base (Fig 1.E) upon which a coating of metal oxide (Fig 1.D) is made, along with surface modification by epoxysilanes, which is the base for the immobilised antibody or peptides or protein markers against specific substrates or antigens (Fig 1.A) introduced as chips and creating a base for the attachment of the antigen (Fig 1.B). A prism (Fig 1.F) is attached to the glass platform (Fig 1.E), through which a monochromatic light of wave length >1400nm is induced from a light source (Fig1.J). The polarised light (Fig 1.G) which is passed through the BK7 prism reflected by the antigen-antibody-activated surface (Fig 1.C), The out coming reflecting light (Fig 1.H) , is detected by a detector (Fig 1.I) which acts as an indicator of venom or substrate load.
4. As per the claim 1. The detection is accomplished by noting the change in dip position of the reflected light, using a photodetector (Fig.2.6) having sensitivity can be used to capture the shift in the dip of the reflected light. The consumed light energy which will be converted into electric current, amplified by an amplifier (Fig.2.7). The dip of the reflected light against antigen-antibody (ligand-substrate) binding compared to the dip of standard unbound coated ligand, detected and analysed by a microcontroller embedded system [Fig.2.8) and which will be displayed in an LCD/LED panel (Fig.2.9) .
5. As per claim 1. This innovative device contains an automated SPR dip adjustor (Fig.2.10) which is for the dip positioning, critical for the detection.
6. As per claim 1. metal oxides-based films developed against the glass substrate is used to create a plasmonic surface which becomes binding factor for the surface modification on which the antibodies are coated.
7. As per claim 1. This innovative device contains introducible chips which enables antigen-antibody interaction to detect the presence of venom load specific to the snakes and other venomous species, which makes the innovative device reusable.
8. As per claim 1. This innovative device contains a specific software capable of analysing the refracted light from the prism and displays the species responsible for the bite and the amount of the reflected light which will be analysed thereby the venom load can be quantified.
9. As per claim 1. This innovative device contains a BK-7 prism which becomes the medium for refracting the light which passes through it, reflected by the activated surface created by the antigen-antibody interaction.
10. As per claim 1. This innovative device contains a detection platform wherein, the light refracted by the antigen-antibody interaction is concentrated, which enables the identification of the presence of specific venomous species and its quantity by deviation of refracted light using a photodetector.