DESC:
COMPLETE SPECIFICATION
Title of Invention-
Electrochemical Biosensor for Venom Detection
Field of the invention
Biotechnology/ Medical device
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 used to detect the presence and/or levels of biomolecules, typically in a fluid sample. For instance, biosensors may be used to determine the levels of particular chemicals in biological fluids, such as blood. Specific sensors can therefore be used to determine the levels of glucose, potassium, calcium, carbon dioxide, and other substances in blood samples. Biosensors such as these often use an electrochemical system to detect a particular substance of interest. The electrochemical system includes substances such as enzymes and redox mediators to react with the substance of interest (the target substance) and to thereby produce ions that can carry a current. A set of electrodes are used to generate an electrical potential that attracts the ions to the electrodes, creating a circuit that can be used to measure the resulting current. In our system, a biosensor includes an enzyme which is immobilized by a membrane. The target substance in a fluid sample migrates through the membrane and reacts with the enzyme. This forms ions within the fluid sample. These ions then migrate through the fluid sample to the system's electrodes. The migration of the ions to the electrodes generates an electrical current that is measured. Because the current depends upon the concentration of the target substance in the sample, the measured current is then translated to a concentration of the target substance.

Though monovalent ASVs are available, lack of a proper diagnostic system preludes this opportunity. Many attempts have been made worldwide to develop species specific diagnostic kit based on antibodies, but have not found successful mainly because of inter-species cross reactivity to the crude venom as also the lack of sensitivity related to the small quantities in which venom is injected.
Conventional approaches in snakebite kit development is to use affinity purified antibodies raised from whole venom/fractions, but there exists significant overlapping of specificity and sensitivity between the species. This would pose problems in cross-reaction and false positive/negatives in the practical application, also due to diversity in the snake species across the country. Hence, there is a need for an effective technology for administration of monovalent anti-snake venom which will avoid the abovementioned drawbacks/disadvantages of the prior arts.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/ species and to monitor the venom clearance from the body using species specific diagnostic biosensor. The present inventors have designed the sensor detector portion under the embedded system, which is a combination of both hardware and software and peripherals additional mechanical parts designed to perform a specific task.
Drawings detailed
Fig. (1) : Electrochemical Biosenor device design
Fig 1 (a)
A- Charging port
B- Liquid Crystal Display (LCD)
C- Microchip insertion site
D- Biosensor strip insertion site
Fig 1(b)
Fig. 2. Biosensor Strip Design
A- Electrodes positioning
B- Inert Polymer matrix
Fig 2 Biosensor Strip external design
A-Electrodes positioning
B-Inert Polymer matrix
Fig 3 Biosensor strip internal design
A. Electrode leads
B. BioPolymer Matrix substrate
C. Counter Electrode
D. Working Electrode
E. Reference Electrode
Fig 4 Working Electrode, antigen antibody bioassembly
A. Working electrode site
B. Primary antibody
C. Bound antigen
D. Enzyme labelled Secondary Antibody
Fig 5 Working Electrode, ligand substrate bioassembly
A. Working electrode site
B. Substrate (Venom specific)
C. Venom components
Fig 6 Electrochemical Biosensor block diagram
Fig 7 Schematic Circuit Diagram
Fig 8 Printed Circuit Board Designs

DETAILED DESCRIPTION OF THE INVENTION:
The present invention relates to an easy to operate quantification platform for the identification of species responsible for the envenomation and to measure the venom present in the victim's body is described herein. The present invention 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.
The present invention relates to an electrochemical biosensor kit, a device for determining the quantitative load of venom toxin in the victim’s blood sample which is essentially important for venomous and non-venomous differentiation and detection. And further, the venom toxins under the venomous families and species-specific detection can also be specifically incorporated into the electrochemical 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 snakebite cases. The quantitative detection kit of the present invention works on electrochemical detection, wherein the immunoassay components are arranged in a defined manner in the screen printed electrode to obtain the detection.
The venom from different snake 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. In this way we can also raise antibodies against major venom components in mildly venomous species, especially in Colubridae and Atractaspididae.
Family specific detection can also be done by pooling together major venomous components under each families and raise the antibodies against components against each families, which will be incorporated/coated in the printed electrodes, especially working electrodes for further detection.
Species-specific proteins like Phospholipase A2, Metalloprotease, Serine protease and L-amino acid oxidase proteins 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 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 the venomous species. The venoms are characterized and isolated and purified venom proteins will be used for antibody raising. For example the identification of Elapidae in the sample, antibodies that are raised against major specific proteins against 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 species 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.
According to the present invention, the secondary antibodies are preferably antibodies raised in specific and required animal models against the primary antibodies. The patient’s samples will be collected from the bite site fluids, the blood, serum, plasma and urine will be collected by novel methodology for further analysis using our kit. The debris and high molecular weight substances will be removed with the support of the novel filtration models. The secondary antibodies are enzyme labelled, mainly responsible for electrochemical reaction, which is essentially important for venom quantification.
Electrochemical Biosensor for Snake Venom Detection:
The present invention is a device which engages 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 venomous 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 snake 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 snakebite 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 snake 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. In the sensor detector portion designed under the embedded system, which is a combination of both hardware and software and peripherals additional mechanical parts designed to perform a specific task.
The current design does not require a mediator and electrons are directly transferred from the electrode to substrate molecule or vice versa. Here, the electron transfer mediator (enzyme itself) shuttles the electrons between the redox-center of the enzyme and the electrodes, having a low oxidation potential. Antibodies used in this technology are having high specificity of antigen-antibody binding which provides accurate and specific result. In order to detect specific antigens, specific antibody fractions are surface mobilized, using micro-contact printing, by adsorption to a conductive polymer matrix. After immobilizing antibodies to a surface, an analyte is introduced to which the antibodies bind specifically. A secondary labelled antibody then binds to the analyte in order to detect its concentration. The detection antibodies re coupled to a detection enzyme, which allows quantitative measurements of the amount of bound antigens by monitoring the electrical signal generated by the enzymatic reaction. In the present venom biosensor, the test strip first applied with the envenomed patients' urine/ bite-site's body fluid/ peripheral blood sample and then treated with wash buffer, which removes the nonspecific components. In order to passively extract serum from whole human blood micro-filters can be placed in sample loading site. This avoids clumping or aggregation of blood cells over the antibody coated strip. After washing, the test strip is then treated with enzyme conjugated secondary antibody and followed by washing, using buffered solution. The fluid flow (automated or capillary action) in micro-channels removes the excess unconjugated antigens, enzyme conjugate and substrate in just few minutes.
The substrate can be measured with three electrode system (WE: Working electrode; CE: Counter electrode; RE: Reference electrode) using a screen printed model. The coating of antibodies is critically important for detection; for venomous and non-venomous detection, antibodies raised against the common proteins in the major venomous species will be coated in the strips, for species-specific quantitative detection, the antibodies raised against the species-specific proteins will be used. The treatment is being done with these specific-antibodies and after incubation, it is then
inserted into separate channels in the detector. A single test strip sensor coated with multiple species-specific antibodies can also be designed. After inserting the test strip in the detector, the electric charge will be automatically generated and channelled. Substrate application to the test strip activates the enzymatic process. This will generate electron (electric signal) accepted by electrode and the detector detects voltage variation. The electric signal produced in the test strip equals the amount of antigen bound to the species specific antibodies coated in the strip. The voltage variation can quantitatively assay the amount of the free antigen circulated in the body fluid of the envenomed victim and it can easily identify the snake responsible for the envenomation. The results will be displayed in the digital monitor of the sensor as species name and envenomation quantity (percentage or ng/ml). The snake identification and envenomation quantity detection using the biosensor replaces the conventional clinical practice with species specific monovalent technology.
The sensor detector portion is designed under the embedded system, which is a novel combination of both hardware and software and peripherals additional mechanical parts designed to perform a specific task, using microcontroller (Fig.1 a) contains a charging port (Fig.1a .A) and results will be displayed in a liquid crystal assembly (Fig.1a. B). The program and software for the analysis will be coded in a microchip is inserted via microchip insertion site (Fig.1a. C) and the strip will be inserted through strip insertion site (Fig.1a.D) as part of detection steps .It’s a hand held, single technique, potentiostat based customized electrochemical reader that is configured to customer’s needs, allowing any layman to use and interpret in a LCD screen where the analyte concentration will be displayed. The venom load will be calculated and results will be displayed as microgram/millilitre against corresponding fluid model. The software in the medical device will analyse the venom load and calculate the amount against each corresponding samples. The insertion of the strip is depicted in the picture, Fig.1b.
This Snake Venom Bio-sensor can work in three electrode configurations made up of novel biopolymer matrix (Fig.2.A) and coated with electrodes (Fig.2.B) with conductive inks.. The rectangular shaped Biosensor strip (Fig.3) consists of reference electrode (Fig.3.E), working electrode (containing immobilized bio-recognition component) electrode (Fig.3.D) and the third counter electrode (Fig.3.C), coated in a biopolymer matrix substrate (Fig.3.B) , in order to solve the limited control of the potential on the working electrode surface with higher currents. The three electrode system is connected to internal circuits in the machine via leads (Fig.3.A). The biosensor strip made of conducting polymer matrix coated with electrodes. The sensor strip can be designed with biomaterial and the biopolymer matrix on strip surface will be activated for further use. The fluid flow in micro-channels of the test strip can be controlled by either capillary action or by automated microfluidics mode. Envenomed animal model/human samples/antigens (Fig.4.C) can be added to the test strip working electrodes functionalized with the capture primary antibody (Fig.4.B) on the working electrode surface (Fig.4.A). After incubation at 37°C, auto wash it with buffer that removes excess/unbound components in the sample. It is then applied by labelled secondary monoclonal antibody (Fig.4.D) and the unbound conjugates removed by washing using buffered solution. The fluid flow in the micro channels can be controlled by either automatic sucking system or by capillary action. This sandwiched test strip is then connected to the detector (apparatus) and the standard substrate solution (concentration specific) is then immediately transferred through substrate channel. The substrate reacts with specifically bounded conjugated antibodies and thereby generates current which equals to the amount of venom; this amperometric measurement is immediately and simultaneously taken at a specific voltage. In addition to the antigen antibody based system, a novel bioassembly, with specific venom specific substrate (Fig.5.B) bound to the working electrode (Fig.5.A) , which will interacts with the sample containing venom components, act as protein/non-protein components (Fig.5.C). The binding of enzyme substrate or protein/non-protein ligand interaction will result in the generation of current flow. This will develop a voltage variation which is detected and analysed by the microcontroller embedded system. The final output from the invention is "an easy to operate multiport biosensor" for the identification of snake envenomation and to monitor the venom quantitatively in the victims of snakebite. The electrochemical biosensor machine’s block diagram was depicted in the picture (Fig.6.) and also the circuit schematic diagram represented in Fig.7. along with the printed circuit board designs (Fig.8) .This invention replaces the conventional non-specific polyvalent technology to specific monovalent technology and thereby significantly reduces the allergen (generally globulins in the polyvalent ASV sera) load on the patient and associated side effects. The present invention brings out novel concept described herein above from bench to bedside, and ensures that the technology, will reach out for the benefit of civilians.
In addition to the antibodies against venom components coated in polymer matrix, we can also use peptides and recombinant proteins as ligands for substrate binding. The substrate against venom components can also be coated in the polymer matrix (working electrode) and the resultant output signals will be detected by the sensor platform when it interacts with the venom components in the snake envenomed victim biological samples.

,CLAIMS:CLAIMS
I Claim:-
1. This novel innovation is an electrochemical biosensor 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 bite 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.1) has a removable biosensor stripattached which has a bioplymer substrate platform as its base (Fig 3.B) upon which a coating of elctrode conducting materials, Counter electrode (Fig.4.C). Working Electrode (Fig.4.D) and Reference Electrode (Fig.4.E) are 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 introduced as strips and creating a base for the attachment of the antigen (Fig 4.B). Venom components or antigens (Fig.5.C) and Fig. 6. C) in the samples are bound to specific antibody (Fig.4.B) or substrates (Fig.5.B) and amperometric measurements are detected and analyzed using a microcontroller based embedded system model (Fig.7).
4. As per the claim 1. The detection is accomplished by amperometric measurement calculation by noting the change in the voltage either by antigen-antibody based biosasmebly model (Fig.5) or by ligand substrate bioassembly model (Fig.6). In antigen antibody interaction, an enzyme labelled secondary monoclonal antibody (Fig.5.D) will binds with the antigen and thereafter it will reacts with the substrate for the generation of electric current, whch is essential for amperometric measurements. In the case of ligand substrate bioassembly (Fig.6), the venom specific substrate (Fig.6. B) will be coated on the working electrode (Fig.6.A) and the electric current or voltage variation will be detected after it binds with venom components (Fig.6.C). The amperometric measurements will bedetected and analysed by a microcontroller embedded system and which will be displayed in an LCD/LED panel (Fig.7)
5. As per claim 1. This innovative device contains introducible electrocehmical strips 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.
6.As per claim 1. This innovative device contains a specific software capable of analysing the amperometric measurements, detects the species responsible for the bite and the amount of the reflected light which will be analysed thereby the venom load can be quantified.
7.As per claim 1. This innovative device contains a detection sensor wherein the amperometric measurements are detected, and then converts the analaog signals into digital signals, which is analyzed in the microcontroller based embedded system and displayed in an LCD/LED panel (Fig.7)