TWI855793B - Biosensor chip and related method - Google Patents

Abstract

The biosensor chip disclosed herein comprises a substrate; a bimetallic layer, which is configured above the substrate; and a detection layer, which is configured above the bimetallic layer; wherein the bimetallic layer is a gold-aluminum bimetallic layer or a gold-chromium bimetallic layer; wherein the detection layer includes a plurality of gold nanoparticles a plurality of nucleic acid aptamers, and the plurality of gold nanoparticles are functionalized by the plurality of nucleic acid aptamers. The biosensor chip and related method provided herein have excellent sensing sensitivity, high detection rate, and easily transportable.

Description

Biosensing chip and related methods

The present invention is related to a biosensor chip and related methods; in particular, it is related to a biosensor chip, a detection kit and an optical biosensor system comprising the same, and a method for evaluating the content of snake venom analytes using the optical biosensor system; however, the present invention is not limited thereto.

It is well known that most members of the Bungarus genus in the family Cobra are venomous. In South Asia and Southeast Asia, Bungarus has caused a considerable number of poisoning cases, and 70% of the cases have caused death; therefore, this type of venomous snake has been regarded as one of the most important public health issues today. In addition, since the venom will cause acute paralysis, respiratory failure and even death in patients within 4 hours after entering the human body, a rapid and accurate detection method is urgently needed to deal with it; if the venom in the human body can be detected and quantified early, there is a chance to extend the patient's expected life as much as possible by giving the patient an appropriate amount of antivenom serum and treatment within a few hours of being bitten.

β-Bungarotoxin (β-btx) in the venom of the genus Bungarotoxin is closely related to the venom of the snake; it is a deadly neurotoxin that can bind to nerve endings and block the transmission of acetylcholine, thereby destroying nerve endings and causing muscle paralysis. Therefore, β-btx can be used as an important biomarker after being bitten by a genus Bungarotoxin, and is an indispensable basis for immediate diagnosis and treatment.

In view of this, the importance of biosensing devices, systems or methods for β-btx is self-evident. Existing technologies include optical immunoassay (OIA), electrochemical immunoassay (EIA), magnetic affinity immunoassay (MAI), enzyme immunoassay (ELISA) and aptamer-based immunoassay (ALISA) designed based on the concept of immunoaffinity; however, most of these immunoassay methods have problems such as complex operation and poor instrument mobility. To address this problem, technologies such as ion-sensitive field-effect transistor (ISFET), liquid crystal sensor (LC) and paper-based sensor element were once considered as solutions; however, these methods all have the problem of poor sensing sensitivity. Later, the lateral flow assay (LFA) was proposed to use labeled nanoparticles to improve sensing sensitivity; unfortunately, this method still lacks results-answers and cannot be quantitatively analyzed.

Surface plasmon resonance (SPR), also known as surface plasmon polariton or surface plasmon propagation, is a phenomenon based on the excitation of surface plasmons at the interface between the metal film and the dielectric layer when light is incident on the surface of the metal film at a specific angle. The incident light passes through the dielectric layer with a high refractive index at a specific angle and is absorbed by the metal film to generate surface plasmons, while the light that is not effectively absorbed by the metal film will be reflected back to the detector and analyzed by the detector to form the SRP reflection spectrum (hereinafter referred to as "SRP spectrum"). The SRP spectrum shows a resonant dip at the wavelength where light is most efficiently absorbed by the metal film and surface plasmons are generated, and at the wavelength where light is not effectively absorbed by the metal film, the SRP spectrum shows a full width at half maximum (FWHM). Based on the aforementioned mechanism, the principle of SRP used in biosensors includes measuring the wavelength shift of the resonant dip caused by the change in refractive index caused by the absorption of biomolecules onto the biosensor surface. By monitoring the resonance shift of the SRP spectrum, the concentration of the analyte can be quantified and measured. Compared with other known snake venom biosensors, this measurement process brings several advantages to the SRP biosensor, including direct and easy installation, instantaneous and excellent sensitivity, making it have great application potential in practice.

Based on the content described in the previous technology, although the biosensor using surface plasmon resonance (SPR) technology has many advantages in use, it has the disadvantage that the detection is not fast enough and the detection results cannot be obtained within a few minutes. Specifically, generally speaking, the accurate determination of the analyte needs to wait until the signal reaches a stable stage (usually It takes tens of minutes). When the sensing time is shortened, the analyte that can be captured on the metal film surface will decrease, resulting in a decrease in signal binding in the SPR spectrum. Therefore, further shortening the detection time to a few minutes may result in the inability of biosensors based on SRP technology to accurately measure the analyte. Therefore, the trade-off between detection time and signal limits the application of SPR technology in rapid analysis. In order to accurately detect analytes at the minute-level measurement scale, it is necessary to improve the sensitivity of SRP biosensors to amplify the signal through improvement strategies. In order to provide a biosensing system using SRP technology (SRP biosensing system), which has the advantages of excellent sensing sensitivity, fast detection and easy portability, the inventors of the present invention propose the following solutions: First, the optical biosensing system proposed in the present invention includes a biosensing chip , is to deposit a double metal layer on a substrate, specifically a gold-aluminum double metal layer or a gold-chromium double metal layer, wherein the gold-aluminum double metal layer includes a gold element layer and an aluminum element layer, the gold element layer is arranged on the aluminum element layer, and the gold-chromium double metal layer is a chromium element layer replacing the aluminum element layer in the gold-aluminum double metal layer. Compared with the chromium element layer, the aluminum element layer can act on a wider wavelength range of light sources, and the surface plasmon resonance effect it generates is more sensitive. The aluminum layer or the chromium layer can be used as an adhesive layer to deposit the gold layer on the substrate, wherein the gold layer has good environmental stability. However, compared with the gold-aluminum bimetallic layer, the gold-aluminum bimetallic layer has more advantages in preparing biosensing chips. Secondly, fixing a plurality of gold nanoparticles on the bimetallic layer can enhance the local surface plasma field of the bimetallic layer, thereby further improving the sensitivity of the optical biosensing system. Finally, each of the gold nanoparticles on the double metal layer is functionalized with multiple nucleic acid aptamers, wherein the multiple nucleic acid aptamers can bind to β-btx with high affinity. More importantly, the multiple nucleic acid aptamers have better environmental tolerance and are easy to reorganize after degradation, so they are beneficial to the optical biosensor system while having high sensitivity and improving the practicality of the optical biosensor system, and also help to reduce costs. Therefore, the biosensor chip in the optical biosensor system proposed by the present invention helps to maintain high sensitivity while having excellent stability, thereby making the application environment of the optical biosensor system more extensive and diverse.

Specifically, the present invention provides a biosensor chip, comprising: a substrate; a gold-aluminum bimetallic layer disposed on the substrate; and a detection layer disposed on the gold-aluminum bimetallic layer; wherein the detection layer comprises a plurality of gold nanoparticles and a plurality of nucleic acid aptamers.

According to one embodiment of the present invention, the substrate is a glass substrate.

According to an embodiment of the present invention, the gold-aluminum bimetallic layer includes a gold element layer and an aluminum element layer, and the gold element layer is disposed above the aluminum element layer; preferably, the thickness of the gold element layer is greater than the thickness of the aluminum element layer.

According to one embodiment of the present invention, an alkane thiol layer is further included between the gold-aluminum bimetallic layer and the detection layer.

According to one embodiment of the present invention, the surface of each of the plurality of gold nanoparticles comprises an avidin coating, and each of the plurality of nucleic acid aptamers comprises a biotin modification, and the plurality of gold nanoparticles and the plurality of nucleic acid aptamers are bonded by avidin-biotin affinity.

According to one embodiment of the present invention, each of the plurality of nucleic acid aptamers comprises the nucleic acid sequence of SEQ ID NO: 1.

According to one embodiment of the present invention, the plurality of nucleic acid aptamers have biological affinity with β-ghost snake toxin.

On the one hand, the present invention also provides a detection kit for detecting snake venom analytes, which includes the biosensor chip as described above.

Another aspect of the present invention provides an optical biosensor system, which includes: the biosensor chip as described above; a sample flow channel connected to the biosensor chip and configured to allow a sample to flow into and contact the biosensor chip; a light source, which is configured to correspond to the biosensor chip and configured to emit a light ray toward the biosensor chip; a dielectric layer, which is configured between the light source and the biosensor chip; and a spectrometer, which is configured to correspond to the biosensor chip and configured to receive the light ray reflected by the biosensor chip and convert it into a response signal.

According to one embodiment of the present invention, the medium layer is a prism and is configured to allow the light to be incident on the biosensor chip at an incident angle of 66 to 79 degrees.

According to one embodiment of the present invention, the spectrometer receives the light with an optical fiber.

According to one embodiment of the present invention, the optical biosensing system is a surface plasmon resonance biosensing system.

Another aspect of the present invention provides a method for evaluating the content of snake venom analytes, which comprises the following steps: providing the optical biosensing system as described above, and inputting a serum sample into the optical biosensing system through the sample flow channel; standing for a reaction time, allowing the optical biosensing system to obtain a snake venom analyte in the serum sample and generate the reaction signal; and quantifying the reaction signal and evaluating the content of the snake venom analyte in the serum sample.

According to one embodiment of the present invention, the snake venom analyte is β-gamma-serpentine toxin.

According to one embodiment of the present invention, the dilution ratio of the serum sample is 1500 times.

According to one embodiment of the present invention, the reaction time is 5 minutes.

Accordingly, the present invention relates to a biosensor chip, a detection kit and an optical biosensor system including the same, and more particularly to a method for evaluating the content of snake venom analytes using the optical biosensor system. The present invention has the advantages of excellent sensing sensitivity, rapid detection and portability; further, the present invention can adopt direct (no labeling required) qualitative and quantitative measurements, and its performance has good reproducibility and is easy to store for a long time in a room temperature environment; in addition, the present invention can also be used to quantify snake venom analytes in serum samples, thereby accurately evaluating the patient's poisoning condition.

100: Biosensor chip

102: substrate

104: Gold-aluminum double metal layer

104A: Aluminum layer

104B: Gold Element Layer

106: Alkane thiol layer

110: Detection layer

110A: Gold nanoparticles

110B: Nucleic acid aptamer

120: Analyte

200:Optical biosensing system

202: Sample flow channel

204: Light source

206: Dielectric layer

208: Spectrometer

210: Optical fiber

212: Collimator

214: Control unit

A: Angle of incidence

L: Light

S102~S106: Steps

In order to make the above and other purposes, features, advantages and embodiments of the present invention more clearly understood, the following diagrams are provided: Figure 1 is a schematic diagram of the combination of a biosensor chip and an analyte in one embodiment of the present invention.

Figure 2 is a schematic diagram of the structure of an optical biosensor system of an embodiment of the present invention.

FIG3 is a flow chart of a method for evaluating the content of snake venom analytes according to an embodiment of the present invention.

Figures 4A and 4B are the results of the relationship between the double metal layer material and the SPR reaction according to an embodiment of the present invention.

FIG. 4C is a p-polarization reflectivity distribution diagram of the interface between water and gold element layer according to an embodiment of the present invention.

Figures 5A to 5F show the effects of double metal layers and gold nanoparticles on the detection limit in an embodiment of the present invention.

Figures 6A to 6D are schematic diagrams showing the improvement in detection speed of the optical biosensor system in an embodiment of the present invention.

Figures 7A to 7C are the measurement results of serum samples in the embodiments of the present invention.

Figures 8A to 8C are the results of reproducibility and storage stability in the embodiments of the present invention.

Sequence Listing

This application is filed together with a sequence listing in electronic format. The sequence listing is named "221095.xml", created on July 25, 2023, and is 1.79KB in size. The relevant information in the sequence listing is incorporated into this article by full reference.

Terminology

Each term used in this specification is generally within the scope of the present invention, and the specific context of each term is the same as its general meaning in the relevant field. The specific terms used in this specification to explain the present invention will be explained below or elsewhere in this specification to help people in the industry understand the relevant description of the present invention. In the same context, the same term has the same scope and meaning. In addition, since there is more than one way to express the same thing; therefore, the terms discussed in this specification can be replaced with alternative terms and synonyms, and whether a term is specified or discussed in this specification does not have any special meaning. Although this specification provides synonyms for some terms, the use of one or more synonyms does not exclude the use of other synonyms.

As used in this specification, "a" and "the" may be interpreted as plural unless the context clearly indicates otherwise. In addition, in this specification and the accompanying patent application, unless the context indicates otherwise, "middle" and "inside" include "located in..."; unless the context indicates otherwise, the direction of the bullet tip is defined as "upper" or "lower". Furthermore, the description may be accompanied by titles and subtitles for ease of reading, but these titles do not affect the scope of the invention.

As used herein, the term "β-bungarotoxin (β-btx)" is a polypeptide and a neurotoxin that is commonly found in the venom of Bungarus snakes. β-btx can bind to nerve endings and block the transmission of acetylcholine, thereby destroying nerve endings and causing muscle paralysis, etc.

As used herein, the term "nucleic acid" or "polynucleotide" refers to a polymer that can correspond to a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) polymer or an analog thereof. This includes nucleotide polymers such as RNA and DNA, as well as synthetic forms, modified (such as chemically or biochemically modified) forms, and mixed polymers (such as including RNA and DNA subunits). Typical modifications include methylation, substitution of one or more naturally occurring nucleotides with analogs, modifications such as uncharged bonds between nucleotides (such as methylphosphonates, phosphotriesters, aminophosphonates, carbamates, etc.), pendant parts (such as polypeptides), intercalants (such as acridine, psoralen, etc.), chelators, alkylating agents, and modified bonds (such as alpha-mutator nucleic acids, etc.). In addition, synthetic molecules that mimic the ability of polynucleotides to bind to a specified sequence through hydrogen bonds and other chemical interactions are also included. Nucleotide monomers are typically linked by phosphodiester bonds, although synthetic forms of nucleic acids may contain other linkages (e.g., peptide nucleic acids as described by Nielsen et al. (Science 254:1497-1500, 1991)). Nucleic acids may be or include, for example, chromosomes or chromosome fragments, vectors (e.g., expression vectors), expression cassettes, naked DNA or RNA polymers, products of polymerase chain reaction (PCR), oligonucleotides, probes, and primers. Nucleic acids may be single-stranded, duplexed, or triple-stranded and are not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence may optionally include or encode complementary sequences in addition to the sequence specifically designated.

As used herein, the term "aptamer" is also called aptamer or aptamer, which refers to oligonucleotides or peptide chains that can bind to various target molecules (small molecule compounds, proteins, nucleic acids, and even cells, tissues and organs, etc.) or targets. Here, aptamers specifically refer to "nucleic acid aptamers", which are commonly used as molecular identification tools in the field of biotechnology. Nucleic acid aptamers are usually selected and cloned from a large number of sequences. The production cost is lower than that of antibodies, and they are easy to modify, and the production has high reproducibility and purity. At the same time, nucleic acid aptamers have better tolerance to the environment and are easy to reassemble after degradation. In addition, nucleic acid aptamers can be functionalized by various feasible technologies in practice; for example, in the field of biological detection to which the present invention belongs, biotin modification (5'biotinylation) can be added to the 5' end of the nucleic acid aptamer.

As used herein, the term "streptavidin", also known as streptavidin, refers to a protein (about 60 kDa) that has a very high affinity for biotin. The binding of biotin to avidin is one of the strongest non-covalent interactions known in nature, with a dissociation constant (Kd) of about 10-14 mol/L. Since the avidin-biotin complex has good tolerance to organic solvents, denaturants (such as guanidine hydrochloride), detergents (such as SDS), proteolytic enzymes, and extreme temperatures and pH values, avidin is widely used in the fields of molecular biology and bio-nanotechnology. Specifically, in the present invention, avidin is fixed on the surface of gold nanoparticles to form an avidin coating, so that the gold nanoparticles can be bonded with nucleic acid aptamers through avidin-biotin affinity.

As used herein, the term "biosensor chip" refers to a micro-device that uses microelectronics technology to place biological materials that can produce specific biochemical reactions with analytes (or specimens) on a substrate, and can be quantitatively detected by a highly sensitive detection system. Biosensor chips are designed using principles of microelectronics, microfluidics, molecular biology, biotechnology, genetic testing, analytical chemistry, etc., and use silicon wafers, glass or polymers as substrates. Combined with micro-electromechanical automation or other precision processing technologies, the high-tech components produced can quickly perform complex calculations like semiconductor chips; biosensor chips have fast, accurate, and low-cost biological analysis and testing capabilities. In molecular biology, biosensor chips enable researchers to quickly screen a large number of biological analytes for various purposes, such as disease diagnosis and bioterrorism detection.

As used herein, the term "surface plasmon resonance (SPR)" is also called surface plasmon polariton or surface plasmon propagation, which is a phenomenon in which polarized electromagnetic waves are generated when a light source passes through a medium layer with a high refractive index (usually glass material) and then enters the surface of a metal film; when SPR occurs, the intensity of the reflected light will be significantly attenuated. Specifically, in the field of biosensing to which the present invention belongs, the surface plasmon resonance biosensing system is based on the fact that once the biomolecules fixed on the surface of the metal film change, their refractive index will also be affected, thereby changing the conditions for generating SPR, and then the sensing effect can be achieved by measuring the signal generated based on the change. In detail, if a biosensor chip is used, the substance that interacts with the analyte is fixed as a ligand on the metal film of the chip, and light is emitted from the back side of the chip to cause total reflection at the interface between the gold film and the glass. At this time, the intensity of the partially reflected light will decrease and generate an SPR signal. Then the analyte flows over the surface of the chip and the ligand binds to the analyte, the mass will increase and cause the refractive index of the chip surface to change, and the position of the SPR signal will also shift accordingly (conversely, if it dissociates, it will return to the original signal position).

As used herein, the term "double metal layer" refers to a layered structure formed by stacking layers of different metal materials.

As used herein, the term "limit of detection (LOD)" refers to the minimum signal strength or minimum physical quantity that can make the detection result meet a certain degree of confidence or have a certain significant difference.

Biosensor Chip

On one hand, the present invention provides a biosensor chip, and FIG1 is a schematic diagram showing the combination of the biosensor chip provided by the present invention and an analyte. Referring to FIG1 , the biosensor chip 100 is a stacked structure, which comprises a substrate 102, a gold-aluminum double metal layer 104, and a detection layer 110 from bottom to top. Specifically, the substrate 102 is a glass material, and its refractive index is different from that of metal; preferably, the material of the substrate 102 is an optical glass with an Abbe number between 50 and 85, such as borosilicate glass (BK7). The gold-aluminum bimetallic layer 104 includes an aluminum layer 104A and a gold layer 104B, and the aluminum layer 104A is disposed below the gold layer 104B, and the thickness of the aluminum layer 104A is less than that of the gold layer 104B. In terms of thickness, the thickness of the aluminum layer 104A is 1 to 10 nanometers, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nanometers; preferably, the thickness is 1 to 5 nanometers, for example, 1, 2, 3, 4 or 5; more preferably, 3 nanometers. The thickness of the gold element layer 104B is 30 to 60 nanometers, for example, 30, 35, 40, 45, 50, 55 or 60 nanometers; preferably, the thickness is 35 to 45 nanometers, for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 nanometers; more preferably, it is 40 nanometers. According to different embodiments of the present invention, the gold-aluminum bimetallic layer 104 can also be replaced by a gold-chromium bimetallic layer, that is, the aluminum element layer 104A is replaced by a chromium element layer, and its thickness is also 1 to 10 nanometers, for example: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nanometers: preferably 1 to 5 nanometers, for example: 1, 2, 3, 4 or 5; more preferably 3 nanometers.

In addition, the detection layer 110 includes a plurality of gold nanoparticles 110A and a plurality of nucleic acid aptamers 110B. According to a preferred embodiment of the present invention, each of the gold nanoparticles 110A includes an avidin coating (not shown in the figure) on its surface. Without being limited by a specific theory, the geometry of the gold nanoparticles 110A is preferably a nanocube with sharp edges or tips to enhance the plasma hot spot. Preferably, the length of any side of each of the gold nanoparticles 110A does not exceed 50 nanometers, for example, 20, 25, 30, 35, 40, 45 or 50 nanometers; more preferably, the length is 40 to 50 nanometers, for example, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nanometers, but the actual implementation is not limited thereto, and the geometric shape of the gold nanoparticle 110A may also be other shapes such as a sphere. Next, the multiple nucleic acid aptamers 110B are a DNA sequence with high specificity and biological affinity for β-btx, and have a biotin modification (not shown in the figure); in detail, each of the multiple nucleic acid aptamers 110B contains the nucleic acid sequence of SEQ ID NO: 1, and the biotin modification is located at its 5' end. Thus, the multiple gold nanoparticles 110A and the multiple nucleic acid aptamers 110B can be bound through avidin-biotin affinity bonding.

Furthermore, an alkanethiol layer 106 is further included between the gold-aluminum bimetallic layer 104 and the detection layer 110; in detail, the alkanethiol layer 106 can be obtained by functionalizing the surface of the gold-aluminum bimetallic layer 104 with alkanethiol, and the ligand in the detection layer 110 is bound through the alkanethiol layer 106. Referring to FIG. 1 again, it can be seen that the biosensor chip 100 of the present invention can bind to multiple analytes 120 (i.e., β-btx) through multiple nucleic acid aptamers 110B on its surface, thereby achieving the purpose of obtaining the analyte in the sample.

According to some embodiments of the present invention, the biosensor chip 100 may also be disposed in a detection kit for detecting snake venom analytes, so as to meet a wider range of practical applications.

Optical biosensing system

On the other hand, the present invention provides an optical biosensing system, and FIG. 2 is a schematic diagram showing the structure of the optical biosensing system 200 provided by the present invention. Please refer to FIG. 1 and FIG. 2 together. The optical biosensing system 200 of the present invention includes the above-mentioned biosensing chip 100; further, the optical biosensing system 200 also includes a sample flow channel 202, a light source 204, a dielectric layer 206 and a spectrometer 208. In detail, the sample flow channel 202 is connected to the biosensing chip 100, and it is configured to allow a sample to flow into and contact the biosensing chip 100; preferably, the sample flow channel 202 is attached to the upper surface of the biosensing chip 100, and the sample flow channel 202 is a fluid structure. In addition, the light source 204 is disposed corresponding to the biosensor chip 100, and the light source 204 is disposed to emit a light ray L toward the biosensor chip 100, and the light ray L is totally reflected at the bottom of the biosensor chip 100. According to a preferred embodiment of the present invention, the light source 204 includes at least one organic light-emitting diode (OLED), and the light ray L is a polychromatic light with a peak wavelength of 580-750 nanometers (nm). In addition, the dielectric layer 206 is disposed between the light source 204 and the biosensor chip 100, and it allows the light ray L to be emitted toward the biosensor chip 100 at an incident angle A. According to a preferred embodiment of the present invention, the medium layer 206 is a prism, and its material is glass; more preferably, its material is an optical glass with an Abbe number between 50 and 85, such as borosilicate glass (BK7). On the other hand, the incident angle A is specifically 66 to 79 degrees, such as 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79 degrees; preferably 75 degrees. Next, the spectrometer 208 is arranged corresponding to the biosensor chip 100, and the spectrometer 208 is arranged to receive the light L reflected by the biosensor chip 100 and convert it into a response signal; preferably, the spectrometer 208 is a miniature spectrometer. Preferably, the spectrometer 208 is a miniature spectrometer that can operate in a wavelength range of 580 to 750 nanometers and has a signal-to-noise ratio (SNR) greater than or equal to 250:1 and a spectral resolution less than or equal to 1.33nm full width at half maximum (FWHM).

According to a preferred embodiment of the present invention, the spectrometer 208 receives the light L through an optical fiber 210; more preferably, the optical fiber 210 and the medium layer 206 are further provided with a collimator 212. In addition, the spectrometer 208 is further connected to a control unit 214 to transmit the response signal generated by the spectrometer 208 to the control unit 214; the control unit 214 is specifically a single board computer, which is used to present the relevant data of the response signal on another computer in real time in the form of wireless connection.

method

The present invention further provides a method for evaluating the content of snake venom analytes, and FIG. 3 is a flow chart of the method for evaluating the content of snake venom analytes of an embodiment of the present invention. Please refer to FIG. 2 and FIG. 3 together. The method for evaluating the content of snake venom analytes includes the following steps: S102: providing the optical biosensing system 200 as described above, and inputting a serum sample into the optical biosensing system 200 through the sample flow channel 202; S104: standing for a reaction time, allowing the optical biosensing system 200 to obtain a snake venom analyte of the serum sample and generate the reaction signal; and S106: quantifying the reaction signal and evaluating the content of the snake venom analyte in the serum sample.

According to a preferred embodiment of the present invention, the snake venom analyte is β-btx. According to a preferred embodiment of the present invention, the dilution ratio of the serum sample is 1000 to 2000 times, for example: 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000; more preferably 1500 times. According to a preferred embodiment of the present invention, the reaction time is 5 minutes.

Embodiment

In this section, the contents of the present invention will be described in detail through the following embodiments. These cases are for illustrative purposes only, and a person with ordinary knowledge in the technical field to which the present case belongs can easily know various modifications and variations. Therefore, various embodiments of the present invention will be described in detail below, but the present invention is not limited to the various embodiments listed in this specification.

Chemical reagents

Here, β-btx was purified from the venom of the parasitic antlers, and was obtained from the Centers for Disease Control, Republic of China. Phosphate buffered saline (PBS), avidin (SA), bovine serum albumin (BSA), human serum (hereinafter referred to as serum sample for testing), immunoglobulin (IgG), Tween-20, 3-butyl-1-propanol (3-MPOH), and 11-butylundecanoic acid (11-MUA) were purchased from Sigma-Aldrich. Standardization buffer (glycerol), 10 mM sodium acetate (pH 5.0), 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 50 mM sodium hydroxide (NaOH), and 1.0 M ethanolamine-HCl (ETH) were purchased from Cytiva. 40 nm SA-coated gold nanoparticles were purchased from Nanopartz and diluted in deionized (DI) water. 40 nm SA-coated gold nanoparticles are collectively referred to as "AuNPs", and multiple 40 nm SA-coated gold nanoparticles are referred to as "AuNPs". The biotin-modified DNA aptamer specific for β-btx (Anand et al., 2021) is SEQ ID NO: 1 with biotin modification at the 5' end, wherein the sequence of SEQ ID NO: 1 is GGA CAG AAA AAA AAA AAG ACA AAG AAG AGA GAG GGA GAT GGG GCT CAT; it was purchased from Integrated DNA Technologies. All other bioaffinity measurements were performed in PBST buffer (containing 1M PBS and 0.005% Tween-20) or diluted serum samples.

SPR Biosensing System

Here, the OLED light source was purchased from Ultimate Image Corp. (Taiwan); the micro-spectrometer was purchased from OceanOptics FLAME (USA); and the single-board computer was purchased from Raspberry Pi (UK). In addition, the relevant data of the SPR reaction signal will be displayed on the computer and stored in a text file for further processing using MATLAB software.

Biosensor Chip Fabrication

Here, a 3nm aluminum layer (or chromium layer) and a 45nm gold layer are first deposited by thermal evaporation to fabricate a gold-aluminum double metal layer (or gold-chromium double metal layer) in the biosensor chip. The double metal layer is then immersed in a mixed monolayer solution of 10mM 3-MPQH and 1mM 11-MUA in ethanol at room temperature overnight and then rinsed with ethanol. The modified biosensor chip is mounted on the SPR fluid system; a gradient standardization buffer (3%, 6%, and 9% wt% glycerol in DI water) is then injected into the reaction chamber for 10 minutes as a calibration ruler for the standardization data. Subsequently, the surface of the biosensor chip was washed with DI water for 8 minutes, and then placed in DI water with 0.1M NHS and 0.4M EDC for 15 minutes to set up an alkane thiol layer capped with NHS esters. Next, the surface of the biosensor chip was rinsed with acetate solution for 8 minutes, and then contacted with 40μg/mL AuNPs in acetate solution for 30 minutes; then the acetate buffer was injected into the reaction chamber for 8 minutes to clean the excess nanoparticles on the surface of the biosensor chip. Afterwards, the surface of the biosensor chip was rinsed with PBST buffer for 8 minutes, and 1M ETH solution was injected for 10 minutes to inactivate the unreacted NHS. The functionalized DNA aptamer and AuNPs were allowed to interact with each other for 40 minutes in PBST buffer at 500 nM to immobilize them, and the PBST buffer was allowed to flow for 8 minutes. For the storage stability test, the modified biosensor chip was removed from the SPR fluid system, covered with a cover glass and placed in a humidity-controlled culture dish; the culture dish was covered with aluminum foil before use and stored at room temperature away from light.

Determine bioaffinity

Here, the biosensor chip of the present invention detects β-btx protein in PBST buffer and serum samples under the SPR system. For the determination of biological affinity, different concentrations of β-btx in PBST buffer were injected onto the surface of the biosensor chip for different reaction times (30 minutes, 20 minutes, 10 minutes and 5 minutes), and then washed for 8 minutes by flowing PBST buffer. Subsequently, the surface of the biosensor chip was restored (regeneration) for 1 minute using NaOH solution and washed with PBST buffer until the SPR signal reached a stable baseline. For serum sample analysis, different β-btx concentrations were added to the serum sample, and the serum sample was diluted in PBST buffer. The β-btx in the diluted serum sample is measured by injecting it separately onto the surface of the biosensor chip and then acting on it with the above reaction time. The washing and recovery process of the serum sample is the same as that in the PBST buffer. The present invention records the bioaffinity and the signal measured by the serum sample by SPR spectroscopy. Thereby, when the serum sample containing β-btx protein contacts the surface of the biosensor chip, the change in its kinetic curve can be used to distinguish healthy people from those bitten by venomous snakes. In addition, the amount of β-btx in the serum can be further evaluated based on the quantitative results of the SPR reaction, thereby distinguishing patients with dry bites or wet bites. In order to quantify and compare the SPR signal, the SPR sensing spectrum is converted into response units (RU) by the following steps. First, the output SPR signal was normalized by subtracting the signal value of the NHS/EDC solution from DI water. Then, the normalized SPR signal was converted to refractive index units (RIU) through a calibration step, as the volume refractive index changes when the standardized buffer solutions with 3%, 6%, and 9% wt% glycerol flow. Finally, the SPR signal in RIU units was converted to RU units by dividing the RIU signal by ^10-6 (Schasfoort, 2017). For the determination of selectivity, the same method as the bioaffinity analysis was used to confirm the interaction of the aptamer with other proteins that can interfere with β-btx binding in serum, including BSA and IgG.

Effects of double metal layers and gold nanoparticles on detection performance

First, in order to further improve the detection performance of the biosensor chip of the present invention, a data simulation is performed to evaluate the relationship between the thickness of an aluminum element layer and a chromium element layer and the minimum reflection peak in the SPR spectrum when the aluminum element layer and the chromium element layer are respectively combined with the gold element layer; the results are shown in Figures 4A and 4B. From Figures 4A and 4B, the darker the color, the smaller the SRP reflection, which means the higher the SRP absorption. When the aluminum element layer is combined with the gold element layer, a higher SPR absorption can be generated at the minimum reflection peak of the SPR compared to the combination of the chromium element layer and the gold element layer. At the same time, it can be seen from FIG. 4A and FIG. 4B that when the thickness of the aluminum element layer or the thickness of the chromium element layer is 3 nanometers and the gold element layer is 40 nanometers, the biosensor chip of the present invention can have the best detection performance. In addition, FIG. 4C shows the p-polarization reflectivity distribution diagram of the interface between water and the gold element layer, wherein the gold element layer is disposed on a substrate, the substrate is borosilicate glass (BK7), and the thickness of the gold element layer is 40 nanometers. The results of FIG. 4C show that the wavelength of the light increases from 580nm (yellow) to 750nm (dark red), while the incident angle decreases from 79° to 66° (black dotted line). For subsequent testing, the wavelength of 615nm and the incident angle of 73° were used for testing. The minimum SRP reflectivity can be obtained under the above parameters. In addition, as shown in Table 1 below, when the spectrometer specifications are poor, such as the spectrometer model USB2000 with a signal-to-noise ratio of 250:1 and a spectral resolution of 1.5nm FWHM, the LOD of the present invention is 2.09× ^10-4 refractive index unit (RIU), indicating that the present invention still has excellent sensitivity when using a low-specification spectrometer. If the spectrometer has a higher signal-to-noise ratio or a lower spectral resolution, the present invention can have a lower LOD, that is, a higher detection sensitivity; wherein the higher signal-to-noise ratio, for example, the QE65 PRO specification in Table 1 has a signal-to-noise ratio of 1,000:1, which is the spectrometer with the highest signal-to-noise ratio in Table 1; wherein the lower spectral resolution, for example, the QE65 PRO in Table 1 has a spectral resolution of 1.2nm FWHM, which is the spectrometer with the lowest spectral resolution in Table 1.

Furthermore, the present invention presents the effect of AuNPs fixed on a gold-aluminum double metal layer on the detection limit (LOD). Figures 5A, 5B and 5C show the baseline noise (expressed as a percentage of baseline error) exhibited by the SPR biosensor system after injection of PBST buffer for 80 minutes under different metal layer and detection layer configurations; to avoid redundant descriptions, the groups used for measurement in the following Figures 5A to 5F and Figures 6B to 6D are respectively labeled as (a) gold-chromium double metal layer; (b) gold-aluminum double metal layer; (c) gold-chromium double metal layer plus AuNPs; and (d) gold-aluminum double metal layer plus AuNPs. Specifically, the baseline error percentage is calculated as (each point RU - average RU) / average RU * 100%.

As shown in Figures 5A and 5B, when AuNPs are not added, the baseline error percentage of the gold-aluminum double metal layer is lower, and the baseline noise standard deviation of the gold-aluminum double metal layer is 1.5 times lower than that of the gold-chromium double metal layer. It is obvious that replacing the chromium element layer with the aluminum element layer can effectively reduce the noise of the sensing system. In addition, as shown in Figures 5A and 5C, when AuNPs are fixed on the surface of the double metal layer, the baseline error percentage of the optical biosensing system of the gold-aluminum double metal layer and the gold-chromium double metal layer increases to 1.58 times and 1.34 times of the standard deviation, respectively; it is speculated that the noise signal amplification of the double metal layer modified by AuNPs should be related to the signal enhancement of the optical biosensing system. As shown in Figure 5D, when the concentration of β-btx is fixed at 500fg/mL, the combination of AuNPs on the double metal layer can amplify the detection signal to 8.31 times higher than that of the double metal layer alone. Comparison of the differences in SPR effects also shows the difference in the shape of the kinetic curves; the optical biosensor system with AuNPs takes the longest time to reach equilibrium (about 25 minutes), followed by the optical biosensor system with only a metal layer, which takes about 20 minutes). This shows that the amplification of signal noise and detection effect comes from the SPR signal enhancement ability of AuNPs.

Figure 5E shows the calibration curves of SPR effect and β-btx concentration, and obtains the 99% correlation coefficient (R ² ) in the SPR effect trend line under the four configurations, which shows that it has a good linear trend. The LOD on the four calibration curves is calculated by inserting the 3σ noise of the blank signal into the fitting curve. For the gold-chromium double metal layer, the LOD of β-btx is 310fg/mL; while the configuration using the gold-aluminum double metal layer can minimize the 3σ noise of the blank signal and reduce the LOD of β-btx to 230fg/mL. On the other hand, modifying the double metal layer with AuNPs can improve the sensitivity of the optical biosensing system; therefore, the LOD of β-btx sensing with AuNPs-modified gold-chromium double metal layer and gold-aluminum double metal layer was reduced to 0.26fg/mL and 0.12fg/mL, respectively.

Figure 5F shows the different reflection spectra generated by the double metal layer depending on whether it is matched with AuNPs. It can be seen that the wavelength of light absorbed by the double metal layer with AuNPs (612nm) is closer to the peak of the SPR light source (615nm) than that of the double metal layer alone (600nm, corresponding to the circle in the figure). Based on the better absorption of the light source and the longer attenuation length of the evanescent wave near the surface of the double metal layer, AuNPs can further help improve the sensitivity of the optical biosensing system.

Improving the detection speed of SPR biosensor system

Timely detection of β-btx is crucial for the implementation of antivenom treatment, because the neurotoxicity produced by β-btx will quickly manifest within 30 minutes after the bite. Therefore, the SRP biosensor used in clinical practice must have the ability to quickly detect β-btx within 30 minutes. However, such a detection time is a challenge for the system architecture of most SRP biosensors, because most SRP biosensors detect the signal when the exponential signal reaches a stable state, hereinafter referred to as the steady-state signal. In order to obtain a stable signal, it usually takes more than 5 times the exponential time constant to make the signal reach within 1% of the final signal value, that is, to achieve 99% analyte coverage. Since the prior art has the above disadvantages for detecting stable signals, the present invention detects transient signals, so there is no need to wait for a long time for the signal to reach a stable state. Since the present invention measures the transient signal of the exponential curve in the initial stage, and does not need to wait for the transient signal to stabilize, the detection time can be reduced. For example, when the transient signal to be detected is a signal of the half-life of the exponential curve, the detection time can be reduced by up to 50% compared to detecting a stable signal. In addition, the detection time can be further minimized by using the present invention to detect the initial curve of the signal. However, when measuring low concentration analytes for the initial curve of the detection signal, since only a small part of the analyte will cover the surface of the biosensor chip and cause the refractive index to change, the initial slope of the transient signal generated will be close to the noise, resulting in a false positive result. Therefore, in order to achieve the transient signal of the initial curve to shorten the detection time and accurately measure the concentration of the analyte, the transient signal must be amplified and the noise reduced, so as to distinguish the transient signal from the noise in a short time.

In the above content, the present invention uses a gold-aluminum double metal layer with AuNPs to reduce noise and amplify the detected signal, and it is found that it has a lower LOD than using a gold-chromium double metal layer. Surface plasmons are generated by the interaction of broadband light with the metal surface, and the remaining light is reflected back to the spectrometer to generate the SRP spectrum. The SRP spectrum presents a resonance dip in the form of FWHM. In this case, the resonance dip means that the surface plasmons are excited most efficiently. FWHM represents the spectral width, which is affected by factors such as incident light scattering, scattered light caused by incomplete absorption of the metal surface, and incident light scattering caused by surface roughness. Since noise and the FWHM of the SRP spectrum are indirectly correlated, the FWHM can be used to help evaluate the quality of the overall SRP signal. Therefore, by replacing the chromium layer with an aluminum layer to enhance the optical properties of the metal surface, better light absorption and a sharper FWHM are achieved, thereby reducing the noise generated. In addition, when light reacts with the free electrons in the AuNPs, resonance occurs at a specific frequency, thereby generating a strong local electromagnetic field around the AuNPs. Plasmonic coupling between surface plasmons and AuNPs close to the surface of the biosensor chip can amplify the SRP signal, so that when there are fewer analytes immobilized on the surface of the biosensor chip, the signal intensity can still be greater than the noise intensity. The combination of AuNPs' signal amplification and gold-aluminum double metal layer's noise reduction is conducive to achieving the purpose of reducing the detection time by detecting transient signals. The following is an experimental proof with further diagrams and text descriptions.

Based on the above, AuNPs combined with a specific metal layer can effectively improve the sensitivity of the optical biosensing system; without being limited by a specific theory, the inventors also believe that it is beneficial to accelerate the detection speed of the optical biosensing system. Specifically, the present invention is based on a fast slope transient measurement strategy to quickly detect β-btx in the sample, thereby improving the detection speed of the optical biosensing system. In detail, as shown in Figure 6A, when the analyte is added to the surface of the biosensing chip, the SPR signal generated by the binding of β-btx (2.5pM) and the aptamer follows the exponential curve of the SPR effect and has a constant half-life (t1 _/2 ); here, the half-life is defined as the time required for the SPR signal to increase from 0% to 50% (about 7 minutes). After the half-life, the SPR signal gradually increases from 65% (10 minutes) to 90% (20 minutes) and approaches the equilibrium state, that is, the situation of 100% reaction (30 minutes, sometimes may exceed, depending on the affinity constant). Therefore, the inventors believe that, without being limited by a specific theory, if β-btx can be measured in the transient phase of the reaction (i.e., the phase of the SPR signal rising sharply before the reaction reaches equilibrium in FIG6A), the detection time requirement can be effectively shortened in various sensing devices or sample forms; and in practice, the above purpose can be achieved by increasing the minimum binding rate of the SPR signal in the transient phase as much as possible within the acceptable signal-to-noise ratio standard (i.e., the minimum value of the slope in FIG6A). According to a preferred embodiment of the present invention, the detection speed of the optical biosensor system is improved through the following process. First, a low concentration of analyte is added to the biosensor chip, and the optical biosensor system is set to measure the SPR signal generated by the interaction between the analyte and the chip surface; after the SPR signal reaches equilibrium, the shortest detection time is taken at the 3σ noise line; then, the fixed minimum analyte concentration is repeatedly measured with the obtained shortest detection time. If it is found that the detection time needs to be extended during the measurement, the detection time is measured again until the SPR reaction signal in the dissociation stage is higher than the 3σ noise line.

Figure 6B and Figure 6C show the SPR response of using a gold-aluminum double metal layer or a gold-chromium double metal layer plus AuNPs (i.e., the above-mentioned groups (b) and (d)) at different detection times. As can be seen from Figure 6B and Figure 6C, regardless of the configuration, reducing the action time of β-btx on the sensing chip actually leads to an increase in the detection limit data, which in turn indicates that the sensitivity of the optical biosensing system is reduced. Specifically, for the single double metal layer, when the sample is exposed to the detection layer surface for more than 10 minutes, the optical biosensor system will generate a detection signal for the lowest concentration of β-btx (500fg/mL); however, when the exposure time is less than 10 minutes or even only 5 minutes, no detection signal will be generated. On the other hand, the optical biosensor system with AuNPs can generate a detection signal for the lowest concentration of β-btx (0.45fg/mL) after 5 minutes of exposure, and it is not lower than the 3σ noise line of the AuNPs with metal layer. It can be seen that the present invention uses AuNPs with a specific metal layer to not only effectively improve the sensitivity of the optical biosensor system, but also help accelerate the detection speed of the optical biosensor system.

In order to find a balance between detection speed and sensitivity, Figure 6D further divides the SPR reaction results into two regions according to the β-btx concentration detected under different configurations and different action times; they are the non-standard area (below the 3σ noise line, marked as the forbidden area in Figure 6D) and the standard area (above the 3σ noise line). In detail, for a single metal layer, for a β-btx concentration of 500fg/mL, after 5 minutes of action, its SPR reaction drops below the 3σ noise line and enters the non-standard area, which means that the detection limit of the single double metal layer is lower than the noise signal of the SPR biosensor system. At the same time, for 0.5fg/mL concentration of β-btx, the combination of AuNPs and metal layer can still keep the SPR reaction within the standard region. The shortest action time that can be adopted by the optical biosensing system with or without AuNPs is estimated by fitting the 3σ noise line with the partition of the SPR reaction; it is estimated that the action time of the double metal layer alone and AuNPs combined with the metal layer can be accelerated from 30 minutes to 12 minutes and 4 minutes, respectively.

Measuring β-BTX in serum

Before measurement, the inventors evaluated the selectivity of the optical biosensor system for proteins abundant in serum (such as IgG and BSA). Figure 7A shows that at a fixed concentration (1 μg/mL), the SPR response of IgG and BSA showed little change relative to the blank signal; while in the presence of the same concentration of β-btx, the SPR response increased significantly, indicating that there is specific recognition between β-btx and the aptamer. In order to determine the selectivity of the SPR biosensor system, the selectivity coefficient (SC) must be defined based on the ratio of non-specific protein and specific protein signals. The SC values of IgG and BSA are 0.14 and 0.07, respectively; the higher SC value of IgG compared to BSA indicates that IgG plays a major role in the selectivity of the optical biosensor system during serum measurement. However, the SC values of the two non-specific proteins are significantly lower than that of β-btx (SC=1), so the optical biosensor system can effectively distinguish β-btx from proteins abundant in serum, thereby improving selectivity and avoiding interference.

According to the preferred embodiment of the present invention, the dilution ratio of the serum sample to be tested is 1500 times, which can make the data of the serum sample detected by the biosensor system highly similar to the true value of the sample measured in PBST buffer, and can provide a sensor recovery rate close to 100%. Moreover, despite such dilution in the case of dry bite, the concentration of β-btx is still within the detectable range of the present invention.

23RU時，可以判別為未咬之情形，即於圖7B中顯示為「沒作用」的區域。當測定的患者血清中β-btx的之SPR反應為23RU

△RU

37RU時，可以判別為乾咬之情形，即於圖7B中顯示為「弱」的區域；此時患者可能會出現局部或全身組織損傷，例如局部咬傷部位腫脹和疼痛，可以採用低劑量的抗蛇毒血清治療。當測定的患者血清中β-btx的之SPR反應為23RU

△RU

37RU時，可以判別為乾咬之 情形。未咬及乾咬引起的環蛇中毒不會對患者造成神經毒性；而當患者被濕咬時則會發生神經毒性效應，其嚴重程度與從蛇注射到受害者體內的β-btx量相關。更進一步而言，濕咬可分為中等，強，以及非常強的咬合力。當患者受到中等咬傷時，會發生輕度神經毒性，其中SPR反應範圍為37RU

△RU

70RU，即於圖7B中顯示為「中間值」的區域。此時，患者可能會出現面部肌肉癱瘓、上瞼下垂或眼肌麻痺。此外，當患者受到強烈的咬合條件(70RU

△RU

87RU)，即於圖7B中顯示為「強」的區域，以及非常強烈的咬合條件(△RU

87RU)，即於圖7B中顯示為「非常強」的區域時，則會引發嚴重的神經毒性；患者可能會產生延髓和呼吸肌的麻痺，此時除了抗蛇毒血清治療外，尚需要額外的醫療護理。有鑑於此，本發明所提出的光學生物感測系統可有效量化樣本中的β-btx，進而利於評估患者的中毒情形。 To demonstrate the utility of the optical biosensor system of the present invention in evaluating the content of snake venom analytes, β-btx neurotoxin was quantified in serum samples at a dilution ratio of 1:1500. FIG7B shows the calibration curve of the SPR response versus β-btx concentration; after measuring the diluted serum samples, the SPR response and kinetic curves were different from those in PBST buffer at all concentrations, which shows the effect of the above-mentioned non-specific binding. Figure 7C shows the recovery rates of individual β-btx concentrations to confirm the accuracy of serum measurement; as shown in Figure 7C, the recovery rates of the optical biosensing system of the present invention are between 96.1% and 103.5%, indicating that it has excellent accuracy because the recovery rates obtained are substantially close to the true value (i.e., 100% recovery rate). In Figure 7B, the present invention provides a quantitative graph based on semi-quantitative measurement of the severity of snake envenoming using the OIA platform, which is divided into three bite types, namely: (1) no bite; (2) dry bite; and (3) wet bite. Prior art has revealed the correlation between each bite type and β-btx concentration. In detail, when the SPR response of β-btx in the patient's serum is measured to be △RU

When the SPR response of β-btx in the patient's serum is 23RU, it can be judged as a non-biting situation, which is shown as the "no effect" area in Figure 7B.

△RU

When the SPR response of β-btx in the patient's serum is 23RU, it can be judged as a dry bite, which is the "weak" area shown in Figure 7B. At this time, the patient may experience local or systemic tissue damage, such as swelling and pain at the local bite site, and can be treated with low-dose antivenom.

△RU

37RU, it can be identified as a dry bite. Ringworm envenomation caused by no bite and dry bite will not cause neurotoxicity to the patient; when the patient is bitten by a wet bite, neurotoxic effects will occur, and the severity is related to the amount of β-BTX injected into the victim from the snake. Further, wet bites can be divided into medium, strong, and very strong bite forces. Mild neurotoxicity occurs when the patient is bitten by a medium bite, where the SPR reaction range is 37RU

△RU

70RU, which is the area shown as the "mid-range" in Figure 7B. At this point, the patient may experience facial muscle paralysis, ptosis, or eye muscle paralysis. In addition, when the patient is subjected to strong occlusal conditions (70RU

△RU

87RU), which is the area shown as “strong” in Figure 7B, and very strong occlusal conditions (△RU

87RU), which is shown as the "very strong" area in Figure 7B, will cause severe neurotoxicity; patients may develop paralysis of the medulla oblongata and respiratory muscles, and at this time, in addition to antivenom treatment, additional medical care is required. In view of this, the optical biosensing system proposed in the present invention can effectively quantify β-btx in the sample, thereby facilitating the assessment of the patient's poisoning condition.

Reproducibility and storage stability

In order to enhance the management efficiency of snake bite poisoning cases in hospitals, and at the same time pursue the ability to be reused to reduce costs, and easy to store so that it can be transported to remote areas, the reproducibility and storage stability of the biosensor chip of the present invention are further tested. Figure 8A shows the reproducibility test of the biosensor chip. After a round of sample analysis, the biosensor chip is restored with NaOH solution for subsequent testing. When the biosensor chip is used to repeatedly detect β-btx at a concentration of 0.5fg/mL, even after 5 cycles, the regeneration signal is still not significantly weakened, and the relative standard deviation is 1.4RU. This shows that the biosensor chip has excellent regeneration performance.

On the other hand, FIG8B and FIG8C show the storage stability of the biosensor chip of the present invention; specifically, after measuring the biological sample, the biosensor chip was dry stored in a culture dish covered with aluminum foil until further measurement. After being stored at room temperature for two weeks and measured on the ^1st day, ^2nd day, ^3rd day, ^4th day, ^7th day and ^14th day, it can be seen that the SPR response on the surface of the biosensor chip remained at 86% of the initial signal change (FIG8B) and still maintained the same reflectivity spectrum (FIG8C).

In summary, the biosensor chip, the detection kit and the optical biosensor system including the same provided in this specification, as well as the method for evaluating the content of snake venom analytes using the optical biosensor system, have the advantages of excellent sensing sensitivity, rapid detection and good mobility; further, the present invention can adopt direct (no labeling required) qualitative and quantitative measurements, and its performance is highly reproducible and can be easily stored for a long time in a room temperature environment; in addition, the present invention can also be used to quantify snake venom analytes in serum samples, thereby accurately evaluating the patient's poisoning condition. Anyone with ordinary knowledge in the technical field to which this case belongs can understand that various changes and modifications can be made without departing from the principles and spirit of the invention. Therefore, the scope of protection of the invention should be based on the scope defined in the attached patent application.

100: Biosensor chip

102: substrate

104: Gold-aluminum double metal layer

104A: Aluminum layer

104B: Gold Element Layer

106: Alkane thiol layer

110: Detection layer

110A: Gold nanoparticles

110B: Nucleic acid aptamer

120: Analyte

Claims (15)

A biosensor chip includes: a substrate; a bimetal layer disposed on the substrate; and a detection layer disposed on the bimetal layer; wherein the bimetal layer is a gold-aluminum bimetal layer; wherein the detection layer includes a plurality of gold nanoparticles and a plurality of nucleic acid aptamers; wherein the bimetal layer is a gold-aluminum bimetal layer, which includes a gold element layer and an aluminum element layer, and the gold element layer is disposed on the aluminum element layer. wherein the thickness of the gold element layer is greater than the thickness of the aluminum element layer. A biosensor chip as described in claim 1, wherein the substrate is a glass substrate. The biosensor chip as described in claim 1, wherein the thickness of the gold element layer is 30 to 60 nanometers, and the thickness of the aluminum element layer is 1 to 10 nanometers. The biosensor chip as described in claim 1, wherein an alkane thiol layer is further included between the double metal layer and the detection layer. The biosensor chip as described in claim 1, wherein the surface of each of the plurality of gold nanoparticles comprises an avidin coating, and each of the plurality of nucleic acid aptamers comprises a biotin modification, and the plurality of gold nanoparticles and the plurality of nucleic acid aptamers are bonded by avidin-biotin affinity. A biosensor chip as described in claim 1, wherein the plurality of nucleic acid aptamers have biological affinity with β-cobra toxin. A detection kit for detecting snake venom analytes, comprising a biosensor chip as described in any one of claims 1 to 6. An optical biosensor system, comprising: a biosensor chip as described in any one of claims 1 to 6; a sample flow channel connected to the biosensor chip and configured to allow a sample to flow into and contact the biosensor chip; a light source, which is configured to correspond to the biosensor chip and is configured to emit a light toward the biosensor chip; a dielectric layer, which is configured between the light source and the biosensor chip; and a spectrometer, which is configured to correspond to the biosensor chip and is configured to receive the light reflected by the biosensor chip and convert it into a response signal. An optical biosensor system as described in claim 8, wherein the medium layer is a prism and is configured to allow the light to be incident on the biosensor chip at an incident angle of 66 to 79 degrees. An optical biosensing system as described in claim 8, wherein the spectrometer receives the light using an optical fiber. The optical biosensor system as described in claim 8 is a surface plasmon resonance biosensor system. A method for evaluating the content of snake venom analyte, comprising the following steps: providing an optical biosensing system as described in claim 8, and inputting a serum sample into the optical biosensing system through the sample flow channel; standing for a reaction time, allowing the optical biosensing system to obtain a snake venom analyte in the serum sample and generate the reaction signal; and quantifying the reaction signal and evaluating the content of the snake venom analyte in the serum sample. The method as described in claim 12, wherein the snake venom analyte is β-gamma-serpentine toxin. The method as described in claim 12, wherein the dilution ratio of the serum sample is 1500 times. The method as described in claim 12, wherein the reaction time is 5 minutes.