TWI583953B - Highly sensitive lspr detection kit and the using method thereof - Google Patents

Description

High-sensitivity LSPR biochemical sensing kit and application method thereof

The invention relates to an LSPR biochemical sensing kit and an application method thereof. More particularly, the present invention relates to a method for modifying bare gold particles on a test object for enhancing LSPR signals when performing LSPR analysis.

The principle of localized surface plasmon resonance (LSPR) is that when metal nanoparticles are fabricated on a transparent substrate, excitation by incident light will cause surface plasma resonance on the surface of the metal nanoparticles. Since the frequency and intensity of the resonance are easily affected by the surrounding environment, and the displacement of the wavelength or the change of the signal intensity is generated, the detection of the analyte can be performed by using the change of the local dielectric constant. Therefore, as long as the analyte is bound to the particle, we can measure the optical change by optical instrument. It is conceivable that the surface of the nanoparticle is like a tiny detector that can measure very high optical changes in the nanometer range.

Previous studies have found that when paired nanoparticle spacing is less than about 2.5 particle radii, plasmonic coupling occurs to produce significant spectral shifts, see Anker, JN et al. Nature Materials 7: 442, 2008. The Au-LSPR wafer, which has its own unique coupling characteristics, has been applied to the LSPR biosensing method, and its signal generation is based on the combination of the absorbance value and the plasma sub-band wavelength. Previously using the LSPR technology of the Golden Nanostructure, the spectral displacement was only 3 nm or less during sensing, so that there was no effective breakthrough in improving the detection sensitivity. Although an Optical Enhancement System (OES) has been proposed in the past, in the previous measurement of the antigen step, the action of the antigen-antibody (the surface of the antibody is calibrated) is additionally performed, and the calibration is complicated with the structure of the gold nanostructure. The plasma coupling effect greatly increases the amount of displacement (Kim, HM et al., Sensors 9 : 2334, 2009). However, the OES technology itself must utilize the calibrated secondary antibody and has lost the advantage of the original LSPR without label-free.

The invention solves the limitation problem of the above-mentioned LSPR biosensing method, and proposes a method for enhancing the LSPR signal by adding bare gold particles to the object to be tested when the detecting method is performed. The method developed by the invention can detect low concentration of the test object by low-cost, short-time analysis process and simple preparation, for example, to detect the anti-α-synuclein antibody (anti-α-synuclein antibody) For example, the sensitivity can reach 3.8 ng/mL, and the overall detection process takes only about 3 hours.

Therefore, in one aspect of the present invention, based on the above object, a high sensitivity localized surface plasmon resonance (LSPR) biochemical sensing set for detecting a low concentration analyte is provided, which comprises a plasmonic metal a nanostructure substrate comprising a substrate, a single layer of nano metal particles embedded on the substrate, and a receptor modified on the surface of the substrate or the nano metal particles; and a bare The gold particle suspension solution comprises bare gold particles suspended in a buffer, wherein the naked gold particles have a particle diameter of 5 to 100 nm. In some embodiments of the invention, the buffer may include PBS buffer, MES buffer, and the like. In one embodiment of the invention, the buffer is 0.1 mM PBS buffer.

In some embodiments of the invention, the substrate is an optical glass or a high transparency plastic.

In some embodiments of the invention, the nano metal particles are selected from the group consisting of gold, silver, copper, platinum, and alloyed nanoparticles thereof. In one embodiment of the invention, the nano metal particles are gold nanoparticles.

In a specific embodiment of the invention, the bare gold particles refer to gold particles whose surface is not modified or functionalized by any chemical molecules. The bare gold particles preferably have a particle diameter of 5 to 50 nm, and most preferably 10 nm.

In some embodiments of the present invention, the receptor is an antibody or an antigen corresponding to the analyte. In a specific embodiment of the present invention, the test substance is a biomarker of Parkinson's disease, which may be α-synuclein or anti-α-synuclein antibody. (anti-α-synuclein antibody).

Another aspect of the invention relates to a localized surface plasmon resonance (LSPR) biosensing method for detecting a low concentration analyte, comprising: providing a plasmonic wafer packaged on a substrate Buried a single layer of nano metal particles; modifying a receptor on the surface of the plasmonic wafer, the receiver is an antibody or an antigen corresponding to the analyte; and then modifying the analyte and simultaneously dropping the naked Gold particles, which modify the bare gold particles on the object to be tested; and detect the LSPR signal of the object to be tested.

In a specific embodiment of the present invention, the test object is a biomarker of Parkinson's disease. In a specific embodiment of the present invention, the analyte is α-synuclein. In a specific embodiment of the present invention, the test substance is an anti-α-synuclein antibody.

Figure 1 is a schematic diagram showing the formation of nanogold clusters on a plasmonic gold nanoparticle wafer. (A) The solid gold nanoparticles (Au NP) embedded on the gold nanoparticle wafer directly bond with the antigen or antigen (A) corresponding to the analyte, and when the analyte (D) is modified, At the same time, a solution of bare gold particles (Au-NPs) is added to bond with the analyte; (B) the bare gold particles modified on the analyte are aggregated on the plasmonic wafer by aggregation. Gold-nanoparticle-based clusters are formed to enhance the LSPR signal.

Fig. 2 is a SEM image showing the nano-gold clusters (red circles) formed on the plasmonic gold nanoparticles wafer by FE-SEM.

Figure 3 is a UV-Vis spectrum of 1 μg/mL IgG antigen detected.

Figure 4 is a UV-Vis extinction spectrum of anti-α-synuclein antibodies, showing that different concentrations of anti-α-synuclein antibodies (1 ppm, 0.1 ppm) were detected using the LSPR biosensing method of the present invention. And the amount of extinction intensity change of 0.0038 ppm).

Fig. 5 is a graph showing changes in the intensity of absorbance of PBS buffer, human anti-IgG (1 ug/ml) and anti-α-synuclein antibody (3.8 ng/mL) by the LSPR biosensing method of the present invention. Error bars indicate the standard deviation produced by performing the three replicate experiments.

The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

Preparation of nano plasma wafer substrate

Nano plasmonic wafer substrates can be prepared by conventional methods, including, for example, laser, microwave, microwave plasma, and the like. The metal film which can be used to prepare the nanoplasm plasma chip substrate of the present invention includes gold, silver, copper, platinum, alloys thereof and the like. In one embodiment of the present invention, the microwave plasma is used as a heating source to perform surface treatment of the material to obtain a plasmonic wafer having a special metal nanostructure. The principle is that the metal film forms a nanostructure due to heat, and due to the action of microwave and microwave plasma, an external high temperature is generated between the metal nanostructure and the glass substrate interface, so that the prepared metal nanometer is produced. In the structural substrate, the bottom of the formed metal nanoparticle is coated with a glass structure.

A method for preparing a gold nano plasma substrate as an embodiment of the present invention is briefly described below. First, a gold film is sputtered on the glass substrate and then placed in a microwave plasma for a treatment time of only 30 seconds, after which the gold nanoparticles can be formed on the substrate. The size of the gold nanoparticles formed on the gold nano plasma substrate of the present invention is about 9 ± 3 nm.

Refining the receiver on the surface of the nano plasma substrate

The "receiver" as used in the present invention means an antigen or an antibody corresponding to an analyte. On the gold nano plasma substrate prepared above, 40 μl of an antigen or antibody solution corresponding to the test substance was added dropwise, and allowed to stand at room temperature for 1 hour, followed by IX PBS buffer and ultrapure water. Rinse several times and dry with nitrogen. Blocking step: 40 μl of 5% BSA solution was added dropwise to the wafer, and allowed to stand at room temperature for 30 minutes, and then washed several times with IX PBS buffer, ultrapure water, and then dried with nitrogen.

Modification of the test object

The method of the present invention is characterized in that the bare gold particle buffer solution is added dropwise while modifying the test object to bond with the test object, and the nano gold cluster is modified on the test object to enhance the LSPR signal. 40 μl of the test solution was added dropwise to the wafer, and after standing at 37 ° C for 1 hour, 40 μl of a 10 nm naked gold particle buffer solution (suspended in 0.1 mM PBS buffer) was added dropwise, and the mixture was allowed to stand at room temperature. After 20 minutes, it was washed several times with 1X PBS buffer, ultrapure water, and then dried with nitrogen. Figure 2 shows an SEM image of a nanogold cluster formed on a gold nanoparticle wafer.

Taking a human IgG antibody as a receptor on the surface of the sensing wafer as an example, we modified the anti-IgG antibody on the plasmonic wafer and then applied different concentrations of IgG antigen. IgG) for detection. Figure 3 is a graph showing the change in UV absorbance intensity of 1 μg/mL IgG. Using a human IgG antibody as a receptor, the resulting sensing wafer will have an absorption peak at 540 ± 2 nm. When the sensing wafer is used to detect human IgG, the amount of optical change caused by the antigen is dropped to about 540 nm.

Detection of Parkinson's disease marker (anti-α-Syn antibody) by the high sensitivity LSPR biosensing method of the present invention

Parkinson's disease marker α-synuclein (α-Syn) is present in our brain, but when there is an excess of α-synuclein, it will cause α-burst The nucleoprotein begins to accumulate and eventually becomes Lewy body and is harmful to nerve cells. At this point, the brain nerve cells begin to produce an anti-α-Syn antibody against α-synuclein and diffuse into the blood. In practice, to detect Parkinson's disease markers, not only can detect α-synuclein (α-Syn) in cerebrospinal fluid, but also detect blood (plasma LSPR biosensing serum). An anti-α-synuclein antibody, or an alpha-synuclein in the form of an oligomer (oligomer) or fibril in blood (plasma or serum). However, in view of the prior art, it has not been possible to detect a marker of Parkinson's disease (α-synuclein or an antibody thereof) by a low-cost, short-time analysis process and simple preparation. Thus, the present invention further provides an example of an antibody that detects a low concentration of Parkinson's disease marker (α-synuclein) using the method and kit of the present invention to demonstrate the advancement of the method of the present invention over conventional detection methods. .

According to the method described above, α-synuclein (α-Syn, purchased from ENZO LIFE SCIENCES) was modified on the plasmonic gold nanoparticle wafer, and then anti-α-Syn antibody (anti-α-Syn, Purchased from SANTA CRUZ BIOTECHNOLOGY, INC). From the results of Fig. 4, the amount of change in absorbance increases as the concentration of the anti-α-synuclein antibody of the test substance increases.

Referring to the results of Fig. 5, it was found by comparison of blank group (PBS buffer), control group (anti-IgG) and experimental group (anti-α-Syn) that the test substance was 3.8 ng/mL anti-α-Syn The amount of change in the absorption intensity of the antibody was greater than the change in the absorbance intensity of the PBS buffer and 1 μg/mL anti-IgG, and the change in the absorption intensity of the 3.8 ng/mL anti-α-Syn antibody was also greater than the triple mark of the PBS buffer. Deviation (refer to the right table of Figure 5), thus successfully detecting 3.8 ng/mL of anti-α-Syn antibody by the detection method of the present invention, and having the opportunity to detect a lower concentration Anti-α-Syn antibody.

The detection limits and the total time-consuming process of the detection process compared to the prior art are summarized in Table 1 below.

The above comparison results show that the high-sensitivity LSPR biosensing method of the present invention can successfully detect low-concentration or small-molecule analytes without additional labeling agents or secondary antibodies, and can effectively reduce high cost. Cost; and the detection time is only about 3 hours, which can greatly shorten the detection time compared to other detection methods.

Claims (17)

A high-sensitivity localized surface plasmon resonance (LSPR) biochemical sensing kit for detecting low-concentration analytes, comprising: a plasmonic nano-structured substrate comprising a substrate; a single-layer nano metal particle embedded on the substrate; a receptor modified on the nano metal particle, the receiver being an antibody or an antigen corresponding to a test object; a bare gold a particle suspension solution, the bare gold particles are suspended in the buffer and have a particle diameter of 5 to 100 nm; the analyte and the naked gold particle suspension solution are dropped on the plasmonic nano-structure substrate, The object to be tested is combined with the receiver, and the surface of the object to be tested is bonded to the gold cluster of bare gold particles; and the amount of change of the extinction intensity of the LSPR signal of the object to be tested is detected by light of a wavelength of 400 to 800 nm. The LSPR biochemical sensing kit of claim 1, wherein the substrate is an optical glass or a high light transmissive plastic. The LSPR biochemical sensing kit of claim 1, wherein the nano metal particles are selected from the group consisting of gold, silver, copper, platinum, and alloy nanoparticle thereof. The LSPR biochemical sensing kit of claim 3, wherein the nano metal particles are gold nanoparticles. The LSPR biochemical sensing kit of claim 1, wherein the analyte is a biomarker of Parkinson's disease. The LSPR biochemical sensing kit of claim 5, wherein the analyte is alpha-synuclein or anti-alpha-synuclein antibody. The LSPR biochemical sensing kit of claim 1, wherein the naked gold particle suspension solution comprises a PBS buffer. The LSPR biochemical sensing kit of claim 1, wherein the naked gold particle suspension solution comprises MES buffer. The LSPR biochemical sensing kit of claim 1, wherein the naked gold particles have a particle size of 5 to 50 nm. The LSPR biochemical sensing kit according to claim 1 or 9, wherein the naked gold particles have a particle diameter of 10 nm. A localized surface plasmon resonance (LSPR) biochemical sensing method for detecting a low concentration analyte, comprising: providing a plasmonic nano wafer, which is embedded on a substrate by heating a layer of nano metal particles; modifying a receptor on the surface of the plasmonic nano wafer, the receiver is an antibody or an antigen corresponding to the analyte; and adding the analyte to the receiver And simultaneously adding the bare gold particles to modify the bare gold particles on the object to be tested; and detecting the change in the extinction intensity of the LSPR signal of the analyte at a wavelength of 400 to 800 nm. The LSPR biosensing method according to claim 11, wherein the nano metal particles are selected from the group consisting of gold, silver, copper, platinum, and alloy nano particles thereof. The LSPR biosensing method according to claim 12, wherein the nano metal particles are gold nanoparticles. The LSPR biosensing method according to claim 11, wherein the naked gold particles have a particle diameter of 5 to 50 nm. The LSPR biosensing method according to claim 14, wherein the naked gold particles have a particle diameter of 10 nm. The LSPR biosensing method according to claim 11, wherein the analyte is a biomarker of Parkinson's disease. The LSPR biosensing method according to claim 16, wherein the analyte is α-synuclein or an anti-α-synuclein antibody.