CN101514986A - A Label-Free Biochemical Detection Method Using Localized Surface Plasmon Enhancement - Google Patents

Abstract

A label-free biochemical detection method utilizing localized surface plasmon enhancement, characterized in that it includes the following steps: (1) preparing an LSPR detection chip according to the detection object; (2) selecting specific biomolecules to activate the chip according to the detection object , test the spectral reference value of the chip by the spectral testing system; (3) then introduce the sample to be tested for detection, and obtain the spectral curve through the test; (4) judge whether the sample to be tested contains the detection object by analyzing the movement of the spectral peak; Realize label-free, high-sensitivity, and fast detection of detection objects. At the same time, arrayed chips can be used to achieve efficient and multi-channel detection. The method of the invention does not require complex equipment, does not need to use radioactive isotopes, enzymes or fluorescence as markers, has the remarkable characteristics of low cost and high sensitivity, and can realize arrayed chips, providing a simple and practical method for rapid detection of biochemical molecules. new method.

Description

A Label-Free Biochemical Detection Method Using Localized Surface Plasmon Enhancement

technical field

The invention belongs to the field of biochemical detection and relates to a novel biochemical detection method, in particular to a high-sensitivity biochemical detection method utilizing localized surface plasmon resonance enhancement technology.

Background technique

Sensing technology is one of the three pillars of modern information technology and plays an important role in national security, scientific experiments, medical and health, and environmental monitoring. Biochemical sensing technology is an important branch of sensing technology, which is related to public safety, virus, bacteria detection, clinical medicine, environmental detection and other fields that are closely related to people's lives. Traditional biochemical sensing technologies usually require labels, are bulky, have low sensitivity (usually at the nanomolar level), cumbersome processes, and low efficiency. 90% of the workload is used for labeling, mainly using radioactive isotopes, enzymes or fluorescence as markers, which are poor in safety and stability, and cannot meet the needs of fast (for real-time processing and control of dangerous substances), sensitive (for detecting traces of severe Toxic substances) and special effects (to exclude the interference and pollution of non-pathogenic components), it is urgent to develop a new generation of highly sensitive, efficient, accurate, convenient, fast and economical biochemical molecular detection methods and technologies. In recent years, label-free detection methods have developed rapidly, and sensing technologies such as elliptically polarized light, optical addressing potential, ion-sensitive field-effect transistors, surface acoustic waves, and quartz crystal oscillator microbalances have emerged, which have improved detection efficiency. Among them, the propagating surface plasmon resonance (SPR) biosensor has been rapidly developed and practically applied. Many units at home and abroad have conducted research for nearly 20 years, and some results have been commercialized. However, SPR propagation chips are difficult to array, integrate, and chip, and the detection technology needs to be further improved.

With the advancement of technology, a new type of sensing technology - Localized Surface Plasmon Resonance Enhancement (LSPR) has been paid more and more attention by researchers from all over the world. It refers to the use of electrons with conductivity in nanoparticles to generate The electromagnetic field, which determines the amount of sensing based on refractive index sensitivity. Since collective oscillations of conduction electrons occur only at specific wavelengths, nanoparticles exhibit selective photon absorption, which can be easily detected with UV-Vis spectrometers. With its convenience, high sensitivity, wide application range, real-time monitoring and many other characteristics, it is favored by researchers and is at the forefront of sensor research. Using this new research method is useful for basic research in life sciences and medical diagnosis. and treatment are of great importance.

Contents of the invention

The problem to be solved by the present invention is: to overcome the shortcomings of existing biochemical sensing technology that require special equipment, high cost, long cycle, and low sensitivity, and utilize LSRP (localized surface plasmon technology) and specific reactions between biochemical molecules to Observing spectral shifts enables high-sensitivity, label-free, and multi-channel efficient detection of specific molecules; the chip can be reused after post-dissociation processing.

The technical solution adopted by the present invention to solve the technical problem is: a label-free biochemical detection method enhanced by localized surface plasmons, characterized in that it includes the following steps:

(1) Prepare a detection chip with a metal micro-nano structure on the surface of the substrate according to the detection object;

(2) Select specific biomolecules to activate the chip according to the detection object, and test the spectrum of the chip through the test system to obtain the chip reference value;

(3) Introduce the sample to be tested for detection, and obtain the spectral curve through the test;

(4) Judging whether the sample to be tested contains the detection object by analyzing the spectral shift condition.

The detection object in the step (1) is bacteria, or toxin, or protein.

The detection chip in the step (1) is prepared through the following steps: (a) select the chip substrate, clean it and perform hydrophilic treatment; (b) make a chip on the surface of the substrate by self-assembly of nanospheres, or photolithography, or nanoimprinting Nanostructure; (c) metallizing the nanostructure to obtain a chip with the nanometer metal structure on the surface of the substrate.

The material of the chip substrate in the above step (a) is glass, or quartz, or silicon, or germanium.

The detection chip in the step (1) is an arrayed chip, the number of arrays is from 1×1 to 150×150, and the size of the subunit is 5 μm×5 μm to 10mm×10mm, which can target various substances to be tested by spotting Activate different subunits to achieve efficient, multi-channel parallel detection.

The metal micro-nano structure on the detection chip in the step (1) is a triangle, or a rhombus, or a five-pointed star, or a cylinder, or a double-layer composite structure, and the metal material is gold, or silver, or silver The surface is compounded with gold, and the characteristic size of the nanostructure is from 30nm to 1000nm.

The detection in the step (3) can be on-line detection or detection after chip reaction, and the reaction time of the analyte is 1 minute to 20 hours.

For the detection in the step (3), the detection method is to use the specific reaction between molecules for testing, and the antibody can be combined on the chip first, and the antigen can be detected by the antibody, or the antigen can be combined on the chip to detect the antibody.

The environment detected in the step (3) is vacuum, or nitrogen environment, or atmospheric environment; meanwhile, the temperature is between -50°C and 80°C.

The analytical spectrum movement status in the step (4) refers to that the spectral peak position movement is greater than 10nm.

Compared with the prior art, the present invention has the advantages that: the method does not require complex equipment, does not need to use radioactive isotopes, enzymes or fluorescence as markers, and has low cost, high sensitivity, wide application range, safety, It has high stability, high spatial resolution (reaching the level of single nanoparticle), good biological affinity, etc., and can realize arrayed chips, providing a simple and practical new method for rapid detection of biochemical molecules.

Description of drawings

Fig. 1 is a schematic flow chart of the present invention to realize label-free biochemical detection;

Fig. 2 is the activation method of the specificity detection adopted in the embodiment 1 of the present invention, first bind the antigen on the chip, and judge whether the analyte contains the analyte in the analyte by detecting the antibody;

Fig. 3 is that the present invention adopts the reference value of the spectrometer test gained and the result figure after the detection object moves;

In the figure: 1. Metal structure, 2. Substrate, 3. Activation molecule, 4. Antibody, 5. Antigen, 6. Chip reference measurement curve, 7. Test curve after binding the tested object.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

The label-free biochemical detection method utilizing local surface plasmon enhancement of the present invention has a specific flow chart as shown in Figure 1, including the following steps:

(1) Prepare a detection chip with a metal micro-nano structure on the surface of the substrate according to the detection object;

(2) Select specific biomolecules to activate the chip according to the detection object, and test the spectrum of the chip through the test system to obtain the chip reference value;

(3) Introduce the sample to be tested for detection, and obtain the spectral curve through the test;

(4) Judging whether the sample to be tested contains the detection object by analyzing the spectral shift condition.

Embodiment 1 In this embodiment, glass is used as the substrate, and the diamond-shaped metal silver structure arranged in a quadrilateral is used as the detection chip; the non-labeled biochemical detection method enhanced by local surface plasmon of the present invention is used to test bacteria, and the specific reaction process adopted is Bind antibodies on the chip.

First, glass is used as the substrate to select the chip substrate, cleaned and treated for hydrophilicity; on the surface of the substrate, a diamond-shaped nano-silver structure with an area of about 5 μm × 5 μm and a quadrilateral arrangement of 1 × 1 is produced by self-assembly of nanospheres. The characteristic size of the structure is 300nm; the nanostructure is metallized by silver plating to obtain a chip with the nanosilver structure on the surface of the substrate. Next, immobilize the active group on the surface of the metal nanostructure (as shown by 3 in Figure 2), and then immobilize the antibody with the target bacterial molecule to be detected (as shown by 4 in Figure 2) on the active group through a zero-distance coupling reagent. on the regiment. Through the above procedures, the process of activating the chip is completed. The reference value of the chip tested by the spectrum testing equipment in the vacuum environment is shown in Figure 3 6 . Finally, the substance to be detected is introduced by dropping, and the target molecule binds to the corresponding antibody through a specific reaction. After fully reacting (the reaction time is about 2 hours), in a vacuum environment, within the temperature range of 40°C to 50°C, the spectral distribution is tested, and the curve shown in 7 in Figure 3 is obtained. Comparing the curves 6 and 7 in Figure 3, it is found that the position of the spectral peak has moved significantly (the amount of movement is about 50nm), then it is judged that the sample to be tested contains the measured bacterial component; the detection chip can be reused after post-dissociation treatment .

Example 2

In this embodiment, fused silica is used as the substrate, and the triangular metal gold structure arranged in hexagons is used as the detection chip; the local surface plasmon-enhanced unlabeled biochemical detection method of the present invention is used to test the toxin, and the specific reaction process adopted is Antigen binding on the chip.

First, fused silica is used as the substrate, cleaned and treated for hydrophilicity; on the surface of the substrate, a triangular nanostructure with a characteristic size of 50nm and hexagonal arrangement is produced by nanolithography; the nanostructure is metallized by gold plating to obtain A chip with a nano-gold structure on the surface of the substrate. Next, active groups are immobilized on the surface of the metal nanostructure, and then the antigen of the detected target bacterial molecule is immobilized on the active groups by a zero-distance coupling reagent. Through the above procedures, the chip activation process is completed. In the atmospheric environment (the temperature is at room temperature), use the spectrum test system built on the experimental bench to test the reference value of the chip, and finally introduce the object to be detected to the surface of the chip through the microfluidic channel. If the object to be tested contains the corresponding antibody, it will be Bind to the corresponding antigen through a specific reaction. In this atmospheric environment, test the spectral distribution online at normal temperature, and analyze whether the peak position of the spectrum moves significantly (the movement is greater than 10nm), so as to determine whether the sample to be tested contains the measured bacterial component.

Example 3

In this embodiment, silicon is used as the substrate, and porous templates are produced by photolithography. The number of chip arrays is 150×150, and 150×150 protein antigens are integrated by the method of spotting, and the localized surface plasmon-enhanced Label-free biochemical detection methods test whether the analyte contains various types of proteins.

First, silicon is used as the substrate, cleaned and treated for hydrophilicity; nanostructure arrays with an array number of 150×150 are produced on the surface of the substrate by nanoimprinting, and the size of each subunit is 1000 μm×1000 μm; the nanostructure is metallized to obtain A chip with a nanometer metal structure on the surface of the substrate. The structure in each subunit can adopt different structures according to the different characteristics of the object to be tested. There are single-layer pentagrams, and there are upper and lower double-layer nano-silver structures composed of upper surface structure and lower surface structure, and there are also composite nano-silver structures. structure, a layer of metal gold is attached to the surface of the silver structure to prevent chip oxidation failure. The characteristic size of the nanostructure is from 30nm to 1000nm. Use a biospotter to spot samples on the chip and bind antibodies (or antigens) to different proteins. Use the integrated equipment to test the benchmark value of the chip in a nitrogen environment, the temperature is -50 ° C ~ 80 ° C, and finally combine the samples to be tested in each sub-area by spotting, and after fully reacting (about 30 minutes) in a nitrogen environment The spectral distribution of each sub-region is tested internally, and the temperature is -50°C to 80°C. Analyze whether the peak position of the spectrum is significantly shifted (the amount of movement is greater than 10nm), so as to determine whether the sample to be tested contains the protein component to be measured.

Claims (10)

1. A label-free biochemical detection method utilizing localized surface plasmon enhancement, characterized in that: the steps are as follows: (1) Prepare a detection chip with a metal micro-nano structure on the surface of the substrate according to the detection object; (2) Select specific biomolecules to activate the chip according to the detection object, and test the spectrum of the chip through the test system to obtain the chip reference value; (3) Introduce the sample to be tested for detection, and obtain the spectral curve through the test; (4) Judging whether the sample to be tested contains the detection object by analyzing the spectral shift condition. 2. A label-free biochemical detection method utilizing localized surface plasmon enhancement according to claim 1, characterized in that: the detection object in the step (1) is bacteria, or toxin, or protein. 3. A label-free biochemical detection method utilizing localized surface plasmon enhancement according to claim 1, characterized in that: the detection chip in the step (1) is prepared by the following steps: (a) selecting a chip substrate, Cleaning and performing hydrophilic treatment; (b) making nanostructures on the surface of the substrate through self-assembly of nanospheres, or photolithography, or nanoimprinting; (c) metallizing the nanostructures to obtain nano-metal structures on the surface of the substrate chip. 4. The preparation of the detection chip according to claim 3, characterized in that: the material of the chip substrate in the step (a) is glass, or quartz, or silicon, or germanium. 5. A label-free biochemical detection method utilizing localized surface plasmon enhancement according to claim 1, characterized in that: the detection chip in the step (1) is an arrayed chip, and the number of arrays ranges from 1× 1～150×150, the subunit size is 5μm×5μm～10mm×10mm, and different subunits can be activated for various substances to be tested by spotting to achieve efficient and multi-channel parallel detection. 6. A label-free biochemical detection method using localized surface plasmon enhancement according to claim 1, characterized in that: the metal micro-nano structure on the detection chip in the step (1) is triangular or rhombus , or pentagram, or cylinder, or a double-layer composite structure, the metal material is gold, or silver, or gold compounded on the surface of silver, and the characteristic size of the nanostructure is from 30nm to 1000nm. 7. A label-free biochemical detection method utilizing localized surface plasmon enhancement according to claim 1, characterized in that: the detection in the step (3) can be on-line detection or detection after chip reaction , the reaction time of the analyte is 1 minute to 20 hours. 8. A label-free biochemical detection method using localized surface plasmon enhancement according to claim 1, characterized in that: the detection in the step (3) is carried out by utilizing the specific reaction between molecules For testing, you can first bind antibodies on the chip, detect antigens through antibodies, or bind antigens on chips for antibody detection. 9. A label-free biochemical detection method using localized surface plasmon enhancement according to claim 1, characterized in that: the environment detected in the step (3) is vacuum, or nitrogen environment, or atmospheric environment ; At the same time, the temperature is between -50°C and 80°C. 10. A label-free biochemical detection method utilizing localized surface plasmon enhancement according to claim 1, characterized in that: the analytical spectrum shift status in the step (4) means that the shift of the spectral peak position is greater than 10nm.

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