CN112067585A

CN112067585A - Spectrum biological sensing device

Info

Publication number: CN112067585A
Application number: CN202010997928.7A
Authority: CN
Inventors: 刘啸虎; 王毅; 张庆文
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-09-21
Filing date: 2020-09-21
Publication date: 2020-12-11

Abstract

The invention relates to the field of molecular detection, in particular to a spectral biosensing device. Based on the optical property of the LSPR nano material and the effective spatial information in spectral imaging, the LSPR nano biochip which only has single-channel single-substance detection capability generally has the capability of simultaneously detecting a plurality of different substances. Meanwhile, the invention can carry out rapid and accurate detection and high-throughput screening only by the nano biochip and the spectral imaging device, thereby greatly reducing the complexity, the use cost and the operation difficulty of a detection system with similar functions.

Description

Spectrum biological sensing device

Technical Field

The invention relates to the field of molecular detection, in particular to a spectral biosensing device.

Background

Since the 80 s of the last century, surface plasmon resonance technology has been widely used to track interactions between biomolecules in real time, and is characterized by the fact that no labeling is required. Then, with the development of micro-nano processing technology and imaging equipment, Local Surface Plasmon Resonance (LSPR) technology is also developed, and support is provided for design of portable related equipment instruments and multifunctional chips. The LSPR phenomenon is an effect in which light energy generated when light of a specific wavelength is incident on a metal material having a nanostructure and resonates with the bulk vibration frequency of electrons on the surface thereof is absorbed. In general, a nano material composed of noble metal such as gold, silver, platinum, etc. has a strong LSPR effect and is embodied in the ultraviolet and visible light band. The resonant wavelength of the nano-material depends on the composition, shape, structure, size and the like of the metal nano-material, and is very sensitive to the surrounding medium. Therefore, the modified LSPR nanomaterials are designed and functionalized to be widely used in chemical and biological sensors based on optical signal changes. The sensor does not need to be marked, has low requirements on spectrum equipment, and can realize real-time and high-sensitivity detection. Has great application prospect in the aspects of environmental monitoring, medicine research, disease diagnosis, food safety and the like.

The existing LSPR biosensing technology is generally single-point detection, detection is needed to be matched with a spectrometer, the output result is the absorption spectrum of the point, and the detection purpose is achieved through the change of the spectrum before and after the absorption of a detection substance. Real-time detection of the detection substance can be achieved by analyzing the intensity of a particular wavelength thereof as a function of time. The specificity is determined by the receptor molecules modified on the surface of the LSPR material.

The spectrometer matched with the spectrometer has been greatly developed in recent years, and the whole spectrometer is developed towards portability, miniaturization, intellectualization, imaging and the like on the basis of the traditional spectrometer for researching application in a laboratory. The Spectral Imaging technology (SI) combines the Imaging technology with spatial resolution and the Spectral technology with Spectral resolution, so as to form the distribution of light intensity along with spatial coordinates and wavelength coordinates: i.e. the spectral data cube. The spectral imaging technology is widely applied to the fields of terrain exploration, target searching, cultural relic identification and the like. Recently, due to the gradual improvement of spectral resolution, the method also plays a role in biomedical fields such as drug testing, cell detection, injury assessment and the like. Compared with color imaging with only RGB three-color channels, spectral imaging has more channels and higher spectral resolution, and can better detect the surface chemical composition and subtle differences of an imaging object. Compared with the traditional spectrometer which can only carry out spectrum acquisition on a single point, the spectrum imaging has the spatial resolution capability, so that the simultaneous spectrum acquisition can be carried out on different sites in a certain space.

Spectral imaging generally takes spatial imaging as a main part, and a spectrum as an auxiliary part, and is a remote sensing detection device mostly. The difference of the surface of the detected substance, which is generated due to different physicochemical properties, does not have strong specificity, and the spectral resolution is low, so the method belongs to the multispectral imaging category. In recent years, identification of cancerous and normal regions (or cells) has been performed by directly analyzing tissue cells using hyperspectral images, and high accuracy has been achieved by identifying them using comprehensive characteristics of biochemical properties of tissue cell surfaces. Spectral imaging has not been widely used in biosensing and detection because of the lack of specificity.

For practical problems, for example, in disease diagnosis, one disease often corresponds to several or even more different indicators, and the purpose of improving the accuracy of disease diagnosis can be achieved by detecting different indicators simultaneously. For example, in drug screening, a few ligand molecules with strong affinity are often required to be rapidly screened in a large-scale molecular library for the same receptor molecule, and a high-throughput screening technology becomes one of the indispensable means. However, the conventional biosensing technology based on LSPR nanomaterials has difficulty in satisfying the above requirements for simultaneous detection of multiple substances or high-throughput screening. By partitioning the detection flow cell in a physical space, the aim of detecting various substances can be fulfilled by utilizing micro-fluidic. The whole microfluidic system undoubtedly greatly increases the detection cost and the operation difficulty. In addition, it is also a solution to collect spectra at a series of sites by using the spatial movement of the spectrometer, but the mechanical movement causes the detection time to be prolonged, and the additional mechanical control device also increases the complexity of the whole detection system (CN208705218U multichannel absorption spectrum detection table and detection system). Therefore, in the field of biosensing, the spatial information of spectral imaging is not effectively utilized.

Disclosure of Invention

In order to solve the above technical problem, the present invention provides a spectral biosensing apparatus, comprising: the LSPR nano biochip comprises an LSPR nano biochip and a spectral imaging module;

wherein the LSPR nano-biochip comprises: the nano material comprises a substrate material, nano materials with an LSPR effect and biomolecules, wherein the nano materials are arranged on the substrate material, and the biomolecules are modified on the surfaces of the nano materials; different biological molecules are modified on the nano materials in different areas, so that the purpose that the different biological molecules are positioned at different sites in a certain known sequence is achieved;

the spectral imaging module collects and records spectral image data of the LSPR nano biochip by a spectral imaging technology, and further analyzes the molecule combination condition of all array sites on the chip simultaneously by image information.

The spectral image data is a reflection spectral image or a transmission spectral image, and a data cube schematic diagram of a spectral imaging graph is shown in figure 1.

Preferably, the nano materials are distributed on the substrate material in an array form, and different biomolecules are modified on the nano materials at different array positions.

Preferably, a multi-channel micro-fluidic device can be arranged in the LSPR nano-biochip for draining the sample to be tested to different arrays. Through the setting, the combination test and the detection of various unknown substances to be tested in various samples can be carried out simultaneously, so that the working efficiency is further improved.

The substrate material refers to a transparent material meeting certain optical requirements, and preferably has a hydrophobic surface, or is a hydrophobic surface after chemical treatment, or can be a flexible material.

Preferably, the substrate material is selected from one of glass, organic glass, quartz, polydimethylsiloxane, polyvinyl alcohol, polyester, polyimide and polyethylene naphthalate.

Preferably, the nano material is divided into a key area and a non-key area, different biomolecules are modified on the nano material in the different key areas, and the nano material in the non-key area has a hydrophobic surface or a hydrophobic surface after chemical treatment, so that the biomolecules can be better modified to corresponding sites.

In some embodiments, the overall vertical configuration of the LSPR nanobead is as in fig. 3.

The device comprises four layers from top to bottom, namely a flow-through layer (part injected with detection solution during detection), a biological molecular layer, a nano material layer (a key hydrophilic part and a non-key hydrophobic part), and a substrate material layer.

The nanometer material layer is divided into key and non-key areas, only the nanometer material in the key area is modified with biological molecules, and the biological molecules modified on the nanometer materials in different key areas are different. The other part is a non-critical area, can be made of other materials and can also be communicated with the flow-through layer of the first layer.

Preferably, the nanomaterial is a metal thin film with periodic nanostructured pores (nanopores), or metal particles with periodic nanostructures (nanodiscs, nanospheres, nanorods, etc.).

According to the adopted metal nano structure on the chip, the spectral imaging module correspondingly adopts a reflection spectrum imaging or transmission spectrum imaging mode.

The same or different nanomaterials can be disposed on different regions (arrays) of the substrate material.

In some embodiments, the nanomaterial is selected from one or more of gold, silver, platinum, copper, and aluminum.

In one embodiment, the LSPR nano-biochip has a design schematic as shown in FIG. 2.

Preferably, the biomolecule is bound to the surface of the nanomaterial through a chemical covalent bond, a coordination bond, or physical adsorption.

In some preferred embodiments, the biomolecule is bound to the nanomaterial surface by a chemical covalent bond (thiol-gold).

Specifically, a uniform self-assembly molecular layer is formed at a key hydrophilic site of the gold nano-film by PEG carboxylic acid molecules with sulfydryl through a sulfydryl-gold covalent bond, and then different biomolecules (with at least one amino group) corresponding to different sites are combined in a mode of forming a peptide bond through EDC/NHS carboxyl activation: specifically, there are specific antibodies or other binding molecules such as polypeptide molecules, chitosan, aptamers, etc.

In some embodiments, the biomolecule is selected from a protein (e.g., an antibody, a cell receptor protein), a polypeptide (e.g., a chain molecule, a cyclic molecule), an oligonucleotide (e.g., a nucleic acid aptamer), an oligosaccharide (e.g., chitosan), a lipid, or a complex molecule consisting of two or more of a protein, a polypeptide, an oligonucleotide, and a lipid in combination (e.g., a glycoprotein, a peptide nucleic acid). Each biomolecule has the ability to specifically bind to a given test substance molecule. The biomolecules modified at different sites can be freely selected according to different requirements of the sensing application.

It is further preferred that the biomolecules at different sites are selected from molecules with smaller molecular weight, such as polypeptide molecules, aptamers, etc., to better match the sensitivity of the LSPR effect.

In addition, the spectral biosensor device further comprises a light source and a sample flow cell to be measured, which are arranged in a conventional manner in the field, according to the common knowledge in the field, and are not further limited herein.

Preferably, the spectrum biosensor device further comprises a data processing module for analyzing the spectrum data to obtain the directional or quantitative result of the sample to be detected.

For example, the directional result of the test sample may be viral or non-viral, and the quantitative result may be the specific concentration of the virus.

When using the spectroscopic biosensing device, the following process is generally involved (see fig. 4):

(I) preparation of respective standard curves of different substances to be measured

Before testing, the spectral image of the LSPR nano-biochip in the flow cell is first recorded by the spectral imaging device as a reference spectral image. The standard substances to be detected with different concentrations and samples of different substances to be detected combined randomly are injected in sequence, and enter the flow cell to perform binding reaction with biomolecules at different sites on the nano biochip. Spectral images of specific sites resulting from different binding reactions are recorded in real time by the spectral imaging device used. After the test is finished, all the spectra obtained from the same position point are differentiated from the reference spectrum to obtain the time-varying information (including the displacement and intensity variation of the spectrum peak) of the spectrum at the position point. And making a standard curve of each substance to be tested according to the change information, the substance to be tested represented by the corresponding site and the concentration injected in each test. Meanwhile, the related binding coefficient between the substance to be detected and the biomolecule modified by the corresponding site can be further calculated through the concentration information of the substance to be detected and the real-time binding curve of the substance to be detected at the corresponding site.

(II) detection of sample to be detected

During detection, a sample containing a plurality of unknown substances to be detected is injected into the flow cell to perform a binding reaction with biomolecules at different sites on the nano-biochip. The spectral image of the specific site resulting from the different binding reactions is recorded in real time by the spectral imaging device used. After the detection is finished, all the spectra obtained from the same position are differentiated from the reference spectrum to obtain the time-dependent changes of the spectrum (including the shift and intensity change of the spectrum peak) of the position (fig. 5). The concentration of different substances to be detected contained in the detection sample can be further calculated according to the changed information and the substances to be detected represented by the corresponding sites and the standard curve made before.

The invention has the beneficial effects that:

the LSPR nano biochip based on the optical property of the LSPR nano material and combined with spectral imaging effectively utilizes the spatial information in the image, so that the LSPR nano biochip which only has single-channel single-substance detection capability generally has the capability of simultaneously detecting a plurality of different substances and has good accuracy and specificity. Meanwhile, the invention can carry out rapid and accurate detection and high-throughput screening only by the nano biochip and the spectral imaging device, thereby greatly reducing the complexity, the use cost and the operation difficulty of a detection system with similar functions.

Drawings

FIG. 1 is a data cube diagram of a spectral imaging plot;

FIG. 2 is a schematic diagram of a design of an LSPR nano-biochip;

FIG. 3 is a vertical configuration diagram of an LSPR nano-biochip; in the figure, 1, a base material; 2. a nanomaterial; 21. a critical area; 22. a non-critical area; 3. a biomolecule;

FIG. 4 is a schematic diagram of spectral biosensing in accordance with the present invention;

FIG. 5 is an example of the change of the absorption spectrum of the specific-site LSPR nano-biochip before and after binding with the substance to be detected;

FIG. 6 is an SEM image of a gold nanopore periodic structure of an LSPR nano biochip, the reduced image being a photograph of the chip;

FIG. 7 is an example of the change of the absorption spectrum before and after the binding of the gold nanopore at a specific site to the substance to be detected.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1

The present embodiments provide a spectroscopic biosensing device comprising: the LSPR nano biochip comprises an LSPR nano biochip, a spectral imaging module and a data processing module;

wherein the LSPR nano-biochip (vertical configuration is shown as A in FIG. 3) comprises: the nano material comprises a substrate material 1, nano materials 2 with LSPR effect arranged on the substrate material, and biomolecules 3 modified on the surfaces of the nano materials.

Specifically, the substrate material is glass.

The nano material is metal particles with periodic nano structures, and is distributed on the substrate material in an array form, and different biological molecules are modified on the nano material at different array positions.

The biological molecules are combined on the surface of the nanometer material through chemical covalent bonds (sulfydryl-gold). Firstly, PEG carboxylic acid molecules with sulfydryl form a uniform self-assembly molecular layer at key hydrophilic sites of a gold nano-film through sulfydryl-gold covalent bonds, and then different biomolecules (with at least one amino group) corresponding to different sites are combined in a mode of forming peptide bonds through EDC/NHS carboxyl activation: specifically, there are specific antibodies or other binding molecules such as polypeptide molecules, chitosan, aptamers, etc.

And according to the adopted metal nano structure on the chip, the spectral imaging module correspondingly adopts a transmission spectrum imaging mode.

And the data processing module is used for analyzing according to the spectral data to obtain an orientation or quantification result of the sample to be detected.

Example 2

wherein the LSPR nano-biochip (vertical configuration is shown as B in FIG. 3) comprises: the nano material comprises a substrate material 1, nano materials 2 with LSPR effect arranged on the substrate material, and biomolecules 3 modified on the surfaces of the nano materials.

The nano material 2 is a gold nano film (nano hole) with periodic nano structure holes (fig. 6), and is divided into a key area 21 and a non-key area 22, wherein only the nano material of the key area 21 is modified with biomolecules, and the modified biomolecules on the nano material of different key areas 21 are different. Another part of the non-critical area 22 may be made of other materials or may be in communication with the flow-through layer.

Specifically, the substrate material is glass.

Example 3

This example provides a method of detection using the spectroscopic biosensing device of example 2 for the detection of different viruses or representative molecules thereof.

When the spectral biosensing device is used for detecting different viruses or representative molecules thereof, the following processes are included (refer to fig. 4):

Before testing, the spectral image of the LSPR nano-biochip in the flow cell is first recorded by the spectral imaging device as a reference spectral image. The standard substances to be detected (including S protein, N protein, specific nucleic acid molecules and the like of new coronavirus, SARS and MERS virus, HA protein, specific nucleic acid molecules and the like of different influenza viruses) with different concentrations (the range covers 0.01 pg/mL-10 mg/mL) and samples of different substances to be detected which are randomly combined are injected into a flow cell to perform a binding reaction with biomolecules at different sites on a nano biochip. The spectral image of the specific site resulting from the different binding reactions is recorded in real time by the spectral imaging device used. After the test is finished, all the spectra obtained from the same position are differentiated from the reference spectrum to obtain the time-dependent change of the spectrum (including the shift and intensity change of the spectrum peak) of the position. And (4) according to the changed information, combining the concentrations of the substances to be tested, which are represented by the corresponding sites, and making a standard curve of each substance to be tested.

(II) detection of sample to be detected

During detection, virus-related samples containing various unknown substances to be detected are injected into the flow cell to perform binding reaction with biomolecules at different sites on the gold nanopore biochip. The spectral image of the specific site resulting from the different binding reactions is recorded in real time by the spectral imaging device used. After the detection is finished, all the spectra obtained from the same position are differentiated from the reference spectrum to obtain the time-dependent changes of the spectrum (including the shift and intensity change of the spectrum peak) of the position (fig. 7). The respective concentrations of different substances to be detected contained in the detected sample can be further calculated according to the changed information and the substances to be detected represented by the corresponding sites and the previously made standard curve, so that the type and the content of the virus-related substances contained in the sample can be judged.

The device of the present invention has been tested for excellent accuracy and specificity in use.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A spectroscopic biosensing device, comprising: the LSPR nano biochip comprises an LSPR nano biochip and a spectral imaging module;

wherein the LSPR nano-biochip comprises: the nano material comprises a substrate material, nano materials with an LSPR effect and biomolecules, wherein the nano materials are arranged on the substrate material, and the biomolecules are modified on the surfaces of the nano materials; different biological molecules are modified on the nano materials in different areas;

the spectral imaging module collects and records spectral image data of the LSPR nano biochip through a spectral imaging technology.

2. The spectroscopic biosensing device of claim 1 wherein said nanomaterials are distributed in an array over a substrate material, wherein different biomolecules are modified on the nanomaterials at different array locations;

preferably, the substrate material has a hydrophobic surface, or has a hydrophobic surface after chemical treatment.

3. The spectroscopic biosensing apparatus of claim 1 wherein the nanomaterials are divided into critical and non-critical regions, wherein different biomolecules are modified in the nanomaterials in the critical regions, and wherein the nanomaterials in the non-critical regions have hydrophobic surfaces or are chemically treated to have hydrophobic surfaces.

4. The spectroscopic biosensing device of claim 1 wherein a multi-channel microfluidics is further provided in the LSPR nanobead for directing the sample to be measured to different arrays.

5. The spectroscopic biosensing device of claim 1 wherein said substrate material is selected from one of glass, plexiglass, quartz, polydimethylsiloxane, polyvinyl alcohol, polyester, polyimide, polyethylene naphthalate.

6. The spectroscopic biosensing device of claim 1, wherein said nanomaterial is a metal thin film with periodic nanostructured pores or metal particles with periodic nanostructures.

7. The spectroscopic biosensing device of claim 6 wherein said nanomaterial is selected from one or more of gold, silver, platinum, copper, aluminum.

8. The spectroscopic biosensing device of claim 1 wherein said biomolecule is bound to said nanomaterial surface by a chemical covalent bond, a coordination bond, or a physisorption.

9. The spectroscopic biosensing device of claim 1 wherein said biomolecule is selected from the group consisting of a protein, a polypeptide, an oligonucleotide, an oligosaccharide, a lipid, and a complex molecule comprising a combination of two or more of a protein, a polypeptide, an oligonucleotide, and a lipid.

10. The spectroscopic biosensing device of claim 1, further comprising a data processing module for analyzing from said spectroscopic data a directional or quantitative result of a sample to be tested.