1. A photosensitive molecule for constructing a photoresponsive soft and hard substrate is characterized by having a structural general formula as follows: r₁-X-R₂

Wherein X is a linking group selected from one of the following structures: - (CH)₂)_mNHCO(OCH₂CH₂)_nO-、(CH₂CH₂O)_m(CH₂)_nNHCO(CH₂)_pO-、-(CH₂)_mCONH(CH₂)_nNHCO(CH₂)_p-, alkylene, - (CH)₂)_mO-；

m is more than or equal to 0, n is more than or equal to 0, and p is more than or equal to 0; m, n and p are integers;

R₁the functional group can be chemically bonded or self-assembled with the soft and hard substrate and is selected from one of the following structures: mercapto, amino, CH₂＝CHCONH-、CH₂＝CHCH₂CONH-, acrylate group, methacrylate group, carboxyl group, and C4-C18 aryl group containing hydroxyl and/or C1-C6 alkyl;

R₂is a photoreactive group that can covalently bond to an antibody and is selected from one of the following structures:

y is selected from one of the following structures: -OH, -OCONH (CH)₂CH₂O)_pCH₃(ii) a p is an integer;

R₃、R₄、R₅、R₆、R₇each independently selected from one of the following structures: hydrogen, halogen, C1-C5 alkyl, C1-C5 alkoxy, hydroxyl, sulfydryl, amido, nitryl and cyano.

2. The photosensitive molecule for constructing optically responsive soft and hard substrates of claim 1, wherein in the formula of the photosensitive molecule for constructing optically responsive soft and hard substrates, R is₁One selected from the following structures:

x is selected from one of the following structures:

wherein m, n and p are integers from 0 to 8;

R₂selecting one of the following structures:

p is an integer of 0 to 8.

3. The photosensitive molecule for constructing a photoresponsive soft or hard substrate according to claim 2, wherein said photosensitive molecule is one of the following structures:

wherein m, n and p are integers from 0 to 8.

4. The photosensitive molecule for constructing a photoresponsive soft or hard substrate according to claim 3, wherein said photosensitive molecule is one of the following structures:

5. a preparation method of a surface enhanced Raman detection substrate based on photo-patterning is characterized by comprising the following steps:

the detection substrate comprises a hard substrate and a soft substrate;

the preparation method of the surface enhanced Raman detection substrate based on the photo-patterning comprises the following steps:

soaking a hard substrate material in 0.01-10mM of photosensitive molecular solution according to any one of claims 1-4 at room temperature for 1-24h, slowly taking out the hard substrate material, soaking and washing the hard substrate material by using a mixed solution of deionized water and acetonitrile in a volume ratio of 1:1, repeatedly washing the hard substrate material for at least three times, and drying the hard substrate material by inert gas to obtain a surface enhanced Raman detection hard substrate based on photo-patterning;

or the like, or, alternatively,

the preparation method of the surface enhanced Raman detection soft substrate based on the photo-patterning comprises the following steps:

dissolving 1-40mg of photosensitive molecule of any one of claims 1 to 4 in 0.1-1mL of nanogold colloid, adding 10-400mg of other crosslinking component, and mixing uniformly to obtain 0.1-40% of solid content hydrogel precursor solution containing 0.1-50mM of photosensitive molecule;

or, dissolving 1-40mg of the water-soluble macromolecule labeled with the photosensitive molecule of any one of claims 1 to 4 in 0.1-1mL of nano gold colloid, and mixing uniformly to obtain a hydrogel precursor solution containing 0.001-10% of the photosensitive molecule;

adding an initiator or an initiation aid into the hydrogel precursor solution, uniformly mixing, injecting the solution into a mold, and carrying out cross-linking polymerization in an inert gas environment at the temperature of 40-60 ℃ to obtain the surface-enhanced Raman detection soft substrate based on the photo-patterning.

6. The method for preparing the substrate for surface enhanced Raman spectroscopy based on photopatterning according to claim 5, wherein the other crosslinking component is a mixture of acrylamide, N-methylenebisacrylamide and acrylic acid;

the water-soluble macromolecules marked with photosensitive molecules are functionalized hyaluronic acid macromolecules marked with photosensitive molecules.

7. The method for preparing the substrate for surface-enhanced Raman spectroscopy based on photopatterning according to claim 6, wherein the method for preparing the functionalized hyaluronic acid macromolecule labeled with the photosensitive molecule comprises the following steps: mixing photosensitive molecules, acrylate functionalized hyaluronic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a molar ratio of 1:1 (1-5), dissolving in anhydrous dichloromethane at 0 ℃, recovering to room temperature for reaction, dialyzing after the reaction is finished, and freeze-drying to obtain the functionalized hyaluronic acid macromolecules marked with the photosensitive molecules;

the initiator or the initiation aid is at least one of potassium persulfate, ammonium persulfate and tetramethyl ethylene diamine.

8. A method for realizing detection of a biomarker by using a sandwich immune sandwich structure is characterized by comprising the following steps:

dissolving the antibody in ultrapure water to prepare an antibody solution;

a first step of wetting the surface of a photo-patterning-based surface enhanced Raman detection substrate prepared by the method according to any one of claims 5 to 7 with deionized water, fixing a photo-mask plate in parallel at a position about 0.5mm above the surface of the substrate, irradiating the substrate with an LED light source, soaking the irradiated substrate in a prepared antibody solution, standing and soaking at normal temperature, taking out the substrate, cleaning the surface with deionized water for three times, slightly blowing and drying water with inert gas, and storing at low temperature for later use; immersing the substrate into 0.5% bovine serum albumin solution for end capping, incubating at room temperature, and finally washing and drying with ultrapure water to obtain a patterned SERS planar substrate for modifying antibody molecules;

illuminating another area of the gold substrate, sequentially programming other antibodies to be illuminated and fixed in different areas on the substrate material according to the first step, and repeating the operation until all the required antibodies are fixed in the corresponding areas;

and thirdly, dropwise adding or immersing the solution to be detected in an area fixed with the antibody, after incubation in a humid and clean environment, gently rinsing the area with deionized water for three times, dropwise adding or immersing the functional nano metal particles modified by the antibody and the Raman signal molecules in the area, after incubation in a humid and clean environment, gently rinsing the area with deionized water for three times, and performing spectrum test analysis by using Raman laser.

9. The method as claimed in claim 8, wherein the wavelength of the LED light source is determined by the absorption of the photoaffinity group of the selected photosensitive molecule, and the wavelength is 250-500nm, and the light intensity is 5-50mW/cm²Irradiating for 1-15 min;

the antibody is a monoclonal or polyclonal antibody of a target detection object, the concentration of the antibody solution is 1 mu g/mL-100 mu g/mL, and the soaking time is 0.1-24 h;

the antibody is a mouse Anti-human C-reactive protein antibody, an aspergillus galactomannan antibody, a human thyroxine antibody, a carcinoembryonic antigen specific monoclonal antibody, an antibody Anti-AFP or an immunoglobulin IgM antibody.

10. The method for detecting biomarkers by using sandwich immune sandwich structure according to claim 8, wherein the method for preparing functional nano metal particles labeled with antibodies and modified by Raman signal molecules comprises the following steps:

adding 0.1-10mg/mL of DMSO solution of Raman signal molecules with the volume of 1-100 muL, 0.1-10mg/mL of hydrosulfuryl polyethylene glycol 3000 monomethyl ether with the volume of 1-100 muL, 0.1-10mg/mL of DMSO solution of polyethylene glycol 2000 monomethyl ether succinimide ester with the volume of 1-100 muL into 0.1-10mL of nano gold colloid, stirring and reacting for 0.5-8h, centrifugally separating nano particles from the solution, ultrasonically dissolving the nano particles in deionized water again, centrifugally separating, dissolving the nano particles in the deionized water again with the same volume, adding 1-10mg of antibody into the solution, stirring and reacting for 1-24h, separating the nano particles from the solution through multiple times of high-speed centrifugation, redispersing the nano particles in the deionized water with the same volume, and storing the mixture in a refrigerator at 4 ℃ for later use to obtain the functional nano metal particles marked with the antibody and modified by Raman signal molecules.

Background

Surface-enhanced Raman scattering (SERS), which is a technology developed on the basis of ordinary Raman scattering, is short. The SERS phenomenon generally refers to an optical phenomenon in which an analyte is adsorbed on a metal sol particle (e.g., gold, silver, or copper) or a metal surface having a rough structure, and in an excitation region, a raman scattering signal of an adsorbed molecule is greatly enhanced due to enhancement of an electromagnetic field on or near the metal surface as compared with ordinary raman scattering. At present, the SERS detection technology has been widely applied in the fields of food safety, environmental pollution detection and the like. Compared with other spectral labeling technologies, SERS has the advantages of high sensitivity and specificity, narrow spectral peak width, multichannel detection, signal immunity from water molecules and the like, and is considered to have great potential application in the field of biomedical in-vitro diagnosis and detection, particularly detection of trace biomarkers in common disease diagnosis.

Clinical qualitative and quantitative analysis of antigens and proteins usually employs immunological techniques, which are based on the specific binding of antigens to antibodies. Currently, the commonly used immunological detection methods include enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), Fluorescent Immunoassay (FIA), and the like. However, these methods have various disadvantages, such as that although the radioimmunoassay and the fluoroimmunoassay have high detection sensitivity, radioactive irradiation and contamination of radioactive substances in the radioimmunoassay are difficult to avoid; the emission lines of conventional fluorophores in the fluoroimmunoassay are broad and susceptible to photodegradation. The conventional enzyme-linked immunosorbent assay can perform low-concentration quantitative analysis on protein, but is expensive, complex in operation and difficult to realize high-throughput protein analysis. Generally, the concentration of the detected biomarker molecules in-vitro diagnosis of diseases is low, meanwhile, components in a serum or urine sample are complex, interference components are multiple, and the selectivity of a Raman spectrum to specific molecules is poor, so that the sensitivity and the anti-interference performance of the traditional SERS sensor are often not met. The surface Raman enhancement technology is combined with immunoassay, so that the application range of SERS in-vitro diagnosis is widened. The currently adopted sandwich method is a common method in immunoassay, and the method is to mark a specific antibody on a carrier, then add a corresponding antigen to be detected and metal nanoparticles marked with the specific antibody and Raman signal reporter molecules to form a double-antibody sandwich detection system, and realize the detection of the antigen in a solution to be detected by detecting the signal of the reporter molecules.

In actual clinical diagnosis, the need for simultaneous detection of multiple disease markers is increasing, and thus the importance of high-throughput integrated detection is becoming more and more prominent. The traditional preparation of SERE substrate materials comprises methods such as chemical deposition, electrochemical deposition, self-assembly of single molecules and the like, but the methods can only realize the preparation of substrate materials with uniform performance. At present, the preparation method of the SERS substrate for realizing high-throughput immunoassay mainly comprises the following steps: obtaining an ordered array metal substrate by electron beam, ion beam and photoetching; the array flexible paper substrate is obtained by ink-jet printing, silk-screen printing, 3D printing and other methods; a bimetallic three-dimensional ordered porous substrate prepared by electrodeposition and ion sputtering coating methods, a surface aldehyde group preset array type solid silicon substrate and the like. However, these substrate materials have the disadvantages of complex structure, uncontrollable surface topography, complex process, high cost, etc. In the field of biomedical materials, high-throughput microarrays or high-throughput screening surfaces prepared by the photo-patterning technology can effectively screen various biomolecule libraries such as DNA, proteins, antibodies and the like.

Disclosure of Invention

The first purpose of the invention is to provide a photosensitive molecule for constructing a photoresponsive soft and hard substrate.

The second purpose of the invention is to provide a preparation method of the surface enhanced Raman detection substrate based on the optical patterning technology.

The third purpose of the invention is to provide a method for detecting biomarkers such as antigens by using a sandwich immune sandwich structure.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the invention provides a photosensitive molecule for constructing a photoresponsive soft and hard substrate, which has the following structural general formula: r₁-X-R₂

Wherein X is a linking group selected from one of the following structures: - (CH)₂)_mNHCO(OCH₂CH₂)_nO-、(CH₂CH₂O)_m(CH₂)_nNHCO(CH₂)_pO-、-(CH₂)_mCONH(CH₂)_nNHCO(CH₂)_p-, alkylene (- (CH)₂)_m-)、-(CH₂)_mO-；

m is more than or equal to 0, n is more than or equal to 0, and p is more than or equal to 0; m, n and p are integers;

R₁the functional group can be chemically bonded or self-assembled with the soft and hard substrate and is selected from one of the following structures: mercapto, amino, CH₂＝CHCONH-、CH₂＝CHCH₃CONH-, acrylate group, methacrylate group, carboxyl group, and C4-C18 aryl group containing hydroxyl and/or C1-C6 alkyl;

R₂is a photoreactive group that can covalently bond to an antibody and is selected from one of the following structures:

y is selected from one of the following structures: -OH, -OCONH (CH)₂CH₂O)_pCH₃(ii) a p is an integer;

R₃、R₄、R₅、R₆、R₇each independently selected from one of the following structures: hydrogen, halogen (fluorine, chlorine, bromine, iodine), C1-C5 alkyl, C1-C5 alkylOxy, hydroxyl, mercapto, amino, nitro, cyano.

Preferably, in the general formula of the photosensitive molecule for constructing the photoresponsive soft and hard substrate, R is₁One selected from the following structures:

x is selected from one of the following structures:

in X, m, n and p are integers from 0 to 8 (including 0 and 8);

R₂selecting one of the following structures:

p is an integer of 0 to 8.

More preferably, the photosensitive molecule is one of the following structures:

wherein m, n and p are integers from 0 to 8.

Most preferably, the photoactive molecule is one of the following structures:

the second aspect of the present invention provides a method for preparing a surface enhanced raman detection substrate based on photo patterning, comprising the following steps:

the detection substrate comprises a hard substrate and a soft substrate;

the preparation method of the surface enhanced Raman detection substrate based on the photo-patterning comprises the following steps:

soaking the hard substrate material in a 0.01-10mM photosensitive molecular solution for 1-24h at room temperature, slowly taking out the hard substrate material, soaking and washing the hard substrate material by using a mixed solution of deionized water and acetonitrile in a volume ratio of 1:1, repeatedly washing the hard substrate material for at least three times, and drying the hard substrate material by inert gas to obtain a surface enhanced Raman detection hard substrate based on photo-patterning;

or the like, or, alternatively,

the preparation method of the surface enhanced Raman detection soft substrate based on the photo-patterning comprises the following steps:

dissolving 1-40mg of photosensitive molecules in 0.1-1mL of nano gold colloid, adding 10-400mg of other crosslinking components, and uniformly mixing to obtain 0.1-40% of hydrogel precursor solution containing 0.1-50mM of photosensitive molecules;

or dissolving 1-40mg of water-soluble macromolecule marked with photosensitive molecules in 0.1-1mL of nano gold colloid, and mixing uniformly to obtain a hydrogel precursor solution of photosensitive molecules with solid content of 0.001-10%;

adding an initiator or an initiation aid into the hydrogel precursor solution, uniformly mixing, injecting the solution into a mold, and carrying out cross-linking polymerization in an inert gas environment at the temperature of 40-60 ℃ to obtain the surface-enhanced Raman detection soft substrate based on the photo-patterning.

The hard substrate comprises a gold/gold-plated substrate, a silver/silver-plated substrate and a gold-silver alloy substrate.

The other crosslinking component is a mixture of acrylamide (AAm), N-Methylene Bisacrylamide (MBA) and acrylic acid.

The water-soluble macromolecules marked with photosensitive molecules are functionalized hyaluronic acid macromolecules marked with photosensitive molecules.

The preparation method of the functionalized hyaluronic acid macromolecule marked with the photosensitive molecule comprises the following steps: mixing photosensitive molecules, acrylate functionalized hyaluronic acid (HAMA), N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a molar ratio of 1:1 (1-5), dissolving in anhydrous dichloromethane at 0 ℃, recovering to room temperature for reaction, dialyzing after the reaction is finished, and freeze-drying to obtain the functionalized hyaluronic acid macromolecules marked with the photosensitive molecules.

The preparation method of the acrylate functionalized hyaluronic acid (HAMA) comprises the following steps:

dissolving a hyaluronic acid compound HA and a methacrylic anhydride compound in a molar ratio of 1:1 in deionized water, dropwise adding a sodium hydroxide solution (4mol/L) to keep the pH value of a hyaluronic acid system at 7-9, reacting at 30-50 ℃ for 1-12 h, dialyzing in deionized water, and freeze-drying to obtain the acrylate functionalized hyaluronic acid (HAMA).

The initiator or the initiation aid is at least one of potassium persulfate, ammonium persulfate and tetramethylethylenediamine, and preferably a mixture of ammonium persulfate and tetramethylethylenediamine with a molar ratio of 1: 1.

The preparation method of the nano gold colloid comprises the following steps:

1g of chloroauric acid HAuCl₄Dissolving in ultrapure water, adding into a 50mL brown volumetric flask to constant volume, shaking thoroughly, mixing, and storing in a refrigerator at 4 deg.C;

adding 98.3mL of ultrapure water accurately measured into a 250mL three-neck flask, accurately measuring 1mL of chloroauric acid solution, adding the chloroauric acid solution into the flask, and heating by an oil bath to enable the water solution in the flask to generate reflux; when the first drop of reflux liquid is generated in the condensation reflux pipe, adding accurately weighed 0.7mL of sodium citrate aqueous solution with the mass fraction of 1% in advance into the vigorously stirred solution, keeping the reflux state, continuously heating, observing that the solution is changed from gray to black within 2-3min, and finally gradually stabilizing into a red solution, when the solution is changed into wine red, continuously refluxing for 15min, stopping heating, when the solution is cooled to room temperature, transferring into a brown reagent bottle, and adding ultrapure water to recover the solution to 100mL, thereby obtaining the nano gold colloid.

The third aspect of the invention provides a method for detecting biomarkers by using a sandwich immune sandwich structure, which comprises the following steps: dissolving the antibody in ultrapure water to prepare an antibody solution;

wetting the surface of the prepared surface-enhanced Raman detection substrate based on photo-patterning with deionized water, fixing a photo-mask plate in a position about 0.5mm above the surface of the substrate in parallel, irradiating the substrate with an LED light source, soaking the irradiated substrate in a prepared antibody solution, standing and soaking at normal temperature, taking out the substrate, cleaning the surface with deionized water for three times, slightly drying water with inert gas, and storing at low temperature for later use; immersing the substrate into 0.5% bovine serum albumin solution for end capping, incubating at room temperature, and finally washing and drying with ultrapure water to obtain a patterned SERS planar substrate for modifying antibody molecules;

illuminating another area of the gold substrate, sequentially programming other antibodies to be illuminated and fixed in different areas on the substrate material according to the first step, and repeating the operation until all the required antibodies are fixed in the corresponding areas;

and thirdly, dropwise adding or immersing the solution to be detected in an area fixed with the antibody, after incubation in a humid and clean environment, gently rinsing the area with deionized water for three times, dropwise adding or immersing the functional nano metal particles modified by the antibody and the Raman signal molecules in the area, after incubation in a humid and clean environment, gently rinsing the area with deionized water for three times, and performing spectrum test analysis by using Raman laser.

The wavelength of the LED light source is determined according to the absorption of the photoaffinity group of the selected photosensitive molecule, and can be 250-500nm, preferably 300-400nm, and the light intensity is preferably 5-50mW/cm²More preferably 10mW/cm²The irradiation time is 1 to 15 minutes, preferably 2 minutes.

The antibody is a monoclonal or polyclonal antibody of a target detection object, the concentration of the antibody solution is 1 mu g/mL-100 mu g/mL, and the soaking time is 0.1-24h, preferably 6 h.

The antibody is specifically a mouse Anti-human C-reactive protein antibody (CPR antibody), an aspergillus galactomannan antibody (EB-A2), a human thyroxine (T4) antibody, a carcinoembryonic antigen (CEA) specific monoclonal antibody (Anti-CEA), an antibody Anti-AFP (alpha-fetoprotein) or an immunoglobulin IgM antibody (Anti-IgM).

The preparation method of the functional nano metal particle marked with the antibody and modified by the Raman signal molecule comprises the following steps:

adding a DMSO solution of Raman signal molecules with the concentration of 0.1-10mg/mL and the volume of 1-100 muL, a water solution of sulfhydryl polyethylene glycol 3000 monomethyl ether (PEG-SH) with the concentration of 0.1-10mg/mL and the volume of 1-100 muL, a DMSO solution of polyethylene glycol 2000 monomethyl ether succinimidyl ester (PEG-NHS) with the concentration of 0.1-10mg/mL and the volume of 1-100 muL into 0.1-10mL of nano gold colloid, stirring and reacting for 0.5-8h, centrifugally separating nano particles and the solution, ultrasonically dissolving the nano particles in deionized water again, centrifugally separating, then dissolving the nano particles in deionized water with the same volume again, adding 1-10mg of antibody into the solution, stirring and reacting for 1-24h, separating the nano particles and the solution through multiple times of high-speed centrifugation, and re-dispersing the nano particles in deionized water with the same volume, and storing the nano particles in a refrigerator at 4 ℃ for later use to obtain the functional nano metal particles marked with the antibodies and modified by the Raman signal molecules.

The Raman signal molecule is preferably 4-mercaptobenzoic acid, 2-mercaptobenzoic acid, 4-mercaptopyridine, p-mercaptoaniline (PATP), 6-mercaptopurine (6-MP).

The liquid to be detected is blood, body fluid and the like.

Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:

the invention adopts the photo-patterning technology to realize the covalent connection between the detection substrate and the antibody by using photosensitive molecules. The labeling of various antibodies on the same substrate material can be realized by accurately regulating and controlling the illumination area, so that the product meets the requirements of high-flux integrated detection of various biomarkers such as antigens, proteins and the like.

The invention belongs to the technical field of in-vitro diagnosis, and the preparation of a detection substrate comprises the following steps: 1) preparing photosensitive molecules for constructing photoresponse soft and hard substrates; 2) the light patterning technology realizes the marking of various antibodies on the surface of the substrate; 3) preparing gold and silver nanoparticles and modifying antibodies and Raman signal molecules; 4) the detection of biomarkers such as antigens and the like is realized through the sandwich immune sandwich structure. SERS detection Using the present inventionThe substrate can simultaneously realize the detection of various biomarkers such as antigens, proteins and the like, and the 2-mercaptobenzoic acid is adopted as a Raman probe molecule to ensure that the detection limit reaches 10^-15And mol, so that the product has higher sensitivity. The SERS detection substrate prepared by the optical patterning technology has the advantages of high resolution, stable signal, high-throughput detection and the like, and has important application value in clinical in-vitro analysis and diagnosis.

The invention realizes antigen immunodetection by utilizing the surface enhanced Raman effect, has low sensitivity to water, can be suitable for aqueous solution systems such as blood, body fluid and the like, does not need sample preparation under most conditions, can meet the requirements of a liquid biopsy technology, and has the advantages of no wound, small amount of required samples, simple and quick operation and the like in the detection process.

The Raman signal detection is carried out by utilizing the surface enhanced Raman scattering effect of the Raman signal molecules and the metal nanoparticles, the Raman scattering signal can be amplified, and the sensitivity of detection and analysis is greatly improved.

Drawings

FIG. 1 is a transmission electron microscopy characterization of the gold nanoparticles labeled with the Raman signal molecules MBA and CPR-antibody in example 11.

FIG. 2 is an XPS (photoelectron spectroscopy) characterization of the photo-responsive gold substrate prepared in example 7.

FIG. 3 is a SERS response curve of the sandwich immuno-sandwich system to CPR (10. mu.g/mL) in example 11.

Figure 4 is a standard curve of the relationship between CPR concentration and raman characteristic peak intensity in example 11.

FIG. 5 is a standard curve of the relationship between CEA concentration and Raman characteristic peak intensity in example 12.

FIG. 6 is a flow chart illustrating the fabrication and application of a photo-patterned surface enhanced Raman detection substrate.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Example 1

Synthetic route of SNB-1:

(1) synthesis and characterization of compound 3:

in a 50mL round-bottom flask, p-nitrophenyl chloroformate (0.4g, 2mmol) was dissolved in 10mL anhydrous DCM. Compound 4 (synthetic methods see: Ming, Z.; Fan, J.; Bao, C.; Xue, Y.; Lin, Q.; Zhu, L., Photogenated Aldehydes for Protein Patterns on Hydrogels and guidelines of Cell behavior.advanced Functional Materials 2018,28 (14)) (0.37g, 1mmol), anhydrous TEA (2mmol, 0.3mL), and a catalytic amount of DMAP were dissolved in 10mL of anhydrous DCM, added dropwise to the flask under argon protection, the solvent was concentrated by a rotary evaporator after stirring at room temperature for 2h, and the intermediate was rapidly purified with a flash silica gel column (100% DCM). The purified intermediate was dissolved in 20mL anhydrous DMF in a 50mL round bottom flask, triethylamine (2mmol, 0.3mL) and cystamine hydrochloride (0.1g, 0.4mmol) were added to it, the solvent was stirred at room temperature for 9h under argon protection, the solvent was dried, redissolved with DCM, the organic phase was washed three times with water (50mL), the organic phase was separated and dried over anhydrous sodium sulfate and purified by silica gel chromatography (dichloromethane: methanol ═ 100:2) to give compound 3.

Nuclear magnetic data for compound 3:¹H NMR(400MHz,Chloroform-d)δ10.23(s,2H),7.81(s,2H),7.08(s,2H),4.31-4.25(m,8H),3.98(s,6H),3.77-3.57(m,24H),3.32(t,J＝4.88Hz,4H),2.79(t,J＝6.28Hz,4H).¹³C NMR(101MHz,Chloroform-d)δ182.3,157.9,156.3,155,9,141.3,123.8,111.8,110.9,69.8,69.7,69.5,68.9,63.1,56.2,40.3,36.6.MS(ESI):m/z:Calcd.for C₃₈H₅₄N₄O₂₀S₂Na⁺[M+Na]⁺:973.3Found:973.3.

(2) synthesis and characterization of compound 2:

compound 3(0.95g, 1mmol) was dissolved in 50mL of methanol and added thereto under ice-bath conditionsAdding NaBH in batches₄(76mg, 2mmol), the reaction was stirred at ambient temperature for 1 h. The solvent was dried by spin-drying with a rotary evaporator, 50mL of ethyl acetate was added to redissolve, the pH of the system was adjusted to neutral with 1N HCl, the organic phase was washed 3 times with water (50mL), the organic phase was dried with anhydrous sodium sulfate and the solvent was spin-dried, and purified by a silica gel column chromatography (dichloromethane: methanol ═ 100:3) to give compound 2.

Nuclear magnetic data for compound 2:¹H NMR(400MHz,Chloroform-d)δ7.78(s,2H),7.02(s,2H),4.61(s,4H),4.28-4.23(m,8H),3.78(s,6H),3.72-3.54(m,24H),3.26(t,J＝4.86Hz,4H),2.78(t,J＝6.29Hz,4H).¹³C NMR(101MHz,Chloroform-d)δ156.6,154.9,148.4,140.6,129.1,126.0,110.4,70.2,69.9,69.4,68.8,62.2,58.3,53.6,40.0,36.3.MS(ESI):m/z:Calcd.for C₃₈H₅₈N₄O₂₀S₂Na⁺[M+Na]⁺:977.3Found:977.3.

(3) synthesis and characterization of Compound SNB-1:

compound 2 was accurately weighed 4mg dissolved in 2mL of a mixed solution of acetonitrile/water (V/V ═ 1:1), tris (2-carboxyethyl) phosphine (TCEP) was weighed 4.98mg added to the solution, the pH was adjusted to 7.2 to 7.4 with 50% aqueous NaOH, and the solution was placed in a shaker at 37 ℃ for 2 hours. After the reaction, acetonitrile was spin-dried using a rotary evaporator, extracted three times with dichloromethane (10 mL. times.3), and the organic phase was dried over anhydrous sodium sulfate and the solvent was spin-dried to give SNB-1, which was stored at 4 ℃ until use.

Nuclear magnetic data for compound SNB-1:¹H NMR(400MHz,Chloroform-d)δ7.57(s,1H),7.12(t,J＝1.1Hz,1H),5.58(t,J＝4.4Hz,1H),4.87(dd,J＝5.9,1.1Hz,2H),4.19(dt,J＝11.9,5.0Hz,4H),4.11(t,J＝5.9Hz,1H),3.80(s,3H),3.76(t,J＝4.9Hz,2H),3.70-3.59(m,10H),3.18(q,J＝4.9Hz,2H),2.59(dt,J＝6.8,4.9Hz,2H).¹³C NMR(101MHz,Chloroform-d)δ156.84,151.79,148.45,141.82,133.13,112.62,110.52,70.74,70.62,70.56,70.47,69.86,69.70,68.57,64.04,60.24,56.11,42.16,28.10.MS(ESI):m/z:Calcd.for C₁₉H₃₀N₂O₁₀SNa⁺[M+Na]⁺:501.1548Found:515.1540.

example 2

Synthetic route of SNB-2:

(1) synthesis and characterization of Compound 2 SNB-2:

in a 50mL round-bottom flask, p-nitrophenyl chloroformate (0.3g, 1.5mmol) was dissolved in 10mL of anhydrous dichloromethane. Compound 2(0.48g, 0.5mmol), anhydrous triethylamine (1.5mmol, 0.22mL) and a catalytic amount of DMAP were dissolved in 10mL anhydrous DCM, added dropwise to the round-bottom flask under argon protection, the reaction was stirred at room temperature for 2h and the solvent was concentrated by rotary evaporator and the intermediate product was purified rapidly using flash silica gel chromatography column (dichloromethane: ethyl acetate ═ 5: 1). The purified intermediate was dissolved in a 50mL round-bottom flask with 20mL anhydrous DCM, triethylamine (1.5mmol, 0.22mL) and aminotetraethyleneglycol monomethyl ether (0.32g, 1.5mmol) were added, and the reaction was stirred overnight at room temperature under argon protection. After the completion of the reaction, the solvent was spin-dried, the organic phase was washed three times with water (50mL), the organic phase was separated and dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (DCM: MeOH ═ 100:3) to obtain compound 2 SNB-2.

Nuclear magnetic data for compound 2 SNB-2:¹H NMR(400MHz,Chloroform-d)δ7.79(s,2H),7.03(s,2H),5.51(s,4H),4.25(m,8H),3.96(s,6H),3.91(t,J＝4.70Hz,4H),3.73-3.52(m,52H),3.49(m,4H),3.42(t,J＝4.91Hz,4H),3.36(s,6H),2.79(t,J＝6.40Hz,4H).¹³C NMR(101MHz,Chloroform-d)δ156.41,153.04,147.30,143.26,129.13,122.75,110.17,71.90,70.94,70.67,70.62,70.55,70.47,70.07,69.55,69.49,69.04,63.43,59.00,56.41,51.34,41.51,41.00,39.71.MS(ESI):m/z:Calcd.for C₅₈H₉₆N₆O₃₀S₂Na⁺[M+Na]⁺:1443.5504.Found:1443.5511.

(3) synthesis of Compound SNB-2

2SNB-2(4.3mg, 0.003mmol) was accurately weighed and dissolved in 3mL of a mixed solution of acetonitrile/water (V/V ═ 1:1), tris (2-carboxyethyl) phosphine (TCEP) (3.5mg, 0.012mmol) was weighed and added to the solution, the pH was adjusted to 7.2-7.4 with 50% aqueous NaOH solution, and the solution was placed in a shaker at 37 ℃ for reaction for 2 hours. After the reaction, acetonitrile was spin-dried using a rotary evaporator, extracted three times with dichloromethane (10 mL. times.3), and the organic phase was dried over anhydrous sodium sulfate and the solvent was spin-dried to give compound SNB-2, which was stored at 4 ℃ until use.

Nuclear magnetic data for compound SNB-2:¹H NMR(400MHz,Chloroform-d)δ7.20(t,J＝1.1Hz,1H),5.58(t,J＝4.4Hz,1H),5.44(d,J＝0.9Hz,2H),5.32(t,J＝5.7Hz,1H),4.19(dt,J＝11.9,5.0Hz,4H),3.88(s,3H),3.76(t,J＝4.9Hz,2H),3.70-3.55(m,24H),3.39-3.31(m,5H),3.18(q,J＝4.9Hz,2H),2.59(dt,J＝6.8,4.9Hz,2H).¹³C NMR(101MHz,Chloroform-d)δ156.84,156.57,151.96,148.59,141.56,130.74,112.95,110.49,71.79,70.74,70.62,70.59,70.57,70.56,70.47,69.86,69.70,69.67,69.43,68.57,64.16,64.04,59.02,56.11,42.16,39.25,28.10.MS(ESI):m/z:Calcd.for C₂₉H₄₉N₃O₁₅S⁺[M+H]⁺:712.3390.Found:712.3423

example 3

Synthetic route of BNBP

(1) Synthesis of Compound 7

Compound 5 (purchased from Shanghai vessel Biotechnology Co., Ltd.) (3.70g, 0.01mol), Compound 6(1.98g, 0.01mol) and potassium carbonate (2g, 0.015mol) were charged into a 250mL single-neck round-bottom flask, 100mL of acetonitrile was added, and the mixture was heated under reflux for 24 hours. After the reaction was completed, the reaction was cooled to room temperature, and the insoluble solid was removed by suction filtration, the filtrate was evaporated under reduced pressure and dried by spin drying, a small amount of dichloromethane was added, and the mixture was purified by a silica gel column chromatography (methanol: dichloromethane ═ 1:100) to obtain compound 7.

Nuclear magnetic data for compound 7:¹H NMR(400MHz,Chloroform-d)δ7.88-7.83(m,2H),7.82-7.77(m,2H),7.58-7.51(m,2H),7.51-7.46(m,1H),7.07-7.01(m,2H),6.50(t,J＝5.1Hz,1H),5.23(t,J＝5.1Hz,1H),4.14(t,J＝5.9Hz,2H),3.63-3.54(m,8H),3.34(dq,J＝5.3,4.3Hz,4H),2.56(t,J＝6.0Hz,2H),1.42(s,9H).¹³C NMR(101MHz,Chloroform-d)δ194.43,172.20,163.11,156.42,138.16,132.60,131.77,131.26,130.33,128.75,114.43,79.54,69.96,69.23,69.61,69.43,63.45,40.08,38.53,37.5,28.30.MS(ESI):m/z:Calcd.for C₂₇H₃₇N₂O₇ ⁺[M+H]⁺:501.25.Found:501.68.

(2) synthesis of Compound BNBP

In a 250mL three-neck round-bottom flask, compound 7(5.0g, 0.01mol) was dissolved in 100mL of a mixed solvent of dichloromethane and trifluoroacetic acid (volume ratio) 5:1, the mixture was stirred at room temperature for 0.5h, and then the solvent was concentrated by rotary evaporation under reduced pressure and purified by a silica gel column (petroleum ether and ethyl acetate 1:1) to obtain compound BNBP.

Nuclear magnetic data of compound BNBP:¹H NMR(400MHz,Chloroform-d)δ7.88-7.83(m,2H),7.83-7.77(m,2H),7.58-7.51(m,2H),7.51-7.46(m,1H),7.07-7.01(m,2H),6.50(t,J＝5.1Hz,1H),6.27(d,J＝14.1Hz,2H),4.14(t,J＝5.9Hz,2H),3.65-3.54(m,6H),3.50(t,J＝3.9Hz,2H),3.35(dt,J＝5.1,4.2Hz,2H),2.84(tt,J＝7.0,3.8Hz,2H),2.56(t,J＝6.0Hz,2H).¹³C NMR(101MHz,Chloroform-d)δ194.20,172.29,163.78,138.32,132.78,131.60,131.77,130.00,128.75,114.67,72.88,69.93,69.65,69.57,63.80,41.75,40.56,37.14.MS(ESI):m/z:Calcd.for C₂₂H₂₈N₂O₅Na⁺[M+Na]⁺:423.1891.Found:423.1945.

example 4

Synthetic route of DBNP:

(1) synthesis and characterization of compound 11:

compound 9 (available from Shanghai vessel Biotechnology Co., Ltd.) (0.412g, 1mmol), compound 8(0.162g, 1.2mmol) and potassium carbonate (0.414g, 3mmol) were charged into a 250mL single neck round bottom flask, 100mL acetonitrile was added, and the reaction was heated under reflux for 24 hours. After completion of the reaction, the reaction was cooled to room temperature, and the insoluble solid was removed by suction filtration, and the filtrate was spin-dried by distillation under reduced pressure, followed by addition of a small amount of dichloromethane and purification by silica gel chromatography (methanol: dichloromethane ═ 1.5:100) to obtain compound 10.

In a 250mL three-neck round-bottom flask, compound 10(0.234g, 0.5mmol) was dissolved in 100mL of a mixed solvent of dichloromethane and trifluoroacetic acid (5: 1), the mixture was reacted with stirring at room temperature for 0.5h, the solvent was concentrated by rotary evaporation under reduced pressure, and the product was purified by silica gel chromatography (petroleum ether, ethyl acetate (1: 1)) to obtain compound 11.

Nuclear magnetic data for compound 11:¹H NMR(400MHz,Chloroform-d)δ7.17-7.08(m,1H),6.85-6.79(m,2H),6.28(d,J＝14.1Hz,2H).4.46(s,2H),3.71-3.61(m,8H),3.59-3.38(m,6H),2.90(tt,J＝7.0,3.9Hz,2H).¹³C NMR(101MHz,Chloroform-d)δ169.34,155.92,136.22,120.14,116.01,72.74,70.60,70.59,69.53,69.67,69.54,67.20,41.31,39.45.MS(ESI):m/z:Calcd.for C₁₆H₂₅N₅O₅Na⁺[M+Na]⁺:390.17.Found:390.24.

(2) synthesis and characterization of compound DBNP:

compound 11(3.67g, 0.01mol) was dissolved in 100mL of redistilled tetrahydrofuran, potassium carbonate (1.4g, 0.015mol) was added, and acryloyl chloride (1.2mL, 0.015mol) was slowly added dropwise to the system under ice bath conditions, and the reaction was carried out for 12 hours. After the reaction was completed, the organic phase was washed with 2X 100mL of water, and dried over anhydrous sodium sulfate. The solvent was removed by distillation under the reduced pressure, and the product was purified by silica gel chromatography (petroleum ether: ethyl acetate 1:1) to obtain compound DBNP.

Nuclear magnetic data of compound DBNP:¹H NMR(400MHz,DMSO-d₆)δ8.15(m,2H),7.12-7.09(m,2H),6.85-6.82(m,2H),6.29(t,J＝10.9Hz,1H),6.08(dd,J＝10.9,3.2Hz,1H),5.96(dd,J＝10.8,3.1Hz,1H),4.46(s,2H),3.71-3.62(m,8H),3.59(td,J＝4.4,2.7Hz,4H),3.38(dt,J＝4.9,4.1Hz,2H),3.20(dt,J＝5.9,4.3Hz,2H).¹³C NMR(101MHz,DMSO-d₆)δ169.24,166.89,155.92,136.22,130.21,126.74,121.14,116.87,70.64,70.59,69.68,69.67,69.35,69.47,67.49,39.86,39.26.MS(ESI):m/z:Calcd.for C₁₉H₂₈N₅O₆ ⁺[M+H]⁺:422.2035.Found:422.2048.

example 5

Synthetic route of CNPA:

(1) synthesis and characterization of compound 14:

HOBt (0.75g, 5.5mmol), HBTU (2.08g, 5.5mmol) were dissolved in anhydrous DMF (20mL) and then compound 13(0.92g, 5mmol) was added followed by compound 12(0.97g, 6mmol), stirred at room temperature for 6h then quenched with water and extracted with ethyl acetate. The organic layer was washed with saturated brine 2 times and then MgSO₄And (5) drying. Purification on a silica gel column (petroleum ether: ethyl acetate ═ 1:4) afforded compound 14.

Nuclear magnetic data for compound 14:¹H NMR(400MHz,Chloroform-d)δ7.30(s,1H),7.08(t,J＝5.1Hz,1H),6.70(m,2H),6.57(m,J＝8.8,2.0,0.9Hz,1H),6.24(s,1H),5.26(t,J＝5.1Hz,1H),3.47(dt,J＝5.3,4.4Hz,2H),3.30(m,2H),2.74(tt,J＝8.2,1.1Hz,2H),2.45(s,1H),2.45(d,J＝16.5Hz,1H),1.42(s,9H).¹³C NMR(101MHz,Chloroform-d)δ173.47,156.41,145.34,144.35,132.79,121.01,116.04,115.34,79.54,40.09,39.25,37.29,29.10,28.10.MS(ESI):m/z:Calcd.for C₁₆H₂₄N₂O₅Na⁺[M+Na]⁺:347.16.Found:347.24.

(2) synthesis and characterization of compound 15:

compound 14(0.648g, 2mmol) was dissolved in 100mL of a mixed solvent of dichloromethane and trifluoroacetic acid in a 250mL three-neck round-bottom flask, the mixture was stirred at room temperature for 1 hour, the solvent was concentrated by rotary evaporation under reduced pressure, and the product was purified by silica gel column chromatography (petroleum ether: ethyl acetate 1:1) to give compound 15.

Nuclear magnetic data for compound 15:¹H NMR(400MHz,Chloroform-d)δ7.30(s,1H),7.09(t,J＝4.9Hz,1H),6.72(m,2H),6.43(ddt,J＝8.8,2.0,0.9Hz,1H),6.10(s,1H),4.08(t,J＝6.3Hz,2H),3.24(dt,J＝4.9,4.2Hz,2H),3.01(tt,J＝6.4,4.3Hz,2H),2.78(tt,J＝8.2,1.1Hz,2H),2.24(t,J＝8.2Hz,2H).¹³C NMR(101MHz,Chloroform-d)δ174.67,145.29,144.35,132.79,121.14,116.04,115.34,40.81,40.12,37.78,30.02.MS(ESI):m/z:Calcd.for C₁₁H₁₆N₂O₃Na⁺[M+Na]⁺:247.11.Found:247.30.

(3) synthesis and characterization of compound 17:

levulinic acid, compound 16(2g, 0.017mol), was charged to a round bottom flask and cooled to 0 ℃ under nitrogen, 7N NH was slowly added₃(dissolved in 5mL of methanol) was added to the flask. After 3 hours, 5mL of an anhydrous methanol solution of hydroxylamine sulfonic acid (3.2g, 0.03mol) was added dropwise to the flask, and after the addition was complete, the resulting solution was allowed to return to room temperature and reacted overnight. The reaction solution was concentrated under vacuum, and the concentrate was resuspended in anhydrous methanol. The white precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure and dissolved in 50mL of anhydrous methanol again. At 0 deg.C, 10mL of anhydrous methanol solution of triethylamine (4.3mL) and iodine (2.1g, 0.008mol) was added until the solution was dark brown and remained unchanged for 10min, indicating that the oxidation of the diazepane intermediate was complete. The solution was diluted with ethyl acetate and washed with 1N hydrochloric acid and a saturated aqueous solution of sodium thiosulfate several times. And separating the system, drying the organic phase by using anhydrous sodium sulfate, concentrating the solvent, and purifying by using a silica gel chromatographic column to obtain the compound 17.

Nuclear magnetic data for compound 17:¹H NMR(400MHz,DMSO-d₆),δ(ppm)，10.44(s,1H,2.30(m,2H,1.85(m,J＝6.4Hz,2H),1.11(s,3H).¹³C NMR(101MHz,Chloroform-d),δ(ppm):178.56,28.73,28.03,24.75,19.74.MS(ESI):m/z:Calcd.for C₅H₉N₂O₂ ⁺[M+H]⁺:129.07.Found:129.10.

(4) synthesis and characterization of compound CNPA:

HOBt (0.75g, 5.5mmol), HBTU (2.08g, 5.5mmol) were dissolved in anhydrous DMF (20mL) and then compound 17(0.64g, 5mmol) was added followed by compound 15(1.35g, 6mmol), stirred at room temperature for 6h then quenched with water and extracted with ethyl acetate. The organic layer was washed with saturated brine 2 times and then MgSO₄And (5) drying. Purification by silica gel chromatography (petroleum ether: ethyl acetate 1:4) afforded compound CNPA.

Nuclear magnetic data of compound CNPA:¹H NMR(400MHz,DMSO-d₆)δ7.11-7.05(m,1H),6.98-6.92(m,1H),6.75-6.68(m,2H),6.57(ddt,J＝8.8,2.0,0.9Hz,1H),6.24(s,1H),3.33-3.25(m,4H),2.78(tt,J＝8.2,1.1Hz,2H),2.51-2.42(m,4H),2.11-2.01(m,2H),1.49(s,3H).

¹³C NMR(101MHz,DMSO-d₆)δ173.84,145.29,144.35,132.79,121.01,116.04,115.34,74.75,49.34,48.83,39.82,37.29,36.06,29.98,24.57,22.83.

MS(ESI):m/z:Calcd.，for C₁₆H₂₂N₄O₄Na⁺[M+Na]⁺:357.1534.Found:357.1560.

example 6

Preparing nano gold colloid:

1g of chloroauric acid HAuCl₄Dissolving in ultrapure water, adding into 50mL brown volumetric flask to constant volume, shaking thoroughly, mixing, and storing in refrigerator at 4 deg.C. 98.3mL of ultrapure water was accurately measured in a 250mL three-necked flask, 1mL of chloroauric acid solution was accurately measured and added to the flask, and the flask was heated in an oil bath to reflux the aqueous solution. When the first drop of reflux liquid is observed to be generated in the reflux condenser pipe, 0.7mL of sodium citrate aqueous solution which is accurately weighed and is prepared in advance with the mass fraction of 1 percent is added into the vigorously stirred solution. The heating was continued while maintaining the reflux state, and it was observed that the solution changed from gray to black within 2-3min, and finally gradually stabilized to a red solution. And when the solution turns to wine red, continuously refluxing for 15min, stopping heating, cooling the solution to room temperature, transferring the solution into a brown reagent bottle, and adding ultrapure water to restore the solution to 100mL to obtain the nano gold colloid.

Example 7

A method for preparing a photoresponse hard substrate capable of realizing labeling of various antibodies, as shown in fig. 6, fig. 6 is a schematic flow chart of a method for preparing a photo-patterned surface enhanced raman detection substrate and an application thereof, and the method comprises the following steps:

the photosensitive molecule SNB-2 prepared in example 2 was modified on a gold substrate by spontaneous formation of a self-assembled monolayer by gold mercaptol. The method comprises the following specific steps: at room temperature, the cleaned gold substrate (size 1 cm. times.3 cm) was immersed in 20mL of a 1mM SNB-2 solution at room temperature for 12 hours. Slowly taking out the gold substrate from the SNB-2 solution at a constant speed, and ensuring that the part just exposed out of the liquid surface is in a clean state. After extracting the SNB-2 solution from all the substrates, gently soaking and washing the gold substrate by using a mixed solution of deionized water and acetonitrile (1:1), repeatedly washing for three times, and drying by inert gas to obtain the photoresponse hard substrate capable of realizing the labeling of various antibodies, namely the gold substrate of the surface-modified functional molecule SNB-2.

FIG. 2 is an XPS (photoelectron spectroscopy) characterization of the photo-responsive gold substrate prepared in example 7. As shown in FIG. 2, the Au surface of the clean Au substrate shows characteristic energy bands of Au element, namely Au4f (84eV, 85eV), Au4d (334eV, 353eV), Au4p (546eV), and relatively lower intensity C1s (248eV) and O1s (532 eV). After the photoresponse molecule is modified, the signal of Au in an XPS energy spectrum is obviously reduced, the signals of C1s (248eV) and O1s (532eV) are relatively enhanced, and a new N1s signal appears at 400eV, so that the photoresponse molecule is successfully modified on the surface of the gold substrate.

Example 8

A method for preparing a photoresponse hard substrate capable of realizing labeling of a plurality of antibodies comprises the following steps:

a clean silver substrate (size 1 cm. times.3 cm) was taken out and immersed in 20mL of the SNB-1 solution prepared in example 1 at a concentration of 1mM for 12 hours at room temperature. Slowly taking out the silver substrate from the SNB-1 solution at a constant speed, and ensuring that the part just exposed out of the liquid surface is in a clean state. After all the substrates are taken out of the SNB-1 solution, the silver substrate is gently soaked and washed by a mixed solution of deionized water and acetonitrile (1:1), the washing is repeated for three times, and inert gas is dried by blowing, so that the photoresponse hard substrate capable of realizing the labeling of various antibodies, namely the silver substrate of the surface modified functional molecule SNB-1 is obtained.

Example 9

A method for preparing a photoresponse soft substrate capable of realizing labeling of a plurality of antibodies comprises the following steps:

the first step, the preparation method of the functionalized hyaluronic acid macromolecule (HA-BNBP) marked with the photosensitive molecule BNBP comprises the following steps:

the preparation method of the acrylate functionalized hyaluronic acid (HA-MA) comprises the following steps: 1g of hyaluronic acid compound HA (purchased from Huaxi Biotechnology Ltd., molecular weight 7.4 ten thousand, cosmetic grade) was dissolved in 100mL of deionized water, 11.5mL of methacrylic anhydride was added to the solution, the molar ratio of the hyaluronic acid compound HA to the methacrylic anhydride compound was 1:1, 4mol/L of sodium hydroxide solution was added dropwise to maintain the pH of the hyaluronic acid system at about 8, and the reaction was carried out at 40 ℃ for 6 hours. Dialyzing the reaction solution in deionized water for 3 days by a dialysis bag (KDa3500), and removing the solvent by a freeze drying technology to obtain HA-MA. The nuclear magnetic characteristic peaks of double bonds on acrylate at δ ═ 5.6 and 6.1ppm, based on the integration of the methyl hydrogen on N-acetamido group at δ ═ 1.8 to 2.0ppm, the marking rate of methacrylate on hyaluronic acid was 60.4% by comparison of the integrated areas.

BNBP (0.4g, 0.001mol) prepared in example 3, acrylate-functionalized hyaluronic acid (HA-MA) prepared above (0.3g), N-hydroxysuccinimide (0.34g, 0.003mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.57g, 0.003mol) were dissolved in 10mL of anhydrous dichloromethane at 0 ℃ and the molar ratio of the photo-sensitive molecule, acrylate-functionalized hyaluronic acid (HAMA), N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was 1:1:3:3 and returned to room temperature for 6 h. After the reaction is finished, the system is dialyzed for 3 days by a dialysis bag (KDa3500), and then is frozen and dried to obtain HA-BNBP. The labeling rate of BNBP was 3.5% by area comparison, based on the nuclear magnetic characteristic peak of hydrogen on the benzene ring in the range of δ 7.85 to 6.50ppm and the integral of methyl hydrogen on the N-acetylamino group in the range of δ 1.8 to 2.0 ppm.

In the second step, the preparation method of the acrylic acid functionalized hyaluronic acid macromolecule (HA-BNBP) hydrogel substrate containing photosensitive molecules BNBP comprises the following steps:

dissolving 20mg of HA-BNBP prepared in the first step into 1mL of nano gold colloid (prepared in example 6) solution to prepare hydrogel precursor solution with the solid content of 2%, adding 50 muL of 2mol/L ammonium persulfate aqueous solution and 50 muL of 2mol/L tetramethylethylenediamine aqueous solution into the solution, swirling to mix uniformly, slowly adding the precursor solution into a substrate mold (a cuboid-shaped polytetrafluoroethylene substrate mold, wherein the precursor solution can become a solid substrate), heating to 50 ℃ under the protection of inert gas to initiate crosslinking (ammonium persulfate is used as a thermal initiator, heating is carried out, and the polymerization is completed to form solid, thus preparing the functionalized hyaluronic acid macromolecule (HA-BP) hydrogel substrate containing photosensitive molecules BNBP.

Example 10

A method for preparing a photoresponse soft substrate capable of realizing labeling of a plurality of antibodies comprises the following steps:

first, 20mg of the photosensitive molecule DBNP prepared in example 4 was dissolved in 0.5mL of the nanogold colloid prepared in example 6, and then 20mg of acrylamide, 20mg of acrylic acid, and 2mg of N, N-methylenebisacrylamide were added and uniformly mixed to obtain a hydrogel precursor solution having a solid content of 12.5%.

And secondly, adding 50 mu L of 2mol/L ammonium persulfate aqueous solution and 50 mu L of 2mol/L tetramethylethylenediamine aqueous solution into the hydrogel precursor solution prepared in the first step, swirling to mix uniformly, slowly adding the precursor solution into a substrate mold, and heating to 50 ℃ under the protection of inert gas to initiate crosslinking to prepare the photoresponse soft substrate containing photosensitive molecules DBNP.

Example 11

A method for realizing multiple antigen immunodetection based on a photoresponse gold substrate of a photosensitive molecule SNB-2 comprises the following steps:

the basic principle is as follows: fixing the antigen to be detected on the surface of the substrate by utilizing immune composite reaction between the antigen to be detected and the antibody fixed on the surface of the substrate, fixing the metal nanoparticles on the surface of the substrate by utilizing immune composite reaction between the antigen to be detected and the antibody modified on the surface of the metal nanoparticles to form a sandwich structure of the substrate antibody-antigen-antibody metal nanoparticles, and detecting the specific antigen by detecting Raman spectrum of Raman labeled molecules modified by the metal nanoparticles. The detection of multiple antigens utilizes the light response reaction characteristic of photosensitive molecules on the surface of a substrate to realize the labeling of multiple antibodies on different areas of the same substrate material. The method specifically comprises the following steps:

the surface of the gold substrate of the surface-modified functional molecule SNB-2 prepared in example 7 was wetted with deionized water, and a photomask plate (illumination area size 2 mm. times.2 mm) was fixed in parallel at a position of about 0.5mm above the surface, with a light intensity of 10mW/cm²After the LED light source with the wavelength of 365nm irradiates for 2min, the gold substrate is soaked in a mouse anti-human C reactive protein antibody (CPR-antibody) solution with the concentration of 100 mu g/mL, stands for 6h at the normal temperature, is taken out, and is washed by deionized water for three times. And slightly blowing the water by inert gas, and storing at low temperature for later use.

And immersing the substrate into a 0.5% bovine serum albumin solution for blocking, incubating at room temperature for 3h, and finally washing and drying with ultrapure water to obtain the patterned SERS planar substrate for modifying the antibody molecules. Then, the other area of the gold substrate is irradiated with light, the operation is repeated, the second antibody aspergillus galactomannan antibody (EB-A2) is labeled, and the labeling of four antibodies, namely CPR-antibody, EB-A2, Anti-CEA and Anti-IgM, on the same substrate is realized through repeated operation for many times.

And (3) dropwise adding 15 mu L of to-be-detected liquid to an area fixed with the antibody, incubating for 1h in a humid and clean environment, and gently rinsing with deionized water for three times. Prepared AuNP solutions respectively labeled with CPR-antibody, EB-A2, Anti-CEA or Anti-IgM and Raman reporter MBA with the concentration of 20 mu g/mL are respectively dripped into corresponding areas by 10 mu L, incubated for 3h in a humid clean environment and gently rinsed by deionized water for three times. And drying the prepared surface-enhanced Raman sandwich gold substrate by using inert gas, and carrying out Raman spectrum test.

FIG. 3 is a SERS response curve of the sandwich immuno-sandwich system to CPR (10. mu.g/mL) in example 11. As can be seen from the figure, under the laser excitation of 633nm wavelength, two obvious Raman signal peaks of C-C ring respiratory vibration mode which is assigned as MBA are presented in the region of SERS sandwich immune response and are respectively positioned at 1075cm^-1And 1585cm^-1To (3). While for substrates without SERS probe added, i.e. antibody modified capture substrate (capture substrate) and substrate after capture CPR (capture substrate + CPR), in phaseUnder the same test conditions, almost no raman signal was detected in the illuminated area. On the basis, the Raman signal test is further carried out on CPR of different concentrations, and figure 4 shows the relationship between the CPR concentration and the Raman characteristic peak intensity in example 11. As can be seen from fig. 4, the raman signal intensity of the signal molecule MBA increases with increasing concentration of CPR in the fluid to be tested, so that the concentration of CPR measured can be inferred by the signal intensity back.

The preparation method of the functional nano metal particles marked with the antibodies and modified by the Raman signal molecules comprises the following steps:

accurately prepared were 2mM Raman signal molecule 2-mercaptobenzoic acid (MBA) in DMSO, 1mM polyethylene glycol 2000 monomethyl ether succinimide ester (PEG-NHS) in DMSO, and 1mM mercaptopolyethylene glycol 3000 monomethyl ether (PEG-SH) in water.

To each ml of nano-gold colloid (nano-gold colloid prepared in example 6, concentration 5.886 × 10) under high-speed stirring^-7mM) and 5 mul of MBA solution and 5 mul of PEG-NHS solution are added, after stirring and reacting for 1 hour, 50 mul of PEG-SH solution is added into each milliliter of nano gold colloid, and the stirring and reacting are continued for 5 hours. And after the reaction is finished, centrifuging the reaction product in a centrifuge at 8000rpm/min for 10min, discarding supernatant, ultrasonically dissolving the precipitated colloidal gold in deionized water again, centrifuging the reaction product in a centrifuge at 7000rpm/min for 10min, discarding supernatant, repeatedly centrifuging the reaction product once again, and ultrasonically dissolving the colloidal gold in deionized water with the same volume to obtain polyethylene glycol coated and modified gold nanoparticles (AuNP-PEG). Under the condition of rapid stirring, 20 mu L of prepared antibody Anti-AFP (alpha fetoprotein) solution with the concentration of 20 mu g/mL is added into the solution, after 12h of stirring reaction, the solution is centrifuged in a centrifuge with 7000rpm/min for 10min, the supernatant is discarded, the operation is repeated for three times, the nanogold is ultrasonically re-dispersed in deionized water with the same volume, and the solution is stored in a refrigerator at 4 ℃ for standby, so that the nanogold particles modified by polyethylene glycol coating are obtained, as shown in the f-AuNP structure in figure 1, and figure 1 is a transmission electron microscope characterization picture of the nanogold particles marked with Raman signal molecules MBA and CPR-antibody in example 11. The functionalized nano-particles are characterized by ultraviolet and particle size, for example, the ultraviolet-visible light absorption spectrum diagram is shown at the lower left in figure 1, and the f-AuNP is 50An absorption peak appears at 0-600nm and a maximum absorption value appears at 542nm, which indicates that the spherical nanoparticles are successfully prepared, the inset shows the corresponding DLS characterization, and the data shows that the average hydrated particle size of the antibody-modified gold nanoparticles is 73.2 nm.

In the same way, mouse Anti-human C-reactive protein antibody (CPR antibody), aspergillus galactomannan antibody (EB-A2), human thyroxine (T4) antibody, carcinoembryonic antigen (CEA) specific monoclonal antibody (Anti-CEA) or immunoglobulin IgM antibody (Anti-IgM) and Raman signal molecule MBA-labeled nano gold particles are respectively prepared.

Example 12

A method for realizing multiple antigen immunodetection based on a photoresponse hydrogel substrate of photosensitive molecules BNBP. The immunoassay principle for various antigens is the same as that in example 9, and specifically comprises the following steps:

the surface of the silver substrate of the surface-modified functional molecule SNB-1 prepared in example 8 was wetted with ultrapure water, and a photomask plate (illumination area size 2 mm. times.2 mm) was fixed in parallel at a position of about 0.5mm above the surface, with a light intensity of 10mW/cm²And after the LED light source with the wavelength of 365nm irradiates for 2min, soaking the hydrogel in Anti-AFP solution with the concentration of 20 mu g/mL, standing for 6h at normal temperature, and washing with deionized water for three times. Then, another area of the hydrogel is irradiated with light, the operation is repeated, the second antibody Anti-CEA is labeled, and the repeated operation realizes the labeling of four antibodies, namely Anti-AFP, CPR-antibody, EB-A2, Anti-CEA and Anti-IgM, on the same substrate.

And (3) dropwise adding 15 mu L of to-be-detected liquid to an area fixed with the antibody, incubating for 1h in a humid and clean environment, and gently rinsing with deionized water for three times. 10 mu L of each of five AuNP solutions respectively marked with antibodies Anti-AFP, CPR-antibody, EB-A2, Anti-CEA, Anti-IgM and Raman reporter MBA are dripped into corresponding areas, incubated for 3h in a humid clean environment and gently rinsed with deionized water for three times. And drying the prepared surface-enhanced Raman sandwich detection hydrogel substrate with inert gas, and performing Raman spectrum test.

FIG. 5 is the relationship between CEA concentration and Raman characteristic peak intensity in example 12. As can be seen from the figure, the Raman signal intensity of the signal molecule MBA is increased along with the increase of the CEA concentration in the liquid to be detected, the detection area covers the clinical detection range, the conventional low-concentration CEA detection can be supported, and the universality of the detection substrate on various antibodies is also proved.

Comparative example 1

Preparing an SERS hard substrate for protein detection by using a stamp printing technology:

the gold-plated silicon wafer is washed by ethanol and dried by inert gas. A Polydimethylsiloxane (PDMS) stamp film with a round hole 3mM in diameter was immersed in a 2mM solution of octadecanethiol for 1min and dried under a stream of high purity nitrogen. And (3) stamping the dried PDMS on the surface of the gold-plated silicon wafer for 30s, so that a 3mm uncovered area is left on the surface of the gold-plated silicon wafer. The substrate was immersed in a 0.1mM dithiosuccinyl propionate (DSP) solution in ethanol for 16h, rinsed with ethanol and dried with inert gas to form a 3mM round coating of DSP. mu.L of Anti-CEA antibody at a concentration of 20. mu.g/mL was added dropwise to the DSP-coated area and reacted in a humid environment for 8h, the antibody being linked to the DSP layer through an amide bond formed by an amino group on the protein and a succinimide ester of DSP. The substrate was rinsed with 10mM PBS and exposed to 20. mu.L of blocking buffer for 16 h. And (3) exposing 20 mu L of the object to be tested in a test area for 8h, exposing the test area in nano gold colloid of functional markers anti-CEA and MBA Raman signal molecules for 16h after washing to form a double-antibody sandwich structure, and carrying out Raman analysis test after washing and drying.

The method can form a protein detection area with a specific rule and realize the concentration detection of a single substance to be detected, but the operation process is complex, and the rule area is difficult to further accurately control. The invention can realize the preparation of controllable patterning multiple and repeated detection areas by using simple illumination, can realize the preparation of micron-sized areas to be detected and can detect multiple objects to be detected on the same substrate material.

Comparative example 2

The SERS soft substrate is used for detecting protein:

the gold colloid encapsulated by the fluorotetraethylene glycol ligand is cast on a silicon oxide substrate cleaned by the piranha solution, and a blue film is formed after the solvent is evaporated. Pouring precursor water solution consisting of 2M acrylic monomer, 10mM tetraethyleneglycol diacrylate cross-linking agent and 8mM 2-ketoglutaric acid initiator onto the gold film protected by the mold, and performing polymerization for 3h under 365nm illumination. The polymerized hydrogel was gently removed and the gold film was transferred to the hydrogel surface. By varying the salt concentration (1-0.001M) of the hydrogel environment, the hydrogel exhibits salt-responsive shrinkage and swelling. The hydrogel is used for detecting cytochrome C and crystal violet, and can obviously detect a signal peak of about 30000counts of an object to be detected due to a surface resonance effect generated by reducing the distance of nanoparticles caused by hydrogel shrinkage in a 1M NaCl environment.

The detection method can obviously embody the Raman signal peak of the object to be detected and realize the identification of the structure of the object to be detected, but the detection environment has high salt concentration and is not beneficial to the detection of a plurality of biomolecules, and although the simultaneous identification of a plurality of objects to be detected can be realized, the increase of the types of the objects to be detected leads to the difficulty in distinguishing the signal peak and is not beneficial to the analysis of the objects to be detected. The detection method designed by the invention can fix different substances in the same solution to be detected in a specific area without mutual interference, and is more beneficial to simultaneous quantitative analysis of multiple substances to be detected.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.