A multi-network gel composite film fluorescence enhancement substrate and a preparation method and application thereof

Abstract

The application discloses a multi-network gel composite film fluorescence enhancement substrate and a preparation method and application thereof. The fluorescence enhancement substrate comprises a multi-gel network-gold particle composite film, and the multi-gel network-gold particle composite film comprises: an adhesive first gel network formed by cross-linking of an adhesive polymer; a reducing second gel network formed by cross-linking of a reducing natural high molecule; a third gel network containing gold particles and formed by in-situ growth of the gold particles in the adhesive first gel network; and a fourth gel network containing gold particles and formed by in-situ growth of the gold particles in the reducing second gel network. The substrate can easily control the size / distribution / gap / density of the gold particles, avoid aggregation of the gold particles, better combine with the substrate, enrich the target, enhance the fluorescence intensity of the labeled target, improve the detection sensitivity and avoid detection deviation caused by the coffee ring.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, in particular to a multi-network gel composite membrane fluorescence enhancement substrate, a preparation method and application thereof. BACKGROUND

[0002] Compared with detection techniques based on electricity, magnetism, mechanics and the like, optical detection techniques based on fluorescence labeling have incomparable advantages in the detection in the fields of biology, chemistry, clinical medicine and the like, and can achieve the detection of target markers (small molecules, nucleic acids, proteins, cells and the like) in samples simply, quickly, reliably and without radioactive pollution. However, when the concentration of the target marker is low, the fluorescence signal is usually very weak and is easily interfered by the fluorescence background of biological molecules in the complex sample, which limits the sensitivity of the detection. Enhancing the fluorescence signal of the target marker is an effective method for improving the sensitivity of the target marker, and is the key to realizing the ultra-sensitivity and high reliability detection of the target marker at a very low concentration.

[0003] At present, the fluorescence signal intensity of the target marker is mainly enhanced by using noble metal nanoparticles such as gold and silver, and the resonance of surface plasmons is formed under the irradiation of excitation light at a specific wavelength range, so as to increase the local electromagnetic field intensity and optical state density to enhance the fluorescence. The matching between the to-be-detected fluorescent molecules and the electromagnetic field enhancement region is the key to improving the detection sensitivity, and the matching degree depends on multiple factors such as the size of the noble metal nanoparticles, the stacking mode and the distance between the noble metal nanoparticles and the fluorescent molecules. Existing researches have shown that large-size and uniformly dense noble metal particles can solve the problem of difficult matching between the to-be-detected fluorescent molecules and the electromagnetic field enhancement region. Surface adsorption, chemical self-assembly or thermal annealing effect is usually used to form a noble metal substrate, but it is difficult to form large-size noble metal particles uniformly and stably, and the effective adjustment mechanism of the gap between the particles is also lacking. The randomness of the plasmon resonance phenomenon of the formed substrate is large, the adsorption of the to-be-detected fluorescent biological molecules is limited and difficult to be uniformly distributed, the repeatability is poor, and it is difficult to be used for quantitative analysis.

[0004] At present, the physical methods for generating noble metal particles, such as laser ablation method, mechanical ball milling method, evaporation condensation method and high-pressure magnetron sputtering method, have strong stability and high purity of the generated particles, but the process is too expensive, complex, time-consuming and requires high requirements for the preparation equipment, so it is difficult to realize the dense arrangement of the demand structure and the efficient and low-cost batch production. The chemical methods such as liquid phase reduction method, photochemical method, microemulsion method and hydrothermal method have good dispersion of the generated particles, simple operation and low requirements for the equipment, but it is difficult to effectively fix the particles on the detection substrate and stabilize them in the detection system. In addition, the particles generated by the existing physical and chemical methods are easy to agglomerate, which affects the matching effect between the to-be-detected fluorescent molecules and the electromagnetic field enhancement region, and leads to the difficulty in repeating and quantifying the detection, which is difficult to meet the application requirements. SUMMARY

[0005] The present application aims to at least solve one of the above technical problems in the prior art. To this end, the present application aims to provide a multi-network gel composite film fluorescence enhancement substrate and a preparation method and application thereof.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present application is:

[0007] In a first aspect of the present application, a fluorescence enhancement substrate is provided, comprising: a multi-gel network-noble metal particle composite film, the multi-gel network-noble metal particle composite film comprising:

[0008] an adhesive first gel network formed by cross-linking of an adhesive polymer;

[0009] a reducing second gel network formed by cross-linking of a reducing natural high molecular;

[0010] a third gel network containing noble metal particles formed by in-situ growth of noble metal particles in the adhesive first gel network;

[0011] a fourth gel network containing noble metal particles formed by in-situ growth of noble metal particles in the reducing second gel network.

[0012] In the present application, the fluorescence-enhancing noble metal particles are grown in-situ in the multi-network gel with adhesion and reduction, which not only facilitates the regulation of the size / distribution / gap / density of the noble metal particles, avoids the aggregation of the noble metal particles, and better binds with the substrate, but also enhances the fluorescence intensity of biological fluorescent molecules, has certain target enrichment characteristics, and prevents the formation of coffee rings by uneven diffusion of the sample solution, thereby improving the sensitivity of the detection while avoiding the detection deviation caused by coffee rings.

[0013] In some embodiments of the present application, in the multi-gel network-noble metal particle composite film, the mass ratio of the noble metal particles, the adhesive polymer and the reducing natural high molecular is 0.15-7:10-60:0.5-4; such as 0.15-5.5:10-60:0.5-4.

[0014] In some embodiments of the present application, the reducing second gel network is formed by cross-linking of the reducing natural high molecular and a second cross-linking agent; the second cross-linking agent comprises at least one of a metal ion salt, a polyphenol cross-linking agent, and an aldehyde cross-linking agent, such as at least one of calcium chloride, magnesium chloride, aluminum chloride, iron chloride, sodium hydroxide, potassium hydroxide, calcium hydroxide, dopamine, tannic acid, genipin, glutaraldehyde, and acetaldehyde; the mass ratio of the reducing natural high molecular and the second cross-linking agent is 0.1-10:0.5-4. The second cross-linking agent can strengthen the cross-linking strength of the reducing second gel network and occupy the ion sites vacated in the cross-linked network after the reduction of the noble metal ions to particles.

[0015] In some embodiments of the present application, the adhesive polymer comprises at least one of polyacrylamide, fibrin, cyanoacrylate, chitosan, natural polysaccharide, and polyurethane. Such polymer can provide better adhesion and has strong adsorption properties to substrates, noble metal particles, and biomolecules.

[0016] In some embodiments of the present application, the reducing natural macromolecule comprises at least one of hyaluronic acid (HA), methacrylated gelatin (GelMa), sodium alginate (SA), natural polysaccharide, and chitosan. Such natural macromolecule has strong reducing property and can form a complex with noble metal cations, which can be reduced to noble metal particles.

[0017] In some embodiments of the present application, the noble metal particles comprise at least one of nano-gold, nano-silver, nano-copper, and nano-platinum. The average particle size of the noble metal particles is 20-200 nm. The ultraviolet absorption range of the noble metal particles is 300-600 nm.

[0018] In some embodiments of the present application, the thickness of the multi-gel network-noble metal particle composite film is 0.04 μm-1.0 mm, such as 0.5 μm-0.8 mm, 0.8 μm-0.6 mm, 1.0 μm-0.5 mm.

[0019] In some embodiments of the present application, the fluorescence-enhanced substrate further comprises a substrate layer, and the multi-gel network-noble metal particle composite film is attached to the substrate layer. The material of the substrate layer comprises any one of glass, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polystyrene (PS), and medical adhesive tape. The thickness of the substrate layer is 1.5-2.5 mm.

[0020] In some embodiments of the present application, the fluorescence-enhanced substrate further comprises a separation layer, and the separation layer is arranged on the multi-gel network-noble metal particle composite film. The separation layer is arranged on the multi-gel network-noble metal particle composite film by physical adsorption and / or chemical bonding. The separation layer comprises one layer, two layers, or multiple layers. The material of the separation layer comprises at least one of anionic polyelectrolyte, cationic polyelectrolyte, and semiconductor medium. The material of the separation layer comprises polyethyleneimine (PEI), polystyrene sulfonate (PSS), polydiallyldimethylammonium chloride (PDADMAC), sodium alginate (SA), hyaluronic acid (HA), and silicon dioxide. The thickness of the separation layer is 5-25 nm.

[0021] In a second aspect of the present application, a preparation method of the fluorescence-enhanced substrate is provided, which comprises the following steps:

[0022] The mixed solution of the adhesive polymer and the reducing natural macromolecule is cross-linked and solidified, and then is reacted in a solution containing noble metal ions to obtain the multi-gel network-noble metal particle composite film after photochemical reduction.

[0023] In some embodiments of the present application, the preparation method of the fluorescence enhancement substrate comprises the following steps: the mixed solution of the adhesive polymer and the reducing natural macromolecule is cross-linked and solidified, and then is reacted in a solution containing noble metal ions, and is cross-linked and encapsulated in an ionic salt solution after photochemical reduction to obtain the multi-gel network-noble metal particle composite film.

[0024] In some embodiments of the present application, the preparation method of the fluorescence enhancement substrate comprises the following steps: the mixed solution of the adhesive polymer and the reducing natural macromolecule is cross-linked and solidified on a substrate layer, and then is reacted in a solution containing noble metal ions, and is cross-linked and encapsulated in an ionic salt solution after photochemical reduction to obtain the fluorescence enhancement substrate.

[0025] In some embodiments of the present application, the mixed solution further comprises a first cross-linking agent and a photoinitiator, the first cross-linking agent comprises at least one of N-methylol acrylamide, N-hydroxy acrylamide, N,N-methylene bisacrylamide (Bis), and N,N'-methylene bisacrylamide (MBA), and the photoinitiator comprises at least one of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), 1-hydroxycyclohexyl phenyl ketone (UV-184), and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methyl benzophenone (I2959); the first cross-linking agent is cross-linked with the adhesive polymer to form an adhesive first gel network; the mass concentration ratio of the first cross-linking agent, the photoinitiator, and the adhesive polymer is 0.15-0.75:0.1-0.5:10-60; the cross-linking reaction is carried out at a temperature of 10-60°C for 10-120s.

[0026] In some embodiments of the present application, the mixed solution further comprises a second cross-linking agent, the second cross-linking agent comprises at least one of metal ion salts such as calcium chloride, magnesium chloride, aluminum chloride, and iron chloride; the second cross-linking agent is cross-linked with the reducing natural macromolecule to form a reducing second gel network; the mass concentration ratio of the second cross-linking agent and the reducing natural macromolecule is 0.1-10:0.5-4, such as 0.5-2:0.5-4; the cross-linking reaction is carried out at a temperature of 10-60°C for 60-600s.

[0027] In some embodiments of the present application, the mass concentration of the adhesive polymer in the mixed solution is 10%-60%, and the mass concentration of the reducing natural macromolecule is 0.15%-7%, such as 0.5%-4%.

[0028] In some embodiments of the present application, the mass concentration of noble metal ions in the solution containing noble metal ions is 0.5% to 2%.

[0029] In some embodiments of the present application, the mass concentration of the ionic salt solution is 0.1% to 10%, such as 0.1% to 5%.

[0030] In some embodiments of the present application, the photochemical reduction includes ultraviolet light irradiation curing, and the intensity of the ultraviolet light irradiation is 70 μW / cm 2 to 1200 mW / cm 2 , and the irradiation time is 10 s to 1000 s, such as 20 s to 800 s, 30 s to 600 s, 50 s, 80 s, 100 s, 120 s, 200 s, 300 s, 400 s, 500 s, etc.

[0031] In some embodiments of the present application, the preparation method of the fluorescence-enhanced substrate further comprises placing the multi-gel network-noble metal particle composite film in a solution of a material of the isolation layer, physically adsorbing and / or chemically cross-linking to form the isolation layer, and optionally placing in an ionic salt solution to cross-link and encapsulate, to obtain the fluorescence-enhanced substrate.

[0032] In a third aspect of the present application, a microfluidic fluorescence detection chip is provided, comprising the fluorescence-enhanced substrate and a structure cover.

[0033] In some embodiments of the present application, the fluorescence-enhanced substrate is bonded to the structure cover.

[0034] In some embodiments of the present application, the material of the structure cover comprises at least one of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), and polystyrene (PS).

[0035] In a fourth aspect of the present application, a target concentration detection method is provided, comprising using the microfluidic fluorescence detection chip for detection.

[0036] In some embodiments of the present application, the detection method comprises contacting the target to be detected with the fluorescence-enhanced substrate for detection.

[0037] In some embodiments of the present application, the detection method comprises detecting the fluorescence intensity, obtaining the fluorescence intensity, and determining the target concentration.

[0038] In a fifth aspect of the present application, the fluorescence-enhanced substrate is used for detecting the target concentration.

[0039] In some embodiments of the present application, the target comprises at least one of a small molecule, a nucleic acid, a protein, a cell.

[0040] In some embodiments of the present application, the detection method comprises at least one of an immunological sandwich method, an immunofluorescence quenching method, and an adsorption method.

[0041] The present application has the following advantages:

[0042] The present application grows fluorescent noble metal particles in situ in a multi-network gel with high adhesion and high reducibility, which not only facilitates the regulation of the size / distribution / gap / density of the noble metal particles, avoids the aggregation of the noble metal particles, and better binds to the substrate, but also has certain fluorescence enhancement characteristics, target enrichment characteristics, and prevents the formation of a coffee ring by uneven diffusion of the sample solution, thereby improving the sensitivity of the detection while avoiding the detection deviation caused by the coffee ring.

[0043] The preparation process of the fluorescent enhancement substrate of the present application is relatively simple, does not depend on expensive equipment and processes, and can simply and quickly form a multi-network gel-noble metal composite film with fluorescence enhancement characteristics on the substrate in batches, thereby providing an effective solution for the high-sensitivity, rapid, wide-linear, and standardized detection of extremely low concentration biomolecules. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 It is a physical map of the chip prepared in Example 4 of the present application.

[0045] Figure 2 It is the ultraviolet-visible light absorption spectrum of the fluorescent enhancement substrate prepared in Example 1 of the present application under different irradiation times.

[0046] Figure 3 It is the ultraviolet-visible light absorption spectrum of the fluorescent enhancement substrate prepared in Example 4 of the present application at different positions.

[0047] Figure 4 It is the ultraviolet-visible light absorption spectrum of the substrate prepared in Comparative Example 1 of the present application under different irradiation times.

[0048] Figure 5 It is a linear relationship diagram of the detection chip of the fluorescent enhancement substrate in Example 4 of the present application for CEA-Fitc detection.

[0049] Figure 6 It is the detection of the fluorescent intensity of the fluorescent enhancement substrate in Example 4 of the present application at the center position and the edge position.

[0050] Figure 7 It is the enhancement effect of the liquid system in Comparative Example 4 of the present application. DETAILED DESCRIPTION

[0051] The application will be further described in detail by specific examples. The raw materials, reagents or devices used in the examples and comparative examples are commercially available or can be obtained by prior art methods unless otherwise specified. The test or test method is the conventional method in the art unless otherwise specified.

[0052] Example 1

[0053] In this example, a solid-state multi-network gel composite film fluorescent enhancement substrate was prepared by the following process:

[0054] A mixture of 0.5 mL of sodium alginate (1 wt%), acrylamide (30 wt%), BIS (0.15 wt%), and LAP (1 wt%) was uniformly coated on a circular glass sheet (55 mm in diameter; 1.1 mm thick). After cross-linking under UV light (405 nm, 1200 mW / cm 2 ) for 10 s, the sample was immersed in 10 mL of silver nitrate solution (0.5 wt%) for 5 min to form a silver alginate complex gel. Then, the sample was irradiated under UV light (405 nm, 1200 mW / cm 2 ) for 30 s, 120 s, 300 s, and 600 s, respectively. After washing with deionized water, the sample was cross-linked with 10 mL of 1 wt% calcium chloride for 5 min, washed with deionized water, and then dried in a 55°C oven to obtain a fluorescent enhancement substrate with a thickness of 1.16 mm. The thickness of the solid-state multi-network gel composite film fluorescent enhancement film was 60 μm.

[0055] Example 2

[0056] In this example, a solid-state multi-network gel composite film fluorescent enhancement substrate film was prepared by the following process:

[0057] A mixture of 0.8 mL of sodium alginate (1 wt%), acrylamide (30 wt%), BIS (0.15 wt%), and LAP (1 wt%) was uniformly coated on a circular glass sheet (55 mm in diameter; 1.1 mm thick). After cross-linking under UV light (405 nm, 1200 mW / cm 2 ) for 10 s, the sample was immersed in 10 mL of silver nitrate solution (0.5 wt%) for 5 min to form a multi-network gel containing noble metal ions. Then, the sample was irradiated under UV light (405 nm, 1200 mW / cm 2 ) for 300 s. After washing with deionized water, the sample was cross-linked with 10 mL of 1 wt% calcium chloride for 5 min, washed with deionized water, and then dried in a 55°C oven to obtain a fluorescent enhancement substrate with a thickness of 1.20 mm. The thickness of the solid-state multi-network gel composite film fluorescent enhancement film was 100 μm.

[0058] Example 3

[0059] A transparent thin sheet-like multi-network gel composite film fluorescent enhancement substrate was prepared in this example, and the specific process was as follows:

[0060] 0.5 mL of a mixed solution of sodium alginate (1 wt%), acrylamide (60 wt%), BIS (0.25 wt%), and LAP (2 wt%) was uniformly coated on a circular glass sheet (55 mm in diameter; 1.1 mm thick), and after being fixed by cross-linking reaction under UV light (405 nm, 1200 mW / cm 2 ) for 10 s, it was immersed in 10 mL of a silver nitrate solution (0.5 wt%) for 5 min to form a multi-network gel containing noble metal ions, and then irradiated under UV light (405 nm, 1200 mW / cm 2 ) for 300 s, washed with deionized water, cross-linked with 10 mL of a 1 wt% calcium chloride solution for 5 min, washed with deionized water again, and then dried in a 55°C oven to obtain a fluorescent enhancement substrate, thereby forming a transparent thin sheet-like multi-network gel composite film fluorescent enhancement film with a thickness of about 160 μm.

[0061] Comparative Example 1

[0062] A solid calcium alginate film substrate was prepared in this comparative example, and the specific process was as follows:

[0063] 0.5 mL of a sodium alginate solution (1 wt%) was uniformly coated on a circular glass sheet (55 mm in diameter; 1.1 mm thick), and then immersed in 10 mL of a silver nitrate solution (0.5 wt%) to form a silver alginate complex gel, and then irradiated under UV light (405 nm, 1200 mW / cm 2 ) for 30 s, 120 s, 300 s, and 600 s, washed with deionized water, cross-linked with 10 mL of a 1 wt% calcium chloride solution for 5 min, washed with deionized water again, and then dried in a 55°C oven to obtain a solid calcium alginate film fluorescent enhancement substrate.

[0064] Comparative Example 2

[0065] A polyacrylamide substrate was prepared in this comparative example, and the specific process was as follows:

[0066] 0.5 mL of a mixture of acrylamide (30 wt%), BIS (0.15 wt%), and LAP (1 wt%) was coated on a circular glass sheet (55 mm in diameter; 1.1 mm thick), but it was found that the mixed solution had a low viscosity and could not be uniformly coated on the glass.

[0067] Example 4

[0068] A chip was prepared in this example, and the specific process was as follows:

[0069] S1: Prepare a PDMS solution at a ratio of 1:100, homogenize and stir to remove air bubbles under vacuum, pour into a porous mold (2 inches), bake in a 75°C oven, and cut to obtain a 2-inch circular multi-well structure cover plate (each cover plate has 8 test holes, each test hole has a diameter of 2.54 mm, which is the same as the diameter and spacing of the 96-well plate).

[0070] S2: The fluorescence-enhancing substrate and the multi-test-hole structure cover sheet prepared by UV irradiation for 300s in Example 1 are bonded together by plasma treatment and then placed in a 75°C oven for 2 hours to obtain a chip.

[0071] chip physical image as shown Figure 1 As shown.

[0072] Test case

[0073] This experimental example tests the ultraviolet absorption intensity of the substrate. The specific procedure is as follows:

[0074] The UV absorption spectra of the substrates in Example 1 and Comparative Example 1 were measured using a microplate reader in the range of 350 nm to 800 nm. The results are as follows: Figure 2 , Figure 3 , Figure 4 As shown.

[0075] from Figure 2 It can be seen that as the UV treatment time increases, the absorption peaks of the substrate in Example 1 are at 390nm, 430nm, 470nm and 500nm respectively, and the absorbance peaks gradually increase, indicating that the size of noble metal nanoparticles on the fluorescence-enhanced substrate of the multi-network gel composite film is adjustable.

[0076] As the volume of the mixed solution coated on the circular glass slide increases, the thickness of the substrate film in Example 2 is 100 μm after 300 s of UV treatment, which is greater than the thickness of the substrate film in Example 1 (60 μm). The thickness of the fluorescence-enhancing substrate of the multi-network gel composite film is adjustable.

[0077] from Figure 3 It can be seen that different locations on the substrate were tested (refer to...) Figure 1 (Sampling at positions 1, 2, 3, 4, and 5 in the sample), after UV treatment for 300s, the absorption peak of the substrate in Example 1 was around 480nm, and the absorbance peak value was around 1.75, which was higher than that in Example 1. The density and distribution of noble metal nanoparticles on the fluorescence-enhanced substrate of the multi-network gel composite film were adjustable.

[0078] from Figure 4It can be seen that with the increase of UV treatment time, the absorption peaks of Comparative Example 1 substrate are at 416 nm, 430 nm, 455 nm and 460 nm respectively, the size of silver nanoparticles generated is limited, and the absorbance peak gradually increases, but the absorbance peak is much lower than that of Example 1 and Example 3.

[0079] Example 5

[0080] In this example, the chip prepared in Example 4 was used to test FITC-labeled carcinoembryonic antigen and FITC-labeled neuron-specific enolase (NES-FITC), and the specific process was as follows:

[0081] 60 μL of 0-500 ng / mL of different concentrations of fluorescent molecule FITC-labeled lung cancer tumor marker carcinoembryonic antigen (CEA-FITC) solution (0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 500 ng / mL) was added to the test hole of the test chip, and the linear relationship between the target molecule on the substrate and the fluorescence intensity was obtained by the enzyme marker under 465 nm excitation light and 535 nm emission light, and the results are shown in Figure 5

[0082] 60 μL of 125 ng / mL of fluorescent molecule FITC-labeled lung cancer tumor marker neuron-specific enolase (NES-FITC) solution was added to the test hole of the test chip, and the center point test and the edge four-point test were selected by the enzyme marker under 465 nm excitation light and 535 nm emission light, and the fluorescence intensity of NES-FITC at the center position and the edge position on the same test hole of the substrate was obtained, and the results are shown in Figure 6

[0083] From Figure 5 It can be seen that CEA-FITC shows a good linear relationship on the substrate at 0-500 ng / mL, and the linearity is also good in the low concentration range of CEA-FITC, R 2 = 0.998, and the detection limit is less than 0.78 ng / mL.

[0084] From Figure 6 It can be seen that the fluorescence intensity of 125 ng / mL NES-FITC at the center position and the edge position on the substrate is small, and the constructed substrate can avoid the detection deviation caused by the coffee ring.

[0085] Comparative Example 3

[0086] ​​The 60 μL of 125 ng / mL concentration fluorescent molecule FITC-labeled lung cancer tumor marker carcinoembryonic antigen (CEA-FITC) solution and 60 μL of PBS were added into the test holes of the control test chip (multi-test hole structure cover sheet + no substrate glass) respectively, and the fluorescence intensity of the target molecules on the control test chip was obtained by the enzyme label instrument under 465 nm excitation light and 535 nm emission light.

[0087] The test found that after removing the background (60 μL of PBS fluorescence intensity), the fluorescence intensity of 125 ng / mL CEA-FITC on the substrate was much higher than that without the enhanced substrate, and the fluorescence intensity of CEA-FITC on the substrate was enhanced by 4.2 times, and the multi-network gel composite film fluorescence enhancement substrate had good fluorescence enhancement characteristics.

[0088] Comparative Example 4

[0089] The enhancement effect of the liquid system was tested in the present comparative example, and the specific process was as follows:

[0090] The 0.1 mg / mL of nano-silver with ultraviolet absorption of about 480 nm was uniformly mixed with 2 wt% of sodium alginate solution, and then the CEA-FITC solution was added to prepare a 0-500 ng / mL CEA-FITC gradient concentration solution containing 0.05 mg / mL nano-silver and 1% sodium alginate (0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 500 ng / mL). Then 100 μL of 0-500 ng / mL CEA-FITC solution with different concentrations was taken out in a 96-well plate, and the fluorescence intensity of the solution was tested by the enzyme label instrument under 465 nm excitation light and 535 nm emission light, and the linear relationship between the target molecule concentration and the fluorescence intensity was analyzed, and the results are shown in Figure 7

[0091] It can be seen from Figure 7 that the CEA-FITC is not linear at a low concentration of 0-50 ng / mL.

[0092] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.​

Claims

1. A fluorescence enhancing substrate, characterized by: The application relates to a multi-gel network-noble metal particle composite film, and a preparation method of a fluorescence enhancement substrate. The fluorescence enhancement substrate further comprises an isolation layer arranged on the multi-gel network-noble metal particle composite film.

2. The fluorescence enhancing substrate of claim 1, wherein: The fluorescence enhancement substrate is prepared by the following steps: placing a mixed solution of sodium alginate, acrylamide, N,N-methylene bisacrylamide and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate salt on a substrate layer for cross-linking and solidification, then placing the substrate layer in a solution containing silver ions for reaction, and then placing the substrate layer in a calcium chloride solution for cross-linking and packaging after photochemical reduction.

3. The method of making a fluorescence enhancing substrate of claim 1 comprising the steps of:

4. A microfluidic fluorescence detection chip comprising the fluorescence enhancement substrate according to any one of claims 1-2 and a structure cover.

5. Application of the fluorescence enhancement substrate according to any one of claims 1-2 in detecting target concentration. ​

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