Micro-fluidic chip for enhancing nucleic acid fluorescence detection by utilizing metal surface plasma resonance effect
The fluorescence signal is enhanced through the structure of Ag@Au@SiO2 nanoparticles, which solves the problem of weak signal in microfluidic detection, and achieves high-sensitivity bacterial nucleic acid detection, which is suitable for clinical diagnosis, environmental monitoring and food safety.
Patent Information
- Application Number
- CN202510519801.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-24
- Publication Date
- 2025-08-01
AI Technical Summary
In the prior art, microfluidic detection methods based on fluorescent signals have weak signals when detecting trace targets, and precious metal nanomaterials have problems such as biotoxicity or low fluorescence intensity, which is difficult to meet the needs of high-throughput detection of bacterial nucleic acids.
Ag@Au@SiO2 nanoparticles were used to adjust the plasmon resonance effect of gold and silver surfaces, and Ag@Au@SiO2 nanoparticles with positive triangular cone were prepared. A microfluidic chip was prepared by combining slides and PDMS, and the fluorescence signal was enhanced by the metal surface plasmon resonance effect.
It significantly enhances the fluorescence intensity, improves detection sensitivity, and achieves efficient bacterial detection at extremely low concentrations, without the need for expensive equipment and complex operations, and is suitable for a variety of nucleic acid detection.
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Figure CN120394859A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of nucleic acid detection, and particularly to a microfluidic chip for enhancing nucleic acid fluorescence detection by using the surface plasmon resonance effect of a metal surface. Background Art
[0002] Currently, the main methods for detecting bacteria are culture methods (plate counting method), protein detection methods (enzyme-linked immunosorbent assay), and nucleic acid detection methods (polymerase chain reaction). The plate counting method is a reliable bacterial detection technology, but this method of culturing colonies is time-consuming and not conducive to rapid diagnosis; the enzyme-linked immunosorbent assay (ELISA) uses the interaction between antibodies and antigens for detection. This method has a fast detection speed, but there is a problem that it is difficult to accurately trace the types of infected bacteria, and the detection specificity is poor; compared with the above detection methods, nucleic acid detection has higher accuracy, and the detection sensitivity is significantly improved by amplification. The polymerase chain reaction is the most widely used nucleic acid detection technology at present, which has the advantages of high sensitivity and specificity, but there are problems such as the need for special equipment and professional operators. Recombinase polymerase amplification (RPA) is a technology for rapidly amplifying DNA or RNA at room temperature, which has the advantages of rapidity, high sensitivity, simple operation, and no need for a thermal cycler, and is widely used in disease detection and environmental monitoring.
[0003] To achieve accurate, sensitive, and rapid diagnosis of bacteria, biosensors based on various signals have been developed. Among them, the detection method based on fluorescence signals has been widely studied due to its advantages such as high sensitivity and rapidity. The microfluidic technology that has emerged in recent years can precisely regulate tiny fluids, improving the experimental efficiency and accuracy, and is also widely used in the biological field. The microfluidic detection technology based on fluorescence signals is currently an important research direction in biosensing and is expected to play an important role in the early diagnosis of bacterial infections and the monitoring of disease progression. Although this detection method has the advantages of high sensitivity and high throughput, there is still a problem of weak signals when detecting trace targets. In view of this, enhancing the intensity of fluorescence signals is a feasible solution to solve this problem. To improve the signal intensity of fluorescence detection, some noble metal nanomaterials have been used to enhance fluorescence signals. For example, Jeong-Woo Choi et al. first developed a nucleic acid fluorescence biosensor based on CRISPR-Cas12a, using DNA-functionalized gold nanoparticles to enhance fluorescence through the surface plasmon resonance (SPR) effect of the metal to detect cfDNA. The method of enhancing the fluorescence signal intensity based on the SPR effect of metal nanomaterials under light excitation is becoming the research forefront of biosensors for detecting trace targets. "Research on the Preparation of Noble Metal@SiO2 Core-Shell Structure Nanoparticles and Their Enhancement of Fluorescence and SERS" (Bian Xiaoying, master's thesis, 2022) discloses a kind of nanoparticles with an Ag / Au alloy as the core and SiO2 as the shell for dual-mode detection of metal-enhanced fluorescence and surface-enhanced Raman scattering. However, Ag is unstable in the gold-silver alloy. If there is no surface-enhanced Raman scattering and only metal-enhanced fluorescence is used to improve the fluorescence signal, it is difficult to meet the requirements of microfluidic chips. The patent with the application number 102764677A discloses a preparation method of a local surface plasmon resonance microfluidic chip, using gold nanorods as LSPR sensing elements for detecting various complex biological samples; but gold has biological toxicity and antibacterial properties, so it cannot be used for bacterial detection. Therefore, how to use only the surface plasmon resonance effect of the metal to achieve metal-enhanced fluorescence, meet the microfluidic chip for high-throughput detection of bacterial nucleic acids, and significantly improve the performance of bacterial detection is a problem to be solved. Summary of the Invention
[0004] In view of the above-mentioned prior art, the object of the present invention is to provide a microfluidic chip that uses the surface plasmon resonance effect of the metal to enhance nucleic acid fluorescence detection. The Ag@Au@SiO2 of the present invention has Ag as the core, Au as the sub-outer layer shell, and SiO2 as the outermost layer shell; the surface plasmon resonance effects of gold and silver in this structure enhance each other. And the Ag@Au@SiO2 is in the shape of a regular triangular pyramid and has a tip hot spot, which can generate a stronger signal. Therefore, the microfluidic chip of the present invention can be used at extremely low concentrations and still has fluorescence intensity, improving the detection sensitivity.
[0005] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect of the present invention, there is provided the use of Ag@Au@SiO2 in improving the detection sensitivity of a microfluidic chip. The structure of the Ag@Au@SiO2 is as follows: with Ag as the core, Au as the second outer shell, and SiO2 as the outermost shell; the shape of the Ag@Au@SiO2 is a regular triangular pyramid; the Ag@Au@SiO2 is prepared by the following method: (1) Add HAuCl4•4H2O solution, NaOH solution and Na2SO3 solution to ultrapure water to prepare an Au growth solution; add AgNO3 solution, trisodium citrate solution and hydrogen peroxide to ultrapure water, and then add NaBH4 solution under rapid stirring to obtain an Ag seed solution; mix ultrapure water, polyvinylpyrrolidone solution, ascorbic acid solution, NaOH solution, Na2SO3 solution and the Au growth solution, and then add the Ag seed solution, heat and react, and after centrifugal washing, obtain a precipitate Ag@Au; (2) Disperse Ag@Au in ultrapure water, adjust the pH to 9.5, add TEOS for reaction, and after centrifugal washing, obtain Ag@Au@SiO2.
[0006] Preferably, the thickness of Au in the Ag@Au@SiO2 is 5 - 10 nm, and the thickness of SiO2 is 8 - 20 nm; the height of the regular triangular pyramid structure is 40 - 60 nm.
[0007] Preferably, in step (1), the volume ratio of the AgNO3 solution, trisodium citrate solution, hydrogen peroxide, ultrapure water and NaBH4 solution is 0.2:12:0.48:187:1.2; the concentration of the AgNO3 solution is 0.1 M; the concentration of the trisodium citrate solution is 75 mM; the mass concentration of the hydrogen peroxide is 30%; the concentration of the NaBH4 solution is 0.1 M; the rotation speed of the rapid stirring is 2000 rpm, and the time is 30 min.
[0008] Preferably, in step (1), the molar ratio of the gold ions contained in the Au growth solution to the silver ions contained in the Ag seed solution is 1:2.
[0009] In the second aspect of the present invention, there is provided a microfluidic chip for enhancing nucleic acid fluorescence detection by using the surface plasmon resonance effect of a metal. The microfluidic chip is prepared by the following method: (1) Treat the glass slide with a silane coupling agent to obtain an amino-modified glass slide; (2) Modify Ag@Au@SiO2 with a silane coupling agent, and then react with a polycarboxylic acid to obtain Ag@Au@SiO2 with carboxyl groups and amide bonds; (3) Attach the PDMS with a flow channel to the amino-modified glass slide. Drop the buffer solution containing Ag@Au@SiO2 with carboxyl and amide bonds into the injection port on the PDMS and fill the flow channel on the PDMS. Let it react at low temperature and stand still, so that it is connected to the amino-modified glass slide through amide bonds. Then drop the buffer solution containing the nucleic acid probe molecular chain modified with amino groups into the injection port again and fill the flow channel on the PDMS. Let it react at low temperature and stand still, so that the nucleic acid probe molecular chain modified with amino groups is connected to Ag@Au@SiO2 with carboxyl groups on the surface through amide bonds. Remove the PDMS to obtain the microfluidic chip.
[0010] Preferably, in steps (1) and (2), the silane coupling agent is 3-aminopropyltriethoxysilane.
[0011] Preferably, in step (2), the polycarboxylic acid is glutaric anhydride.
[0012] Preferably, in step (3), the temperature of the low-temperature standing reaction is 4 °C and the time is 12 h.
[0013] In the third aspect of the present invention, there is provided the use of the microfluidic chip in improving the sensitivity of bacterial detection, and the concentration of the bacteria is at the ppm level.
[0014] In the fourth aspect of the present invention, there is provided a method for detecting bacteria using the microfluidic chip, and the method is as follows: Attach the PDMS with several sample addition holes to the above-mentioned microfluidic chip. Drop the solution containing the bacteria to be detected into the sample addition holes. After incubation, drop the solution of the nucleic acid reporter molecular chain labeled with fluorescent molecules into the sample addition holes. After incubation, remove the PDMS, and perform fluorescence detection on the microfluidic chip using a microarray chip scanner.
[0015] The beneficial effects of the present invention are as follows: (1) The present invention utilizes the strong surface plasmon resonance effect of Ag nanoparticles and the excellent stability of Au nanomaterials to adjust the coincidence of the ultraviolet absorption peak of the synthesized core-shell metal nanoparticles and the fluorescence excitation peak of the fluorescent molecule. When the excitation light irradiates the surface of the Ag / Au core-shell nanostructure, it can significantly enhance the excitation light field of the fluorescent molecules within a certain distance range nearby, resulting in a strong change in the reflection signal and enhancing the emission intensity of the fluorescent molecules, thereby improving the detection sensitivity.
[0016] (2) The present invention uses a microfluidic chip with sample addition holes containing multiple detection lines inside. This chip can precisely process trace amounts of liquid in channels with a width of micrometers and has the advantages of reagent micro-quantification, high sensitivity, and high throughput.
[0017] (3) The present invention combines a glass slide and PDMS to prepare a microfluidic chip, which does not require the use of expensive equipment and complex operation processes during the detection process, and has the advantages of low cost, easy operation, and large-scale preparation.
[0018] (4) The microfluidic chip of the present invention is applicable to the detection of various nucleic acids and has broad application prospects in many fields such as clinical diagnosis, environmental monitoring, and food safety. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 : Schematic diagram of the detection principle of the microfluidic chip in the specific embodiment; Figure 2 : TEM and EDS characterization diagrams of the Ag@Au@SiO2 nanoparticle structure in Example 1; Figure 3 : Ultraviolet absorption spectrum diagram of the Ag@Au nanoparticle solution and fluorescence emission spectrum diagram of Cy3 in Example 1; Figure 4 : Schematic diagram of the PDMS channel design in Example 2; Figure 5 : Schematic diagram of the detection of the microfluidic chip in Example 3; Figure 6 : Schematic diagram of the sample loading hole of the microfluidic chip in Example 3; Figure 7 : Comparison diagram of the fluorescence detection intensities of Example 3 and Comparative Examples 1 to 4; Figure 8 : Comparison diagram of the average difference in fluorescence signal intensities between Example 3 and Comparative Examples 1 to 4. SPECIFIC EMBODIMENTS <><<>
[0020] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.
[0021] As introduced in the background art section, whether it is the plasmon resonance effect of gold nanoions or gold-silver alloy nanoparticles to enhance the fluorescence intensity, there are problems such as biotoxicity or low fluorescence intensity.
[0022] Based on this, the objective of the present invention is to provide a microfluidic chip that utilizes the surface plasmon resonance effect of metals to enhance the fluorescence detection of nucleic acids. In the present invention, Ag@Au@SiO2 is used to prepare the detection line of the microfluidic chip, and the surface plasmon resonance effects of gold and silver in Ag@Au@SiO2 enhance each other. By adjusting the addition amount of hydrogen peroxide in the Ag solution, as well as the stirring speed and stirring time, etc., an Ag core with a regular triangular pyramid structure is obtained, and then an Au shell and an SiO2 shell are coated. By adding Na2SO3, the etching effect of gold on the inside of silver is avoided, and gold is grown on the edge of the silver surface to maintain the triangular pyramid structure of the Ag core, and Ag@Au@SiO2 with a regular triangular pyramid structure is obtained. Ag@Au@SiO2 has a tip hot spot, which can generate a stronger signal and further enhance the fluorescence intensity.
[0023] At the detection line of the glass slide, the amino amide bond connects a part of the carboxyl groups on the surface of Ag@Au@SiO2, and another part of the carboxyl groups of Ag@Au@SiO2 connects the nucleic acid probe molecular chain modified with amino groups. A gene sequence of a target nucleic acid molecular chain is complementarily connected to the nucleic acid probe molecular chain, and another gene sequence is complementarily connected to the nucleic acid reporter molecular chain labeled with fluorescence, so as to detect the target substance. The basic principle is as Figure 1 shown.
[0024] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in combination with specific embodiments.
[0025] Note: The PDMS containing channels and the PDMS containing several sample addition holes in the present invention are both prepared by the casting method. The specific method is as follows: (1) Use a photoresist with a protruding channel or sample addition hole as a template to prepare PDMS, heat the mold, and heat it at 120 °C for 30 min.
[0026] (2) Mix PDMS and RTV615 curing agent in a volume ratio of 15:1, stir evenly, and then place it in a vacuum drying oven to remove air bubbles by vacuum to obtain a mixed solution.
[0027] (3) Fix the silicon wafer on the mold fixture, pour the evacuated mixed solution into the mold, and heat the mold at 70 °C for 2 h until it is cured.
[0028] (4) Peel the cured product from the mold to obtain the required PDMS.
[0029] (5) The PDMS containing channels needs to be vertically punched at its inlet and outlet positions using a puncher.
[0030] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels.
[0031] Example 1: Synthesis of Ag@Au@SiO2 (1)Au growth solution: 40 μL of HAuCl4•4H2O (0.25 M) solution, 240 μL of NaOH solution (0.2 M), and 3 mL of Na2SO3 solution (0.01 M) were added to 4.72 mL of ultrapure water. The solution was left undisturbed overnight before use. (2)Ag seed solution: 200 μL of AgNO3 solution (0.1 M), 12 mL of trisodium citrate solution (75 mM), 480 μL of H2O2 solution (30 wt%) were added to 187 mL of ultrapure water. 1.2 mL of NaBH4 solution (0.1 M) was added under rapid stirring at 2000 rpm. After 30 min, the resulting solution was used as the stock solution of Ag seeds. The Ag seeds were centrifuged at 10000 rpm for 15 min from the stock solution, washed 3 times with ultrapure water, and then redispersed in 10 mL of ultrapure water to obtain the Ag seed solution.
[0032] (3)5.10 mL of ultrapure water, 2 mL of polyvinylpyrrolidone (PVP) solution (5 wt%, average molecular weight ~40000), 400 μL of ascorbic acid (AA) solution (500 mM), 1 mL of NaOH solution (0.2 M), 100 μL of Na2SO3 solution (0.1 M), and 8 mL of Au growth solution were placed in a 50 mL glass bottle and combined with 4 mL of Ag seed solution to initiate seed growth. The reaction was allowed to proceed at 60 °C for 1 h. Finally, it was centrifuged at 10000 rpm for 15 min and washed 3 times with water. Finally, all the water was aspirated out, leaving only the precipitate, which was Ag@Au nanoparticles.
[0033] (4)Under alkaline conditions, a SiO2 layer was coated on the surface of the Ag@Au core-shell nanostructure through the hydrolysis and condensation reaction of TEOS: Disperse 400 μL of the Ag@Au nanoparticle structure into 8 mL of ultrapure water, add 320 μL of NaOH solution to adjust the pH to about 9.5, add 80 μL of TEOS solution, react for 12 h, centrifuge at 10000 rpm for 15 min, wash 3 times with water and ethanol respectively, and centrifuge at 10000 rpm for 10 min each time to obtain Ag@Au@SiO2. Add Ag@Au@SiO2 to a 10 mL mixture of 3-aminopropyltriethoxysilane (APTES) and ethanol (volume ratio = 1:10) and react for 12 h. Modify the amino group on the SiO2 surface through the chemical bonding reaction of APTES, wash 3 times with ethanol solution, and centrifuge at 10000 rpm for 10 min each time. Finally, disperse it into 10 mL of glutaric anhydride ethanol solution with a concentration of 10 mg / mL, place it at 37 °C and react for 5 h. The glutaric anhydride undergoes an addition reaction with the modified amino group to modify the carboxyl group, and obtain Ag@Au@SiO2 with carboxyl and amide bonds.
[0034] Example 1 synthesized nano-Ag triangular pyramids by the seed-mediated method. Then, in the presence of SO3 2- , by complexing tetrachloroaurate with sulfite to prevent the oxidative etching of the Ag core, a triangular pyramid with an Ag core and an Au shell was synthesized, and then coated with a SiO2 structure. The morphology is as Figure 2 shown. The thickness of Au in the synthesized Ag@Au@SiO2 is 8 nm, and the thickness of SiO2 is 10 nm; the height of the regular triangular pyramid structure is 50 nm. And the ultraviolet absorption peak of Ag@Au@SiO2 was adjusted to coincide with the excitation peak of Cy3 fluorescent molecules, as Figure 3 shown.
[0035] Example 2: Preparation of a microfluidic chip S01. Slide treatment (1) A common glass slide with a length of 76 mm, a width of 25 mm, and a thickness of 1 mm is used as the substrate. Ultrasonically clean it with water, isopropanol, ethanol, and water in turn for 5 min. Prepare a piranha solution by mixing concentrated sulfuric acid and 30% H2O2 solution in a ratio of 7:3. Add the cleaned and dried glass slide to the piranha solution and treat it at 95 °C for 10 min. (2) Washing and drying: Add the boiled glass slide to ultrapure water and ultrasonically wash it, then repeatedly wash it 3 times with ultrapure water and dry it at 60 °C.
[0036] (3) Treat the dried glass slide with oxygen plasma using a Diener Atto-BLS plasma cleaner for 10 min.
[0037] (4)Amination: Immerse the treated glass slides in a mixed solution of APTES and ethanol (volume ratio = 1:20) and soak for 12 h at room temperature to modify the amino groups on the glass slide surface.
[0038] (5)Take out the glass slides and wash them successively with ethanol and ultrapure water three times, and dry them at 60 °C for subsequent use.
[0039] S02. On-chip modification (1)Attach the PDMS with two non-connected meandering channels tightly to the glass slide substrate. There are two inlets and outlets with meandering channels at the leftmost and rightmost sides of the PDMS. Mark the positioning points on the back of the glass slide substrate with a marker pen. The width of each meandering channel is 100 µm and the height is 20 µm, as Figure 4 shown.
[0040] (2)Mix 20 μL of the carboxyl- and amide bond-containing Ag@Au@SiO2 prepared in Example 1, 4 μL of 10 mg / mL EDC solution, 4 μL of 10 mg / mL NHS solution, and 12 μL of MES buffer with pH = 6 and a concentration of 0.5 M evenly. Then take 5 μL of each and add them to the two inlets of the PDMS respectively, so as to activate the carboxyl groups on the surface of Ag@Au@SiO2 to connect with the amino groups on the glass slide substrate through amide bonds. Use a vacuum pump to draw the solution at the inlet into the two meandering channels for 1 h. After stopping the vacuum pump, let it stand at 4 °C for 12 h.
[0041] (3)Mix 5 μL of the nucleic acid probe molecular chain of amino-modified Staphylococcus aureus (its DNA sequence is shown in SEQ ID NO:1), 5 μL of the nucleic acid probe molecular chain of amino-modified Klebsiella pneumoniae (its DNA sequence is shown in SEQ ID NO:4), 1 μL of 10 mg / mL EDC solution, 1 μL of 10 mg / mL NHS solution, and 3 μL of MES buffer with pH 6 and 0.5 M evenly, and then add them to the two inlets of the PDMS, so as to activate the carboxyl groups on the surface of Ag@Au@SiO2 to connect with the amino-modified nucleic acid probe molecular chains. Use a vacuum pump to draw the solution at the inlet into the two meandering channels for 1 h. After stopping the vacuum pump, let it stand at 4 °C for 12 h. The number of channels can be set according to the types of samples to be detected. For example, when simultaneously detecting Staphylococcus aureus and Klebsiella pneumoniae, add the nucleic acid probe molecular chain of amino-modified Staphylococcus aureus or the nucleic acid probe molecular chain of amino-modified Klebsiella pneumoniae to different channels respectively, so that the finally prepared microfluidic chip can simultaneously detect these two bacteria.
[0042] (4)Remove the remaining nucleic acid probe molecular chain solution from the sample inlet and outlet, remove the PDMS in 3% BSA / PBS solution and let it stand for 30 min to block non-specific binding sites. Wash the glass slide substrate successively with undiluted PBS solution, 1-fold diluted PBS solution, and twice with ultrapure water, and centrifuge to dry to obtain the microfluidic chip.
[0043] Example 3: Detection using the microfluidic chip prepared in Example 2 (1)Attach the PDMS with several sample addition holes to the front of the microfluidic chip prepared in Example 2, and note that the positioning holes coincide with the marked positioning points. The PDMS with several sample addition holes contains 5 rows and 12 columns of 2.5×2.5×0.5 mm sample addition holes, and there are four positioning holes around it. Before the two are attached, ensure that the front of the glass slide substrate is dust-free. Attach the PDMS with several sample addition holes tightly to the glass slide substrate without bubbles to avoid liquid leakage, as Figure 5 shown.
[0044] Add 4 µL of a solution of Staphylococcus aureus DNA to be detected (NCBI Reference Sequence: NZ_LFUU01000018.1) with a concentration of 10 pM (10 -11 M) and a solution of Klebsiella pneumoniae DNA to be detected (NCBI Reference Sequence: NZ_JBMHLP010000030.1) to the sample addition holes respectively. The gene sequence of a nucleic acid target molecular chain contained in Staphylococcus aureus (as shown in SEQ ID NO: 2) is complementary to its nucleic acid probe molecular chain (as shown in SEQ ID NO: 1), and the gene sequence of a nucleic acid target molecular chain contained in Klebsiella pneumoniae (as shown in SEQ ID NO: 5) is complementary to its nucleic acid probe molecular chain (as shown in SEQ ID NO: 4). Each sample is in parallel 2 times, incubated for 30 min to capture the nucleic acid target molecular chain onto the nucleic acid probe molecular chain, and then the sample addition holes are washed 3 times with 3 wt% BSA / PBS solution. (2)Add 4 µL of a solution of a Staphylococcus aureus reporter molecular chain labeled with Cy3 fluorescent molecules (its DNA sequence is as shown in SEQ ID NO: 3) with a concentration of 0.1 µM and a solution of a Klebsiella pneumoniae reporter molecular chain labeled with Cy3 fluorescent molecules (its DNA sequence is as shown in SEQ ID NO: 6) to the corresponding nucleic acid sample addition holes respectively. This molecular chain can be complementary to another gene sequence of the nucleic acid target molecular chain, so as to achieve the detection of a specific sequence. Incubate for 30 min to connect the fluorescently labeled reporter molecular chain to the nucleic acid target molecular chain, and wash the sample addition holes 3 times with 3 wt% BSA / PBS solution.
[0045] (3) Peel off the PDMS containing several sample wells. On the glass slide substrate corresponding to each sample well, there are 2 sets of detection lines, and each set of detection lines consists of 2 detection lines for different bacteria (Staphylococcus aureus and Klebsiella pneumoniae). The width of each detection line is 100 µm, as Figure 6 shown. Fluorescence detection was performed using a LuxScan 10K A microarray chip scanner.
[0046] The DNA sequences involved in the examples are shown in Table 1.
[0047] Table 1 DNA sequences involved in the examples Comparative Example 1 (1) The carboxyl-modified glass slide substrate is directly connected to the amino-modified nucleic acid probe molecular chain. Different from Example 2, the glass slide substrate after APTES treatment is added to a 10 mg / mL glutaric anhydride ethanol solution and reacted at 37 °C for 12 h. Glutaric anhydride undergoes an addition reaction with the modified amino group to modify the carboxyl group, and the modified glass slide substrate is directly used to connect the amino-modified nucleic acid probe molecular chain. The subsequent operations are the same as steps (3) and (4) in Example 2 to prepare the microfluidic chip.
[0048] (2) Use the microfluidic chip prepared in step (1) to detect Staphylococcus aureus and Klebsiella pneumoniae according to the method of Example 3.
[0049] Comparative Example 2 (1) Prepare Au@SiO2 with a triangular pyramid structure: Add 1 mL of HAuCl4 solution (25 × 10 -3 M) to 50 mL of BDAC aqueous solution (5 × 10 -3 M). Then, use 100 μL of 3BA mild reducing agent and stir gently at 75 °C for 24 min. To remove the residual reactants, centrifuge the obtained product at 8000 rpm for 5 min to wash it. Disperse the obtained sample in 10 mL of CTAC aqueous solution (100 × 10 -3 M) and treat it for 10 h. Collect the supernatant and extract the obtained supernatant by centrifuging at 9000 rpm for 10 min, and redisperse it in 10 mL of CTAC aqueous solution (300 × 10 -3 M) and treat it for another 10 h. Collect the precipitate and purify it by centrifuging at 9000 rpm for further use.
[0050] Add 250 μL of APTES (1 × 10 -3 M) to 10 mL of the pre-prepared aqueous solution of Au triangular pyramid (containing 4 × 10-7 In 50 mL of chloroauric acid solution (0.5 mM, 100 mmol), 20 mL of sodium citrate solution (38.8 mM, 776 mmol) was added, and the mixture was vigorously stirred for 20 min. Then, 3 mL of Na2SiO3 (0.54 wt%, pH 8.0) was added to the above solution, and the mixture was reacted at 90 °C for 1 h. The reaction was stopped under ice bath conditions. The obtained Au@SiO2 solution was centrifuged at 10000 rpm for 5 min for purification. The final Au@SiO2 was dispersed in 1 mL of ultrapure water for further use.
[0051] (2) According to the method of Example 1, Au@SiO2 with carboxyl groups and amide bonds was prepared; according to the method of Example 2, the Ag@Au@SiO2 with carboxyl groups and amide bonds was replaced with an equal amount of Au@SiO2 with carboxyl groups and amide bonds. Finally, a microfluidic chip was prepared.
[0052] (3) Using the microfluidic chip prepared in step (2), according to the method of Example 3, Staphylococcus aureus and Klebsiella pneumoniae were detected.
[0053] Comparative Example 3 (1) Preparation of Ag@SiO2 with a triangular pyramid structure: To 248 mL of ultrapure water, 500 μL of AgNO3 solution (50 mM), 5 mL of trisodium citrate solution (75 mM), 1 mL of PVP solution (17.5 mM), and 600 μL of H2O2 solution (30 wt%) were added. Finally, 2.5 mL of aqueous NaBH4 solution (0.1 M) was rapidly injected into the solution. After the formation of a blue solution, Ag nanopyramids were collected by centrifugation at 10000 rmp for 10 min.
[0054] After centrifugation, the Ag nanopyramids were dispersed in 100 mL of ultrapure water, 1.5 mL of MHA ethanol solution (5 mM) was added and stirred for 10 min. The MHA-functionalized Ag nanopyramids were centrifuged at 10000 rpm for 10 min and redispersed in 20 mL of TEOS ethanol solution (16 mM). After stirring for 2 min, 2 mL of aqueous dimethylamine solution (20% w / v) was added and the solution was stirred for 12 h. The Ag@SiO2 nanopyramids were collected by centrifugation at 10000 rpm for 10 min.
[0055] (2) According to the method of Example 1, Ag@SiO2 with carboxyl groups on the surface was prepared; according to the method of Example 2, the Ag@Au@SiO2 with carboxyl groups was replaced with an equal amount of Ag@SiO2 with carboxyl groups. Finally, a microfluidic chip was prepared.
[0056] (3)Using the microfluidic chip prepared in step (2), detect Staphylococcus aureus and Klebsiella pneumoniae according to the method of Example 3.
[0057] Comparative Example 4 (1)Prepare Ag / Au alloy@SiO2 Add 38 mL of ultrapure water, 6 mL of ethanol and 0.07 g of cetyltrimethylammonium chloride (CTAC) into a 100 mL beaker in sequence. After ultrasonic dissolution, add 2 mL of ammonia water (2.5 wt%) and 1 mL of formaldehyde (3.7 wt%), and then add 0.5 mL of HAuCl4 (8.14 mM) solution and 0.51 mL of AgNO3 (24 mM) solution. Place the beaker in an oil bath at 80 °C and start stirring. After 20 min, add 100 μL of TEOS solution dropwise to the reaction solution. Continue heating and stirring for 40 min, and then cool to room temperature. Centrifuge at 10000 rpm for 20 min, and then wash once with water and ethanol as solvents respectively to obtain Ag / Au alloy@SiO2. Finally, disperse Ag / Au alloy@SiO2 in ethanol for standby.
[0058] (2)According to the method of Example 1, prepare Ag / Au alloy@SiO2 with carboxyl and amide bonds; according to the method of Example 2, replace Ag@Au@SiO2 with carboxyl and amide bonds with an equal amount of Ag / Au alloy@SiO2 with carboxyl and amide bonds. Finally, a microfluidic chip is prepared.
[0059] (3)Using the microfluidic chip prepared in step (2), detect Staphylococcus aureus and Klebsiella pneumoniae according to the method of Example 3.
[0060] Compare the fluorescence intensities obtained from the detection of Example 3 and Comparative Examples 1-4. As Figure 7 shown, it can be seen that in Example 3, strong fluorescence can still be detected at a concentration of 10 pM. The fluorescence intensities of Comparative Examples 1-4 are all lower than that of Example 3. According to Figure 8 the fluorescence intensities of each group can be directly observed, it can be seen that in the structure of the present invention with Ag as the core and Au as the secondary outer layer shell, the fluorescence intensity is much higher than the sum of Comparative Example 2 and Comparative Example 3, indicating that the surface plasmon resonance effects of gold and silver in the structure of the present invention enhance each other.
[0061] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. Application of Ag@Au@SiO2 in improving the detection sensitivity of a microfluidic chip, characterized in that, The structure of the Ag@Au@SiO2 is as follows: Ag serves as the core, Au serves as the secondary outer shell, and SiO2 serves as the outermost shell; the shape of the Ag@Au@SiO2 is a regular triangular pyramid; the Ag@Au@SiO2 is prepared by the following method: (1) Add HAuCl4•4H2O solution, NaOH solution, and Na2SO3 solution to ultrapure water to prepare an Au growth solution; add AgNO3 solution, trisodium citrate solution, and hydrogen peroxide to ultrapure water, and then add NaBH4 solution under rapid stirring to obtain an Ag seed solution; mix ultrapure water, polyvinylpyrrolidone solution, ascorbic acid solution, NaOH solution, Na2SO3 solution, and the Au growth solution, and then add the Ag seed solution, heat and react, and after centrifugal washing, obtain a precipitate of Ag@Au; (2) Disperse Ag@Au in ultrapure water, adjust the pH to 9.5, add tetraethyl orthosilicate for reaction, and after centrifugal washing, obtain Ag@Au@SiO2.
2. The application according to claim 1, wherein In the Ag@Au@SiO2, the thickness of Au is 5 - 10 nm, and the thickness of SiO2 is 8 - 20 nm; the height of the regular triangular pyramid structure is 40 - 60 nm.
3. The application according to claim 1, wherein In step (1), the volume ratio of the AgNO3 solution, trisodium citrate solution, hydrogen peroxide, ultrapure water, and NaBH4 solution is 0.2:12:0.48:187:1.2; the concentration of the AgNO3 solution is 0.1 M; the concentration of the trisodium citrate solution is 75 mM; the mass concentration of the hydrogen peroxide is 30%; the concentration of the NaBH4 solution is 0.1 M; the rotation speed of the rapid stirring is 2000 rpm, and the time is 30 min.
4. The application according to claim 1, characterized in that, In step (1), the molar ratio of the gold ions contained in the Au growth solution to the silver ions contained in the Ag seed solution is 1:
2.
5. A microfluidic chip for enhancing nucleic acid fluorescence detection by utilizing the surface plasmon resonance effect of metal, characterized in that, The microfluidic chip is prepared by the following method: (1) Treat the glass slide with a silane coupling agent to obtain an amino-modified glass slide; (2) Modify the Ag@Au@SiO2 according to any one of claims 1 - 4 with a silane coupling agent, and then react with a polycarboxylic acid to obtain an Ag@Au@SiO2 with carboxyl groups and amide bonds; (3) Attach the PDMS containing channels to the amino-modified glass slide, drop a buffer solution containing the Ag@Au@SiO2 with carboxyl groups and amide bonds at the injection port on the PDMS to fill the channels on the PDMS, perform a low-temperature static reaction, then drop a buffer solution containing nucleic acid probe molecular chains at the injection port on the PDMS to fill the channels on the PDMS, perform a low-temperature static reaction, and remove the PDMS to obtain the microfluidic chip.
6. The microfluidic chip according to claim 5, characterized in that, In steps (1) and (2), the silane coupling agent is 3-aminopropyltriethoxysilane.
7. The microfluidic chip according to claim 5, characterized in that, In step (2), the polycarboxylic acid is glutaric anhydride.
8. The microfluidic chip according to claim 5, wherein, In step (3), the temperature of the low-temperature static reaction is 4°C, and the time is 12 h.
9. Use of the microfluidic chip according to any one of claims 5 to 8 in improving the sensitivity of bacterial detection, characterized in that, The concentration of the bacteria is at the pM level.
10. A method for detecting bacteria using the microfluidic chip according to any one of claims 5 to 8, characterized in that, The method is as follows: Attach the PDMS with a number of sample addition holes to the microfluidic chip described in any one of claims 5 to 8. Drop a solution containing the bacteria to be tested into the sample addition holes. After incubation, drop a solution containing a reporter molecule chain labeled with a fluorescent molecule into the sample addition holes. After incubation, remove the PDMS and perform fluorescence detection on the microfluidic chip using a microarray chip scanner.