CRISPR (clustered regularly interspaced short palindromic repeats)-mediated liposome-based micro-fluidic chip sensor as well as preparation method and application thereof

Abstract

The invention belongs to the field of detection, and particularly relates to a micro-fluidic chip sensor based on CRISPR (clustered regularly interspaced short palindromic repeats) mediated liposome as well as a preparation method and application of the micro-fluidic chip sensor. The invention develops a method capable of sensitively detecting the ampicillin, which comprises the following steps: converting the concentration of the ampicillin into a DNA (Deoxyribonucleic Acid) signal, realizing primary signal amplification by utilizing a catalytic hairpin reaction, and then carrying out secondary signal amplification on a 4-MPBA-liposome marker by utilizing an Au-Ag noble metal substrate in combination with a surface enhanced Raman scattering technology. Therefore, the ultra-sensitive detection of the ampicillin is realized. Experimental results show that the sensor has a good linear relation within 1 fM to 1 nM, and the detection limit is as low as 0.74 fM. The method shows good stability, selectivity and sensitivity, realizes sensitive detection of ampicillin residues in environmental water and milk samples, provides a new technical means for environmental monitoring and food safety, and has important practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of detection, and specifically relates to a microfluidic chip sensor based on CRISPR-mediated liposomes, and a preparation method and application thereof. Background Art

[0002] In recent years, the overuse of antibiotics has led to the emergence and spread of bacterial resistance, posing a significant challenge to global public health. Among common antibiotics, ampicillin belongs to the aminopenicillin class, a β-lactam antibiotic primarily used to treat a variety of infections caused by susceptible bacteria, such as streptococcal infections, Escherichia coli infections, meningitis, and sepsis. However, excessive or inappropriate use of ampicillin not only harms soil and water environments but can also spread through the food chain, posing serious risks to human health and causing symptoms such as allergies and respiratory distress. The European Union sets a maximum residue limit (MRL) of 50 μg / kg in animal tissues and 4 μg / kg in milk.

[0003] Therefore, the development of a rapid, sensitive and portable ampicillin detection method is crucial for controlling its abuse and drug resistance detection. There are many methods for detecting ampicillin, such as high performance liquid chromatography (HPLC), spectrophotometry, fluorescence, electrochemical surface plasmon resonance, colorimetry, liquid chromatography-mass spectrometry, etc. Although these methods are accurate and reliable, they have disadvantages such as cumbersome operation, long time consumption, and the need for expensive instruments and professional technicians, making it difficult to meet the needs of rapid on-site detection. In recent years, detection methods based on nucleic acid amplification, such as polymerase chain reaction (PCR), have received widespread attention due to their high sensitivity and specificity. However, PCR technology has problems such as expensive equipment, complex operation, and easy contamination.

[0004] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a method for preparing a microfluidic chip sensor based on CRISPR-mediated liposomes, the preparation method comprising the following steps:

[0006] (1) cleaning and drying the silicon wafer to obtain a dry silicon wafer;

[0007] (2) spin-coating photoresist on the dry silicon wafer and laminating it with a mask plate, and then sequentially exposing, drying, and entering a developer to obtain a template;

[0008] (3) The template is dried to form a hard film, and then silanized, cured, and cleaned in sequence.

[0009] Preferably, in step (1), the cleaning method is: ultrasonic cleaning with anhydrous ethanol, acetone and ultrapure water in sequence; the drying temperature is 60-65°C;

[0010] In step (2), the exposure time is 55 to 65 seconds; the drying method is: first drying at 60 to 65° C. for 2 to 3 minutes and then drying at 90 to 95° C. for 12 to 15 minutes;

[0011] In step (3), the drying method is: drying at 90-95° C. for 2-2.5 hours; the silanization method is: silanization at 0.08-0.1 Pa for 2-3 hours.

[0012] Based on the same technical concept, another embodiment of the present invention is to provide a CRISPR-mediated liposome-based microfluidic chip sensor obtained by the above-mentioned preparation method.

[0013] Based on the same technical concept, another embodiment of the present invention provides a CRISPR-mediated liposome-based microfluidic chip sensor for use in detecting ampicillin. The detection method comprises the following steps:

[0014] (I) Preparation of Au@Ag nanoparticles

[0015] (I-1) Preparation of Au nanoparticles: HAuCl4 solution was mixed with water and heated to boiling. Trisodium citrate was then added. The mixture was stirred while boiling until the color of the solution changed from colorless to wine red. After completion, the mixture was cooled to obtain an Au nanoparticle solution.

[0016] (I-2) Preparation of Au@Ag nanoparticles: Au nanoparticle solution, Tollens reagent, HCHO, and water were mixed and stirred to react, thereby obtaining Au@Ag nanoparticles;

[0017] (II) Preparation of liposomes

[0018] Phosphatidylcholine, cholesterol and 4-mercaptophenylboronic acid were dispersed in ethanol solution respectively, and 4-mercaptophenylboronic acid-loaded liposomes were prepared by using a microfluidic device and setting the total flow rate and flow rate ratio.

[0019] (III) Microfluidic chip sensor grafted with ssDNA

[0020] The microfluidic chip sensor was treated with 3-aminopropyltrimethoxysilane to introduce amine groups, then washed and 20 μL of 5'-Biotin-3'NH2 ssDNA was added to the Raman detection chamber for reaction;

[0021] (IV) CRISPR / Cas12a-mediated ssDNA cleavage and signal output

[0022] The presence of ampicillin is converted into a DNA signal by triggering a catalytic hairpin reaction, and ssDNA is cut through the cleavage activity of the CRISPR / Cas12a system on the target DNA; at the same time, 4-MPBA is encapsulated in liposomes, and then the Raman signal is further amplified using the Au@Ag nanoparticles, and finally the product is detected.

[0023] Preferably, in step (I-1), the time of maintaining boiling and stirring is 30 to 40 minutes.

[0024] Preferably, in step (I-2), the reaction temperature is 20-25° C., and the reaction time is 10-15 min.

[0025] Preferably, in step (II), the total flow rate is 120 μL / min and the flow rate ratio is 1:11.

[0026] Preferably, in step (III), the treatment time is 25 to 30 minutes; the reaction temperature is 35 to 37° C., and the reaction time is 2 to 3 hours.

[0027] The beneficial effects of the present invention are:

[0028] The present invention explores a CRISPR / Cas12a amplification strategy based on a microfluidic chip that integrates liposome preparation and ampicillin separation, combined with SERS as a signal output, for rapid, sensitive, and specific detection of ampicillin. This strategy uses ampicillin-specific aptamers as recognition elements, converts the presence of ampicillin into a DNA signal by triggering the CHA reaction, and cuts ssDNA through the cutting activity of the CRISPR / Cas12a system on the target DNA. At the same time, the excellent encapsulation ability of liposomes is utilized to encapsulate a large amount of Raman reporter molecules 4-MPBA, which are evenly fixed in the Raman detection chamber, and then the Au@Ag noble metal substrate is used to further amplify the Raman signal. Subsequently, the SERS technology is used to detect its product, and 4-MPBA is detected at 1000 cm -1 , 1060cm -1 、1597cm -1 The characteristic peak is generated at the DNA concentration, which is converted back into a SERS signal, achieving highly sensitive and specific signal output. The integration of microfluidic chips can realize the integration of sample separation, nucleic acid amplification and signal detection, greatly improving detection efficiency and portability.

[0029] Experimental results demonstrate that the sensor exhibits excellent linearity within the range of 1 fM to 1 nM, with a detection limit as low as 0.74 fM (S / N = 3). Furthermore, the method demonstrates excellent stability, selectivity, and sensitivity, enabling sensitive detection of ampicillin residues in environmental water and milk samples. This provides a new technical approach for food safety and environmental monitoring, with significant practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 Schematic diagram of AMP detection using the CRISPR / Cas12a-mediated liposome amplification strategy based on a microfluidic device.

[0032] Figure 2 This is a diagram of the CRISPR / Cas12a system triggered by the catalytic hairpin self-assembly reaction (CHA).

[0033] Figure 3 A is a schematic diagram of liposome self-assembly.

[0034] Figure 3 B is the TEM image of blank liposomes.

[0035] Figure 3 C~ Figure 3 D is the TEM image of liposomes loaded with 4-MPBA.

[0036] Figure 3 E is the UV spectra of 4-MPBA, 4-MPBA@liposome and liposome.

[0037] Figure 3 F is the particle size diagram of blank liposomes and 4-MPBA loaded liposomes.

[0038] Figure 3 G is the TEM image of Au@Ag.

[0039] Figure 3 H is the HAADF-TEM image of Au@Ag.

[0040] Figure 3 I~ Figure 3 K is Au( Figure 3 I), Ag( Figure 3 J) TEM-EDS element curve and its superimposed image ( Figure 3K).

[0041] Figure 3 L is the SERS spectra of 4-MPBA, liposome@4-MPBA, and 4-MPBA@liposome+Au@Ag.

[0042] Figure 4 is the lattice spacing diagram of Au@Ag nanoparticles.

[0043] Figure 5 A is the XPS image of the unmodified PDMS chip.

[0044] Figure 5 B is the XPS image of the APTMS-modified PDMS chip.

[0045] Figure 5 C is the XPS image of single-stranded DNA immobilized on the APTMS-modified PDMS chip.

[0046] Figure 6 A is a schematic diagram of the chip preparation of liposomes.

[0047] Figure 6 B is the liposome particle size diagram at different flow rate ratios.

[0048] Figure 6 C is the Raman intensity graph at different 4-MPBA concentrations.

[0049] Figure 6 D is the Raman spectra at different 4-MPBA concentrations.

[0050] Figure 6 E is the Raman intensity diagram under different cholesterol ratio conditions.

[0051] Figure 6 F is the Raman spectrum under different cholesterol ratio conditions.

[0052] Figure 7 A is the Raman spectra of 4-MPBA at different locations on the chip.

[0053] Figure 7 B is the stability of 4-MPBA at different locations on the chip.

[0054] Figure 7 C is the Raman spectra of 4-MPBA recorded from chips stored for different days.

[0055] Figure 7 D is the chip recorded at 1597cm at different storage times -1 The Raman intensity at .

[0056] Figure 7E is the Raman spectra of 10 batches of chips.

[0057] Figure 7 F is the stability of 10 batches of chips.

[0058] Figure 8 A is the Raman spectrum of different logarithmic concentrations of ampicillin.

[0059] Figure 8 B is the linear relationship between Raman intensity and the logarithm of ampicillin concentration, and the error bars are obtained from three sets of data. DETAILED DESCRIPTION

[0060] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0061] Example 1

[0062] This embodiment provides a method for preparing a microfluidic chip sensor based on CRISPR-mediated liposomes, the preparation method comprising the following steps:

[0063] (1) The channel pattern was designed using AutoCAD software and then masked; the silicon wafer was ultrasonically cleaned with anhydrous ethanol, acetone, and ultrapure water in sequence, dried at 65°C, and naturally cooled for use to obtain a dry silicon wafer.

[0064] (2) After spin coating SU-82050 photoresist on the dry silicon wafer using a desktop coating machine (KW-4A), it was laminated with a mask plate, placed on a photolithography table for exposure for 1 minute, and then the mask plate was peeled off. The wafer was dried at 65°C for 2 minutes and then dried at 95°C for 15 minutes. The photolithographic silicon wafer was immersed in a developer for 15 seconds and rinsed with isopropyl alcohol until no white color was produced to obtain a template.

[0065] (3) The template was dried at 95°C for 2 hours to harden the film, and then the chip was placed in a drying dish. 20 μL of silanization reagent was transferred to the drying dish, and the vacuum was evacuated to 0.08 Pa and silanized for 2 hours. The cross-linking agent and curing agent were mixed in a ratio of 10:1 to obtain PDMS (chip material) glue, which was then poured on the template and dried at 55°C for 1 hour. After demolding, the chip was obtained, and the obtained channel was cut with a knife and bonded using a plasma cleaning machine to obtain a microfluidic chip.

[0066] Example 2

[0067] This embodiment provides a method for detecting ampicillin using a microfluidic chip sensor, the detection method comprising the following steps:

[0068] (I) Preparation of Au@Ag nanoparticles

[0069] (I-1) Preparation of Au nanoparticles: 50 μL of 10% HAuCl4 solution was added to a flask containing 50 mL of water and heated to boiling. Then, 4 mL of 1% (w / v) trisodium citrate was quickly added with rapid stirring. After a few minutes, the color of the solution changed from colorless to wine red. The solution was kept boiling and stirred for 30 minutes, cooled to room temperature, and the prepared Au nanoparticles (AuNPs) were stored in a refrigerator at 4°C until use.

[0070] (I-2) Preparation of Au@Ag Nanoparticles: First, prepare Tollens' reagent (6 mL AgNO₃ (0.1 M), 1.28 mL NH₃·H₂O, and 3 mL KOH (0.8 M). Then, add 2 mL AuNPs, 7.22 mL H₂O, 0.3 mL Tollens' reagent, and 0.48 mL HCHO under stirring. The mixture reacts at room temperature for 10 minutes to obtain Au@Ag nanoparticles.

[0071] (II) Preparation of liposomes

[0072] Using ethanol as the solvent, a 5 mg / mL phosphatidylcholine solution, a 5 mg / mL cholesterol solution, a 1 mg / mL distearoylphosphatidylethanolamine-polyethylene glycol-biotin (DSPE-PEG-Biotin) solution, and a 1 mg / mL 4-mercaptophenylboronic acid solution were prepared as the organic phase, and the aqueous phase was a phosphate buffer solution with a pH of 7.4.

[0073] In order to prepare 4-mercaptophenylboronic acid-loaded liposomes, phosphatidylcholine, cholesterol and 4-mercaptophenylboronic acid with a molar ratio of 66:100:66 were dispersed in 1 mL of ethanol solution respectively. Using a microfluidic device, the total flow rate (TFR) was set to 120 μL / min and the flow rate ratio (FRR) was set to 1:11 to prepare 4-mercaptophenylboronic acid-loaded liposomes, and the liposomes were stored in a refrigerator at 4°C.

[0074] (III) Microfluidic chip sensor grafted with ssDNA

[0075] After plasma cleaning, the microfluidic chip sensor was treated with 5% 3-aminopropyltrimethoxysilane (APTMS) for 30 minutes. After introducing amine groups onto the surface, the sensor was washed three times with ethanol and then deionized water. Subsequently, 20 μL of 1 μM 5'-Biotin-3'NH2 ssDNA was added to the Raman (SERS) detection chamber. The reaction was incubated at 37°C for 3 hours and then washed three times with deionized water.

[0076] (IV) CRISPR / Cas12a-mediated ssDNA cleavage and signal output

[0077] First, trigger the CHA reaction and anneal all hairpin DNAs within 1 hour before use. The ssDNA primer (Pr) containing the CHA trigger sequence was annealed with the AMP adapter (Apt) sequence at a concentration ratio of 20:21 to form a Pr / Apt complex; a mixture containing H1 (1 μM), H2 (1 μM), Pr / Apt (1 μM) and different concentrations of AMP was incubated at 25°C for 3 hours. In the blank group, AMP was replaced with TM buffer. Prepare Cas12a / gRNA-AMP solution, add 2 μL 1 μM Cas, 2 μL 1 μM r2.1 and 2 μL 1 μM gRNA, and react at room temperature for 30 minutes. Mix the CHA reaction product with cas12a / gRNA-amp and add TM buffer to a total volume of 20 μL.

[0078] 20 μL of streptavidin solution (1.25 μg / mL) was added thereto, incubated at room temperature for 30 minutes, washed three times with deionized water, and 20 μL of 4-mercaptophenylboronic acid-loaded liposomes was added thereto, incubated at room temperature for 30 minutes, washed three times with deionized water to remove excess liposomes.

[0079] (V) SERS signal detection

[0080] The microfluidic chip detection chamber was measured using a confocal Raman microscope at ambient temperature, using a 532nm laser excitation wavelength and an 1800 grating to disperse the scattered light. All spectra were generated using Origin software.

[0081] (VI) Results and Discussion

[0082] (VI-1) Working Principle

[0083] refer to Figure 1 , which shows the principle of CRISPR / Cas12a-mediated liposome amplification strategy based on microfluidic device for SERS detection of ampicillin. First, the surface of the PDMS chip is modified by a plasma cleaner to introduce a large amount of -OH, and then the surface is amino-modified with 5% APTMS, and then the 5'-Biotin-3'NH2ssDNA is fixed by electrostatic adsorption. Then, the CRISPR / Cas12a reaction system (Cas12a, r2.1, gRNA) is added. When the target detection substance ampicillin is added, the CHA reaction is triggered, and the trans-cleavage activity of Cas12a is activated (such as Figure 2After the supernatant was completely removed, 4-MPBA-loaded liposomes were introduced. Streptavidin was used to form a sandwich structure between the biotin-modified liposomes and the DNA, thereby fixing them on the chip. Finally, Au@Ag was added to enhance the Raman signal using hot spots. The calculated EF was 1.22×10 5 Compared with the case where no target substance is added, the liposome concentration will be reduced. -1 、1036cm -1 and 1597cm -1 The Raman intensity of the characteristic peak ΔI = I0-I (I0 and I represent the Raman intensity without and with the target ampicillin, respectively) can be quantitatively detected. This method can cleverly convert AMP concentration into Raman intensity, achieving sensitive detection of trace AMP.

[0084] (VI-2) Material Characterization and SERS Enhancement Mechanism Study

[0085] Blank liposomes and 4-MPBA-loaded liposomes were prepared by microfluidic flow focusing, and the liposomes were characterized by TEM, DLS and UV-vis. Figure 3 As shown in A, during the liposome self-assembly process, the particle size of the liposomes encapsulated with 4-MPBA increases. TEM also proves that the particle size of the liposomes loaded with 4-MPBA increases significantly. At the same time, it is observed that the synthesized 4-MPBA@liposome has a quasi-spherical shape and a thin shell with a clear boundary ( Figure 3 B, C, D). Analysis by UV spectrophotometer showed that 4-MPBA absorbed at 255 nm, and the liposomes loaded with 4-MPBA also showed a corresponding peak, while the blank liposomes had no peak, proving that 4-MPBA was successfully encapsulated in the liposomes ( Figure 3 E) In addition, DLS was used to measure the particle size of blank liposomes and 4-MPBA loaded liposomes. Figure 3 As shown in Figure F, the hydrated particle size of the liposomes without encapsulated 4-MPBA is 100.79 nm, and the hydrated particle size of the liposomes after loading with 4-MPBA is 212 nm. The increase in particle size also proves the successful encapsulation of 4-MPBA.

[0086] In addition, it was found that the synthesized Au@Ag particle size was about 50nm, with obvious core-shell structure characteristics ( Figure 3 G~K). In order to evaluate the enhancement effect of Au@Ag on SERS, the Raman spectra of 4-MPBA and 4-MPBA after adding Au@Ag were tested respectively ( Figure 3 L), and the enhancement factor of the SERS substrate was calculated using the following formula.

[0087]

[0088] I SERS and I Raman are the Raman intensities of 4-MPBA with and without Au@Ag, respectively. N refers to the number of molecules, which can be calculated by adjusting the laser spot diameter, 4-MPBA concentration, and Au@Ag lattice gap ( Figure 4 ) and other data were substituted into the following formula to calculate. The calculation shows that the enhancement factor EF is 1.22×10 5 .

[0089] N SERS =σ.A

[0090] N Raman =CAhN A

[0091] X-ray photoelectron spectroscopy (XPS) revealed that the PDMS chip was successfully modified and connected to single-stranded DNA. Figure 5 As shown in Figure 3, the N content increased to 2.17%. Subsequently, 5'-Bintio-3'NH2ssDNA was grafted onto APTMS-modified PDMS, and the N content increased to 3.12%. This demonstrates the successful modification of APTMS and the successful grafting of 5'-Bintio-3'NH2ssDNA onto PDMS.

[0092] (VI-3) Experimental parameter optimization

[0093] Liposomes were prepared using flow focusing, and the total flow rate (TFR) was set to 120 μL / min. Since the flow rate ratio (FRR) affects the size of the liposomes, the FRR was optimized, and five sets of organic phase and aqueous phase ratios of 1:2, 1:5, 1:11, 1:14 and 1:19 were set. It was found that the size of the liposomes became smaller with increasing FRR. This is because the FRR changes the concentration of the organic phase. When the FRR is large, the concentration of phospholipids in the microfluidic channel is high. At this time, the phospholipids tend to form larger spheres after self-assembly, forming liposome disks in the channel. However, when the flow rate is large enough, it will cause the phospholipids to aggregate and sink again, making the PDI larger. As Figure 6 As shown in A and B, the liposome size decreases with decreasing FRR, but when the flow rate ratio reaches a certain value, the liposome size does not change significantly. Therefore, an FRR of 1:11 was selected for subsequent experiments.

[0094] The concentration of 4-MPBA in liposomes significantly affects the signal intensity of Raman spectroscopy and improves the Raman detection performance of 4-MPBA@liposomes. Therefore, the concentration of liposomes loaded with signal molecules was optimized. Figure 6As shown in C and D, the best Raman enhancement can be achieved when the 4-MPBA concentration is 5mM in the range of 0.5mM-6.5mM. In addition, the encapsulation efficiency of liposomes is also an important factor that needs to be examined. Cholesterol is usually added to the preparation of liposomes to increase the fluidity of their membranes, making the liposomes less likely to rupture and also adjusting the encapsulation efficiency. Therefore, five groups of cholesterol: soy lecithin ratios of 0.1, 0.25, 0.5, 0.75, and 0.9 were prepared respectively. It was found that the cholesterol: soy lecithin ratio of 1:1 had the strongest Raman signal and the best effect (as shown in Figure 2). Figure 6 E and F).

[0095] (VI-4) Raman characterization

[0096] The uniformity of the SERS substrate is an important factor in quantitative Raman measurement. Liposomes loaded with 4-MPBA can overcome this problem. In order to test the uniformity of the PDMS chip based on liposome growth, Raman signal testing was performed on random areas of the chip detection chamber. Based on Raman mapping, 4-MPBA at 1000 cm -1 、1036cm -1 and 1597cm -1 The signal peak intensity distribution is as follows Figure 7 As shown in A, Figure 7 The Raman spectra of 9 random points were selected from A. The relative standard deviation (RSD) of the Raman intensity was 1000 cm -1 、1036cm -1 and 1597cm -1 The peak values ​​are 6.89%, 8.33% and 6.71% respectively ( Figure 7 B), indicating that the SERS signal uniformity of the PDMS chip based on liposome growth is very good. In addition, its stability was also checked, such as Figure 7 As shown in C and D, under ambient conditions, the Raman signal remains stable after 0, 10, and 50 days of storage. The reproducibility was also explored. Figure 3 E shows that the Raman spectra recorded by the ten batches of chips are consistent. They have high reproducibility and the RSD is 4.06% ( Figure 7 E, F). Therefore, microfluidic chips with excellent reproducibility have great advantages in the quantitative detection of AMP.

[0097] (VI-5) SERS Detection of Ampicillin

[0098] In order to study the performance of microfluidic chip in detecting AMP, the concentration of AMP was prepared in the range of 1fM to 1nM. -1 The △I at the position was used to quantitatively analyze AMP. Figure 8A shows the SERS spectra of AMP at different concentrations and blank samples without AMP. -1 As the main peak. The results show that at 1597cm -1 At the characteristic peak, the peak intensity increases as the concentration of added AMP decreases, and ΔI decreases. ΔI and AMP concentration show a good linear relationship in the concentration range of 1fM to 1nM, as shown in the following formula: y = 925.91lg(x)-143.72( Figure 8 B), the limit of detection (LOD) was 0.74 fM.

[0099] The results of the present invention compared with other methods are shown in Table 1.

[0100] Table 1

[0101]

[0102] (VI-6) Determination of Ampicillin in Reagent Samples

[0103] In order to test the practicality of this method in actual samples, the developed biosensor was used to test samples such as milk, landfill leachate, anaerobic algae liquid from restaurant kitchens, anaerobic tanks for urban sewage, and fish pond water. The results are shown in Table 2 (real samples were tested using the spiked method).

[0104] Table 2

[0105]

[0106] In addition, the oligonucleotide sequences used in the present invention are shown in Table 3.

[0107] Table 3

[0108]

[0109] in conclusion:

[0110] This study successfully developed a microfluidic chip sensor for the detection of ampicillin based on a CRISPR / Cas12a-mediated liposome-based SERS signal amplification strategy. This method combines a catalytic hairpin (CHA) with SERS to achieve dual signal amplification, improving sensitivity while also achieving ultra-high specificity and selectivity through the design of ampicillin aptamers.

[0111] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing a microfluidic chip sensor based on CRISPR-mediated liposomes, characterized in that: The preparation method comprises the following steps: (1) cleaning and drying the silicon wafer to obtain a dry silicon wafer; (2) spin-coating photoresist on the dry silicon wafer and laminating it with a mask plate, and then sequentially exposing, drying, and entering a developer to obtain a template; (3) The template is dried to form a hard film, and then silanized, cured, and cleaned in sequence.

2. The method for preparing a CRISPR-mediated liposome-based microfluidic chip sensor according to claim 1, characterized in that: In step (1), the cleaning method is: ultrasonic cleaning with anhydrous ethanol, acetone and ultrapure water in sequence; the drying temperature is 60-65°C; In step (2), the exposure time is 55 to 65 seconds; the drying method is: first drying at 60 to 65° C. for 2 to 3 minutes and then drying at 90 to 95° C. for 12 to 15 minutes; In step (3), the drying method is: drying at 90-95° C. for 2-2.5 hours; the silanization method is: silanization at 0.08-0.1 Pa for 2-3 hours.

3. A CRISPR-mediated liposome-based microfluidic chip sensor obtained by the preparation method according to claim 1 or 2.

4. The use of the CRISPR-mediated liposome-based microfluidic chip sensor in the detection of ampicillin according to claim 3, characterized in that: The detection method includes the following steps: (I) Preparation of Au@Ag nanoparticles (I-1) Preparation of Au nanoparticles: HAuCl4 solution was mixed with water and heated to boiling. Trisodium citrate was then added. The mixture was stirred while boiling until the color of the solution changed from colorless to wine red. After completion, the mixture was cooled to obtain an Au nanoparticle solution. (I-2) Preparation of Au@Ag nanoparticles: Au nanoparticle solution, Tollens reagent, HCHO, and water were mixed and stirred to react, thereby obtaining Au@Ag nanoparticles; (II) Preparation of liposomes Phosphatidylcholine, cholesterol and 4-mercaptophenylboronic acid were dispersed in ethanol solution respectively, and 4-mercaptophenylboronic acid-loaded liposomes were prepared by using a microfluidic device and setting the total flow rate and flow rate ratio. (III) Microfluidic chip sensor grafted with ssDNA The microfluidic chip sensor was treated with 3-aminopropyltrimethoxysilane to introduce amine groups, then washed and 20 μL of 5'-Biotin-3'NH2 ssDNA was added to the Raman detection chamber for reaction; (IV) CRISPR / Cas12a-mediated ssDNA cleavage and signal output The presence of ampicillin is converted into a DNA signal by triggering a catalytic hairpin reaction, and ssDNA is cut through the cleavage activity of the CRISPR / Cas12a system on the target DNA; at the same time, 4-MPBA is encapsulated in liposomes, and then the Raman signal is further amplified using the Au@Ag nanoparticles, and finally the product is detected.

5. The use of the CRISPR-mediated liposome-based microfluidic chip sensor in the detection of ampicillin according to claim 4, characterized in that: In step (I-1), the boiling and stirring time is 30 to 40 minutes.

6. The use of the CRISPR-mediated liposome-based microfluidic chip sensor in detecting ampicillin according to claim 4, characterized in that: In step (I-2), the reaction temperature is 20-25° C. and the reaction time is 10-15 minutes.

7. The use of the CRISPR-mediated liposome-based microfluidic chip sensor in detecting ampicillin according to claim 4, characterized in that: In step (II), the total flow rate is 120 μL / min, and the flow rate ratio is 1:

11.

8. The use of the CRISPR-mediated liposome-based microfluidic chip sensor in detecting ampicillin according to claim 4, characterized in that: In step (III), the treatment time is 25 to 30 minutes; the reaction temperature is 35 to 37° C., and the reaction time is 2 to 3 hours.

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