Method and detection kit for detecting lead ions using paper-based microfluidic chip based on CRISPR-Cas12a system

Abstract

The present invention relates to the field of bioanalysis detection technology, and specifically discloses a method and a detection kit for detecting lead ions using a paper-based microfluidic chip based on a CRISPR-Cas12a system. The detection kit comprises: a G-quadruplex chain, crRNA, ssDNA, a circular probe CP, a connection probe LP, a capture probe CAP, a buffer system and a paper-based microfluidic chip. The detection method comprises: preparation of DNA nanoflowers, preparation of ssDNA-AuNPs, preparation of a paper-based microfluidic chip, establishment of a standard curve, and sample detection. The detection kit of the present invention is used to detect the lead content in a liquid solution, and has the characteristics of high efficiency, rapidity, specificity, sensitivity, portability, etc., and the method is easy to operate, has low detection cost, and can be applied to on-site detection of lead ions.

Description

Technical Field

[0001] The present invention relates to the technical field of bioanalysis and detection, and in particular to a colorimetric method and a detection kit for detecting lead ions using a paper-based microfluidic chip based on a CRISPR-Cas12a system. Background Art

[0002] Lead ions are one of the most serious heavy metal ions that are harmful to human health. Lead ions enter the human body through food intake, tap water drinking and other ways. Most of the lead that enters the human body is stored in the bones, and a small part flows to various tissues and organs of the body through blood circulation. Since lead ions are not biodegradable, they will accumulate in the human body and cause serious damage to the brain, kidneys, cardiovascular function and nervous system of the human body. Lead ingested by infants and young children may cause permanent adverse effects, and lead poisoning is still one of the most common pediatric health problems today. The most important harm of lead to children is the impact on children's brain development. It can affect many functions of the nervous system, causing children to have symptoms such as decreased intelligence, lowered IQ, delayed or impaired neurobehavioral development, decreased hearing acuity, speech and language disorders, and growth retardation. In addition, lead can also have adverse effects on children's hematopoietic system, respiratory system and immune system. Lead pollution and the health problems caused by lead pollution are increasingly attracting people's attention. Therefore, it is very important to design and develop highly sensitive and selective lead determination methods.

[0003] At present, many traditional and reliable methods for detecting lead ions have been developed, such as atomic fluorescence spectrometry (AFS), atomic absorption spectrometry (AAS), anodic stripping voltammetry (ASV), high performance liquid chromatography and inductively coupled plasma mass spectrometry (ICP-MS), but these methods have some common disadvantages: they require large instruments and equipment, well-trained staff and complex sample pretreatment procedures, and most of these methods are expensive and cannot be used on site. Therefore, it is still urgent to develop a lead ion detection method with high sensitivity, good specificity, low detection limit, rapid detection, strong portability, and can be used on site.

[0004] China CN110286107A discloses a method for detecting lead ions, wherein nanogold is modified on the surface of aminated magnetic beads to form a fluorescent quencher, the nanogold is connected to the DNA enzyme chain modified with red quantum dots and combined with the DNA substrate chain modified with green quantum dots through base complementary pairing, the lead ions are used to recognize and cut the substrate chain, the distance between the green quantum dots and the nanogold is changed, the quenching and recovery of green fluorescence is achieved, and the change in the red-green fluorescence ratio caused by the change in the green fluorescence intensity is used to achieve quantitative detection of lead ions. This method adopts a dual-signal detection mode, which effectively reduces the impact of the background environment on the detection results, but the preparation conditions of the surface modification of the magnetic beads with nanogold and then connected to the DNA enzyme are harsh, the reaction steps are complicated, and the reagent cost is high. Summary of the invention

[0005] In view of the shortcomings of existing detection technologies, the present invention proposes a colorimetric method and detection kit for detecting lead ions using a paper-based microfluidic chip based on the CRISPR-Cas12a system. The colorimetric method is used to detect the lead content in a liquid solution, and has the characteristics of high efficiency, rapidity, specificity, sensitivity, and portability. The method is easy to operate and has low detection cost, and can be applied to on-site detection of lead ions.

[0006] The invention provides a kit for detecting lead ions using a paper-based microfluidic chip based on a CRISPR-Cas12a system, comprising: a G-quadruplex chain, crRNA, ssDNA, a circular probe CP, a connection probe LP, a capture probe CAP, a buffer system and a paper-based microfluidic chip.

[0007] Furthermore, the chain sequence of the circular probe CP is shown as SEQ ID No.1; the chain sequence of the connecting probe LP is shown as SEQ ID No.2; and the chain sequence of the capture probe CAP is shown as SEQ ID No.3.

[0008] Furthermore, the G-quadruplex chain sequence is shown as SEQ ID No.4; the crRNA sequence is shown as SEQ ID No.5; and the ssDNA sequence is shown as SEQ ID No.6.

[0009] Furthermore, the buffer system is a Tris buffer system, which is composed of 10 mM Tris-HAc and 10 mM MgCl2 (PH=7.4) in a volume ratio of 1:1.

[0010] The present invention also provides a method for detecting lead ions using a paper-based microfluidic chip based on the CRISPR-Cas12a system, comprising the following steps:

[0011] S1. Preparation of DNA nanoflowers: The circular probe CP undergoes a rolling circle amplification reaction in the presence of the connection probe LP to form a DNA nanoflower, and then the DNA nanoflower undergoes a connection reaction with the capture probe CAP to obtain a DNA nanoflower with a capture probe;

[0012] S2. Preparation of ssDNA-AuNPs: Gold nanoparticles AuNPs were prepared by sodium citrate reduction method. The thiol-modified ssDNA and Tris-HCl with a pH of 7.4 were mixed evenly, and TCEP was added to react at 37°C. The prepared ssDNA was added to the AuNPs solution and mixed evenly, and sodium chloride was repeatedly added. After standing at room temperature for 24 hours, ssDNA-AuNPs were obtained.

[0013] S3. Preparation of paper-based microfluidic chip: Use Adobe Illustrator software to draw graphics, use a laser engraving machine to cut paper and splice the cut paper on a PVC board, fix DNA nanoflowers with capture probes on the detection area paper to obtain a paper-based microfluidic chip;

[0014] S4. Establishment of standard curve: dilute the G quadruplex chain with Tris buffer system and then anneal it. After cooling, mix it with lead ion solution of determined concentration to obtain a reaction system. At the same time, mix and incubate the Cas12a-crRNA system. Mix the Cas12a-crRNA system with the reaction system, then add ssDNA-AuNPs for cutting reaction, inactivate the activity of cas12a protein, take the reaction product and add it dropwise to the chip, take a picture of the color of the detection area with a smart phone, analyze the RGB value with software, and make a linear graph of the target substance lead ion and R / G value and the corresponding linear equation.

[0015] S5. Sample detection: Input the linear equation obtained in S4 into the software. Under the same conditions as in S4, dilute the G quadruplex chain with a Tris buffer system and then anneal it. After cooling, mix it with the test solution to obtain a reaction system. At the same time, mix and incubate the Cas12a-crRNA system. Mix the Cas12a-crRNA system with the reaction system, and then add ssDNA-AuNPs for a cutting reaction. Take the reaction product and drop it onto the chip. Use a smart phone to take a picture of the color of the detection area. The software analyzes the RGB value and automatically analyzes to obtain the content of lead ions in the test solution.

[0016] Furthermore, the specific steps of step S1 are as follows:

[0017] S101. The circular probe CP, the ligation probe LP and 1×T4 DNA ligase buffer were reacted at 95°C for 5 minutes, allowed to stand for 1 hour and gradually cooled to room temperature, and T4 DNA ligase was added and incubated at 37°C for 3 hours; phi 29 DNA polymerase, dNTPs and 1×phi 29 DNA polymerase buffer were added to the circularization reaction product, incubated at 37°C for 48 hours, and then heated at 75°C for 10 minutes;

[0018] S102. The product obtained in step S101 is centrifuged at 12000r / min, the supernatant is removed, and then ultrapure water is added, and the centrifugation is washed 3 times. The obtained DNA nanoflower is re-dissolved in ultrapure water, reacted at 35-40°C, 300-700rpm for 1-5h, the supernatant is removed by washing, deionized water is added, and stored in a -20°C refrigerator;

[0019] S103. The DNA nanoflower obtained in step S102 is subjected to a ligation reaction with the capture probe CAP. The reaction conditions are incubation at room temperature for 20 minutes to obtain a DNA nanoflower with a capture probe.

[0020] Furthermore, the chain sequence of the circular probe in step S1 is shown as SEQ ID No.1; the chain sequence of the connecting probe is shown as SEQ ID No.2; and the chain sequence of the capture probe is shown as SEQ ID No.3.

[0021] Furthermore, in step S1, the volume ratio of the circular probe CP to the connection probe LP is 1:2; the volume ratio of the DNA nanoflower to the capture probe CAP is 1:10.

[0022] Furthermore, in step S2, the mass ratio of TCEP to ssDNA is 10:1; and the volume ratio of AuNPs to ssDNA is 4:1.

[0023] Furthermore, the reaction conditions of ssDNA and AuNPs in step S2 are shaking at 30°C for 16 hours; sodium chloride is added at a concentration of 2M and repeated 9 times; ssDNA-AuNPs are stored in PBS in a 4°C refrigerator away from light.

[0024] Furthermore, the paper-based microfluidic chip in step S3 includes a sample loading area, a sample channel, a detection area, a water absorption channel and a water absorption area; wherein the paper base of the sample loading area and the sample channel adopts a sample pad and is treated with Tween 20, the detection area adopts a nitrocellulose membrane and fixes 3 μL of DNA nanoflowers with capture probes, and the water absorption channel and the water absorption area adopt a water absorption pad.

[0025] Furthermore, the parameters of the laser engraving machine in step S3 are set to a speed of 90 mm / s and a power of 10%.

[0026] Furthermore, the annealing treatment in step S4 is performed at a temperature of 95° C. and for a time of 5 minutes.

[0027] Furthermore, in step S4, the reaction temperature of the G-quadruplex chain and the lead ions is 37° C. and the reaction time is 30 min.

[0028] Furthermore, the temperature of the mixed incubation of the Cas12a-crRNA system described in step S4 is 37° C., and the time is 20 min; the temperature of the Cas12a-crRNA cleavage reaction is 37° C., and the time is 30 min; and the treatment condition for inactivating the activity of the cas12a protein is 65° C. for 10 min.

[0029] Furthermore, the Cas12a-crRNA system in step S4 is prepared by mixing 9.75 μL buffer, 4 μL, 200 nM Cas12a and 1.25 μL, 200 nM crRNA; the volume ratio of the reaction system to the Cas12a-crRNA system is 4:1.

[0030] Furthermore, the volume ratio of the ssDNA-AuNPs to Cas12a-crRNA system in step S4 is 2:1.

[0031] Furthermore, the concentration range of the lead ion solution determined in step S4 is 0.1 μM-10 μM.

[0032] Furthermore, the Tris buffer system in step S4 or S5 is composed of 10 mM Tris-HAc and 10 mM MgCl2 (PH=7.4) in a volume ratio of 1:1.

[0033] Furthermore, the G quadruplex chain sequence in step S4 or S5 is shown as SEQ ID No.4; the crRNA sequence is shown as SEQ ID No.5; and the ssDNA sequence is shown as SEQ ID No.6.

[0034] Furthermore, the linear equation in step S4 or S5 is: R / G=0.0082C+0.9722(R 2 =0.9923).

[0035] Furthermore, the sample detection in step S5 has a linear detection range of 0.1 μM-10 μM and a detection limit of 18.3 nM.

[0036] The present invention has the following beneficial effects:

[0037] 1. The present invention makes the analysis method have good specificity through the specific combination of lead ions and G quadruplexes, eliminating the interference of other substances;

[0038] 2. The present invention has developed a multi-channel paper-based microfluidic chip, which fixes DNA nanoflowers in the detection area to capture gold nanoparticles for color development, has high capture efficiency, and the multi-channel design of the chip can be used for simultaneous detection of multiple samples;

[0039] 3. The present invention uses CRISPR-Cas12a to trans-cut ssDNA on AuNPs as signal output, which can amplify the signal and enhance the sensitivity of the sensor;

[0040] 4. The colorimetric analysis method based on gold nanoparticles of the present invention has the advantages of simple synthesis, high sensitivity, and simple detection method, and can efficiently, sensitively and quickly detect lead ions in liquid solutions;

[0041] 5. The smartphone-assisted paper-based microfluidic chip designed in the present invention has the advantages of simple operation and convenient carrying, and can be applied to on-site detection of lead ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a schematic diagram of the principle of the colorimetric method for detecting lead ions using a paper-based microfluidic chip based on the CRISPR-Cas12a system of the present invention;

[0043] Figure 2 Schematic diagram of paper-based microfluidic chip parameters;

[0044] Figure 3 Optimization of time for cleavage of ssDNA on gold nanoparticles by the CRISPR-Cas12a system;

[0045] Figure 4 The R / G value diagram of the lead ion detection method provided by the present invention;

[0046] Figure 5 A standard curve diagram of the lead ion detection method provided by the present invention;

[0047] Figure 6 A schematic diagram of the selectivity test results of the lead ion detection method provided by the present invention. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0049] Example 1: Colorimetric method for detecting lead ions using a paper-based microfluidic chip based on the CRISPR-Cas12a system

[0050] The colorimetric method for detecting lead ions using a paper-based microfluidic chip based on the CRISPR-Cas12a system provided by the present invention comprises the following steps:

[0051] S1. Preparation of DNA nanoflowers:

[0052] S101. React 5 μL, 1 μM circular probe CP, 10 μL, 1 μM ligation probe LP and 5 μL 1×T4 DNA ligase buffer at 95°C for 5 minutes, let stand for 1 hour and gradually cool to room temperature, add 1.6 μL, 20 U / μL T4 DNA ligase and incubate at 37°C for 3 hours; add 1.5 μL 10 U / μL phi 29 DNA polymerase, 4 μL 10 mM dNTPs and 12 μL 1×phi 29 DNA polymerase buffer to the circularization reaction product, add ultrapure water to make the rolling circle amplification reaction system volume 80 μL, incubate at 37°C for 48 hours, and then heat at 75°C for 10 minutes;

[0053] S102. The product obtained in step S101 is centrifuged at 12000r / min, the supernatant is removed, and then ultrapure water is added, and the centrifugation is washed 3 times. The obtained DNA nanoflower is re-dissolved in ultrapure water, reacted at 35-40°C, 300-700rpm for 1-5h, the supernatant is removed by washing, deionized water is added, and stored in a -20°C refrigerator;

[0054] S103. The 80 μL DNA nanoflower obtained in step S102 is subjected to a ligation reaction with 800 μL capture probe CAP. The reaction conditions are incubation at room temperature for 20 minutes to obtain a DNA nanoflower with a capture probe.

[0055] S2. Preparation of ssDNA-AuNPs: Gold nanoparticles (AuNPs) were prepared by sodium citrate reduction method. 16 μL of 100 μM thiol-modified ssDNA and 144 μL of Tris-HCl with a pH of 7.4 were mixed evenly, and 2 μL of 100 μM TCEP was added to react at 37°C. 40 μL of the prepared ssDNA was added to 260 μL of 10 nM AuNPs solution and mixed evenly. The mixture was shaken at 30°C for 16 hours, and 2 μL of Tween 20 and 3.75 μL of 2M sodium chloride were added. The mixture was ultrasonically incubated for 10 seconds and incubated at room temperature for 1 hour. After adding sodium chloride three times and ultrasonically incubating, the amount of sodium chloride was increased to 7.5 μL, and sodium chloride was added and ultrasonically incubated for 6 times. After standing at room temperature for 24 hours, ssDNA-AuNPs were obtained. The ssDNA-AuNPs were stored in PBS in a 4°C refrigerator protected from light.

[0056] S3. Preparation of paper-based microfluidic chip: draw graphics using Adobe Illustrator software, cut paper with a laser engraving machine and splice the cut paper on a PVC board, fix DNA nanoflowers on the paper in the detection area to obtain a paper-based microfluidic chip; wherein the paper-based microfluidic chip includes a sample loading area, a sample channel, a detection area, a water absorption channel and a water absorption area; the paper base of the sample loading area and the sample channel adopts a sample pad and is treated with Tween20, the detection area adopts a nitrocellulose membrane and fixes 3μL of DNA nanoflowers, and the water absorption channel and the water absorption area adopt a water absorption pad; the parameters of the laser engraving machine are set to a speed of 90mm / s and a power of 10%.

[0057] S4. Establishment of standard curve: 10 μL, 10 μM G quadruplex chain was diluted with 190 μL Tris buffer system (10 mM Tris-Hac, 10 mM MgCl2, PH = 7.4) and annealed at 95 ° C for 5 min. After cooling, it was divided into ten parts, and mixed with 20 μL of lead ion solution with a determined concentration (0 μM, 0.1 μM, 0.7 μM, 1.5 μM, 4 μM, 5 μM, 8 μM, 10 μM, 15 μM, 20 μM) and 20 μL ultrapure water at 37 ° C for 30 min to obtain a lead ion reaction system. At the same time, ten parts of Cas12a-crRNA system (mixed by 9.75 μL buffer, 4 μL, 200 nM Cas12a and 1.25 μL, 200 nM crRNA) were prepared and mixed and incubated at 37 ° C for 20 min; 15 μL The Cas12a-crRNA system was mixed with ten 60 μL lead ion reaction systems to obtain an activated reaction system, and then 30 μL and 1 nM ssDNA-AuNPs were added to the activated reaction system for cutting reaction. The reaction temperature was 37 ° C and the time was 30 min. Then, the cas12a protein activity was inactivated at 65 ° C for 10 min. 5 μL of the reaction product was added dropwise to the chip, and the sample was waited to flow to the water absorption area. The color of the detection area was photographed with a smartphone, and the software analyzed the RGB value, and a linear graph of the target substance lead ion and R / G value and the corresponding linear equation were made.

[0058] S5. Sample detection: Enter the linear equation obtained in S4 into the software. Under the same conditions as S4, dilute 2 μL, 5 μM G quadruplex chain with 18 μL Tris buffer system (10 mM Tris-Hac, 10 mM MgCl2, PH = 7.4) and anneal at 95 ° C for 5 min. After cooling, mix with 20 μL of test solution and 20 μL ultrapure water to obtain a reaction system. At the same time, the Cas12a-crRNA system (prepared by mixing 9.75 μL buffer, 4 μL, 200 nM Cas12a and 1.25 μL, 200 nM crRNA) was mixed and incubated at 37 ° C for 20 min; the Cas12a-crRNA system was mixed with the reaction system, and then 30 μL, 1 nM ssDNA-AuNPs undergo a cutting reaction, and 5 μL of the reaction product is dripped onto the chip. After the sample flows to the water absorption area, a smartphone is used to take a picture of the color of the detection area. The software analyzes the RGB value and automatically analyzes the content of lead ions in the test liquid.

[0059] like Figure 1, a schematic diagram of the detection principle of the paper-based microfluidic chip based on the CRISPR-Cas12a system provided by the present invention for detecting lead ion colorimetry. When there is no lead ion in the reaction system, the G-quadruplex chain can bind to crRNA to activate the CRISPR-Cas12a system, and the activated CRISPR-Cas12a system has cutting activity and can trans-cut the ssDNA on the gold nanoparticles. The reaction system is added to the paper-based microfluidic chip, and the gold nanoparticles in the sample flow through the detection area and flow to the water absorption area through the water absorption channel, and the R / G value of the detection area is low. When lead ions are present in the reaction system, the lead ions can induce the G-quadruplex chain to form a G-quadruplex structure, and thus cannot bind to crRNA to activate the CRISPR-Cas12a system. The AuNP-ssDNA in the sample is captured by the DNA nanoflower fixed in the detection area, and the captured gold nanoparticles are colored in the detection area, and the R / G value of the detection area is high.

[0060] A paper-based microfluidic chip was produced by AI drawing and laser cutting technology to obtain Example 2.

[0061] By adjusting the time for the CRISPR-Cas12a system to cut the ssDNA on the gold nanoparticles, the optimal experimental conditions for the reaction system were obtained to obtain Example 3.

[0062] Example 2: Paper-based microfluidic chip

[0063] like Figure 2 As shown, all graphics are drawn using Adobe Illustrator software. The paper base of the sample loading area and sample channel uses a sample pad, the detection area uses a nitrocellulose membrane, and the water absorption channel and water absorption area use a water absorption pad. The paper in the detection area is made by punching with a hole puncher with a diameter of 3 mm. The sample loading area, sample channel, water absorption channel and water absorption area are cut using a laser engraving machine. The parameters of the laser engraving machine are set to a speed of 90 mm / s and a power of 10%. The cut paper is washed in ultrapure water and then placed in a 40°C oven for drying. All prepared paper parts are connected to a square hydrophobic PVC board with a side length of 6 cm as shown in the figure, DNA nanoflowers are dripped into the detection area, and fixed at room temperature to obtain a paper-based microfluidic device to be used.

[0064] Example 3: Time optimization of CRISPR-Cas12a system cutting ssDNA on gold nanoparticles

[0065] The time for CRISPR-Cas12a system to cut ssDNA on gold nanoparticles was 10min, 20min, 30min, 40min, and 50min respectively. The concentration of added lead ions was 1μM; the other parameters were referred to Example 1.

[0066] R / G value detection: Place the paper-based microfluidic chip into the sample area of ​​the dark box, use a smart phone to photograph the detection area of ​​the paper-based microfluidic chip, analyze the color of the detection area to obtain the RGB value, and the SmartIons software will automatically process it to obtain the R / G value.

[0067] like Figure 3 , which is the R / G value diagram of the CRISPR-Cas12a system cutting ssDNA on gold nanoparticles at different reaction times. Figure 3 It can be seen that when the reaction time is 30 minutes, the corresponding R / G value reaches the lowest. Therefore, under this condition, the CRISPR-Cas12a system can cut more ssDNA, and the detection sensitivity is higher when detecting lead ions.

[0068] In summary, in the lead ion detection method provided by the present invention, the optimal reaction time for the CRISPR-Cas12a system to cut ssDNA on gold nanoparticles is 30 minutes.

[0069] Example 4: Drawing a standard curve

[0070] The volume of the reaction system is 90 μL, the reaction time of the CRISPR-Cas12a system cutting ssDNA on the gold nanoparticles is 30 min, the lead ion concentrations of the test solutions are 0 μM, 0.1 μM, 0.7 μM, 1.5 μM, 4 μM, 5 μM, 8 μM, 10 μM, 15 μM, 20 μM, and the remaining parameters are referred to Example 1. By measuring the R / G value of the chip detection area, a linear graph of lead ions and R / G values ​​is made to determine the content level of the detected lead ions.

[0071] like Figure 4 and Figure 5 ,in Figure 4 It is the R / G value diagram of the reaction system with different concentrations of lead ions; Figure 5 This is a standard curve diagram of the lead ion detection method provided by the present invention. Figure 4 and Figure 5 It can be seen that the R / G value of the chip detection area increases with the increase of lead ion concentration. Figure 5 It can be seen that the linear equation of the lead ion detection method provided by the present invention is: R / G=0.0082C+0.9722(R 2 =0.9923), the linear relationship is good, the linear detection range is 0.1μM-10μM, and the detection limit is 18.3nM. This shows that the lead ion detection method provided by the present invention has a lower detection limit and can meet the actual detection requirements.

[0072] Example 5: Specificity test of the detection method of the present invention

[0073] In order to test the specificity of the lead ion detection method provided by the present invention, several common metal ions Ni 2+ 、Zn 2+ Cr 3+ Mg 2+ , Mn 2+ , Ba 2+ 、Cd 2+ The specificity of the method was verified by following the steps of Example 1; similarly, Ni 2+ 、Zn 2+ Cr 3+ Mg 2+ , Mn 2+ , Ba 2+ 、Cd 2+ and Pb 2+ Added together into the detection system, and processed according to the operating steps of Example 1 to verify the anti-interference ability of the method; wherein Ni 2+ 、Zn 2+ Cr 3+ Mg 2+ , Mn 2+ , Ba 2+ 、Cd 2+ The concentration of Pb was 100 μM. 2 + The concentration was 10μM; a blank group was added for comparative experiments.

[0074] like Figure 6 , which is a schematic diagram of the specific experimental results of the lead ion detection method provided by the present invention. Figure 6 It can be seen that even if Ni 2+ 、Zn 2+ Cr 3+ Mg 2+ , Mn 2+ , Ba 2+ 、Cd 2+ The concentration of Pb 2+ The concentration was 10 times higher, but it still did not cause a significant change in the R / G value; and other metal ions had no significant effect on Pb 2+ The detection of lead ions does not cause much interference, which is mainly due to the high specificity of the binding of lead ions to G-quadruplexes, which further indicates that the lead ion detection method provided by the present invention has good selectivity and anti-interference.

[0075] Application Example: Actual Sample Detection Research

[0076] 2μL, 5μM G quadruplex chain was diluted with 18μL Tris buffer system (10mM Tris-Hac, 10mM MgCl2, PH=7.4) and annealed at 95°C for 5min. After cooling, it was mixed with the sample to be tested and reacted. At the same time, the Cas12a-crRNA system (9.75μL buffer, 4μL, 200nM Cas12a and 1.25μL, 200nM crRNA) was mixed and incubated; 15μL of Cas12a-crRNA system was added to the 60μL reaction system, and 30μL, 1nM ssDNA-AuNPs were added after the reaction to continue the reaction. The product after the reaction was added dropwise to the chip, and the color of the detection area was photographed with a smartphone. The software analyzed the RGB value and automatically analyzed the content level of the detected lead ions. The linear equation is: R / G=0.0082C+0.9722(R 2 =0.9923).

[0077] The concentration of lead ions in the liquid solution was determined by the colorimetric method of the present invention using the sample recovery method. All samples were measured three times to obtain the average value, as shown in the following table. It can be seen that the colorimetric sensor of this embodiment can accurately determine the concentration of lead ions in the actual sample. The results are shown in Table 1.

[0078] Table 1

[0079]

[0080]

[0081] As shown in Table 1, the lead ion detection method provided by the present invention utilizes the specific binding of lead ions to G-quadruplexes to inhibit the activation of the CRISPR-Cas12a system, and cannot trans-cut the ssDNA on the gold nanoparticles. The DNA nanoflowers in the chip detection area capture the gold nanoparticles, and finally use a smart phone to shoot and analyze the R / G value through software to achieve lead ion detection. The lead ion detection method provided by the present invention has the advantages of simple operation, high selectivity, high sensitivity, and easy carrying.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for detecting lead ions using a paper-based microfluidic chip based on a CRISPR-Cas12a system, comprising: G-quadruplex chain, crRNA, ssDNA, circular probe CP, connection probe LP, capture probe CAP, buffer system and paper-based microfluidic chip; Among them, the chain sequence of the circular probe CP is shown as SEQ ID No.1; the chain sequence of the connecting probe LP is shown as SEQ ID No.2; the chain sequence of the capture probe CAP is shown as SEQ ID No.3; the G-quadruplex chain sequence is shown as SEQ ID No.4; the crRNA sequence is shown as SEQ ID No.5; and the ssDNA sequence is shown as SEQ ID No.

6.

2. The kit according to claim 1, characterized in that: The buffer system is a Tris buffer system, which is composed of 10 mM Tris-HAc and 10 mM MgCl2 in a volume ratio of 1:1, and has a pH of 7.

4.

3. A method for detecting lead ions using a paper-based microfluidic chip based on a CRISPR-Cas12a system, comprising: S1. Preparation of DNA nanoflowers: The circular probe CP undergoes a rolling circle amplification reaction in the presence of the connection probe LP to form a DNA nanoflower, and then the DNA nanoflower undergoes a connection reaction with the capture probe CAP to obtain a DNA nanoflower with a capture probe; S2. Preparation of ssDNA-AuNPs: Gold nanoparticles AuNPs were prepared by sodium citrate reduction method. The thiol-modified ssDNA and Tris-HCl with a pH of 7.4 were mixed evenly, and TCEP was added to react at 37°C. The prepared ssDNA was added to the AuNPs solution and mixed evenly, and sodium chloride was repeatedly added. After standing at room temperature for 24 hours, ssDNA-AuNPs were obtained. S3. Preparation of paper-based microfluidic chip: Use Adobe Illustrator software to draw graphics, use a laser engraving machine to cut paper and splice the cut paper on a PVC board, fix DNA nanoflowers with capture probes on the detection area paper to obtain a paper-based microfluidic chip; S4. Establishment of standard curve: dilute the G quadruplex chain with Tris buffer system and then anneal it. After cooling, mix it with lead ion solution of determined concentration to obtain a reaction system. At the same time, mix and incubate the Cas12a-crRNA system. Mix the Cas12a-crRNA system with the reaction system, then add ssDNA-AuNPs for cutting reaction, inactivate the activity of cas12a protein, take the reaction product and add it dropwise to the chip, take a picture of the color of the detection area with a smart phone, analyze the RGB value with software, and make a linear graph of the target substance lead ion and R / G value and the corresponding linear equation. S5. Sample detection: Input the linear equation obtained in S4 into the software. Under the same conditions as in S4, dilute the G quadruplex chain with a Tris buffer system and perform annealing treatment. After cooling, mix it with the test solution to obtain a reaction system. At the same time, mix and incubate the Cas12a-crRNA system; mix the Cas12a-crRNA system with the reaction system, and then add ssDNA-AuNPs for cutting reaction. Take the product after the reaction and add it dropwise to the chip. Use a smart phone to take a picture of the color of the detection area. The software analyzes the RGB value and automatically analyzes the content of lead ions in the test solution. Among them, the chain sequence of the circular probe CP is shown as SEQ ID No.1; the chain sequence of the connecting probe LP is shown as SEQ ID No.2; the chain sequence of the capture probe CAP is shown as SEQ ID No.3; the G-quadruplex chain sequence is shown as SEQ ID No.4; the crRNA sequence is shown as SEQ ID No.5; and the ssDNA sequence is shown as SEQ ID No.

6.

4. The method according to claim 3, characterized in that: The specific steps of step S1 are as follows: S101. The circular probe CP, the ligation probe LP and 1×T4 DNA ligase buffer were reacted at 95°C for 5 minutes, allowed to stand for 1 hour and gradually cooled to room temperature, and T4 DNA ligase was added and incubated at 37°C for 3 hours; phi 29 DNA polymerase, dNTPs and 1×phi 29 DNA polymerase buffer were added to the circularization reaction product, incubated at 37°C for 48 hours, and then heated at 75°C for 10 minutes; S102. The product obtained in step S101 is centrifuged at 12000r / min, the supernatant is removed, and then ultrapure water is added, and the centrifugation is washed 3 times. The obtained DNA nanoflower is re-dissolved in ultrapure water, reacted at 35-40°C, 300-700 rpm for 1-5h, the supernatant is removed by washing, deionized water is added, and stored in a -20°C refrigerator; S103. The DNA nanoflower obtained in step S102 is subjected to a ligation reaction with the capture probe CAP. The reaction conditions are incubation at room temperature for 20 minutes to obtain a DNA nanoflower with a capture probe.

5. The method according to claim 3, characterized in that: In step S1, the volume ratio of the circular probe CP to the connection probe LP is 1:2; the volume ratio of the DNA nanoflower to the capture probe CAP is 1:

10.

6. The method according to claim 3, characterized in that: In step S2, the mass ratio of TCEP to ssDNA is 10:1; the volume ratio of AuNPs to ssDNA is 4:

1.

7. The method according to claim 3, characterized in that: The reaction conditions of the ssDNA and the AuNPs in step S2 are shaking at 30° C. for 16 hours; sodium chloride is added at a concentration of 2 M and is added repeatedly 9 times; ssDNA-AuNPs are stored in PBS in a 4° C. refrigerator protected from light.

8. The method according to claim 3, characterized in that: The paper-based microfluidic chip in step S3 includes a sample loading area, a sample channel, a detection area, a water absorption channel and a water absorption area; wherein the paper base of the sample loading area and the sample channel adopts a sample pad and is treated with Tween 20, the detection area adopts a nitrocellulose membrane and fixes 3 μL of DNA nanoflowers with capture probes, and the water absorption channel and the water absorption area adopt a water absorption pad.

9. The method according to claim 3, characterized in that: The parameters of the laser engraving machine in step S3 are set to a speed of 90 mm / s and a power of 10%.

10. The method according to claim 3, characterized in that: The annealing treatment in step S4 is performed at a temperature of 95° C. and for a time of 5 minutes.

11. The method according to claim 3, characterized in that: The reaction temperature of the G-quadruplex chain and lead ions in step S4 is 37° C. and the reaction time is 30 min.

12. The method according to claim 3, characterized in that: The temperature of the mixed incubation of the Cas12a-crRNA system described in step S4 is 37° C. for 20 min; the temperature of the Cas12a-crRNA cleavage reaction is 37° C. for 30 min; and the treatment condition for inactivating the activity of the cas12a protein is 65° C. for 10 min.

13. The method according to claim 3, characterized in that: The Cas12a-crRNA system described in step S4 is prepared by mixing 9.75 μL buffer, 4 μL, 200 nM Cas12a, and 1.25 μL, 200 nM crRNA; the volume ratio of the reaction system to the Cas12a-crRNA system is 4:

1.

14. The method according to claim 3, characterized in that: The volume ratio of ssDNA-AuNPs to Cas12a-crRNA system described in step S4 is 2:

1.

15. The method according to claim 3, characterized in that: The concentration of the lead ion solution determined in step S4 ranges from 0.1 µM to 10 µM.

16. The method according to claim 3, characterized in that: The linear equation in step S4 or S5 is: R / G = 0.0082 C + 0.9722, R 2 = 0.9923.

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