CN117420299A - Wearable biosensor based on CRISPR-Cas12a system and application thereof - Google Patents
Wearable biosensor based on CRISPR-Cas12a system and application thereof Download PDFInfo
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- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention relates to the technical field of biomedical detection, and discloses a wearable biosensor based on a CRISPR-Cas12a system and application thereof, wherein the wearable biosensor based on the CRISPR-Cas12a system comprises a cover layer, an adhesive layer and a micro-channel micro-fluidic layer arranged between the cover layer and the adhesive layer, a detection unit is arranged on the micro-channel micro-fluidic layer, the detection unit comprises a first reaction chamber, a second reaction chamber and an outlet which are sequentially communicated, a component nucleic acid aptamer triggering probe to be detected is embedded in the first reaction chamber, a CRISPR/Cas12a system reagent is arranged in the second reaction chamber, an inlet is embedded in the adhesive layer, and the inlet penetrates through the adhesive layer and is communicated with the first reaction chamber. The invention combines the microfluidic technology with the CRISPR detection technology, realizes high-sensitivity and automatic detection of fluorescent signals of components to be detected on the wearable biosensor, and can be used for detecting chemical substances.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a wearable biosensor based on a CRISPR-Cas12a system and application thereof.
Background
Methamphetamine and cocaine are the most commonly abused drugs worldwide. Traditional detection methods for methamphetamine and cocaine include High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). While these methods provide excellent sensitivity, they rely heavily on expensive equipment and complex detection protocols. Furthermore, they are still susceptible to interference by biological matrices, leading to inaccurate results.
In recent years, sweat has become a valuable biological matrix for the study of compounds of interest due to its many advantages. For example, sweat collection is non-invasive, straightforward, safe, ensures strong privacy protection, and its composition is relatively uncomplicated. Studies have shown that traces of cocaine and methamphetamine appear in their sweat in one to two hours, peaking in about 24 hours. With this feature, a non-invasive test can be performed using the sweat sample. However, sweat sample analysis for illegal drug detection is currently performed using GC-MS or LC-MS, and still relies on complex and expensive detection instruments. Although enzyme-linked immunosorbent assays (ELISA) can also be performed in such matrices, a validated analysis by conventional techniques is also required.
In summary, a new method is explored for detecting illegal drugs in sweat samples, and a microfluidic patch (biosensor) is used as one of the current mainstream research directions, has the advantages of high detection speed and low cost, and can be used for detecting illegal drugs in sweat, but in the research process, two cavities are generally required to be arranged when the microfluidic patch is adopted for detection, illegal substances in sweat in one cavity react with an aptamer thereof and release cDNA, and the released cDNA then enters the other cavity to react and generate fluorescent signals. Although the detection of illegal substances can be realized through the characterization of fluorescent signals in the mode, the following problems exist in the detection process: if the reactants in the first reaction chamber do not react sufficiently, then the entry into the second reaction chamber will affect the generation of the fluorescent signal and the intensity of the fluorescent signal, thereby affecting the accuracy and sensitivity of the overall assay.
Disclosure of Invention
The invention aims to provide a wearable biosensor based on a CRISPR-Cas12a system and application thereof, and the method combines microfluidics and CRISPR detection technology to realize high-sensitivity and automatic detection of fluorescent signals of components to be detected on the wearable biosensor.
The invention provides a wearable biosensor based on a CRISPR-Cas12a system, which comprises a cover layer, an adhesive layer and a micro-channel micro-fluidic layer arranged between the cover layer and the adhesive layer, wherein a detection unit is arranged on the micro-channel micro-fluidic layer, the detection unit comprises a first reaction chamber, a second reaction chamber and an outlet which are sequentially communicated, a component nucleic acid aptamer trigger probe to be detected is embedded in the first reaction chamber, a CRISPR/Cas12a system reagent is embedded in the second reaction chamber, sucrose is filled in a communication pipeline of the first reaction chamber and the second reaction chamber, the communication pipeline is opened after sweat dissolves the sucrose, and an inlet is arranged on the adhesive layer, penetrates through the adhesive layer and is communicated with the first reaction chamber.
Further, the inlet is communicated with the first reaction chamber through a micro-channel, and a capillary blast valve is arranged on the micro-channel.
Further, an immunomagnetic bead is arranged in the first reaction chamber, and the aptamer trigger probe of the component to be detected is marked on the immunomagnetic bead.
Further, a magnetic structure is arranged between the microchannel microfluidic layer and the adhesion layer, and the magnetic structure is used for fixing the immunomagnetic beads in the first reaction chamber.
Further, the detection units are provided with at least two groups, and the first reaction chambers in all the detection units are connected with the inlet.
Further, the detection units are provided with two groups, and the first reaction chambers in the two groups of detection units are respectively embedded with immune magnetic beads marked by cocaine aptamer trigger probes and immune magnetic beads marked by methamphetamine aptamer trigger probes.
Further, the CRISPR/Cas12a system reagent is embedded in the second reaction chamber in the form of freeze-dried powder after quick freezing and low-temperature drying, the CRISPR/Cas12a system reagent comprises Cas12a-CrRNA, NEBuffer 2.1.1 buffer solution and fluorescent probes, and the NEBuffer 2.1 buffer solution mainly comprises NaCl, tris-HCl and MgCl 2 And BSA, wherein after the second reaction chamber is filled with sweat, the concentration of Cas12a-CrRNA in the second reaction chamber is 20nM, the concentration of fluorescent probe is 4 μM, the concentration of NaCl is 10mM, the concentration of Tris-HCl is 5mM, and MgCl 2 1mM, and BSA at 10. Mu.g/mL.
Further, the volume of the first reaction chamber is 10 mu L, the volume of the second reaction chamber is 25 mu L, and the diameter of a communication pipeline between the first reaction chamber and the second reaction chamber is 0.05mm;
the sucrose is filled on the communicating pipe by the following way:
2 mu L of sucrose solution with the concentration of 2.5mg/mL is buried in the connecting channel, and the sucrose is obtained after drying.
The invention also provides application of the wearable biosensor based on the CRISPR-Cas12a system, and the wearable biosensor based on the CRISPR-Cas12a system is worn on the surface of human skin and is used for detecting chemical substances in sweat.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention designs a wearable biosensor based on a CRISPR-Cas12a system by utilizing the CRISPR-Cas12a system, and the biosensor which can meet the reaction requirement of the CRISPR/Cas12a system is designed, then the aptamer and cDNA of cocaine and methamphetamine are selected, crRNA is designed, and then the concentration of the reagent of the CRISPR/Cas12a system is optimized, so that various components to be detected can be analyzed simultaneously, and the analyte in sweat is monitored in a non-invasive mode, thereby greatly simplifying the operation, and having the characteristics of high detection speed, high sensitivity, strong specificity, low cost, high reliability and the like;
(2) According to the invention, the communication pipelines are opened at fixed time in a mode of filling sucrose in the communication pipelines of the first reaction chamber and the second reaction chamber, so that the first reaction chamber has enough reaction time, the second reaction chamber can generate fluorescent signals with enough intensity, the reaction accuracy and sensitivity are improved, and compared with the method of directly setting a valve, the method is simple to operate, low in cost and higher in practicability;
(3) The biosensor in the invention utilizes the capillary burst valve, and sweat is driven by natural sweat pressure generated by permeation to sample sweat through the microfluidic structure, so that the biosensor can collect sweat in a timing and quantitative manner;
(4) The actual optimal incubation temperature of the CRISPR/Cas12a system is 37 ℃, so that the biosensor has wearable property, becomes an ideal platform for personal health monitoring by sweat analysis, and can collect, store and analyze sweat on site by using the biosensor, thereby realizing on-site real-time detection of cocaine and methamphetamine and preventing counterfeiting in the detection process.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic plan view of a wearable biosensor provided in an embodiment of the present invention;
fig. 2 is a schematic perspective view of a wearable biosensor provided in an embodiment of the present invention;
FIG. 3 is a graph showing the comparison of fluorescence intensity of methamphetamine at different concentrations provided in the examples of the present invention;
FIG. 4 is a graph showing the comparison of fluorescence intensity of cocaine at different concentrations according to the present invention;
FIG. 5 is a line graph of the log of concentration versus ΔF for a wearable biosensor provided in an embodiment of the present invention for detecting methamphetamine;
FIG. 6 is a line graph of the log of the concentration versus ΔF for a wearable biosensor according to an embodiment of the present invention;
FIG. 7 is a graph of Cas12a-CrRNA concentration versus ΔF for a wearable biosensor provided by an embodiment of the invention when methamphetamine is detected;
FIG. 8 is a graph of the relationship between the concentration of Cas12a-CrRNA and ΔF when the wearable biosensor provided by the embodiment of the invention detects cocaine;
FIG. 9 is a graph of CRISPR/Cas12a system reagent incubation temperature versus ΔF for a wearable biosensor provided by an embodiment of the present invention detecting methamphetamine;
FIG. 10 is a graph of CRISPR/Cas12a system reagent incubation temperature versus ΔF for a wearable biosensor provided by an embodiment of the present invention to detect cocaine;
FIG. 11 is a graph showing the comparison of ΔF for different substances using a wearable biosensor to detect methamphetamine according to an embodiment of the present invention;
FIG. 12 is a graph showing the comparison of the detection of ΔF of different substances using a wearable biosensor for detecting cocaine according to an embodiment of the present invention;
FIG. 13 is a graph showing the effect of embedding sucrose solutions with different concentrations on the pipeline opening time on the communication pipelines of the first reaction chamber and the second reaction chamber according to the embodiment of the present invention.
Reference numerals:
1. an inlet; 2. a first reaction chamber; 3. a second reaction chamber; 4. an outlet; 7. a cover layer; 8. a microchannel microfluidic layer; 9. an adhesive layer; 10. triggering a probe by a component nucleic acid aptamer to be detected; 11. CRISPR/Cas12a system reagent; 12. a magnetic structure.
Detailed Description
The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown.
The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention.
All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Wearable sensing technology can continuously monitor the health condition of individuals, and is important to realizing personalized medicine. The sensors in combination with wearable sensing technology can monitor analytes in sweat in a non-invasive manner to dynamically monitor the physiological health status of an individual. Human sweat is rich in a large number of potential health and disease related markers, and compared with conventional blood and urine detection, the human sweat has the advantages of non-invasive and real-time continuous monitoring, and the like, so that research on in-situ sweat sensors becomes an important branch of development in the field of wearable equipment.
However, the CRISPR-Cas system has received a great deal of attention in the detection field due to its remarkable sensitivity and specificity. Microfluidic is a technology for researching precise control and analysis of biochemical microfluidics in a micrometer scale isolation channel, can miniaturize functions of biological and chemical laboratories to patches of several square centimeters, has the characteristics of trace, low cost, high flux, integration, microminiaturization and the like, and presents a plurality of unique advantages which are incomparable with conventional macroscopic systems in research and application processes. The invention combines the microfluidic and CRISPR detection technologies, and realizes multi-target, high-sensitivity and automatic signal detection on a single patch.
Specifically, as shown in fig. 1 and 2, the embodiment of the invention provides a wearable biosensor based on a CRISPR-Cas12a system, the actual size of which can be freely adjusted, and the wearable property of which is considered, the wearable biosensor can be designed to be 60mm in diameter and 2mm in thickness. The biosensor comprises three layers, namely a cover layer 7, an adhesive layer 9 and a micro-channel micro-fluidic layer 8 arranged between the cover layer 7 and the adhesive layer 9, wherein the micro-channel micro-fluidic layer 8 is provided with a detection unit, the detection unit comprises a first reaction chamber 2, a second reaction chamber 3 and an outlet 4 which are sequentially communicated, and check valves are arranged between the adjacent first reaction chamber 2 and the second reaction chamber 3 and between the second reaction chamber 3 and the outlet 4 to avoid backflow. An inlet 1 is provided in the adhesive layer 9, the inlet 1 penetrating the adhesive layer 9 and communicating with the first reaction chamber 2.
In the process of preparing a wearable biosensor based on a CRISPR-Cas12a system, one of the important points is to construct a microfluidic layer with a complex microchannel design using PDMS materials and to layout the microchannels using AutoCAD software. In summary, the main templates for the micro-channels are created by wire-cutting of copper plates, then wire-cutting of electrodes, and finally pulse processing. Then, an optically transparent soft elastomer PDMS was cast on the master template of the template to prepare a PDMS microchannel plate. PDMS substrate and curing agent were mixed in a ratio of 10:1 (w/w) and vigorously stirred for about 5 minutes to eliminate any gas or foam formation. The PDMS prepolymer solution was poured into a master and cured at 80 ℃ for 2 hours. The sample was placed in a vacuum chamber for about 30 minutes to remove any trapped air bubbles prior to curing. Subsequently, the PDMS layer was carefully peeled off from the master template, and then precisely cut and perforated.
Secondly, it is also necessary to embed the aptamer trigger probe 10 of the component to be detected in the first reaction chamber 2, and the CRISPR/Cas12a system reagent 11 in the second reaction chamber 3 by flash freezing and cryogenic drying techniques.
Specifically, the CRISPR/Cas12a system reagent 11 is embedded in the second reaction chamber in the form of a lyophilized powder after being rapidly frozen and dried at a low temperature, which is different from a direct liquid embedded form, and the lyophilized powder can effectively prevent the biological characteristics of Cas12a protein from being changed. The CRISPR/Cas12a system reagent 11 comprises Cas12a-CrRNA, a buffer solution and a fluorescent probe, wherein in order to improve the cleavage activity of the Cas12a-CrRNA, the buffer solution is preferably NEBuffer 2.1, and the NEBuffer 2.1 buffer solution mainly comprises NaCl, tris-HCl and MgCl 2 And BSA.
Wherein, the mechanism of action of CRISPR/Cas12a system reagent 11 is: the CRISPR-Cas12a system utilizes crRNA to specifically recognize target double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), generating a fluorescent signal through the trans-cleavage activity of Cas12a, enabling signal amplification.
In Cas12a-CrRNA, CRISPR/Cas12 belongs to a class 2V RNA-guided endonuclease, crRNA is a shorthand for CRISPR RNA, crRNA is transcribed in the form of a precursor pre-CrRNA, tracrRNA is transcribed separately, tracrRNA and pre-CrRNA are combined by base pairing, sheared by RNaseIII, and combined with Cas12a into a complex with DNA cleavage capability.
Considering that the reaction time of the cleavage reporter probe of the first reaction chamber 2 and the second reaction chamber 3 cas12a system is 20-25min, this time may show a macroscopic fluorescent signal. In order to ensure that sweat can fill the first reaction chamber 2 and enter the second reaction chamber 3 within 20-25min, the volume of the first reaction chamber 2 is 10 μl and the volume of the second reaction chamber 3 is 25 μl in order to allow sweat to fill the second reaction chamber 3 within 20-25min, starting with a first drop of liquid entering the second reaction chamber 3.
If the target in the sweat entering the first reaction chamber 2 does not react with the aptamer of the first reaction chamber 2 sufficiently, the target enters the second reaction chamber 3, so that the amount of cDNA entering the second reaction chamber 3 is too small, and thus the amount of cDNA combined with CrRNA is too small, resulting in low fluorescence intensity, and thus negative reaction may be generated, and the accuracy and sensitivity of detection are affected.
Therefore, the invention also sets a timing opening and closing structure on the communication pipe of the first reaction chamber 2 and the second reaction chamber 3, the timing opening and closing structure can be a valve structure in the prior art, the control unit of the valve can be integrated with the control unit of the biosensor, but the built-in valve is more complex to operate and has higher cost, therefore, in the invention, preferably, the opening and closing structure is directly set as sucrose, the sucrose has a certain viscosity, the communication pipe can be blocked, and sweat and the like in the first reaction chamber 2 can be slowly dissolved when entering the communication pipe, so that the sweat enters the second reaction chamber 3.
Specifically, how to fill the sucrose in the communicating pipe may be performed by burying the sucrose in a sucrose solution, and evaporating the moisture in the sucrose solution by heating in an oven, in a specific embodiment:
for a first reaction chamber volume of 10 μl and a second reaction chamber volume of 25 μl, and a diameter of the communication line between the first reaction chamber 2 and the second reaction chamber 3 of 0.05mm, filling 2 μl of sucrose solution is preferable, and the concentration of sucrose solution is selected to affect the final opening time of the communication line, as shown in fig. 13, the present invention has also been studied, and as can be seen from fig. 13: the opening time of the communication line may be set at 20 to 25 minutes on the basis of ensuring that the first reaction chamber 2 has a sufficient reaction time (20 to 25 minutes), and in addition, a sucrose solution having a concentration of 2.5mg/mL is preferably selected in view of shortening the time for on-site detection.
Thus, sucrose is filled in the communicating pipe by: 2 mu L of sucrose solution with the concentration of 2.5mg/mL is buried in the connecting channel, and then the sucrose is obtained after drying, wherein the specific drying temperature and the drying time can be set by themselves as long as the evaporation of water in the sucrose solution can be realized, the temperature is generally not more than 65 ℃, otherwise, the sucrose can turn yellow, for example, the sucrose can be heated in an oven at 50+/-2 ℃ for 1h. Finally, the cover layer and the micro-channel micro-fluidic layer are connected; in use, sweat is allowed to flow into the patch and time is recorded.
The concentration of each component in a particular CRISPR/Cas12a system reagent 11 is preferably such that the following conditions are met: after the second reaction chamber was filled with sweat, the concentration of fluorescent probe was 4. Mu.M, the concentration of NaCl was 10mM, the concentration of Tris-HCl was 5mM, mgCl 2 The concentration of (B) was 1mM, and the concentration of BSA was 10. Mu.g/mL.
And after the second reaction chamber is filled with sweat, considering that the concentration of the critical component Cas12a-CrRNA in the CRISPR/Cas12a system reagent 11 is too low, the number of cdnas that a small amount of CrRNA recognizes through Cas12a protein is small, the ability of Cas12a protein to activate cleavage is small, and the number of cleaved reporter probes is small, resulting in a decrease in fluorescence signal. On the other hand, since cas12a protein is expensive, the fluorescence signal is not increased even if the concentration is too high. Specifically, as shown in fig. 7 and 8, for different components to be detected (cocaine and methamphetamine), the concentrations of the other components are kept unchanged, the Cas12a-CrRNA concentrations are compared by gradient experiments from 5 nM, 10 nM, 15 nM, 20nM, 25 nM, and finally 20nM is determined to be the optimal concentration.
Here, the component to be detected may be cocaine, methamphetamine, or chemical substances such as dopamine, and the like, and is mainly characterized in that an appropriate aptamer and cDNA chain can be provided, and the selection of the aptamer is performed according to practical application.
In addition, cas12a protein activity is temperature dependent, and too low or too high a temperature can affect Cas12a protein activity, thereby reducing fluorescence signal. Therefore, the invention also carries out optimal design on the incubation temperature of the CRISPR/Cas12a system, as shown in fig. 9 and 10, carries out comparison experiments aiming at different incubation temperatures of 30 ℃, 37 ℃, 40 ℃, 45 ℃ and 50 ℃, selects the optimal reaction temperature at 37 ℃ to be exactly consistent with the temperature of a human body, so that the biosensor in the invention has wearable property, and can be worn on the skin surface of the human body for detecting the chemical substances in sweat aiming at the detection field of the chemical substances.
The method for detecting by using the wearable biosensor based on the CRISPR-Cas12a system comprises the following steps:
step 1, attaching a wearable epidermis microfluidic patch to human skin;
step 2, utilizing a valve channel to flow human sweat from an inlet 1 into a first reaction chamber 2 so that a component to be detected in the sweat and a corresponding nucleic acid aptamer trigger a probe to react and release cDNA;
step 3, enabling the cDNA released in the step 2 to flow into a second reaction chamber 3 along with sweat, wherein the optimal temperature of a CRISPR reaction system is 37 ℃, the CRISPR reaction system is close to the skin temperature of a human body, and the temperature provided by the human body is used as the reaction temperature so that the cDNA and the CRISPR/Cas12a system reagent 11 are mixed and react and generate a fluorescent signal;
and 4, checking a fluorescent signal under an ultraviolet lamp, and judging the content of the component to be detected according to the fluorescent intensity.
Further, by disposing an immunomagnetic bead in the first reaction chamber 2, the aptamer trigger probe 10 of the component to be detected is labeled on the immunomagnetic bead. And a magnetic structure 12, such as a small magnetic sheet, is arranged between the microchannel microfluidic layer 8 and the adhesion layer 9, so that the small magnetic sheet can fix the immunomagnetic beads in the first reaction chamber 2, and the strand displacement reaction between cDNA and CrRNA is prevented.
For different numbers of components to be detected, a specific number of detection units may be provided, for example the detection units are provided with at least two groups, all the first reaction chambers 2 in the detection units being connected to the inlet 1.
In order to ensure that the release of cDNA and the reaction of CRISPR/cas12a can be sequentially carried out, the invention also communicates with the first reaction chamber 2 through a micro-channel at the inlet 1, a capillary burst valve is arranged on the micro-channel, and sweat is driven to be sampled through a micro-fluidic structure by virtue of natural sweat pressure generated by permeation, so that the biosensor can collect sweat in a timed and quantitative manner.
In a specific embodiment, the CRISPR/Cas12a system is used in combination with a microfluidic patch, using methamphetamine and cocaine specific aptamers as signal-mediated intermediaries to convert methamphetamine and cocaine into nucleic acid signals, followed by single-stranded DNA generation to activate Cas12 protein. This method allows sweat to be collected, stored and analyzed in situ. Thereby realizing the on-site real-time detection of cocaine and methamphetamine and preventing the counterfeits in the detection process.
Specifically, the nucleotide sequence of the cocaine aptamer is shown as SEQ ID No.1, the nucleotide sequence of the cocaine cDNA is shown as SEQ ID No.2, the nucleotide sequence of the cocaine CrRNA is shown as SEQ ID No.3, the nucleotide sequence of the methamphetamine aptamer is shown as SEQ ID No.4, the nucleotide sequence of the methamphetamine cDNA is shown as SEQ ID No.5, and the nucleotide sequence of the methamphetamine CrRNA is shown as SEQ ID No. 6.
The detection units are provided with two groups, and the first reaction chambers 2 in the two groups of detection units are respectively embedded with immune magnetic beads marked by cocaine aptamer trigger probes and immune magnetic beads marked by methamphetamine aptamer trigger probes, so that the cocaine and methamphetamine can be analyzed simultaneously.
The cocaine aptamer trigger probe labeled magnetic bead or methamphetamine aptamer trigger probe labeled magnetic bead is prepared by the following steps:
suspending 10 mu L of streptavidin-modified immunomagnetic beads in 10 mu L of 0.1M PBS buffer solution to obtain diluted immunomagnetic bead solution;
isocratic mixing cocaine aptamer or methamphetamine aptamer with blocking-chain DNA (cDNA) at a concentration of 1 mu M, heating the mixture to 95 ℃ for 5 minutes, and gradually cooling to room temperature to form double chains to obtain a cocaine or methamphetamine aptamer trigger probe;
adding the obtained cocaine or methamphetamine aptamer trigger probe into diluted immunomagnetic bead solution, fully mixing for 2 hours at room temperature, and then performing magnetic separation and washing to obtain immunomagnetic beads (APTPL-MBs) with a final concentration of 1 mu M, which are marked by the cocaine or methamphetamine aptamer trigger probe.
The method for detecting cocaine and methamphetamine by using the wearable biosensor based on the CRISPR-Cas12a system comprises the following steps:
(a) Trace amounts of methamphetamine or cocaine in the sweat sample interact with its aptamer, resulting in release of cDNA of the probe triggered by cocaine or methamphetamine aptamer into solution.
(b) After magnetic separation, the cDNA released in solution was transferred into CRISPR-Cas12a reporter cell solution containing Cas12a, crRNA and fluorescent probes. The programmability of the CRISPR-Cas12a system is utilized to design cDNA as activator of Cas12a, complementary to the crRNA portion.
(c) After activating DNase activity of the Cas12a-crRNA complex, activating cleavage of the reporter gene, recovering fluorescence of the reporter gene, and increasing COC/METH in the sample, increasing released blocking strand DNA, so that the stronger the activity of the Cas12-crRNA complex, the stronger the fluorescence signal.
Using this protocol, the invention selects for an aptamer of cocaine and methamphetamine and a cDNA, specifically, the aptamer is selected to have a high affinity for the target, and the cDNA is selected to hybridize to the aptamer to form a stable duplex with a weaker affinity than the aptamer to the target. Then, crRNA, which is at least 18bp in base pairing length with cDNA, is designed to form an R-loop structure to a stable checkpointed state, where Cas12a adopts an active conformation in preparation for subsequent cleavage of the target DNA. The base pairing length of crRNA and cDNA is best about 20-23bp, too little can not react, and too much increases the synthesis difficulty of RNA.
Further, the invention also carries out quantitative detection on cocaine and methamphetamine, and the primer sequences, the crRNA sequences and the fluorescent probe sequences are shown in table 1:
table 1 primer sequence, crRNA sequence and fluorescent probe sequence table
Under the aforementioned experimental conditions, the present invention incubates samples containing different concentrations of methamphetamine and cocaine in sweat with cocaine or methamphetamine aptamer-triggered probe-labeled immunomagnetic beads and CRISPR/Cas12a systems to assess the quantitative analytical ability of the method on these substances. Transfer of released blocker DNA in these samples into the reporter solution as shown in fig. 3 and 4, we observed a proportional increase in fluorescence intensity at 510 nm with increasing methamphetamine and cocaine concentrations. This observation suggests that higher concentrations of methamphetamine and cocaine lead to more blocker DNA release. As shown in fig. 5 and 6, regression equations of concentrations and Δf (%) for methamphetamine and cocaine were y= 23.67651 lg c-25.247.7 (R, respectively 2 = 0.9869) and y= 3.79107 lg c+ 24.21123 (R) 2 = 0.9854). Wherein, Δf (%): percent change in relative fluorescence intensity; the specific calculation mode is as follows: the average value of the fluorescence intensities of the groups is calculated by experiments, and then the maximum average value of the group is taken as 100 percent, and the percentages of other values are calculated.
The detection by using the method shows good detection performance, and the biosensor provided by the invention can be used for rapidly detecting methamphetamine and cocaine in sweat. A considerable fluorescent signal can be detected by reacting in the second reaction chamber 3 for about 10 minutes, so that the end user can complete the analysis in about 35 minutes. The capillary burst valve of the biosensor has no reflux, ensures the release of cDNA and the easy sequential combination of CRISPR/cas12a, greatly simplifies the operation and achieves the aim of experiments.
To evaluate the specificity and sensitivity of the biosensor of the present invention, we performed specificity experiments for four different compounds other than methamphetamine and Cocaine, glucose (GLU), dopamine (DA), amoxicillin and Propranolol (PR), as shown in FIGS. 11 and 12, FIG. 11, methyl benzolactam (METH, 100 ng/mL), glucose (GLU, 1000 ng/mL), dopamine (DA, 1000 ng/mL), cocaine (Cocaine, 1000 ng/mL), amoxicillin (Amoxil, 1000 ng/mL), propranolol (PR, 1000 ng/mL), mixtures (METH, 100ng/mL, GLUDA, cocaine, amoxil, PR 1000 ng/mL); fig. 12: cocaine (Cocaine, 100 ng/mL), glucose (GLU, 1000 ng/mL), dopamine (DA, 1000 ng/mL), methyl-phenyllactam (METH, 1000 ng/mL), amoxic (Amoxil, 1000 ng/mL), propranolol (PR, 1000 ng/mL), mix (Cocaine, 100ng/mL, glada, METH, amoxil, PR 1000 ng/mL). The biosensor in the invention has strong detection specificity to methamphetamine and cocaine by taking delta F larger than 25% as a detection judgment standard. Furthermore, it can be seen from a combination of FIGS. 3 and 4 that the ΔF fluorescence intensity is 2-3 times that of the blank at a solution concentration of 0.1ng/mL, and is almost close to the blank at 0.05, so that the sensitivity of 0.1ng/mL can be used to determine whether or not there is any drug in sweat.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (9)
1. Wearable biosensor based on CRISPR-Cas12a system, characterized by, including overburden (7), adhesion layer (9) and set up microchannel microfluidic layer (8) between overburden (7) and adhesion layer (9) microchannel microfluidic layer (8) are provided with detecting element, detecting element is including first reaction chamber (2), second reaction chamber (3) and export (4) that communicate in order, first reaction chamber (2) have embedded in it to wait to detect ingredient nucleic acid aptamer trigger probe (10), have embedded in second reaction chamber (3) CRISPR/Cas12a system reagent (11) in the intercommunication pipeline of first reaction chamber (2) and second reaction chamber (3) is filled with the sucrose, after the sweat is dissolved the sucrose, the intercommunication pipeline is opened, be provided with entry (1) in adhesion layer (9), entry (1) run through adhesion layer (9) and with first reaction chamber (2) intercommunication.
2. The wearable biosensor based on the CRISPR-Cas12a system according to claim 1, characterized in that the inlet (1) communicates with the first reaction chamber (2) through a micro-channel on which a capillary burst valve is provided.
3. The wearable biosensor based on the CRISPR-Cas12a system according to claim 1, characterized in that an immunomagnetic bead is provided within the first reaction chamber (2), on which immunomagnetic bead the component nucleic acid aptamer trigger probe (10) to be detected is labeled.
4. Wearable biosensor based on the CRISPR-Cas12a system according to claim 1, characterized in that a magnetic structure (12) is provided between the microchannel microfluidic layer (8) and the adhesion layer (9), the magnetic structure (12) being used to immobilize the immunomagnetic beads within the first reaction chamber (2).
5. Wearable biosensor based on CRISPR-Cas12a system according to claim 1, characterized in that the detection units are provided with at least two sets, all the first reaction chambers (2) in the detection units being connected with the inlet (1).
6. The wearable biosensor based on the CRISPR-Cas12a system according to claim 5, wherein the detection units are provided with two groups, and the first reaction chambers (2) in the two groups of detection units are respectively embedded with cocaine aptamer trigger probe-labeled immunomagnetic beads and methamphetamine aptamer trigger probe-labeled immunomagnetic beads.
7. According to claimThe wearable biosensor based on the CRISPR-Cas12a system according to claim 1, wherein the CRISPR/Cas12a system reagent (11) is embedded in the second reaction chamber in the form of a lyophilized powder after being flash frozen and dried at low temperature, the CRISPR/Cas12a system reagent (11) comprises Cas12a-CrRNA, NEBuffer 2.1 buffer and fluorescent probe, the NEBuffer 2.1 buffer is mainly composed of NaCl, tris-HCl, mgCl 2 And BSA, wherein after the second reaction chamber is filled with sweat, the concentration of Cas12a-CrRNA in the second reaction chamber is 20nM, the concentration of fluorescent probe is 4 μM, the concentration of NaCl is 10mM, the concentration of Tris-HCl is 5mM, and MgCl 2 1mM, and BSA at 10. Mu.g/mL.
8. The wearable biosensor based on the CRISPR-Cas12a system according to claim 1, characterized in that the volume of the first reaction chamber (2) is 10 μl, the volume of the second reaction chamber (3) is 25 μl, the diameter of the communication line of the first reaction chamber (2) and the second reaction chamber (3) is 0.05mm;
the sucrose is filled on the communicating pipe by the following way:
2 mu L of sucrose solution with the concentration of 2.5mg/mL is buried in the connecting channel, and the sucrose is obtained after drying.
9. Use of the CRISPR-Cas12a system based wearable biosensor according to any one of claims 1-8, characterized in that the CRISPR-Cas12a system based wearable biosensor is worn on the skin surface of a human body for detecting chemicals in sweat.
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