CN115112741B - Method for detecting RNA by graphene field effect transistor and application - Google Patents
Method for detecting RNA by graphene field effect transistor and application Download PDFInfo
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Abstract
The invention provides a method for detecting RNA by a graphene field effect transistor and application thereof, comprising the following steps: s1: growing graphene on a copper substrate by a chemical vapor deposition method; s2: the graphene prepared in the step S1 is transferred onto a glass substrate plated with an ITO electrode by a wet method; s3: a preset liquid reaction tank is additionally arranged on the glass substrate in the S2, so that a graphene field effect transistor with the liquid reaction tank is formed; s4: functionalizing the graphene in the graphene field effect transistor in the S3 through a functional solution, so as to anchor a report probe with a specific design; s5: extracting RNA to be detected, mixing with a nucleic acid shearing system, and incubating to obtain a mixed solution; s6: adding the mixed solution into a liquid reaction tank in the graphene field effect transistor for reaction; s7: and flushing the liquid reaction tank, analyzing the transfer characteristic curve of the field effect transistor of the graphene, and reading the detection signal. The invention has the advantages of high principle breakthrough, simple device preparation and low cost.
Description
[ Field of technology ]
The invention relates to the technical field of biological genes, in particular to a method for detecting RNA by a graphene field effect transistor and application thereof.
[ Background Art ]
The graphene field effect transistor has the advantages of high sensitivity, easy integration and the like, and is widely focused by scientific researchers in the fields of biochemical analysis and molecular diagnosis. The Alzheimer's disease, down's three-body syndrome, and the high-sensitivity detection of various cancers are successively analyzed and detected by a field effect transistor based on graphene. Usually, researchers usually fix probes of disease molecules, such as nucleic acid molecules or proteins, on the surface of graphene, and capture target molecules in an analysis solution by utilizing the specific binding between the nucleic acid molecules and the nucleic acid molecules or between antibodies and antigens, and the captured target molecules influence certain electrical properties of the graphene due to electrification or scattering effect on the graphene, so that effective detection signals for analysis can be obtained. The detection mechanism can be classified as a graphene surface fixed gripper, and the accumulation change of signals is realized by continuously grabbing target molecules, so that the detection mechanism belongs to 'small addition' signals. The properties of small addition signals make it difficult for graphene field effect transistors to achieve efficient detection of low concentration target molecules. In addition, the movement of the dirac point of graphene is an analysis signal which is most commonly selected in the use of a graphene field effect transistor for biochemical analysis and molecular diagnosis, and generally the dirac point moves leftwards along with the increase of target molecules gripped by probe molecules. However, in actual detection, due to instability of graphene in an analyte solution, even if no target molecule is captured, the dirac point of graphene spontaneously shifts to the left over time. The large noise signal is caused to the graphene field effect transistor for detecting the biomolecules, so that the detection signal has large nonspecific property, and the detection result cannot be effectively convinced. The graphene field effect transistor is required to be improved and innovated for biochemical analysis and molecular diagnosis from the aspect of signal generation mechanism to further improve the detection limit and improve the signal reliability.
Accordingly, there is a need to investigate methods and applications of graphene field effect transistors to detect RNA to address the deficiencies of the prior art, to solve or mitigate one or more of the problems described above.
[ Invention ]
In view of the above, the invention provides a method for detecting RNA by using a graphene field effect transistor and application thereof, and the method can effectively realize high-precision and high-specificity detection of novel coronavirus RNA by preparing the graphene field effect transistor capable of generating a large subtraction signal and a high-reliability signal in detection of disease RNA.
In one aspect, the present invention provides a method for detecting RNA by using a graphene field effect transistor, the method for detecting RNA comprising:
S1: preparing a graphene field effect transistor with a liquid reaction tank;
S2: functionalizing graphene in the graphene field effect transistor in the S1 through a functional solution, so as to anchor a report probe with a specific design;
S3: extracting RNA to be detected, mixing with a nucleic acid shearing system, and incubating to obtain a mixed solution;
s4: adding the mixed solution into a liquid reaction tank in the graphene field effect transistor for reaction;
S5: and flushing the liquid reaction tank, analyzing the transfer characteristic curve of the field effect transistor of the graphene, and reading the detection signal.
In the aspect and any possible implementation manner as described above, there is further provided an implementation manner, where the S1 specifically includes:
s11: growing graphene on a copper substrate by a chemical vapor deposition method;
s12: wet-transferring the graphene prepared in S11 onto a glass substrate plated with an ITO electrode;
s13: and (3) adding a preset liquid reaction tank on the glass substrate in the step (S12) to form the graphene field effect transistor with the liquid reaction tank.
In the aspect and any possible implementation manner described above, there is further provided an implementation manner, wherein the liquid reaction tank in S13 is polymethyl methacrylate material PMMA, the adding method is ultraviolet glue curing for half an hour, and the graphene field effect transistor is a liquid gate type graphene field effect transistor.
In aspects and any one of the possible implementations described above, there is further provided an implementation, wherein the nucleic acid cleavage system in S3 is a CRISPR/Cas13a cleavage system.
In the aspect and any possible implementation manner described above, there is further provided an implementation manner, wherein the specifically designed reporter probe in S2 is an rna+dna composite structure, the solute of the functional solution is 1-pyrene butyrate succinimide ester PBASE, the solvent is dimethylformamide, the concentration is 100mM/L, and the functionalization time is 1 hour.
In aspects and any of the possible implementations described above, there is further provided an application of the graphene field effect transistor for detecting RNA, in particular for detecting novel coronavirus RNA.
In aspects and any of the possible implementations described above, further provided is an implementation in which the specifically designed reporter probe sequence is NH 2 -5-UUUUUU + TTTTTTTTTTTTTTTT-3, in which the 5' end modified amino NH 2 is used to bind to 1-pyrene butyrate succinimide ester and thereby be anchored to the graphene surface, six RNAs are cleavage sites for activated CRISPR/Cas13a, and 16 DNAs are signal generation sites.
In aspects and any of the possible implementations described above, there is further provided an implementation in which the CRISPR/Cas13a cleavage system designs four different CRISPRs, wherein the four Guide RNAs correspond to three specific sites of the N region gene and one specific site of the E region gene in the new coronavirus RNA sequence, respectively.
Compared with the prior art, the invention can obtain the following technical effects:
1. According to the method, the nonspecific signal caused by spontaneous left shift of the Dirac point along with the time due to instability of graphene in the analysis liquid in the traditional method is avoided, and compared with the left shift signal generation mechanism in the traditional method, the generation mechanism of the right shift signal in the method enables the obtained signal to have high reliability and purity;
2. According to the invention, tens of thousands of specially designed report probes can be sheared at will by activating a CRISPR/Cas13a nucleic acid shearing system by a new coronavirus RNA, so that the obtained accumulation and subtraction signals belong to the large subtraction signals, and the addition of the CRISPR/Cas13a nucleic acid shearing system enables the effect of generating signals of the new coronavirus RNA to be tens of thousands times greater than the effect of generating signals under a mechanism of simple grabbing in a traditional detection method;
3. The invention is not only limited to the quantitative detection of the novel coronavirus RNA, but also can be popularized to the detection of any virus RNA molecule;
4. the invention discloses a method for realizing 'large subtraction' and obtaining a novel coronavirus RNA detection signal with high specificity based on a CRISPR/Cas13a nucleic acid shearing system composite field effect transistor for the first time.
Of course, it is not necessary for any of the products embodying the invention to achieve all of the technical effects described above at the same time.
[ Description of the drawings ]
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a liquid gate type graphene field effect transistor with an anchored reporter probe according to the present invention;
FIG. 2 is a schematic representation of the CRISPR/Cas13a cleavage system of the invention specifically recognizing novel coronavirus RNA and being activated;
FIG. 3 is a schematic diagram of a CRISPR/Cas13a cleavage system cleavage graphene surface reporter probe after activation by a novel coronavirus RNA of the present invention;
FIG. 4 is a graph showing the transfer characteristic curves of the graphene field effect transistor of the present invention for implementing a novel coronavirus RNA detection process;
FIG. 5 is a schematic diagram and a result diagram of detection of RNA in N region of a novel coronavirus by a CRISPR/Cas13a shear system composite graphene field effect transistor;
fig. 6 is a graph of the results of full-length RNA detection of a CRISPR/Cas13a shear system composite graphene field effect transistor versus a new coronavirus actual sample of the present invention.
In the figure, glass is a Glass substrate, and ITO is an indium tin oxide electrode; source is drain; drain is the source; gate is the Gate; PMMA is a polymethyl methacrylate material reaction tank; ΔPre-cut signal is the amount of Pre-cut signal change; RNP is short for CRISPR and Cas13a protein complex; PBASE is 1-pyrene butyric acid succinimidyl ester; probes are reporter probes.
[ Detailed description ] of the invention
For a better understanding of the technical solution of the present invention, the following detailed description of the embodiments of the present invention refers to the accompanying drawings.
It should be understood that the described embodiments are merely some, but not all, 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.
The terminology used in the embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The invention provides a method for detecting RNA by a graphene field effect transistor and application thereof, wherein the method for detecting RNA comprises the following steps:
S1: preparing a graphene field effect transistor with a liquid reaction tank;
S2: functionalizing graphene in the graphene field effect transistor in the S1 through a functional solution, so as to anchor a report probe with a specific design;
S3: extracting RNA to be detected, mixing with a nucleic acid shearing system, and incubating to obtain a mixed solution;
s4: adding the mixed solution into a liquid reaction tank in the graphene field effect transistor for reaction;
S5: and flushing the liquid reaction tank, analyzing the transfer characteristic curve of the field effect transistor of the graphene, and reading the detection signal.
The S1 specifically comprises the following steps:
s11: growing graphene on a copper substrate by a chemical vapor deposition method;
s12: wet-transferring the graphene prepared in S11 onto a glass substrate plated with an ITO electrode;
s13: and (3) adding a preset liquid reaction tank on the glass substrate in the step (S12) to form the graphene field effect transistor with the liquid reaction tank.
The liquid reaction tank in the step S13 is made of polymethyl methacrylate (PMMA), the adding method is ultraviolet glue curing for half an hour, and the graphene field effect transistor is a liquid gate type graphene field effect transistor.
The nucleic acid cleavage system in S3 is a CRISPR/Cas13a cleavage system.
The report probe specifically designed in the S2 is of an RNA+DNA composite structure, the solute of the functional solution is 1-pyrene butyrate succinimide ester PBASE, the solvent is dimethylformamide, the concentration is 100mM/L, and the functionalization time is 1 hour.
The invention also provides an application of the graphene field effect transistor in RNA detection, and the application is particularly used for detecting novel coronavirus RNA. The specifically designed reporter probe sequence is NH 2 -5-UUUUUU + TTTTTTTTTTTTTTTT-3, wherein the 5' -end modified amino NH 2 is used for combining with 1-pyrene butyrate succinimide ester so as to be anchored on the surface of graphene, six RNAs are activated CRISPR/Cas13a cleavage sites, and 16 DNAs are signal generation sites. The CRISPR/Cas13a shearing system designs four different CRISPRs, wherein four gRNAs respectively correspond to three specific sites of an N region gene and one specific site of an E region gene in a novel coronavirus RNA sequence.
Example 1
The invention provides a method for detecting RNA by a graphene field effect transistor and application thereof, which are realized based on a CRISPR/Cas13a shearing system and the graphene field effect transistor, wherein the method specifically comprises the following steps:
step 1, growing graphene on a copper substrate by using a chemical vapor deposition method;
Step 2, transferring graphene onto a glass substrate plated with an ITO electrode through a wet method;
Step 3, adding a customized liquid reaction tank to form a liquid grid type graphene field effect transistor;
step 4, functionalizing graphene through 1-pyrene butyrate succinimide ester;
Step 5, anchoring a report probe with specific design; fig. 1 is a schematic diagram of a liquid gate type graphene field effect transistor with an anchored reporting probe according to the present invention.
Step 6, according to the mass 2:1, preparing a CRISPR/Cas13a shear system mixed solution;
step 7, mixing the novel coronavirus RNA with a CRISPR/Cas13a shearing system mixed solution, and incubating at 37 ℃ for one hour;
Step 8, dripping the mixed solution into a reaction tank of a field effect transistor after incubation is completed, and reacting for two hours at 37 ℃;
And 9, flushing the reaction tank by using 0.1X phosphate buffer solution, and then testing the transfer characteristic curve of the field effect transistor of the graphene by using a semiconductor characteristic analysis tester to read the detection signal.
FIG. 2 (a) is a schematic diagram of the CRISPR-CAS13a system of the invention, FIG. 2 (b) is a schematic diagram of the Guide RNA in the CRISPR-CAS13a system of the invention recognizing a specific site of a novel coronavirus RNA, and FIG. 2 (c) is a schematic diagram of the CRISPR-CAS13a system of the invention activated by a specific novel coronavirus RNA.
FIG. 3 is a schematic diagram of a CRISPR-Cas13a system of the invention after activation by a target novel coronavirus RNA cleaving a reporter probe anchored to a graphene surface. (a) The CRISPR-CAS13a system is in a non-shearing active state under the condition that no target new coronavirus RNA exists, and the CRISPR-CAS13a system is in an active state under the condition that new coronavirus RNA exists, so that RNA sites of a report probe can be effectively sheared.
FIG. 4 is a schematic representation of the detection of novel coronavirus RNA at a concentration of 0.5aM according to the present invention. Wherein the red line represents the graphene transfer characteristic curve after being functionalized by PBASE; wherein the blue line represents the graphene transfer characteristic curve anchored to the reporter probe; wherein the green line represents the graphene transfer characteristic curve of the CRISPR-Cas13a system after being sheared by the anchored reporter probe after activation with a concentration of 0.5aM of novel coronavirus RNA; it is clear that the novel coronavirus RNA detection signal is a signal that shifts to the right. Injecting; the lowest point of the transfer characteristic curve is a graphene dirac point, and the graphene dirac point is used as a characterization signal.
FIG. 5 (a) is a schematic representation of three different CRISPR-Cas13a systems of the invention recognizing three different specific sites of a novel coronavirus N region gene. FIG. 5 (b) is a graph showing the detection results of the CRISPR/Cas13a nucleic acid cleavage system of the present invention in a 50 microliter system for the concentration of novel coronavirus N-region RNA from 0.1aM to 10aM in a composite graphene field effect transistor.
Fig. 6 (a) is a graph showing the detection result of the CRISPR/Cas13a nucleic acid cleavage system composite graphene field effect transistor of the present invention on the actual sample new coronavirus full-length RNA in a 50 microliter system at a concentration of 0.1aM to 10aM, and fig. 6 (b) is a graph showing the detection result of the CRISPR/Cas13a nucleic acid cleavage system composite graphene field effect transistor of the present invention on the other five actual patient sample new coronavirus full-length RNA at a concentration of 0.25aM in a 50 microliter system.
Compared with the prior art, the CRISPR/Cas13a nucleic acid shearing system composite graphene field effect transistor is used for detecting novel coronavirus RNA, combines graphene and CRISPR/Cas13a nucleic acid shearing systems, and can fully exert the advantages of the two: since in this system for detecting novel coronavirus RNA the specifically designed reporter probe is pre-anchored, CRISPR/Cas13a will be activated after detecting novel coronavirus RNA and then cleave the pre-anchored specifically designed reporter probe, which is the cleavage of signal molecules, unlike the capture of traditional methods, the signal obtained is not an accumulated signal, but rather an subtracted signal. The detected dirac point is continuously shifted to the right instead of the traditional one, which completely and thoroughly avoids the nonspecific signal caused by spontaneous left shift of the dirac point over time due to instability of graphene in the analysis solution in the traditional method. The generation mechanism of the right shift signal in the method is higher in reliability and pureness than that of the left shift signal in the traditional method. At the same time, the CRISPR/Cas13a nucleic acid cleavage system can be activated by a new coronavirus RNA to randomly cleave tens of thousands of specially designed reporter probes, so that the obtained subtraction signal belongs to the subtraction signal of 'large subtraction'. The addition of the CRISPR/Cas13 nucleic acid cleavage system allows a new coronavirus RNA to generate a signal several tens of thousands of times greater than the signal generated by the mechanism of simple capture in conventional detection methods.
The key point is that the invention is a method for realizing 'large subtraction' and obtaining a novel coronavirus RNA detection signal with high specificity based on a CRISPR/Cas13 nucleic acid shearing system composite field effect transistor for the first time.
The method for detecting RNA by the graphene field effect transistor and the application thereof provided by the embodiment of the application are described in detail. The above description of embodiments is only for aiding in the understanding of the method of the present application and its core ideas; meanwhile, as those skilled in the art will have variations in the specific embodiments and application scope in accordance with the ideas of the present application, the present description should not be construed as limiting the present application in view of the above.
Certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will appreciate that a hardware manufacturer may refer to the same component by different names. The description and claims do not take the form of an element differentiated by name, but rather by functionality. As referred to throughout the specification and claims, the terms "comprising," including, "and" includes "are intended to be interpreted as" including/comprising, but not limited to. By "substantially" is meant that within an acceptable error range, a person skilled in the art is able to solve the technical problem within a certain error range, substantially achieving the technical effect. The description hereinafter sets forth a preferred embodiment for practicing the application, but is not intended to limit the scope of the application, as the description is given for the purpose of illustrating the general principles of the application. The scope of the application is defined by the appended claims.
It should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a product or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such product or system. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a commodity or system comprising such elements.
It should be understood that the term "and/or" as used herein is merely one relationship describing the association of the associated objects, meaning that there may be three relationships, e.g., a and/or B, may represent: a exists alone, A and B exist together, and B exists alone. In addition, the character "/" herein generally indicates that the front and rear associated objects are an "or" relationship.
While the foregoing description illustrates and describes the preferred embodiments of the present application, it is to be understood that the application is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of numerous other combinations, modifications and environments and is capable of changes or modifications within the scope of the inventive concept as expressed herein, either as a result of the foregoing teachings or as a result of the knowledge or technology of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the application are intended to be within the scope of the appended claims.
Claims (3)
1. A method for detecting RNA by a graphene field effect transistor, the method comprising: s1: preparing a graphene field effect transistor with a liquid reaction tank;
S2: functionalizing graphene in the graphene field effect transistor in the S1 through a functional solution, so as to anchor a report probe with a specific design;
S3: extracting RNA to be detected, mixing with a nucleic acid shearing system, and incubating to obtain a mixed solution;
s4: adding the mixed solution into a liquid reaction tank in the graphene field effect transistor for reaction;
s5: flushing a liquid reaction tank, analyzing a transfer characteristic curve of a field effect transistor of graphene, and reading a detection signal;
The S1 specifically comprises the following steps: s11: growing graphene on a copper substrate by a chemical vapor deposition method;
s12: wet-transferring the graphene prepared in S11 onto a glass substrate plated with an ITO electrode;
s13: a preset liquid reaction tank is additionally arranged on the glass substrate in the S12, and a graphene field effect transistor with the liquid reaction tank is formed;
the liquid reaction tank in the step S13 is made of polymethyl methacrylate (PMMA), the adding method is ultraviolet glue curing for half an hour, and the graphene field effect transistor is a liquid gate type graphene field effect transistor;
the nucleic acid cleavage system in S3 is a CRISPR/Cas13a cleavage system;
The report probe specifically designed in the S2 is of an RNA+DNA composite structure, the solute of the functional solution is 1-pyrene butyrate succinimide ester PBASE, the solvent is dimethylformamide, the concentration is 100mM/L, and the functionalization time is 1 hour;
The reaction time in the step S4 is 2 hours, and the reaction temperature is 37 ℃;
the incubation time in the step S3 is 1 hour, and the temperature is 37 ℃;
The sequence of the report probe anchored in the S2 is NH2-5-UUUUUU + TTTTTTTTTTTTTTTT-3, wherein the amino NH2 modified at the 5' end is used for being combined with 1-pyrene butyrate succinimide ester so that the report probe is anchored on the surface of graphene, six RNAs are activated CRISPR/Cas13a shearing sites, and 16 DNAs are signal generating sites.
2. Use of a graphene field effect transistor for detecting RNA by the method of detecting RNA according to claim 1, in particular for the detection of novel coronavirus RNA.
3. The use according to claim 2, wherein the CRISPR/Cas13a cleavage system is designed with four different CRISPRs, four grnas corresponding to three specific sites of the N region gene and one specific site of the E region gene in the new coronavirus RNA sequence, respectively.
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