CN114634974A - Nucleic acid detection system, nucleic acid detection system array, nucleic acid detection method, and method for screening candidate guide nucleic acids - Google Patents
Nucleic acid detection system, nucleic acid detection system array, nucleic acid detection method, and method for screening candidate guide nucleic acids Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
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Abstract
The present invention relates to a nucleic acid detection system, a nucleic acid detection system array, a nucleic acid detection method, and a method for screening candidate guide nucleic acids. The nucleic acid detection system comprises: a substrate comprising a substrate surface and at least two electrodes; a linker molecule having a first ligand bound to the substrate surface and a second ligand bound to a Cas enzyme; a Cas enzyme that binds to the second ligand of the linker molecule and forms an enzyme complex with the guide nucleic acid, the Cas enzyme comprising at least one selected from Cas12 (type VA), Cas13 (type VI), and Cas14 (type VF); a guide nucleic acid comprising sequences capable of binding to the enzyme and the target nucleic acid, respectively; and a reporter nucleic acid comprising a non-specific sequence. The nucleic acid detection system has excellent signal-to-noise ratio, sensitivity reaching aM level and greatly shortened detection time.
Description
Technical Field
The present invention relates to a nucleic acid detection system, a nucleic acid detection system array, a nucleic acid detection method, and a method for screening candidate guide nucleic acids for detecting a target nucleic acid in a sample. The nucleic acid detection system is a graphene-based amplification-free nucleic acid detection system.
Background
Most of the existing nucleic acid detection methods require a large amount of reagents and expensive and heavy instruments. Conventional nucleic acid detection techniques such as PCR require multi-step reactions for amplifying a sample, and further improvement in terms of cost and integration is required. In view of this, a graphene-based field effect transistor (GFET) has been developed in the prior art, which implements digital detection of a target sequence in a complete genome by means of a CRISPR (Clustered regulated Short Palindromic Repeats) technique (see non-patent document 1 and patent document 1). This biosensor called CRISPR chip utilizes the gene targeting ability of catalytically inactivated CRISPR-associated protein 9(Cas9) complexed with a specific single guide RNA and immobilized on a transistor, forming a label-free nucleic acid detection device whose output signal can be measured by a simple reader. In the presence of genomic DNA containing a target gene, the CRISPR chip was able to generate a current signal with a sensitivity of 1.7fM within 15 minutes, no amplification was required in the detection, and the output signal was significantly enhanced compared to samples without target sequence. The development of the CRISPR chip greatly expands the application of the CRISPR-Cas9 technology in the electrical detection of nucleic acid chips.
Documents of the prior art
Patent document
Patent document 1: US20190112643A
Non-patent document
Non-patent document 1: hajian, Reza, et al, "Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field-effect transmitter" Nature biological engineering 3.6(2019):427-437.
Disclosure of Invention
Problems to be solved by the invention
However, the CRISPR chip described above has the following problems: (ii) SIGNAL-to-NOISE RATIO (SNR) is not sufficiently high, and for example, non-patent document 1 describes that the SIGNAL-to-NOISE RATIO when the target gene BFP (blue fluorescent protein) is detected using its CRISPR-Cas9 chip, and shows about 4-fold SIGNAL-to-NOISE RATIO when exposed to HEK-BFP containing a BFP target gene as compared with a sample without the BFP target gene. Detection limit is to be improved: as described above, the sensitivity of dCas9 is at the fM (about 1.7fM) level, resulting in a limitation in the sensitivity of the entire system. The detection time is to be shortened: the CRISPR chip detection in non-patent document 1 requires about 15 minutes, and is not suitable for rapid detection in large quantities.
As a result of intensive studies in view of the above-described problems in the prior art, the present inventors have found that by binding Cas12(VA type), Cas13(VI type), Cas14(VF type) or another enzyme having a concomitant cleavage activity to GFET, amplification-free nucleic acid detection with a high signal-to-noise ratio and high sensitivity can be achieved, and POCT (point-of-care testing) is expected to be achieved. Specifically, the invention utilizes the programmable sequence specificity and the associated cleavage activity of Cas12, Cas13 or Cas14 and the like combined with GFET to detect the target sequence, thereby obtaining the effects of excellent signal-to-noise ratio, sensitivity reaching aM level and shortening detection time.
Means for solving the problems
The present invention provides the following technical means.
[1] A nucleic acid detection system, comprising:
a substrate comprising a substrate surface and at least two electrodes;
a linker molecule having a first ligand bound to the substrate surface and a second ligand bound to a Cas enzyme;
a Cas enzyme that binds to the second ligand of the linker molecule and forms an enzyme complex with the guide nucleic acid, the Cas enzyme comprising at least one selected from Cas12 (type VA), Cas13 (type VI), and Cas14 (type VF);
a guide nucleic acid comprising sequences capable of binding to the enzyme and the target nucleic acid, respectively; and
a reporter nucleic acid comprising a non-specific sequence.
[2] The nucleic acid detection system according to [1], wherein the Cas enzyme has a target nucleic acid cleavage activity.
[3] The nucleic acid detection system according to [2], wherein the Cas enzyme comprises at least one selected from Cas12a, Cas13a, Cas13b, Cas14a, Cas14b and Cas14 c.
[4] The nucleic acid detection system according to [1], wherein the Cas enzyme has no target nucleic acid cleavage activity.
[5] The nucleic acid detection system according to [4], wherein the Cas enzyme comprises at least one selected from dCas12a, dCas13a, dCas13b, dCas14a, dCas14b, and dCas14 c.
[6]According to [1]]The nucleic acid detecting system, wherein the substrate surface is selected from graphene, silicone, germanium alkene, Graphene Nanoribbon (GNR), double-layer graphene (BLG), phosphorus alkene, tin forest, graphene oxide, reduced graphene, fluorine graphene, MoS2At least one of gold and carbon.
[7] The nucleic acid detecting system according to [1], wherein the substrate is at least one selected from the group consisting of silicon, silicon oxide, silicon dioxide and paper.
[8] The nucleic acid detecting system according to [1], wherein the substrate is a Field Effect Transistor (FET).
[9] The nucleic acid detection system of [1], wherein the linker molecule not bound to the Cas enzyme is blocked.
[10] The nucleic acid detecting system according to [1], wherein the electrode is selected from a source electrode, a drain electrode and a gate electrode.
[11] The nucleic acid detecting system according to [1], wherein the substrate surface is graphene.
[12] The nucleic acid detecting system according to [1], wherein the first ligand is a pyrene ring or a cyclodextrin ring.
[13] The nucleic acid detecting system according to [1], wherein the second ligand is selected from a hydroxyl group, a carboxyl group, an amine group, an ester group, an amino group, a diethylamino group, an imide ester group, a boronic acid group, or an N-hydroxysuccinimide (NHS) group.
[14] The nucleic acid detection system according to [1], wherein the linker molecule is 1-pyrenebutanoic acid succinimidyl ester (PBSE).
[15] The nucleic acid detecting system according to [1], wherein a buffer solution is kept consistent before and after the manufacturing process of the nucleic acid detecting system.
[16] A method for detecting a nucleic acid using the nucleic acid detection system according to any one of [1] to [15], the method comprising:
adding a sample to be tested to the surface of a substrate of the nucleic acid detection system;
applying a current to the substrate; and
and measuring the current change on the surface of the substrate, and judging whether the target nucleic acid exists in the sample to be detected according to the current change.
[17] The method according to [16], wherein the sample to be tested is urine, blood, serum, cerebrospinal fluid or saliva.
[18] An array of nucleic acid detection systems, comprising:
a substrate comprising a plurality of substrate surfaces and at least two electrodes;
a plurality of linker molecules having a first ligand bound to the substrate surface and a second ligand bound to a Cas enzyme;
a plurality of Cas enzymes bound to the second ligand of the linker molecule and forming an enzyme complex with the guide nucleic acid, the Cas enzymes comprising at least one selected from Cas12 (type VA), Cas13 (type VI), and Cas14 (type VF);
a plurality of guide nucleic acids comprising sequences capable of binding to the Cas enzyme and the target nucleic acid, respectively, the plurality of guide nucleic acids binding to a plurality of the same or different target nucleic acids, respectively; and
a reporter nucleic acid comprising a non-specific sequence.
[19] The nucleic acid detecting system array according to [18], wherein: a plurality of the linker molecules may be the same or different from each other.
[20] A method for screening candidate guide nucleic acids using the nucleic acid detection system array according to [18], wherein the candidate guide nucleic acids include a plurality of different candidate guide nucleic acids, the method comprising:
adding a composition comprising a target nucleic acid to the array of nucleic acid detection systems;
applying a current to the substrate; and
measuring a change in current on the surface of the substrate, and screening the guide nucleic acid based on the change in current.
Effects of the invention
The invention can provide a nucleic acid detection system with excellent signal-to-noise ratio, sensitivity reaching aM level and greatly shortened detection time.
Drawings
Figure 1 is a schematic diagram showing the comparison of the cleavage activity of Cas12, Cas13 and Cas 9.
FIG. 2 is a schematic diagram showing the design of the nucleic acid detecting system of the present invention.
FIG. 3 is a schematic diagram showing an array of nucleic acid detecting systems according to a second embodiment of the present invention.
Fig. 4 is a graph showing the temporal change of response efficiency when the sample to be tested and the control sample are added to the CRISPR-Cas12 chip and the control CRISPR-Cas9 chip in example 1 of the present invention in example 2.
Fig. 5 is a graph showing the signal-to-noise ratio when the sample to be tested and the control sample are added to the CRISPR-Cas12 chip and the control CRISPR-Cas9 chip in example 1 of the present invention in example 2.
Reference numerals
1 source electrode
2 drain electrode
3 gate electrode
4 graphene
5 Cas + GFET chip
Detailed Description
Embodiments of the present invention will be described in detail with reference to the drawings, but the present invention is not limited to the embodiments shown in the drawings, and includes all embodiments satisfying the features recited in the claims of the present application.
First embodiment
The first embodiment of the present invention relates to a nucleic acid detection system comprising:
a substrate comprising a substrate surface and at least two electrodes;
a linker molecule having a first ligand bound to the substrate surface and a second ligand bound to a Cas enzyme;
a Cas enzyme that binds to the second ligand of the linker molecule and forms an enzyme complex with the guide nucleic acid, the Cas enzyme comprising at least one selected from Cas12 (type VA), Cas13 (type VI), and Cas14 (type VF);
a guide nucleic acid comprising sequences capable of binding to the enzyme and the target nucleic acid, respectively; and
a reporter nucleic acid comprising a non-specific sequence.
The above Cas enzyme comprises at least one Cas enzyme selected from Cas12(VA type), Cas13(VI type), and Cas14(VF type), which may have target nucleic acid cleavage activity, for example, at least one selected from Cas12a, Cas13a, Cas13b, Cas14a, Cas14b, and Cas14c, and may not have target nucleic acid cleavage activity, for example, at least one selected from dCas12a, dCas13a, dCas13b, dCas14a, dCas14b, and dCas14 c.
The following description will be made by taking Cas12 and Cas13 as examples.
Cas12, Cas13 are enzymes with cognate cleavage activity that, upon binding to a target sequence, Cas12, 13 activates the cognate cleavage activity under the guidance of a guide RNA sequence, which will simultaneously cleave the surrounding reporter sequence, thereby amplifying the signal. Figure 1 is a schematic diagram showing the comparison of the cleavage activity of Cas12, Cas13 and Cas 9. As shown in fig. 1, Cas9 cleaves (binds) one target gene at a time to generate one signal, whereas Cas12 and Cas13 have a concomitant cleavage activity, and cleave the surrounding reporter sequence after cleaving (binding) one target gene at a time, thereby generating a large amount of signal.
Table 1 shows the characteristics of Cas12, Cas13 compared to Cas9, from which Cas12, Cas13 can be expected to have sensitivity at the aM level.
TABLE 1
| LwaCas13a | LbaCas13a | CcaCas13b | PsmCas13b | AsCas12a | SpyCas9 | |
| Microorganisms | L.wadei | L.bacterium | C.canimorsus | P.sp. | A.sp. | S.pygenes |
| Target | ssRNA | ssRNA | ssRNA | ssRNA | ssRNA/dsDNA | dsDNA |
| tracrRNA | No | No | No | No | No | Yes |
| Shear properties | Associated activity | Associated activity | Associated activity | Associated activity | Associated activity | Blunt |
| Sensitivity of the probe | aM | aM | aM | aM | aM | fM |
FIG. 2 is a schematic diagram showing the design of the nucleic acid detecting system of the present invention. The figure shows an exemplary graphene Field Effect Transistor (FET) biosensor in which a source electrode 1 and a drain electrode 2 are formed on graphene 4 with a gate electrode 3 disposed therebetween. The biosensor can capture target nucleic acids bound to an enzyme complex from a sample by functionalizing the biosensor with a Cas12 enzyme or/and a Cas13 enzyme complexed with a guide ribonucleic acid (gRNA). When the target sequence is captured, Cas12 or/and Cas13 will cleave the reporter nucleic acid, causing a field effect, generating a signal that can be read by a reader, such as a lock-in amplifier or semiconductor parameter analyzer.
A Field Effect Transistor (FET) is a transistor that controls the flow of current by an electric field. The FET has three terminals: a source electrode, a gate electrode, and a drain electrode. FETs control the flow of current by applying a voltage to the gate electrode, which in turn changes the conductivity between the drain and source electrodes.
The three terminals of the FET are:
terminal 1: and a source electrode (S) through which carriers enter the channel. In general, the current entering the channel at S is represented by ISAnd (4) showing.
A terminal 2: and a drain electrode (D) through which carriers leave the channel. Typically, the current entering the channel at D is IDAnd (4) showing. Drain-source voltage of VDS。
And (3) a terminal: a gate electrode (G) that modulates channel conductivity. By applying a voltage to G, I can be controlledD。
The nucleic acid detection system of the present invention is comprised of a three terminal graphene-based Field Effect Transistor (FET) that utilizes graphene as the channel between the source and drain electrodes, with a liquid gate electrode in contact with the nucleic acid sample.
The substrate surface, substrate, and the like in the nucleic acid detecting system of the present invention are not particularly limited, and those described in the prior art can be used.
For example, the substrate surface may be selected from graphene, silicone, germanium-alkene, Graphene Nanoribbons (GNRs), double-layer graphene (BLG), phosphorus-alkene, tin-forest, graphene oxide, reduced graphene, fluorine-graphene, MoS2At least one of gold and carbon, preferably graphene.
The substrate may be at least one selected from silicon, silicon oxide, silicon dioxide and paper, and is preferably a Field Effect Transistor (FET).
As the linker molecule used in the present invention, a pyrene derivative or a cyclodextrin derivative can be used from the viewpoint of the efficiency of the linkage with the substrate and the Cas enzyme and the maintenance of an appropriate distance from the graphene surface after the linkage. The first ligand connected with the substrate surface by the connecting molecule can be pyrene ring or cyclodextrin ring, and the second ligand combined with the enzyme can be hydroxyl, carboxyl, amino, ester group, amino, diethylamino, imide ester group, boric acid group or N-hydroxysuccinimide (NHS) group.
Specific examples of the linker molecule usable in the present invention include 1-pyrenebutyric acid succinimidyl ester (PBSE), 1-pyrenebutyric acid (1-PBA), and perylenebutyric acidBenzylated alpha-cyclodextrin (alpha-CDBn)18) Perbenzylated beta-cyclodextrin (beta-CDBn)21) Or perbenzylated gamma-cyclodextrin (gamma-CDBn)24) Etc., wherein PBSE is further preferably used.
The guide nucleic acid in the nucleic acid detection system of the invention comprises sequences capable of binding to the Cas enzyme and the target nucleic acid, respectively, which can be sequence designed depending on the specific Cas enzyme and target nucleic acid being used in the system.
The reporter sequence in the nucleic acid detection system of the invention comprises a non-specific sequence, and for Cas enzymes with associated cleavage activity, the reporter nucleic acid can be any sequence. The timing of adding the reporter nucleic acid to the system is not particularly limited, and for example, the reporter nucleic acid may be added to the sample before the sample is added to the nucleic acid detecting system of the present application, or the reporter nucleic acid may be added to the nucleic acid detecting system after the sample is added to the nucleic acid detecting system of the present application.
The method for producing the nucleic acid detection system of the present invention is not particularly limited, but generally includes a graphene-FET sensor production step and a graphene-FET sensor functionalization step. In the graphene-FET sensor manufacturing process, graphene is patterned, a source electrode and a drain electrode are manufactured and then are incorporated into a prepared substrate chip, so that the graphene-FET sensor is obtained. In the graphene-FET sensor functionalization process, a connecting molecule is added on the surface of graphene to functionalize the graphene, after the connecting molecule is activated, Cas enzyme is transferred to the surface of the graphene, and then the unreacted connecting molecule is sealed, so that the Cas enzyme chip, namely the nucleic acid detection system, is obtained.
In addition, any technique that can pattern graphene may be used for the patterning of graphene, and for example, a photolithography technique that is conventional in the art may be used. The source electrode and the drain electrode are not particularly limited, and for example, electrodes commonly used in the art, such as Ti/Pt or Cr/Au electrodes, may be used. In the above step, the purpose of activating the linker molecule is to activate the linker group at the terminus of the linker molecule, thereby allowing the linker molecule to be easily linked to the Cas enzyme. The compound for activating the linker molecule may be selected from any suitable compounds known in the art according to the specific second ligand in the linker molecule, and is not particularly limited. In addition, the purpose of blocking the unreacted linker molecules in the above step is to prevent the unreacted linker molecules from non-specifically binding to the substance in the sample to be tested and interfering with the subsequent detection of the target nucleic acid. The compound to be blocked may be selected from any suitable compounds known in the art according to the specific second ligand in the linker molecule, and is not particularly limited.
In addition, in the nucleic acid detection system of the present invention, the Cas enzyme may be added to the functionalized graphene surface in the form of an enzyme complex after forming an enzyme complex with the guide nucleic acid, or the Cas enzyme may be added only to the functionalized graphene surface, and the Cas enzyme may be bound to the linker molecule and then the guide nucleic acid may be added to form the enzyme complex.
In addition, in the manufacturing process of the nucleic acid detecting system of the present invention, it is preferable to maintain the buffer solution to be uniform throughout the manufacturing process to ensure the activity of the enzyme, thereby avoiding interference with the subsequent detection of the target nucleic acid.
The nucleic acid detection system of the present invention may further comprise an electronic controller for measuring a change in current on the surface of the substrate.
The nucleic acid detection system of the present invention can be used for detecting a target nucleic acid in a sample to be detected. The detection may specifically comprise the steps of: adding a sample to be tested to the surface of a substrate of the nucleic acid detection system of the invention; applying a current to the substrate; then, the current change on the surface of the substrate is measured, and the presence or absence of the target nucleic acid in the sample to be tested can be judged from the current change. The sample to be tested is not particularly limited, and may be urine, blood, serum, cerebrospinal fluid, saliva, or the like.
Second embodiment
A second embodiment of the present invention is directed to an array of nucleic acid detection systems comprising:
a substrate comprising a plurality of substrate surfaces and at least two electrodes;
a plurality of linker molecules having a first ligand bound to the substrate surface and a second ligand bound to a Cas enzyme;
a plurality of Cas enzymes that bind to the second ligand of the linker molecule and form an enzyme complex with the guide nucleic acid, the Cas enzymes comprising at least one selected from Cas12 (type VA), Cas13 (type VI), and Cas14 (type VF);
a plurality of guide nucleic acids comprising sequences capable of binding to the enzyme and the target nucleic acid, respectively, the plurality of guide nucleic acids binding to a plurality of the same or different target nucleic acids, respectively; and
a reporter nucleic acid comprising a non-specific sequence.
Fig. 3 shows an array of nucleic acid detection systems, in which a plurality of independent Cas + GEFT chips 5 are arranged between a source electrode 1 and a drain electrode 2 of a substrate, and in each chip 5, enzyme complexes, which may be the same or different, are bound to the surface of the substrate by linker molecules. For example, as shown in FIG. 3, the guide nucleic acid in the chip of row 1 is designed for the 1 st target nucleic acid, and the chip of row 1 is connected to the 1 st reader; the guide nucleic acid in the chip of row 2 is designed for the 2 nd target nucleic acid, and the chip of row 2 is connected to the 2 nd reader and the like. The nucleic acid detecting system array can detect a plurality of target nucleic acids in a sample at the same time. The arrangement of the array of nucleic acid detection systems of the present invention and the target nucleic acid sequence to which the enzyme complex is directed can be specifically designed by those skilled in the art according to specific detection needs.
The nucleic acid detection system array of the present invention can be used for screening candidate guide nucleic acids in addition to simultaneously detecting a plurality of target nucleic acids in a sample to be detected. Specifically, for example, a plurality of different candidate guide nucleic acids are contained in a nucleic acid detection system array, a composition containing a target nucleic acid is added to the system array, an electric current is applied to a substrate, a change in the electric current on the surface of the substrate is measured, and then the guide nucleic acids are screened for the change in the electric current.
Examples
The nucleic acid detecting system and the nucleic acid detecting method of the present invention are specifically exemplified below by taking a novel coronavirus (hereinafter, also referred to as COVID19) as an example, but the present invention is not limited to these examples.
Example 1: preparation of CRISPR-Cas12 chip of nucleic acid detection system
The CRISPR-Cas12 chip of the nucleic acid detection system of the present invention was prepared as follows.
1. graphene-FET sensor fabrication
Firstly, patterning graphene on a 4-6 inch silicon wafer transferred with high-quality graphene by adopting a lifting process, and manufacturing a Ti/Pt source electrode and a drain electrode. Wherein, the silicon wafer adopts piranha cleaning process in advance to remove all organic residues which can be used as final dopants; the patterning of the graphene is achieved by Reactive Ion Etching (RIE). The patterned graphene chip, including the wire bonds of the Printed Circuit Board (PCB), was then connected into the custom PCB package using epoxy, leaving only an open cavity above the bare graphene transistor for placement of the solution gate electrode and introduction of the sample to be tested. Data was collected using a semiconductor parameter analyzer and current changes were analyzed.
Preparation of Cas12 enzyme complex:
mu.M of LbaCas12a (M0653S, NEB), 2. mu.M of crRNA sequence prepared as described below and 2mM MgCl2Mixed and left to stand at room temperature for 15 minutes.
3. Functionalization of graphene-FET sensors
The graphene-FET sensor fabricated as above was washed twice with acetone and once with deionized water. Subsequently, pbse (sigma aldrich) (structural formula below) was added on the graphene surface, and incubated at room temperature for 2 hours. The surface of the sensor was then rinsed twice with Dimethylformamide (DMF) and once with deionized water (DIW) and allowed to dry completely. PBSE was then activated using N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (S-NHS).
The Cas12 enzyme complex prepared above was transferred to the graphene surface and incubated at 37 ℃ for 30 minutes. This process is continuously monitored by measuring the current change (Ids) of the chip between the drain and source electrodes. The change of the graphene conductivity and the chip Ids can ensure that the Cas12 enzyme complex is fixed on the graphene surface. After fixation of Cas12 enzyme complex, unreacted PBSE molecules were blocked by addition of blocking agents, amino-PEGS alcohol and ethanolamine hydrochloride, which rapidly react with unbound activated PBSE molecules to block them. After blocking, the graphene surface was coated with 2mM MgCl2The solution was washed and incubated until the Ids reading stabilized. Thus, the CRISPR-Cas12 chip of example 1 (hereinafter also referred to as "CRISPR-Cas 12 chip of the present invention") was obtained.
The crRNA was prepared as follows:
according to the genome sequence of the novel coronavirus, aiming at the N gene, a guide sequence crRNA is designed.
A plasmid containing an N gene fragment with a template of COVID19, wherein the N gene is as follows (SEQ ID NO: 1):
CCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAACTCAGGCCTAA
the designed crRNA sequence (SEQ ID NO: 2) was as follows:
UAAUUUCUACUAAGUGUAGAUUUGAACUGUUGCGACUACGU
the reporter sequence used was the following sequence (SEQ ID NO: 3):
5’TTTTTTTTTTTTTTTT 3’
example 2: determination of sample to be detected by using CRISPR-Cas12 chip
A plasmid containing the N gene fragment of COVID19 purchased from tsingkasei corporation in south kyo was used as a sample to be tested. After concentration determination, the plasmid was diluted to 10 pg/. mu.L (about 2.3 aM).
Deionized water was used as a control solution (control).
The CRISPR-Cas9 chip prepared in example 1 of patent document 1 was used as a control chip (hereinafter referred to as "control CRISPR-Cas9 chip").
Before adding a sample to be detected into the CRISPR-Cas12 chip, the CRISPR-Cas12 chip is treated by using 2mM MgCl2The chip was calibrated by incubating at 37 ℃ for 5 minutes. Dissolving the report nucleic acid in deionized water, adding the solution with the final concentration of 1 mu M into a sample to be detected, then respectively adding 1 mu L of sample solution to be detected and 1 mu L of control solution into the CRISPR-Cas12 chip and the control CRISPR-Cas9 chip of the invention, incubating for 20 minutes at 37 ℃, and continuously monitoring the current change in the chips.
The detection results are shown in fig. 4 and 5. FIG. 4 shows a graph of response efficiency of a test sample and a control sample added to the chip, which changes with time, and it can be seen from FIG. 4 that the signal output of the CRISPR-Cas12 chip of the invention is exponential, while the signal of the control CRISPR-Cas9 chip is displayed after a period of time. FIG. 5 shows the SNR of the test sample and the control sample after they are added to the chip. The results of fig. 4 and 5 show that the CRISPR-Cas12 chip of the present invention has shorter detection time (fig. 4, about 10 minutes) and higher signal-to-noise ratio (fig. 5, about 15 times).
As can be seen from the above-described detection experiments, the present invention provides a nucleic acid detection system having an excellent signal-to-noise ratio, a sensitivity up to an aM level, and a greatly shortened detection time.
Sequence listing
<110> Canon medical systems corporation
<120> nucleic acid detection system, nucleic acid detection system array, nucleic acid detection method, and method for screening candidate guide nucleic acids
<130> PTMA-20093-CN(98G81001040-CN-A)
<160> 3
<170> PatentIn version 3.5
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<211> 1000
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<400> 1
ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa atgaaagatc tcagtccaag 60
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agacggcatc atatgggttg caactgaggg agccttgaat acaccaaaag atcacattgg 180
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gccaaaaggc ttctacgcag aagggagcag aggcggcagt caagcctctt ctcgttcctc 300
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taagaaatct gctgctgagg cttctaagaa gcctcggcaa aaacgtactg ccactaaagc 540
atacaatgta acacaagctt tcggcagacg tggtccagaa caaacccaag gaaattttgg 600
ggaccaggaa ctaatcagac aaggaactga ttacaaacat tggccgcaaa ttgcacaatt 660
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agatcaagtc attttgctga ataagcatat tgacgcatac aaaacattcc caccaacaga 840
gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa gccttaccgc agagacagaa 900
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tttttttttt tttttt 16
Claims (20)
1. A nucleic acid detection system, comprising:
a substrate comprising a substrate surface and at least two electrodes;
a linker molecule having a first ligand bound to the substrate surface and a second ligand bound to a Cas enzyme;
a Cas enzyme that binds to the second ligand of the linker molecule and forms an enzyme complex with the guide nucleic acid, the Cas enzyme comprising at least one selected from Cas12 (type VA), Cas13 (type VI), and Cas14 (type VF);
a guide nucleic acid comprising sequences capable of binding to the Cas enzyme and the target nucleic acid, respectively; and
a reporter nucleic acid comprising a non-specific sequence.
2. The nucleic acid detection system of claim 1, wherein the Cas enzyme has a target nucleic acid cleavage activity.
3. A nucleic acid detection system according to claim 2 wherein the Cas enzyme comprises at least one selected from Cas12a, Cas13a, Cas13b, Cas14a, Cas14b and Cas14 c.
4. The nucleic acid detection system of claim 1, wherein the Cas enzyme has no target nucleic acid cleavage activity.
5. The nucleic acid detection system of claim 4, wherein the Cas enzyme comprises at least one selected from dCas12a, dCas13a, dCas13b, dCas14a, dCas14b, and dCas14 c.
6. The nucleic acid detection system of claim 1, wherein the substrate surface is selected from graphene, silicone, germane, Graphene Nanoribbons (GNRs), double-layer graphene (BLG), phospholene, tin forest, graphene oxide, reduced graphene, fluorographene, MoS2At least one of gold and carbon.
7. The nucleic acid detecting system according to claim 1, wherein the substrate is at least one selected from the group consisting of silicon, silicon oxide, silicon dioxide, and paper.
8. The nucleic acid detection system of claim 1, wherein the substrate is a Field Effect Transistor (FET).
9. The nucleic acid detection system of claim 1, wherein the linker molecule not bound to the Cas enzyme is blocked.
10. The nucleic acid detection system of claim 1, wherein the electrode is selected from the group consisting of a source electrode, a drain electrode, and a gate electrode.
11. The nucleic acid detection system of claim 1, wherein the substrate surface is graphene.
12. The nucleic acid detecting system according to claim 1, wherein the first ligand is a pyrene ring or a cyclodextrin ring.
13. A nucleic acid detection system according to claim 1, wherein the second ligand is selected from a hydroxyl, carboxyl, amine, ester, amino, diethylamino, imidoester, boronic acid or N-hydroxysuccinimide (NHS) group.
14. The nucleic acid detection system of claim 1, wherein the linker molecule is 1-pyrenebutanoic acid succinimidyl ester (PBSE).
15. The nucleic acid detecting system according to claim 1, wherein a buffer solution is kept consistent before and after a manufacturing process of the nucleic acid detecting system.
16. A method for detecting a target nucleic acid in a sample to be tested using the nucleic acid detection system according to any one of claims 1 to 15, the method comprising:
adding a sample to be tested to the substrate surface of the detection system;
applying a current to the substrate; and
and measuring the current change on the surface of the substrate, and judging whether the target nucleic acid exists in the sample to be detected according to the current change.
17. The method of claim 16, wherein the test sample is urine, blood, serum, cerebrospinal fluid, or saliva.
18. An array of nucleic acid detection systems, comprising:
a substrate comprising a plurality of substrate surfaces and at least two electrodes;
a plurality of linker molecules having a first ligand bound to the substrate surface and a second ligand bound to a Cas enzyme;
a plurality of Cas enzymes that bind to the second ligand of the linker molecule and form an enzyme complex with the guide nucleic acid, the Cas enzymes comprising at least one selected from Cas12 (type VA), Cas13 (type VI), and Cas14 (type VF);
a plurality of guide nucleic acids comprising sequences capable of binding to the Cas enzyme and the target nucleic acid, respectively, the plurality of guide nucleic acids binding to a plurality of the same or different target nucleic acids, respectively; and
a reporter nucleic acid comprising a non-specific sequence.
19. The nucleic acid detection system array of claim 18, wherein: a plurality of the linker molecules may be the same or different from each other.
20. A method of screening candidate guide nucleic acids using the nucleic acid detection system array of claim 18, wherein a plurality of different candidate guide nucleic acids are included, the method comprising:
adding a composition comprising target nucleic acids to the array of nucleic acid detection systems;
applying a current to the substrate; and
measuring a change in current on the surface of the substrate, and screening the guide nucleic acid based on the change in current.
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