CN116987770A - Method and system for ultrasensitive detection of target analyte in sample - Google Patents
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Abstract
The invention discloses a method and a system for ultrasensitive detection of target analytes in a sample, wherein a capture reagent fixed on a solid phase carrier can be specifically combined with a target to be detected in the sample through a first binding site of the target, so that target enrichment is realized; and adding a detection reagent capable of combining with the target second binding site, wherein the detection reagent contains a specific nucleic acid sequence which is complementarily combined with the guide RNA, activating the activity of Cas protein along with the addition of a CRISPR/Cas reaction system, cutting an excitation sequence, simultaneously cutting ssDNA, releasing a reporter group, and carrying out fluorescence detection after light excitation. The intensity of the fluorescence signal is proportional to the concentration of the detected target, and the concentration of the target in the original sample is calculated by detecting the fluorescence signal. Through the enzyme digestion signal amplification technology of the Cas protein, targets in trace amounts in a sample can be detected, and the detection sensitivity is improved.
Description
Technical Field
The invention belongs to the technical field of detection, relates to detection of proteins, and particularly relates to a method and a system for ultrasensitive detection of target analytes in a sample based on a CRISPR/Cas system.
Background
The change of protein in human body has close relation with the occurrence and development of human health and diseases. The method can accurately measure the change of the protein content related to the diseases in human bodies, and has important significance in the aspects of disease prevention, screening, accurate diagnosis, treatment and the like.
Detection methods based on immunological techniques are currently the main method for protein detection, with enzyme-linked immunosorbent assay being the most widely used immunological detection method. The method is to label catalytic enzyme on the secondary antibody, combine the specific reaction of antigen and antibody with the action of enzyme catalytic substrate, and judge the detection result according to the chromogenic reaction of catalytic substrate. The method does not need special instruments, is simple in detection and wide in application. However, due to the limitations of enzyme-catalyzed substrate signal amplification, this approach has low sensitivity to target detection, reaching ng-level only, and has difficulty in detecting lower levels of protein (e.g., pg-level).
With the development of technology, quanterix developed a digital single-molecule immune array analyzer (Simoa technology), which captures and seals immune complex in a chip containing 20 ten thousand flying-level pores for reaction, and can directly detect proteins, nucleic acids and the like in serum and plasma with ultra-high sensitivity, but the method needs expensive detection equipment, has higher detection operation technical requirements, and is unfavorable for clinical popularization and detection. Therefore, there is a need to develop a detection method for large-scale clinical detection that is easy to operate and low in cost.
The CRISPR/Cas system is an adaptive immune system of prokaryotes for combating the invasion of foreign genetic elements present in phages or plasmids. Is a defense mechanism that exists in most bacteria and all archaea to destroy foreign plastid or phage DNA. The system is composed of two parts: CRISPR sequences and CAS (CRISPR associate system). CRISPR sequences are derived from phage DNA fragments that can infect prokaryotes. It can detect and destroy similar DNA in other phages that can cause similar infections. CAS is an endonuclease that recognizes and cleaves a DNA strand that is specifically complementary to its sequence, using RNA guidelines corresponding to the spacer sequence (spacer) in the CRISPR sequence. CRISPR-Cas systems can be divided into two classes, class1 (comprising typeI, III, and iv) and Class2 (comprising typeII and typeV and typeVI) according to whether the effector is a multi-subunit or a single subunit. In Class1, one large Cas protein complex (consisting of more than one Cas protein) and guide RNA are required for cleavage of the exogenous genome; in Class2, only a single cleavage protein is required for cleavage of the foreign gene, e.g., cas9 protein in TypeII and cpf protein in TypeV. In the practical application process, the system of class2 is widely applied in the aspects of nucleic acid detection and diagnosis, gene editing, gene screening and the like due to the simplicity and high efficiency of the system.
It was found that Cas12 and Cas13 families have collateral cleavage capability (or cleavage in trans), i.e., cas protein-crRNA binary complexes, after recognizing and binding substrates, can cleave not only substrates, but also any single-stranded nucleic acid sequences (e.g., ssDNA) that are free in the environment. The ssDNA can be modified (for example, fluorescent dye emission pair is modified, one end is a fluorescent group, the other end is a quenching group), signals are generated through the cleavage of the Cas protein on the ssDNA, and the signals are detected by a simple signal detection system; meanwhile, by means of enzyme digestion signal amplification of the CRISPR/Cas system, trace target signals can be amplified, and detection sensitivity is improved. The method only needs to be slightly changed on the basis of the traditional ELISA method, does not need expensive equipment, is simpler to operate, and is more suitable for clinical popularization and detection.
Disclosure of Invention
In order to solve the problems that the existing protein detection is time-consuming, high in cost (special instrument is needed), not sensitive enough (signal amplification cannot be performed), not automatic and the like, the invention provides a method for performing ultrasensitive detection on a target analyte in a sample based on a CRISPR/Cas system.
The capture reagent fixed on the solid phase carrier can specifically bind with a target to be detected in a sample through a first binding site of the target, so that target enrichment is realized; then adding a detection reagent capable of combining with the target second combining site, wherein the detection reagent contains a specific nucleic acid sequence (hereinafter referred to as an "excitation sequence") which is complementarily combined with the guide RNA, and can activate the activity of Cas protein, cut the excitation sequence and simultaneously cut ssDNA (deoxyribonucleic acid) to release a reporting group (such as a fluorescent group) combined on the ssDNA, and can perform fluorescence detection after light excitation. Because the cleavage reaction is ongoing and the intensity of the fluorescent signal is proportional to the concentration of the detection target. The concentration of the target in the original sample can thus be calculated by detecting the fluorescent signal. In addition, through the enzyme digestion signal amplification technology of the Cas protein, targets in trace amounts in a sample can be detected, and the detection sensitivity is improved.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for ultrasensitive detection of an analyte of interest in a sample, said method comprising the steps of:
1) Immobilization of capture reagent: immobilizing a capture reagent capable of specifically binding to a first binding site of a substance to be detected in a sample on a carrier to form a carrier-capture reagent complex;
2) Closing: after fixing the capture reagent on the carrier, sealing the surface of the carrier;
3) Capture antigen: mixing the carrier-capture reagent complex after the sealing in the step 2) with a sample to capture an antigen;
4) Detection reagent binding: after the incubation is finished, removing the supernatant, washing the supernatant for 3 to 6 times by using a washing buffer solution, and then adding a detection reagent for incubation, so that the detection reagent is specifically combined with the captured target to form a capture reagent-target-detection reagent complex; the detection reagent is capable of specifically binding to the target second binding site and contains a specific nucleic acid sequence that binds complementarily to the guide RNA;
5) Cas protein reaction system: the kit mainly comprises Cas protein, a guide RNA sequence, a report probe and a buffer system; before the reaction, the Cas protein and the guide RNA sequence are assembled into a Cas-guide RNA complex in a buffer system, and a report probe can be added into a Cas protein reaction system at the same time or can be added separately;
6) And (3) signal detection: after the reaction is completed, the signal generated by the detection is detected, and a result is obtained.
As a preferred embodiment of the present invention, in step 1), the sample comprises blood, serum, plasma, cerebrospinal fluid, interstitial fluid, urine, lymph fluid, aqueous humor or a treatment fluid; the substance to be detected comprises protein, polysaccharide or small molecules with biological activity and a complex of the small molecules and the protein; the capture reagent is a reagent capable of specifically binding to a substance to be detected; the carrier comprises nylon, cellulose, a micro-pore plate, magnetic beads, a chip, a centrifuge tube or nano microspheres.
As a preferred embodiment of the invention, the capture reagent is covalently or non-covalently attached to the carrier; wherein, covalent bonding means that one or more functional groups of amino, hydroxyl, carboxyl, sulfhydryl, sulfonyl and epoxy groups are carried on the surface of the carrier or modified, and the covalent bonding is carried out with a capture reagent or the modified capture reagent; non-covalent binding refers to the adsorption of the capture reagent or modified capture reagent to the surface of the support by hydrogen bonding, electrostatic interactions, hydrophobic forces, van der Waals forces.
As a preferred embodiment of the present invention, in step 2), the blocking agent comprises BSA, casein or tween.
As a preferable scheme of the invention, in the step 3), the antigen capturing is carried out at the temperature of 25-37 ℃ for 10-180min.
As a preferred embodiment of the present invention, in step 4), the detection reagent includes an aptamer or an antibody;
when the detection reagent is an aptamer, one structure of the aptamer is as follows:
NNNN......NNNN[AA...AA XXX......XXX]n；
wherein: nnnn..nnnn..nnnn is an aptamer sequence that specifically binds to a target, xxx..xxx..xxx is a specific nucleic acid sequence that can complementarily bind to a guide RNA, aa..aa is a spacer sequence;
alternatively, one structure of the aptamer is as follows:
NNNN......NNNN...NNN...NNN[AA...AA NNN...NNN]n；
nnnn..nnnn..nnn..nnn..nnn is an aptamer sequence that specifically binds to a target, nnn..nnn..nnn is a specific nucleic acid sequence that binds complementarily to a guide RNA while the sequence is part of an aptamer, aa..aa is a spacer sequence;
the spacer sequence and the specific nucleic acid sequence are repeated a plurality of times; the spacer sequence and the specific nucleic acid sequence may be located at the 3 'end of the aptamer or at the 5' end of the aptamer.
As a preferred embodiment of the invention, when the detection reagent is an antibody, the specific nucleic acid sequence is bound directly or indirectly to the antibody by covalent or non-covalent means; or directly or indirectly by covalent or non-covalent forms to a secondary antibody that can specifically bind to the detection antibody.
As a preferred scheme of the invention, in the step 5), the concentration of Cas protein is 50-500nM, the concentration of guide RNA is 50-500nM, the concentration of report probe is 10-1000nM, the reaction temperature is 25-37 ℃ and the reaction time is 10-60min; cas proteins include V-type CRISPR/Cas effector proteins; the concentration of magnesium ions in the buffer system is 0.5-50mM, and the concentration of DTT is 0.5-100mM;
the reporter probe is a sequence which is not specifically cut by the activated Cas protein, and the cut probe can generate a detectable signal; when the report probe is a single-stranded DNA sequence, one end is modified with a detectable signal, and the fragment length is 5-40 nucleotides;
the reporter group or the marker molecule comprises a fluorescent group and a quenching group, wherein the fluorescent group is selected from one or more of FAM, VIC, HEX, FITC, JOE, TET, CY3, CY5, ROX, texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.
The detection mode can be different according to the different modification signals, and the detection signals can be fluorescence, rare metals and the like.
If the detection signal is a fluorescent signal, the fluorescent signal can be detected by a fluorescence spectrophotometer, a fluorescence microplate reader, or the like. The fluorescent group is modified at one end of a single-stranded DNA sequence, the other end of the single-stranded DNA sequence is a quenching group, no fluorescent signal is generated when the sequence is complete, and the fluorescent signal can be generated after the fluorescent group is cut. In some embodiments, the other end is modified on a microsphere such as a magnetic bead, the microsphere is precipitated by centrifugation after reaction, the supernatant is the signal which is cut off, and the concentration of the supernatant signal intensity reaction system is detected.
The invention also provides a system for ultrasensitive detection of target analytes in samples, which comprises the capture reagent, a blocking agent for blocking, a detection reagent and a Cas protein reaction system.
Compared with the prior art, the invention has the following beneficial effects:
the invention has high sensitivity; the operation is simple and the automation can be realized; can be directly used for the original ELISA detection system; the reagent cost and the detection cost are low; multiplex detection (multiple detection by optimizing the enzyme, selecting specific cleavage sites).
Drawings
FIG. 1 shows the standard curve test results of example 1.
FIG. 2 is the standard curve test results of example 2.
FIG. 3 is the standard curve test results of example 3.
Detailed Description
In order to facilitate understanding of the technical means, the creation characteristics, the achievement of the objects and the effects achieved by the present invention, the present invention is further described below with reference to specific examples, but the following examples are only preferred examples of the present invention, not all of which are described in detail below. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
The invention relates to a method for ultrasensitive detection of target analytes in samples based on a CRISPR/Cas system, which comprises the following specific steps:
1) Immobilization of capture reagent: a capture reagent capable of specifically binding to a first binding site of a substance to be detected in a sample is immobilized on a carrier to form a carrier-capture reagent complex.
The sample comprises blood, serum, plasma, cerebrospinal fluid, tissue fluid, urine, lymph fluid, aqueous humor and other body fluids and treatment fluids thereof; the substance to be detected comprises protein, polysaccharide or small molecules with biological activity and a complex of the small molecules and the protein.
The capture reagent refers to a reagent which can specifically bind with a substance to be detected, such as an antibody, an aptamer and the like;
the solid phase carrier is used for separating detection samples and reagents and can be divided into nylon, cellulose, a micro-pore plate, magnetic beads, chips, centrifuge tubes, nano-microspheres and the like according to the form;
the capture reagent may be covalently or non-covalently attached to the carrier. Wherein covalent bonding means that one or more functional groups such as amino, hydroxyl, carboxyl, sulfhydryl, sulfonyl, epoxy and the like are carried on the surface of the carrier or modified, and the carrier can be bonded with a capture reagent or the modified capture reagent through covalent bonds, so that the bonding is stable. Non-covalent binding means that the capture reagent or modified capture reagent can be adsorbed on the surface of the carrier directly through non-covalent interactions such as hydrogen bonding, electrostatic interactions, hydrophobic forces, van der Waals forces, and the like.
2) Closing: after the capture reagent is immobilized on the carrier, the surface of the carrier needs to be subjected to sealing treatment so as to reduce the nonspecific adsorption of components in the sample and the subsequent detection reagent on the surface of the carrier.
The blocking agent comprises BSA, casein and Tween.
3) Capture antigen: the blocked carrier-capture reagent complex is mixed with a sample to capture an antigen. The incubation temperature is 25-37 ℃, and the incubation time is 10-180min, preferably 30min.
4) Detection reagent binding: after the incubation is finished, removing the supernatant, washing the supernatant for 3 to 6 times by using a washing buffer solution, and then adding a detection reagent for incubation, so that the detection reagent is specifically combined with the captured target to form a capture reagent-target-detection reagent complex.
The detection reagent is capable of specifically binding to the target second binding site and contains a specific nucleic acid sequence (priming sequence) that binds complementarily to the guide RNA.
The detection reagent is an aptamer, an antibody and other components capable of specifically binding with the target.
When the detection reagent is an aptamer (abbreviated as "detection aptamer"), the excitation sequence may be the sequence of the aptamer or a sequence covalently attached at one end of the aptamer.
When the detection reagent is an aptamer, one structure of the aptamer is as follows:
NNNN......NNNN[AA...AA XXX......XXX]n
wherein:
nnnn..nnnn is an aptamer sequence that specifically binds to a target;
xxx..xxx. XXX is a specific nucleic acid sequence (priming sequence) that can bind complementarily to the guide RNA;
aa. AA is a spacer sequence.
When the detection reagent is an aptamer, one structure of the aptamer is as follows:
NNNN......NNNN...NNN...NNN[AA...AA NNN...NNN]n
NNNN......NNNN...NNN...NNNan aptamer sequence that specifically binds to a target;
NNN...NNNa specific nucleic acid sequence (priming sequence) that is complementary to the guide RNA, while the sequence is part of an aptamer;
aa. AA is a spacer sequence;
the spacer sequence and the excitation sequence may be repeated a number of times, such as 2-10 times, or more; the spacer and excitation sequences may be located at the 3 'end of the aptamer or at the 5' end of the aptamer.
When the detection reagent is an antibody (abbreviated as "detection antibody"), the excitation sequence may be directly or indirectly bound to the antibody by covalent or non-covalent means; or may be bound directly or indirectly by covalent or non-covalent means to a secondary antibody which specifically binds to the detection antibody. When the priming sequence is linked to the second antibody, this step can be further performed in two steps, first incubation binding of the primary antibody to the captured target, followed by addition of the modified secondary antibody for incubation binding.
The priming sequence refers to a sequence that is capable of specific binding to complementary binding to the guide RNA to activate Cas protease activity, which may be a double-stranded or single-stranded oligonucleotide sequence.
The complementary pairing of the guide RNA with the target nucleic acid determines the specificity of the reaction. Mismatches between bases may occur when two nucleic acid sequences hybridize complementarily. Although the corresponding reaction can also be activated, the specificity of the method is significantly reduced, and the occurrence of mismatch events should be strictly avoided in practice. Hybridization between two nucleic acid sequences depends on the length of the nucleic acid fragment, the base composition, the buffer system, etc. Nucleic acid fragments generally useful for hybridization are 8 nucleotides or more in length, such as12, 20, 30, etc. Or repetitive sequences of complementary nucleotides, with intervals of several bases between the repetitive sequences, and repeating the test 2 times or more, such as 5 times, 10 times, etc.
5) Cas protein reaction system: the kit mainly comprises Cas protein, a guide RNA sequence, a report probe and a buffer system. Before the reaction, an appropriate amount of Cas protein and guide RNA sequence are first assembled into a Cas-guide RNA complex in a buffer system. The reporter probes may be added simultaneously to the Cas protein reaction system or separately.
The Cas protein reaction system has important influence on the sending of detection signals, and the concentration of each component is optimized. Wherein the concentration of the Cas protein is 50-500nM, the concentration of the guide RNA is 50-500nM, the concentration of the report probe is 10-1000nM, the reaction temperature is 25-37 ℃ and the reaction time is 10-60 min.
The Cas proteins comprise V-type CRISPR/Cas effect proteins, including protein families such as Cas12, cas14 and the like. Preferably, the Cas12 protein comprises Cas12a, cas12b, cas12i, cas12j, etc.; cas14 protein families include Cas14a, cas14b, and the like.
The magnesium ion concentration in the buffer system has a great influence on the enzyme activity, and the experiment optimizes the magnesium ion concentration, preferably 0.5-50mM, preferably 1-20 mM, more preferably 5-20 mM; in addition, the inactivation is remarkably improved by adding additives such as DTT and the like into the buffer system, and the concentration of the DTT is 0.5 mM-100 mM.
The reporter probe refers to a sequence which can be activated and has no specific cleavage by the Cas protein, and the cleaved probe can generate a detectable signal. The reporter probe may be a single stranded DNA sequence modified at one end with a detectable signal and a fragment of 5 to 40 nucleotides, preferably 2 to 20 bases in length. Wherein the T base accounts for 20% -80% or the C base accounts for 30% -50%.
In one embodiment, the reporter or marker molecule comprises a fluorophore and a quencher, wherein the fluorophore is selected from one or more of FAM, VIC, HEX, FITC, JOE, TET, CY, CY5, ROX, texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.
6) Signal detection
The root can detect the signal generated by the detection through the above reaction. The signal is a signal modified by the reporter probe. The detection mode can be different according to the different modification signals, and the detection signals can be fluorescence, rare metals and the like.
If the detection signal is a fluorescent signal, the fluorescent signal can be detected by a fluorescence spectrophotometer, a fluorescence microplate reader, or the like. The fluorescent group is modified at one end of a single-stranded DNA sequence, the other end of the single-stranded DNA sequence is a quenching group, no fluorescent signal is generated when the sequence is complete, and the fluorescent signal can be generated after the fluorescent group is cut. In some embodiments, the other end is modified on a microsphere such as a magnetic bead, the microsphere is precipitated by centrifugation after reaction, the supernatant is the signal which is cut off, and the concentration of the supernatant signal intensity reaction system is detected.
Example 1
This example provides aptamer-CRISPR/Cas for IGFBP3 protein detection
1. Reagent(s)
Capture antibody, aptamer, PBS, blocking buffer, binding buffer, wash buffer, cas protein reaction system.
The IGFBP3 aptamer sequence is: 5'
-GGGACCAGCACACGCATAACCCTCACGGTATGTGCTGTATGTTCCTGTTGCCCTCCGGACCAGCACA CGCATAACG-3’，SEQ ID No.1。
Wherein the cross-line portion is the complement of the sequence paired with the gRNA, which is part of the aptamer.
The gRNA sequence is: 5'-TAATTTCTACTAAGTGTAGATCGTTATGCGTGTGCTGGTCC-3', SEQ ID No.2.
The fluorescence quenching report probe sequence is as follows: 5'-FAM-TTATT-BHQ1-3'.
2. Cas protein response system configuration
Cas12a protein (30 pmol) and guide RNA (30 pmol) were added to 1mL of 1 XNEB 2.1Buffer and gently mixed, followed by 180pmol of fluorescence-modified ssDNA reporter probe. Mixing well for standby.
3. Experimental procedure
The concentrations of IGFBP3 antigen were diluted to 0, 0.01, 0.1, 0.5, 1, 5, 10 and 100pg/mL, respectively, using fetal bovine serum.
To the ELISA plate coupled with the capture antibody, 100. Mu.L of the above-mentioned diluent was added, and incubated at 37℃for 60 minutes. The residual sample was washed three times with 150. Mu.L of wash buffer and the supernatant was blotted.
mu.L of aptamer (1 nM) was added and incubated at 37℃for 30min. The residual sample was washed off six times using 150. Mu.L of wash buffer and the supernatant was blotted.
100 μl of Cas protein reaction solution was added.
And (3) placing the reaction plate into a fluorescence enzyme-labeled instrument, and performing fluorescence detection under the room temperature condition. The fluorescent signal was detected every 5 minutes for 90 minutes continuously.
4. Analysis of results
Referring to FIG. 1, the linear range is 1 pg/mL-1 ug/mL, and the detection sensitivity is about 1pg/mL, which is nearly 100 times higher than the sensitivity in the conventional detection.
Example 2
This example discloses antibody-CRISPR/Cas for IL-6 protein detection:
1. reagent(s)
Capture antibody, detection antibody, PBS, blocking buffer, binding buffer, wash buffer, cas protein reaction system. The gRNA sequence is: 5'-TAATTTCTACTAAGTGTAGATCCCCCAGCGCTTCAGCGTTC-3', SEQ ID No.3.
The biotin-modified priming DNA sequences were: 5'
-Biozin-AAAAAAGAACGCTGAAGCGCTGGGGGAAAAAAGAACGCTGAAGCGCTGGGGG-3’，SEQ ID No.4。
The reporter probe is as in example 1.
2. Coupling of the excitation DNA to the detection antibody
The biotin-modified priming DNA sequence was synthesized and purified by HPLC for subsequent ligation reactions.
Antibody-coupled SA: specific methods refer to kits (Abcam, ab 102921). Briefly, 1. Mu.L of the modifying reagent in the coupling kit was gently mixed with 10. Mu.L of the antibody (1 mg/mL,66.7 pmol), followed by addition of 10. Mu.L of streptavidin, gently mixed, and reacted at room temperature for 3 hours. After the completion of the reaction, 1. Mu.L of the completion buffer was added thereto, and the reaction was completed by reacting at room temperature for 30 minutes.
Excitation DNA assembly, purification: 6.7pmol of the SA-conjugated antibody was added with synthetic biotin-modified trigger DNA (10. Mu.L, 10. Mu.M), and the mixture was reacted at room temperature for 3 hours to conjugate the trigger DNA to the antibody. The free priming DNA that did not participate in the reaction can be removed by centrifugation and the antibody conjugated with priming DNA can be redissolved in PBS (containing 1% BSA) for further use after washing.
Cas protein reaction system configuration: as in example 1.
Experimental procedure
The concentrations of IL-6 antigen were diluted to 0, 0.001, 0.01, 0.1, 1, 10, 100, 1000pg/mL using fetal bovine serum, respectively. Other steps, after the completion of capturing the antigen by the antibody, the reaction was performed by adding the antibody coupled to the trigger DNA, and the other steps were the same as those in example 1.
4. Analysis of results
Referring to FIG. 2, the linear range is 0.001pg/mL to 1000pg/mL, and the detection sensitivity is 0.001pg/mL.
Example 3
The present example provides anti-antibody-CRISPR/Cas for IL-6 protein detection:
the excitation DNA was conjugated to the antibody (secondary antibody) of the detection antibody in the same manner as in example 2.
Other reactions were the same as in example 2, except that in the reaction stage of the detection reagent, the detection antibody was added first to form an antibody-target-detection antibody complex, and then a secondary antibody binding to the detection antibody was added, and the secondary antibody modified to excite DNA.
Referring to FIG. 3, the linear range is 0.001 to 1000pg/mL, and the detection sensitivity is close to 0.001pg/mL.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims (9)
1. A method for ultrasensitive detection of an analyte of interest in a sample, said method comprising the steps of:
1) Immobilization of capture reagent: immobilizing a capture reagent capable of specifically binding to a first binding site of a substance to be detected in a sample on a carrier to form a carrier-capture reagent complex;
2) Closing: after fixing the capture reagent on the carrier, sealing the surface of the carrier;
3) Capture antigen: mixing the carrier-capture reagent complex after the sealing in the step 2) with a sample to capture an antigen;
4) Detection reagent binding: after the incubation is finished, removing the supernatant, washing the supernatant for 3 to 6 times by using a washing buffer solution, and then adding a detection reagent for incubation, so that the detection reagent is specifically combined with the captured target to form a capture reagent-target-detection reagent complex; the detection reagent is capable of specifically binding to the target second binding site and contains a specific nucleic acid sequence that binds complementarily to the guide RNA;
5) Cas protein reaction system: the kit mainly comprises Cas protein, a guide RNA sequence, a report probe and a buffer system; before the reaction, the Cas protein and the guide RNA sequence are assembled into a Cas-guide RNA complex in a buffer system, and a report probe can be added into a Cas protein reaction system at the same time or can be added separately;
6) And (3) signal detection: after the reaction is completed, the signal generated by the detection is detected, and a result is obtained.
2. The method of claim 1, wherein in step 1) the sample comprises blood, serum, plasma, cerebrospinal fluid, interstitial fluid, urine, lymph, aqueous humor, or treatment fluid; the substance to be detected comprises protein, polysaccharide or small molecules with biological activity and a complex of the small molecules and the protein; the capture reagent is a reagent capable of specifically binding to a substance to be detected; the carrier comprises nylon, cellulose, a micro-pore plate, magnetic beads, a chip, a centrifuge tube or nano microspheres.
3. A method of ultrasensitive detection of an analyte of interest in a sample according to claim 1 or 2, wherein the attachment of the capture reagent to the carrier is covalent or non-covalent; wherein, covalent bonding means that one or more functional groups of amino, hydroxyl, carboxyl, sulfhydryl, sulfonyl and epoxy groups are carried on the surface of the carrier or modified, and the covalent bonding is carried out with a capture reagent or the modified capture reagent; non-covalent binding refers to the adsorption of the capture reagent or modified capture reagent to the surface of the support by hydrogen bonding, electrostatic interactions, hydrophobic forces, van der Waals forces.
4. The method of claim 1, wherein in step 2) the blocking agent comprises BSA, casein or tween.
5. The method according to claim 1, wherein in step 3), antigen capture is performed at a temperature of 25 to 37℃for a period of 10 to 180 minutes.
6. The method of claim 1, wherein in step 4) the detection reagent comprises an aptamer or an antibody;
when the detection reagent is an aptamer, one structure of the aptamer is as follows:
NNNN......NNNN[AA...AA XXX......XXX]n；
wherein: nnnn..nnnn..nnnn is an aptamer sequence that specifically binds to a target, xxx..xxx..xxx is a specific nucleic acid sequence that can complementarily bind to a guide RNA, aa..aa is a spacer sequence;
alternatively, one structure of the aptamer is as follows:
NNNN......NNNN...NNN...NNN[AA...AA NNN...NNN]n；
nnnn..nnnn..nnn..nnn..nnn is an aptamer sequence that specifically binds to a target, nnn..nnn..nnn is a specific nucleic acid sequence that binds complementarily to a guide RNA while the sequence is part of an aptamer, aa..aa is a spacer sequence;
the spacer sequence and the specific nucleic acid sequence are repeated a plurality of times; the spacer sequence and the specific nucleic acid sequence may be located at the 3 'end of the aptamer or at the 5' end of the aptamer.
7. The method of claim 6, wherein, when the detection reagent is an antibody, the specific nucleic acid sequence is bound directly or indirectly to the antibody in a covalent or non-covalent form; or directly or indirectly by covalent or non-covalent forms to a secondary antibody that can specifically bind to the detection antibody.
8. The method of claim 1, wherein in step 5), the Cas protein concentration is 50-500nM, the guide RNA concentration is 50-500nM, the reporter probe concentration is 10-1000nM, the reaction temperature is 25-37 ℃ and the reaction time is 10-60min; cas proteins include V-type CRISPR/Cas effector proteins; the concentration of magnesium ions in the buffer system is 0.5-50mM, and the concentration of DTT is 0.5-100mM;
the reporter probe is a sequence which is not specifically cut by the activated Cas protein, and the cut probe can generate a detectable signal; when the report probe is a single-stranded DNA sequence, one end is modified with a detectable signal, and the fragment length is 5-40 nucleotides;
the reporter group or the marker molecule comprises a fluorescent group and a quenching group, wherein the fluorescent group is selected from one or more of FAM, VIC, HEX, FITC, JOE, TET, CY3, CY5, ROX, texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.
9. A system for ultrasensitive detection of an analyte of interest in a sample, comprising a capture reagent according to any one of claims 1 to 8, a blocking agent for blocking, a detection reagent, and a Cas protein reaction system.
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