CN107419005A - A kind of QCM detection methods and application based on multi-signal amplifying technique detection lysozyme - Google Patents
A kind of QCM detection methods and application based on multi-signal amplifying technique detection lysozyme Download PDFInfo
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Abstract
The present invention discloses a kind of QCM detection methods based on multi-signal amplifying technique detection lysozyme and application, DNA hybridize with the fit partial complementarity of lysozyme, reacted by lysozyme and fit specific binding, DNA is discharged.The hairpin dna and assistant DNA Complementary hybridizations modified on the DNA and gold plaque of release form Y-shaped structure, in the presence of restriction enzyme, sheared by specific recognition site and open hairpin dna, in the presence of DNA ligase and archaeal dna polymerase, to latch cyclic DNA as template strand, along the hairpin dna aggregation growth being opened, forming one has the single-stranded of a large amount of repetitive sequences.Signal probe and the repetitive sequence hybridization of generation for being marked with biotin are complementary, and rear catalyzing hydrogen peroxide is combined with the streptavidin of HRP marks and aoxidizes the generation precipitation reaction of 4 chloronaphthols, so as to add chip surface quality, high-sensitivity detections of the QCM to lysozyme is realized.
Description
Technical Field
The invention relates to a QCM detection method for detecting lysozyme based on multiple signal amplification technologies and application thereof, belonging to the technical field of biomolecule detection.
Background
Tumors are one of the serious life and health threatening malignancies of humans. At present, the clinical diagnosis method mainly depends on the imaging to detect the morphology of tumor cells, and the resolution is low. The tumor marker is used as an important index for tumor diagnosis, and the detection of the tumor marker has important significance for early diagnosis and treatment of diseases and prognosis. More than 100 tumor markers are found, but the number of tumor markers in early stage of cancer patients is very small, and the conventional detection method is difficult to perform accurate quantitative analysis on the tumor markers. Therefore, a novel method for searching for an identification probe capable of selecting a tumor marker and establishing the tumor marker with high sensitivity and good specificity has very important scientific significance and clinical value for early diagnosis and treatment of cancer.
Disclosure of Invention
In order to improve the sensitivity of detecting lysozyme, the present inventors previously proposed a technical solution, which includes: the specific binding effect of the aptamer and the target protein is utilized, DNA S1 hybridized and complemented with the aptamer is released to be further combined with a hairpin probe fixed on the surface of an electrode, an initiation chain is released through shearing of a specific recognition site under the action of restriction endonuclease, complementary DNA S1 is released to form cyclic amplification, rolling circle replication amplification reaction is carried out under the action of T4 ligase and DNA polymerase, a single chain with a large number of repetitive sequences is formed, an avidin-labeled HRP is captured to the surface of a chip by utilizing a biotin-labeled signal probe, then a biocatalytic precipitation reaction is initiated, the quality of the chip is increased, and therefore the protein is detected.
However, the inventors have analyzed that DNA S1 hybridizes to hairpin DNA, and when restriction endonuclease exists, the hairpin DNA is cleaved at the recognition site, the hairpin DNA is opened, and the cleaved DNA S1 fragment is released, and although it can hybridize to other hairpin DNA, it does not have the recognition site of restriction endonuclease and thus cannot be cleaved, so that the cycle reaction performance is not excellent enough, resulting in low sensitivity.
The inventor finds that after the problem, various improvements are made on the prior technical scheme, however, the technical scheme of 'complementary hybridization of main DNA, assistant DNA and hairpin DNA to form Y-shaped structure' is adopted in the detection method, and the detection method is unexpectedly found that 1.0 × 10 can be detected by lysozyme-16mol/L, the sensitivity is improved by 10 times compared with the prior work, and the method has outstanding beneficial effects. The analysis of the inventor can be carried out, and the signal is enhanced due to the enhanced hybridization process for forming the Y-shaped structure, so that the detection sensitivity is improved.
The technical scheme adopted by the invention is as follows:
the invention aims to provide a QCM detection method for detecting lysozyme based on multiple signal amplification technologies, which comprises the following steps: complementary hybridization is carried out on the lysozyme aptamer connected with the magnetic bead carrier and the main DNA part, and the main DNA is released through the specific binding reaction of the lysozyme and the lysozyme aptamer; the main DNA, the assistant DNA and the hairpin DNA connected with the gold sheet are complementarily hybridized to form a Y-shaped structure; under the action of restriction endonuclease, opening hairpin DNA by shearing specific recognition site, and dissociating complete main DNA for recycling, wherein the complete main DNA forms a template with other hairpin DNA and assistant DNA to enhance hybridization; under the action of DNA ligase and DNA polymerase, taking the latch circular DNA as a template chain, and carrying out polymerization growth along the opened hairpin DNA to form a DNA single chain with a plurality of sections of repetitive sequences; the signal probe marked with biotin is hybridized and complemented with a repetitive sequence in a generated DNA single chain, and is combined with HRP-marked chain enzyme avidin to catalyze hydrogen peroxide to oxidize 4-chloronaphthol to generate precipitation reaction, so that the surface quality of gold flakes is increased, and the high-sensitivity detection of the QCM on lysozyme is realized; wherein,
the main DNA comprises a first section of main DNA and a second section of main DNA which are mutually connected, and the first section of main DNA is complementarily hybridized with a partial lysozyme aptamer sequence;
the assistant DNA comprises a first assistant DNA and a second assistant DNA which are connected with each other, and the second assistant DNA is complementarily hybridized with part of the first main DNA sequence;
the hairpin DNA comprises a specific recognition site of restriction enzyme in a circular sequence, a part of the circular sequence can be complementarily hybridized with the second section of main DNA, and a part of the circular sequence can be complementarily hybridized with the first section of assistant DNA, thereby forming a Y-shaped structure.
The method is a non-disease diagnostic and therapeutic method.
The second object of the present invention is to provide a QCM assay kit for lysozyme assay based on various signal amplification techniques, comprising:
lysozyme aptamer connected with a magnetic bead carrier, main DNA, assistant DNA, hairpin DNA connected with a gold sheet, and endonuclease;
rolling circle replication amplification reaction system: comprises lock loop circular DNA for rolling circle replication amplification, DNA ligase, DNA polymerase and amplification raw material dNTPs.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
(1) the invention provides a QCM sensing system and a detection method for detecting lysozyme with high sensitivity by utilizing various signal amplification technologies, firstly, main DNA, assistant DNA and hairpin DNA are complementarily hybridized to form a Y-shaped structure, and the hybridization process is enhanced to enhance signals; secondly, the invention adopts rolling circle replication amplification reaction to obtain a DNA long chain with a large number of repetitive sequences, so as to further enhance signals; furthermore, HRP is modified on a DNA long chain through the specific combination of biotin and avidin; finally, the QCM is used for detection after a series of signal method technologies.
Compared with the previous work, the invention has the core invention that the complementary hybridization of the main DNA, the assistant DNA and the hairpin DNA is adopted to form a Y-shaped structure, when the restriction endonuclease exists, the hairpin DNA is cut and opened at the recognition site, the complete main DNA is released, the hybridization process is enhanced, so that the signal is enhanced, and the detection sensitivity is improved by one order of magnitude-16mol/L, sensitivity is improved by 10 times compared with the previous work.
(2) The detection method provided by the invention is simple to operate, low in cost, rapid and efficient.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of the design process of the present invention.
FIG. 2 is a lysozyme curve, the left graph is a concentration gradient curve, the concentrations of a-f are respectively 0 and 1.0 × 10-16mol/L、1.0×10-15mol/L、1.0×10-14mol/L、1.0×10-13mol/L、1.0×10-12mol/L; the right graph is a working curve.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Interpretation of terms:
QCM (quartz crystal microbalance) is a high-precision quality detection instrument, has the advantages of real-time monitoring, high precision and the like, and the precision can reach 10-9The gram rating.
HRP, horseradish peroxidase.
As introduced in the background art, the detection of lysozyme in the prior art has certain defects, and in order to solve the technical problems, the invention provides the QCM detection method for detecting lysozyme based on various signal amplification technologies.
The detection method comprises the following steps: complementary hybridization is carried out on the lysozyme aptamer connected with the magnetic bead carrier and the main DNA part, the main DNA is released through the specific binding reaction of the lysozyme and the lysozyme aptamer, and the main DNA and the assistant DNA are hybridized with the hairpin DNA connected with the gold sheet to form a Y-shaped structure; when the restriction endonuclease exists, cutting open the hairpin DNA at the recognition site, and dissociating the complete main DNA for recycling, wherein the complete main DNA forms a template for enhancing the hybridization with other hairpin DNA and assistant DNA; under the action of DNA ligase and DNA polymerase, the opened hairpin DNA grows by polymerization by taking the latch circular DNA as a template to form a DNA single chain with a plurality of sections of repetitive sequences; the signal probe marked with biotin is hybridized and complemented with a repetitive sequence in a generated DNA single chain, and is combined with HRP-marked chain enzyme avidin to catalyze hydrogen peroxide to oxidize 4-chloronaphthol to generate precipitation reaction, so that the surface quality of gold flakes is increased, and the high-sensitivity detection of the QCM on lysozyme is realized; wherein,
the main DNA comprises a first section of main DNA and a second section of main DNA which are mutually connected, and the first section of main DNA is complementarily hybridized with the lysozyme aptamer;
the assistant DNA comprises a first assistant DNA and a second assistant DNA which are connected with each other, and the second assistant DNA is complementarily hybridized with a part of the second main DNA sequence;
the hairpin DNA comprises a specific recognition site of restriction enzyme in a circular sequence, a part of the circular sequence can be complementarily hybridized with the second section of main DNA, and a part of the circular sequence can be complementarily hybridized with the first section of assistant DNA, thereby forming a Y-shaped structure.
The detection method specifically comprises the following steps:
(1) activation of magnetic beads:
the carboxyl modified magnetic beads were washed with imidazole hydrochloride buffer, added to imidazole buffer containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and shaken.
(2) Modification of magnetic bead carrier:
adding lysozyme aptamer into the activated magnetic beads for reaction; magnetic separation, washing, adding main DNA solution for reaction; performing magnetic separation, washing, and adding a sample solution to be detected containing lysozyme for reaction; performing magnetic separation, and taking supernatant;
(3) gold plate modified DNA:
adding hairpin DNA to the gold plate for reaction, washing, drying, fixing the hairpin DNA, and passivating the gold plate; adding assistant DNA, the supernatant obtained in the step (2) and restriction endonuclease for reaction, washing and drying;
(4) rolling circle replication amplification reaction:
uniformly mixing DNA ligase, ligase buffer solution and lock catch circular DNA, adding the mixture into the gold plate obtained by the treatment in the step (3) for reaction, washing and drying; then mixing DNA polymerase, polymerase buffer solution, dNTPs and water uniformly, adding the mixture to a gold sheet for reaction, washing and drying;
(5) HRP modification:
adding a biotin-modified probe into the gold sheet obtained by the treatment in the step (4) for reaction, washing and drying; then adding streptavidin modified HRP for reaction;
(6) QCM detection:
adding H into the gold flakes obtained by the treatment in the step (5)2O2And detecting the frequency change value in real time by a quartz crystal microbalance.
The following is a detailed description of the above steps:
in the step (1), the solution is added into an imidazole buffer solution containing EDC for reaction for 2 h.
For better activating the magnetic beads and facilitating subsequent modification of magnetic bead carriers, the specific steps of magnetic bead activation are as follows: the carboxyl group-modified magnetic beads were washed several times with imidazole hydrochloride buffer, added to 100. mu.L of imidazole buffer containing EDC (0.2M), and shaken at 37 ℃ for 2 hours. Wherein the concentration of the imidazole hydrochloric acid buffer solution is 0.1M, and the pH value is 6.8.
And (2) adding lysozyme aptamer into the activated magnetic beads, and reacting for 16 h.
The reaction time after the addition of the master DNA solution was 1 h.
The reaction time for adding the sample solution containing lysozyme to be detected is 1 h.
In order to better modify the lysozyme aptamer into a magnetic bead carrier, the specific steps of modification of the magnetic bead carrier are as follows: adding lysozyme aptamer into the activated magnetic beads, and reacting for 16h at 37 ℃; magnetic separation, Tris-HCl washing, adding main DNA solution, and incubation for 1 h; performing magnetic separation, washing with Tris-HCl, adding a sample solution to be detected containing lysozyme, and oscillating for 1h at 37 ℃; performing magnetic separation, and collecting supernatant for storage.
In the step (3), the reaction time for combining the hairpin DNA and the gold plate is 16 h.
The time for adding the assistant DNA, the supernatant in the step (2) and the restriction endonuclease to carry out the reaction is 1.5 h.
The method comprises the following specific steps: adding the hairpin DNA onto a gold chip, standing for 16h at 37 ℃, washing with Tris-HCl and ultrapure water for 3 times respectively, and drying with nitrogen; after hairpin DNA is fixed, 1% bovine serum albumin (BSA, w/w) is added to the gold plate for passivation for 2 hours; adding assistant DNA, the supernatant in the step (2) and restriction endonuclease, standing for 1.5h at 37 ℃, washing with Tris-HCl and ultrapure water for several times respectively, and drying with nitrogen.
In the step (4), the reaction time of the DNA ligase and the locking circular DNA is 1 h.
Mixing DNA polymerase, polymerase buffer solution, dNTPs and water uniformly, and adding the mixture onto a gold plate for reaction for 2 hours.
In order to better perform the rolling circle replication amplification reaction and enhance the signal, the specific steps are as follows: uniformly mixing DNA ligase, ligase buffer solution and a lock catch probe, dropwise adding the mixture on the gold plate obtained by the treatment in the step (3), standing the mixture for 1h at 37 ℃, respectively washing the mixture for 3 times by using Tris-HCl and ultrapure water, and drying the mixture by using nitrogen; uniformly mixing DNA polymerase, polymerase buffer solution, dNTPs and ultrapure water, then dropwise adding the mixture on the surface of the gold plate, standing for 2 hours at 37 ℃, respectively washing for 3 times by Tris-HCl and ultrapure water, and drying by nitrogen.
In the step (5), the biotin-modified probe is added into the gold plate obtained by the treatment in the step (4) to react for 1 h.
The time for reaction by adding streptavidin modified HRP was 0.5 h.
In order to better realize QCM detection, the specific steps are as follows: dropwise adding the biotin-modified probe to the surface of the gold plate, standing for 1h at 37 ℃, washing with Tris-HCl and ultrapure water for 3 times respectively, and drying with nitrogen; then streptavidin modified HRP is added and the mixture is kept stand for 0.5h at 37 ℃.
The HRP is modified on a DNA single strand through the specific binding of biotin and avidin.
In the present invention, the sequence of the lysozyme aptamer is not particularly limited, and can be various lysozyme aptamers in the prior art, and in a preferred embodiment of the present invention, the sequence of the lysozyme aptamer is 5' -TTTTATC TAC GAA TTC ATC AGGGCT AAA GAG TGC AGA GTT ACT TAG-NH2-3', as represented by seq id NO: 1 is shown.
In the present invention, the sequence of the host DNA is not particularly limited as long as it can be partially complementarily hybridized with the lysozyme aptamer. In a preferred embodiment of the invention, the sequence of the main DNA is 5-ATG AAT TCG TAG ATC CGT TCG AC-3', as represented by seq id NO: 2, respectively. The main DNA comprises a first section of main DNA (shown as a single-dashed line) and a second section of main DNA (shown as a double-dashed line) which are mutually connected, wherein the first section of main DNA is complementarily hybridized with a part of lysozyme aptamer sequence (shown as a single-dashed line);
in the present invention, the sequence of the assistant DNA is not particularly limited as long as it can form a Y-shaped structure with the above-mentioned main DNA and hairpin DNA. In a preferred embodiment of the invention, the helper DNA sequence is 5-CCT CAG CAA AAT CTA CGA ATT CAT-3', as represented by seq id NO: 3, respectively. The assistant DNA comprises a first assistant DNA (shown as a double-dashed line) and a second assistant DNA (shown as a single-dashed line) which are linked to each other, and which complementarily hybridize to the first main DNA sequence (i.e., to a portion of the lysozyme aptamer sequence, as shown by a single-dashed line in the lysozyme aptamer).
In the present invention, hairpin DNA, i.e., DNA stem-loop structure, the design of conventional stem-loop structure is well known to those skilled in the art and includes a stem portion and a loop portion, the stem portion usually being composed ofThe loop portion is usually a single-stranded sequence formed by an oligonucleotide. The person skilled in the art will know the conventional sequences constituting the stem in the stem-loop structure, as long as the stalk formation is satisfied. The loop sequence in the hairpin DNA of the invention contains a recognition site specific for a restriction endonuclease (sequences shown in italics and bold)) In a preferred embodiment of the invention, the hairpin DNA has a sequence which is complementary to the second main DNA segment (sequence in bold) and complementary to the first auxiliary DNA segment (sequence in italics) Such as SEQ ID NO: 4, respectively. The hybridization complement of the stem is shown in single-dashed lines. The sequence of the ring portions is shown by double-dashed lines.
In the present invention, the shackle circular DNA is also called a shackle probe, a padlock probe or a padlock probe, an artificially synthesized oligonucleotide chain molecule has about 70-100 bases, and the shackle probe after forming the circular molecule can be used as a specific signal source to be rapidly amplified and detected by a rolling circle replication method reaction method. In a preferred embodiment of the present invention, the locking circular DNA has the sequence of 5' -PO4-GAC CCT CTA AAA CCC AACCCG CCC TAC CCA AAA CCC AAC CCG CCC TAC CCA AAA CCC AAC CCG CCC TAC CCA AGCACC GTT C-3', as shown in seq id NO: 5, respectively.
In the present invention, the probe sequence of the biotin-modified signaling probe is not particularly limited, and is designed according to the following principle: so long as it is complementary to the repeat sequence portion on the single-stranded DNA by hybridization. In a preferred technical scheme of the invention, the sequence of the biotin-modified probe is 5 '-biotin-TTTTTT CCC AAC CCG CCC TAC C-3', such as SEQ ID NO: and 6.
The invention also provides a QCM detection kit for detecting lysozyme based on multiple signal amplification technologies, which comprises:
lysozyme aptamer connected with a magnetic bead carrier, main DNA, assistant DNA, hairpin DNA connected with a gold sheet, and endonuclease;
rolling circle replication amplification reaction system: comprises lock loop circular DNA for rolling circle replication amplification, DNA ligase, DNA polymerase and amplification raw material dNTPs.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Example 1
Ultra-sensitive detection lysozyme based on multiple signal amplification technologies
Modification of magnetic bead carrier
(1) Activated magnetic bead
The beads were washed three times with imidazole HCl buffer (0.1M, pH 6.8), added to 100. mu.L of imidazole buffer containing 0.2mol/LEDC, and shaken at 37 ℃ for 2 h.
(2) Modification of magnetic bead vector
To the activated beads, 15. mu.L of 1.0 × 10 concentration was added-7mol/L lysozyme aptamer DNA, shaking for 16h at 37 ℃ for magnetic separation to remove DNA not connected to magnetic beads, washing with Tris-HCl, adding main DNA solution, incubating for 1h, performing magnetic separation, washing with Tris-HCl, and adding 30 μ L of different concentrations (1.0 × 10)-15mol/L、1.0×10-14mol/L、1.0×10-13mol/L、1.0×10-12mol/L、1.0×10-11mol/L) lysozyme, incubation for 1h at 37 ℃, and magnetic treatmentSeparating, collecting supernatant, and storing.
Second, gold plate modified DNA
30 μ L of 1.0 × 10-7Dropwise adding mol/L hairpin DNA onto the gold plate, incubating at constant temperature for 16h, washing with Tris-HCl buffer solution and ultrapure water for 3 times, blowing with nitrogen, adding 1% BSA onto the gold plate for passivation for 2h, and adding 15 μ L of 1.0 × 10-7mol/L assistant DNA, supernatant after magnetic separation and 1. mu.L 10U/. mu.L restriction endonuclease Nb. BbvCI, incubation for 1.5h at constant temperature, washing with Tris-HCl buffer and ultrapure water for 3 times respectively, and blowing with nitrogen gas.1. mu.L 5U/. mu.LT 4 ligase, 2.5. mu. L T4buffer and 21.5. mu.L 1.0 × 10-7Uniformly mixing and dripping a mol/L lock catch probe on a gold plate, incubating at constant temperature for 1h, respectively washing with Tris-HCl buffer solution and ultrapure water for 3 times, drying by nitrogen, mixing and dripping 1 mu L of 0.2U/mu L phi29 polymerase, 2.5 mu L phi29buffer, 2 mu L of 10mmol/LdNTPs and 19 mu L of ultrapure water on the surface of the gold plate, incubating at constant temperature for 2h, respectively washing with Tris-HCl buffer solution and ultrapure water for 3 times, drying by nitrogen, 30 mu L of 1.0 × 10-7And dropwise adding the mol/L biotin modified probe to the surface of the gold plate, incubating for 1h at constant temperature, washing for 3 times with Tris-HCl buffer solution and ultrapure water respectively, and drying by nitrogen. Then 30 mu L of streptavidin modified HRP is added, incubation is carried out for 0.5h at constant temperature, Tris-HCl buffer solution and ultrapure water are respectively washed for 3 times, and nitrogen is blown dry.
Three, QCM detection
Introducing 500 mu L of 1mmol/LH into the gold flakes2O2And 1mmol/L tetrachloronaphthol, and detecting the frequency change value.
The detection result is that the detection signal of the lysozyme is between-209 Hz and-81 Hz, and the detection range is about 1.0 × 10- 16mol/L～1.0×10-12mol/L。
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Qingdao university
<120> QCM detection method for detecting lysozyme based on multiple signal amplification technologies and application
<130>2017
<160>6
<170>PatentIn version 3.5
<210>1
<211>46
<212>DNA
<213> Artificial sequence
<400>1
ttttatctac gaattcatca gggctaaaga gtgcagagtt acttag 46
<210>2
<211>23
<212>DNA
<213> Artificial sequence
<400>2
atgaattcgt agatccgttc gac 23
<210>3
<211>24
<212>DNA
<213> Artificial sequence
<400>3
cctcagcaaa atctacgaat tcat 24
<210>4
<211>53
<212>DNA
<213> Artificial sequence
<400>4
ttttttctgt gtccgtattt agagggtcga acggtgctga ggtacggaca cag 53
<210>5
<211>82
<212>DNA
<213> Artificial sequence
<400>5
gaccctctaa aacccaaccc gccctaccca aaacccaacc cgccctaccc aaaacccaac 60
ccgccctacc caagcaccgt tc 82
<210>6
<211>22
<212>DNA
<213> Artificial sequence
<400>6
ttttttccca acccgcccta cc 22
Claims (10)
1. A QCM detection method for detecting lysozyme based on multiple signal amplification technologies is characterized by comprising the following steps: complementary hybridization is carried out on the lysozyme aptamer connected with the magnetic bead carrier and the main DNA part, and the main DNA is released through the specific binding reaction of the lysozyme and the lysozyme aptamer; the main DNA, the assistant DNA and the hairpin DNA connected with the gold sheet are complementarily hybridized to form a Y-shaped structure; under the action of restriction endonuclease, opening hairpin DNA by shearing specific recognition site, and dissociating complete main DNA for recycling, wherein the complete main DNA forms a template with other hairpin DNA and assistant DNA to enhance hybridization; under the action of DNA ligase and DNA polymerase, taking the latch circular DNA as a template chain, and carrying out polymerization growth along the opened hairpin DNA to form a DNA single chain with a plurality of sections of repetitive sequences; the signal probe marked with biotin is hybridized and complemented with a repetitive sequence in a generated DNA single chain, and is combined with HRP-marked chain enzyme avidin to catalyze hydrogen peroxide to oxidize 4-chloronaphthol to generate precipitation reaction, so that the surface quality of gold flakes is increased, and the detection of lysozyme by QCM is realized; wherein,
the main DNA comprises a first section of main DNA and a second section of main DNA which are mutually connected, and the first section of main DNA is complementarily hybridized with a partial lysozyme aptamer sequence;
the assistant DNA comprises a first assistant DNA and a second assistant DNA which are connected with each other, and the second assistant DNA is complementarily hybridized with part of the first main DNA sequence;
the hairpin DNA comprises a specific recognition site of restriction enzyme in a circular sequence, a part of the circular sequence can be complementarily hybridized with the second section of main DNA, and a part of the circular sequence can be complementarily hybridized with the first section of assistant DNA, thereby forming a Y-shaped structure.
2. The detection method according to claim 1, comprising in particular the following steps:
(1) activation of magnetic beads:
washing carboxyl modified magnetic beads with imidazole hydrochloric acid buffer solution, adding the carboxyl modified magnetic beads into imidazole buffer solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and oscillating;
(2) modification of magnetic bead carrier:
adding lysozyme aptamer into the activated magnetic beads for reaction; magnetic separation, washing, adding main DNA solution for reaction; performing magnetic separation, washing, and adding a sample solution to be detected containing lysozyme for reaction; performing magnetic separation, and taking supernatant;
(3) gold plate modified DNA:
adding hairpin DNA to the gold plate for reaction, washing, drying, fixing the hairpin DNA, and passivating the gold plate; adding assistant DNA, the supernatant obtained in the step (2) and restriction endonuclease for reaction, washing and drying;
(4) rolling circle replication amplification reaction:
uniformly mixing DNA ligase, ligase buffer solution and lock catch circular DNA, adding the mixture into the gold plate obtained by the treatment in the step (3) for reaction, washing and drying; then mixing DNA polymerase, polymerase buffer solution, dNTPs and water uniformly, adding the mixture to a gold sheet for reaction, washing and drying;
(5) HRP modification:
adding a biotin-modified probe into the gold sheet obtained by the treatment in the step (4) for reaction, washing and drying; then adding streptavidin modified HRP for reaction;
(6) QCM detection:
adding H into the gold flakes obtained by the treatment in the step (5)2O2And detecting the frequency change value in real time by a quartz crystal microbalance.
3. The detection method according to claim 2, wherein in the step (1), the reaction time is 2 hours when the solution is added into an imidazole buffer solution containing EDC;
preferably, the magnetic bead activation comprises the following specific steps: washing carboxyl modified magnetic beads for several times with imidazole hydrochloric acid buffer solution, adding into 100 μ L imidazole buffer solution containing EDC, and oscillating for 2h at 37 deg.C; wherein the concentration of the imidazole hydrochloric acid buffer solution is 0.1M, and the pH value is 6.8.
4. The detection method according to claim 2, wherein in the step (2), the lysozyme aptamer is added to the activated magnetic beads, and then the reaction is carried out for 16 hours;
the reaction time is 1h after the main DNA solution is added;
adding a sample solution to be detected containing lysozyme for 1 h;
preferably, the specific steps of modifying the magnetic bead carrier are as follows: adding lysozyme aptamer into the activated magnetic beads, and reacting for 16h at 37 ℃; magnetic separation, Tris-HCl washing, adding main DNA solution, and incubation for 1 h; performing magnetic separation, washing with Tris-HCl, adding a sample solution to be detected containing lysozyme, and oscillating for 1h at 37 ℃; performing magnetic separation, and collecting supernatant for storage.
5. The detecting method according to claim 2, wherein in the step (3), the reaction time for the combination of the hairpin DNA and the gold plate is 16 hours;
adding assistant DNA, the supernatant obtained in the step (2) and restriction endonuclease for reaction for 1.5 h;
the method comprises the following specific steps: adding the hairpin DNA onto a gold chip, standing for 16h at 37 ℃, washing with Tris-HCl and ultrapure water for 3 times respectively, and drying with nitrogen; after fixing the hairpin DNA, adding 1% bovine serum albumin to the gold plate for passivation for 2 h; adding assistant DNA, the supernatant in the step (2) and restriction endonuclease, standing for 1.5h at 37 ℃, washing with Tris-HCl and ultrapure water for several times respectively, and drying with nitrogen.
6. The detecting method according to claim 2, wherein in the step (4), the reaction time of the DNA ligase and the locking circular DNA is 1 h;
mixing DNA polymerase, polymerase buffer solution, dNTPs and water uniformly, and adding the mixture onto a gold plate for reaction for 2 hours;
the method comprises the following specific steps: uniformly mixing DNA ligase, ligase buffer solution and a lock catch probe, dropwise adding the mixture on the gold plate obtained by the treatment in the step (3), standing the mixture for 1h at 37 ℃, respectively washing the mixture for 3 times by using Tris-HCl and ultrapure water, and drying the mixture by using nitrogen; uniformly mixing DNA polymerase, polymerase buffer solution, dNTPs and ultrapure water, then dropwise adding the mixture on the surface of the gold plate, standing for 2 hours at 37 ℃, respectively washing for 3 times by Tris-HCl and ultrapure water, and drying by nitrogen.
7. The detection method according to claim 2, wherein in the step (5), the biotin-modified probe is added to the gold plate processed in the step (4) for reaction for 1 hour;
adding streptavidin modified HRP for reaction for 0.5 h;
the method comprises the following specific steps: dropwise adding the biotin-modified probe to the surface of the gold plate, standing for 1h at 37 ℃, washing with Tris-HCl and ultrapure water for 3 times respectively, and drying with nitrogen; then streptavidin modified HRP is added and the mixture is kept stand for 0.5h at 37 ℃.
8. The method of claim 1 or 2, wherein said lysozyme aptamer has the sequence 5' -TTTT ATCTAC GAA TTC ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG-NH2-3’；
The sequence of the main DNA is 5'-ATG AAT TCG TAG ATC CGT TCG AC-3';
the assistant DNA sequence is 5'-CCT CAG CAA AAT CTA CGA ATT CAT-3';
the hairpin DNA sequence is 5 '-SH-TTTTTT CTG TGT CCG TAT TTA GAG GGT CGA ACGGTG CTGAGG TAC GGA CAC AG-3'.
9. The method according to claim 1 or 2, wherein the sequence of the locked circular DNA is 5' -PO4-GACCCT CTA AAA CCC AAC CCG CCC TAC CCA AAA CCC AAC CCG CCC TAC CCA AAA CCC AACCCG CCC TAC CCA AGC ACC GTT C-3’；
The sequence of the biotin-modified probe is 5 '-biotin-TTTTTT CCC AAC CCG CCC TAC C-3'.
10. The QCM detection method detection kit for detecting lysozyme based on multiple signal amplification techniques according to any one of claims 1 to 9, characterized in that it comprises:
lysozyme aptamer connected with a magnetic bead carrier, main DNA, assistant DNA, hairpin DNA connected with a gold sheet, and endonuclease;
rolling circle replication amplification reaction system: comprises lock loop circular DNA for rolling circle replication amplification, DNA ligase, DNA polymerase and amplification raw material dNTPs.
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