CN117757901A - SERS detection kit for detecting acute myocardial infarction micro-nucleic acid markers, and preparation method and application thereof - Google Patents
SERS detection kit for detecting acute myocardial infarction micro-nucleic acid markers, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a SERS detection kit for detecting acute myocardial infarction micro nucleic acid markers, and a preparation method and application thereof. The kit comprises: a SERS detection chip with a tetrahedral DNA structure is modified on the surface of the silver nanorod array substrate; hairpin DNA single strand H2; the SERS Probe is used in combination with a substrate, and is gold nanoparticles with surface modified Probe single chains and Raman molecules. (1) Preparing a SERS detection chip with a tetrahedral DNA structure modified on the surface of a silver nano rod array substrate; (2) Under the conditions that the target micro nucleic acid marker is triggered and the hairpin DNA single-strand H2 is assisted, a Catalytic Hairpin Assembly (CHA) reaction occurs, and a SERS probe is captured to a SERS detection chip; (3) The quantitative detection of the acute myocardial infarction micro nucleic acid marker is realized by collecting the Raman signal of the SERS probe on the SERS detection chip. The method has the advantages of rapid detection (40 minutes), high sensitivity (the detection limit is as low as the aM level) and good specificity.
Description
Technical Field
The invention belongs to the field of functional nano materials and biological detection, and particularly relates to a SERS detection kit for detecting acute myocardial infarction micro nucleic acid markers, and a preparation method and application thereof.
Background
Numerous studies have shown that micronucleic acid markers (mirnas) are closely related to cardiac fate. They can regulate the death and regeneration of cardiac cells following Acute Myocardial Infarction (AMI). Some mirnas, such as miR-208a and miR-499, are rapidly expressed after the onset of AMI and are therefore considered potential markers for AMI. Thus, analysis of AMI-related mirnas can improve the accuracy of diagnosing AMI. However, mirnas have unique features of small size, high sequence similarity, low abundance, etc. The actual environment of the heart is more complex, various cell types exist, unnecessary miRNA and low-content target miRNA can appear in the blood of a patient, and various interferents in the blood can lead the miRNA to be easily degraded. Thus, detection of only a single AMI-related miRNA inevitably results in false positive or false negative results. In order to realize accurate screening and diagnosis of AMI, a sensing strategy capable of accurately detecting various AMI-related mirnas needs to be constructed. Most of the currently developed technologies for determining AMI-related mirnas, such as fluorescence, electrochemiluminescence (ECL), electrochemistry, etc., are time-consuming and can only perform single-index detection, and lack accuracy. Therefore, surface Enhanced Raman Scattering (SERS) technology is one of the most powerful spectroscopic tools for determining AMI-related mirnas due to advantages such as specific fingerprint, multiplexing, and rapid data acquisition. The sensing chip is constructed based on the unique advantages of the SERS technology, so that the accurate determination of the AMI related miRNA can be realized.
In addition to accuracy, the sensitivity of the biosensing chip is very important for detecting biological samples such as low abundance mirnas. To increase sensitivity, probe immobilization is typically optimized and/or signal amplification methods are employed. However, maldistribution of probes, crowding effects and cross-reactions can negatively impact stability and sensitivity of the sensor chip, and thus, there is a need to explore an optimized method of immobilization of probes. Currently, probes with modified substrate surfaces mainly take three forms: single-stranded DNA, hairpin DNA single-stranded and three-dimensional DNA structures. However, when single-stranded DNA is immobilized, problems of maldistribution and entanglement are likely to occur, and there is a problem that the hairpin DNA single strand has large steric hindrance on the surface of the substrate. In contrast, tetrahedral DNA structured probes (TSPs) are an ideal method for immobilization of probes due to their simple synthesis, high mechanical rigidity, ordered structure, and high stability. Modification of the TSP sensing interface not only increases the accessibility and selectivity of the probe, but also reduces non-specific adsorption, thereby increasing the sensitivity of detecting target molecules. In addition, TSP, as a three-dimensional DNA structure, can be exposed to a solution to have hybridization efficiency similar to that of a homogeneous solution, further improving the sensitivity of detection. Catalytic Hairpin Assembly (CHA) is a highly efficient isothermal enzyme-free amplification technique that is widely used to increase the sensitivity of biosensing chips.
Aiming at the problem of insufficient sensitivity and accuracy of the existing detection of the micro nucleic acid marker, a SERS detection kit for detecting the acute myocardial infarction micro nucleic acid marker needs to be developed, and the SERS detection kit is simple in preparation and application, rapid in detection, high in sensitivity and high in accuracy.
Disclosure of Invention
The invention aims to: aiming at the problem of insufficient sensitivity and accuracy of the existing detection of the micro nucleic acid marker, the invention discloses a SERS detection kit for detecting the acute myocardial infarction micro nucleic acid marker, and a preparation method and application thereof.
The SERS detection kit comprises a SERS detection chip, hairpin DNA single-stranded H2 and a SERS probe. The SERS detection chip is prepared by modifying a tetrahedral DNA structure on the surface of a silver nano-rod array substrate, the hairpin DNA single-strand H2 is designed according to a hairpin DNA sequence extending from the 5' -end of a D chain in the tetrahedral DNA structure, and the SERS Probe is prepared by modifying a Probe single-strand and Raman molecules on the surface of gold nano-particles.
The SERS detection kit for detecting the acute myocardial infarction micro-nucleic acid marker disclosed by the invention is simple to prepare, rapid in detection (40 minutes), high in sensitivity (the detection limit is as low as an aM level), and good in specificity (single base difference can be identified), can realize various biomarkers related to acute myocardial infarction by modifying the base sequence of a nucleic acid probe, and provides a simple and practical detection technology for effective diagnosis of the acute myocardial infarction.
In order to solve the technical problems, the invention adopts the following technical scheme:
the SERS detection kit for detecting the acute myocardial infarction micro nucleic acid marker comprises a SERS detection chip, a hairpin DNA single-strand H2 and a SERS probe matched with a substrate.
The SERS detection chip is prepared by modifying a tetrahedral DNA structure on the surface of a silver nano rod array substrate;
the hairpin type DNA single-chain H2 is designed according to a hairpin type DNA sequence extending from the 5' -end of a D chain in a tetrahedral DNA structure;
the SERS Probe is prepared by modifying a Probe single chain and a Raman molecule on the surface of a gold nanoparticle.
The silver nano rod array is prepared by adopting a vacuum electron beam evaporation coating technology, and 3X 10 array type small holes are prepared on the surface of the silver nano rod array by using a Polydimethylsiloxane (PDMS) film, wherein the aperture of each small hole is 3-5 mm, and the depth is 0.8-1.2 mm. Preferably, the aperture of the small hole is 4mm and the depth is 1mm.
The particle size of the gold nano particles (AuNP) is 15-100 nm. Preferably, the gold nanoparticles have a particle size of 15nm.
Taking miR-208a as an example of a micro nucleic acid marker for detecting acute myocardial infarction;
the SERS detection chip is characterized in that the surface of the SERS detection chip is modified by a sequence shown as SEQ ID NO:1-4 and D-208a to form a tetrahedral DNA structure TSP-208a;
the hairpin type DNA single-stranded H2 used for detecting the miR-208a SERS detection kit is a DNA single-stranded H2 with a sequence shown as SEQ ID NO:5, wherein the SERS probe for miR-208a detection is a surface modification sequence shown in SEQ ID NO:6 and a Raman molecule of 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB).
Taking a micro nucleic acid marker miR-499 for detecting acute myocardial infarction as an example;
the SERS detection chip is characterized in that the surface of the SERS detection chip is modified by a sequence shown as SEQ ID NO: a, B, C and the sequence shown as SEQ ID NO:8 and D-499, and a silver nanorod array substrate of a tetrahedral DNA structure TSP-499 formed by assembling the same;
the hairpin type DNA single-stranded H2 used for detecting the miR-499SERS detection kit is provided with a sequence shown in SEQ ID NO:9, wherein the SERS probe for miR-499 detection is a surface modification sequence shown in SEQ ID NO:10 and a Raman molecule of 4-mercaptobenzoic acid (4 MBA).
The working concentration of the tetrahedral DNA structure is 0.1-5 mu M, and preferably, the concentration of the tetrahedral DNA structure is 1 mu M;
the working concentration of hairpin DNA single-strand H2 is 5-20. Mu.M, preferably H2 concentration is 10. Mu.M;
the concentration of the SERS probe is 1-10 nM, and preferably, the concentration of the SERS probe is 2.3nM. Specifically, in the process of preparing a SERS Probe, the dosage of gold nanoparticles (AuNP) is 500 mu L of 2.3nM, the single strand of Probe-208a (or Probe-499) is 10 mu L of 50 mu M, the 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) (or 4-mercaptobenzoic acid (4 MBA)) of Raman molecule is 10 mu L of 100 mu M, and the gold nanoparticles are obtained after centrifugal purification and volume fixation to 75 mu L.
The preparation method of the SERS detection kit for detecting the acute myocardial infarction micro nucleic acid marker comprises the steps of mixing A, B, C and D with equal molar ratio to form a tetrahedral DNA structure TSP, and co-culturing with a silver nanorod array substrate to prepare the SERS detection chip; adding Raman molecules into the AuNP solution with single-chain functionalization of the Probe, so as to prepare the SERS Probe, namely: adding 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) into a Probe-208a single-chain functionalized AuNP solution to prepare a SERS Probe for miR-208a detection; SERS probes for miR-499 detection can be prepared by adding 4-mercaptobenzoic acid (4 MBA) to a Probe-499 single-stranded functionalized AuNP solution.
Specifically, the method comprises the following steps:
taking the detection of acute myocardial infarction micro nucleic acid marker miR-208a as an example,
1) SERS detection chip preparation:
(1) Preparing a silver nano rod array and keeping the surface clean;
(2) A, B, C and D-208a with equal molar ratio are mixed and heated to 90-95 ℃ for 5-10 min, then cooled to 25-37 ℃ in ice water bath, and assembled to form a tetrahedral DNA structure TSP-208a;
(3) Placing 10-20 mu L of 0.1-5 mu M tetrahedral DNA structure TSP-208a and silver nanorod array substrate in 25-37 ℃ and 60-80% humidity environment for co-culturing for 3-5 hours;
(4) Sequentially with reaction buffer (10 mM tris-HCl and 1mM MgCl) 2 pH 8.0) cleaning the substrate to obtain a SERS detection chip;
2) Hairpin DNA single-strand H2 preparation:
designing and synthesizing hairpin DNA single-chain H2-208a with the sequence shown as SEQ ID NO.5 according to the hairpin DNA sequence extended at the 5' end in D-208 a;
3) SERS probe preparation:
(1) Mixing 1-10 mu L of 10-100 mu M Probe-208a single strand with 100-500 mu L of 1-10 nM AuNP solution in 0.5 XTBE solution and culturing at 200-400 rpm at 25-37 ℃ overnight;
(2) The molar concentration 1 was slowly added 4 times every 30 minutes: 2:3:4, and placing the solution in 10-50 mu L of 1-3M NaCl solution at the temperature of 25-37 ℃ for culturing overnight at 200-400 rpm, wherein the final concentration of NaCl is 100-300 mM;
(3) Adding 10-100 mu L of 10-100 mu M Raman molecule DTNB for reacting for 2-4 hours;
(4) Finally centrifuging to remove the supernatant, dispersing the centrifugal sediment by using 0.5 XTBE solution and fixing the volume to 10-100 mu L to obtain the SERS probe.
Wherein, the working concentration of the tetrahedral DNA structure in the steps 1) -3) is 0.1-5 mu M, the working concentration of the hairpin DNA single-chain H2 is 5-20 mu M, and the working concentration of the SERS probe is 1-10 nM.
The application of the SERS detection kit for detecting the acute myocardial infarction micro nucleic acid marker is as follows (taking the detection of the acute myocardial infarction micro nucleic acid marker miR-208a as an example, the nucleotide sequence is shown as SEQ ID NO: 7):
1) Mixing hairpin DNA single-stranded H2-208a and SERS probes with sample solutions containing target acute myocardial infarction micro-nucleic acid markers miR-208a with different concentrations (the concentration range is 100 aM-1 nM), and dripping the mixed solutions onto the surface of an SERS detection chip for co-culture;
2) After cleaning SERS detection chips for multiple times, carrying out SERS test to obtain SERS spectrums and characteristic signal intensity values thereof corresponding to target miR-208a with different concentrations, taking logarithm of the target miR-208a concentration as an abscissa, and taking SERS characteristic peak intensity value as an ordinate, obtaining a working curve of the SERS detection kit, and calculating the detection limit of the SERS detection kit for detecting miR-208a according to the working curve;
3) Mixing a sample to be detected, hairpin DNA single-stranded H2-208a and a SERS probe, then dripping the mixture onto the surface of a SERS detection chip for co-culture, cleaning the chip for multiple times, and then carrying out SERS test to obtain a SERS spectrum and a characteristic signal intensity value thereof, and calculating according to a working curve to obtain the concentration of the target miR-208a in the sample to be detected.
Wherein, the co-culture conditions in the steps 1) and 3) are that the culture is carried out for 30 to 60 minutes at the temperature of 25 to 37 ℃ at 200 to 400 rpm.
Detecting another acute myocardial infarction micro nucleic acid marker miR-499, wherein the nucleotide sequence of the marker is shown in SEQ ID NO: 11.
The detection principle of the invention (taking the detection of acute myocardial infarction micro nucleic acid marker miR-208a as an example) is as follows:
a, B, C and D-208a are mixed and assembled to form a tetrahedral DNA structure TSP-208a, and the tetrahedral DNA structure TSP-208a is fixed on the surface of a silver nanorod array through a sulfydryl and silver to obtain the SERS detection chip.
When the target miR-208a is present, it can hybridize to hairpin DNA designed at the apex of the tetrahedral DNA structure TSP-208a, triggering a Catalytic Hairpin Assembly (CHA) reaction. Then, after simultaneously adding hairpin DNA single-stranded H2-208a and SERS probes on the SERS detection chip, the Probe single-stranded on the SERS probes hybridizes with the exposed sticky ends of the H2-208a, and the SERS probes are captured on the SERS detection chip.
The released miR-208a can trigger a new cycle, so that target circulation is realized, and after SERS probes are further specifically combined, raman signals from the SERS probes can be collected from an SERS detection chip, so that quick, high-sensitivity and accurate detection of the acute myocardial infarction micro nucleic acid marker miR-208a is realized.
The beneficial effects are that: the invention has the following advantages:
the invention discloses a SERS detection kit for detecting acute myocardial infarction micro nucleic acid markers, and a preparation method and application thereof. The SERS detection kit comprises a SERS detection chip, hairpin DNA single-stranded H2 and a SERS probe. The SERS detection chip is prepared by modifying a tetrahedral DNA structure on the surface of a silver nano-rod array substrate, the hairpin DNA single-strand H2 is designed according to a hairpin DNA sequence extending from the 5' -end of a D chain in the tetrahedral DNA structure, and the SERS Probe is prepared by modifying a Probe single-strand and a Raman molecule on the surface of a gold nano-particle.
The SERS detection kit for detecting the acute myocardial infarction micro-nucleic acid marker disclosed by the invention is simple to prepare, rapid in detection (40 minutes), high in sensitivity (the detection limit is as low as an aM level), and good in specificity (single base difference can be identified), can realize various biomarkers related to acute myocardial infarction by modifying the base sequence of a nucleic acid probe, and provides a simple and practical detection technology for effective diagnosis of the acute myocardial infarction.
Drawings
FIG. 1 is a schematic diagram of the preparation of a SERS detection kit for detecting acute myocardial infarction micronucleic acid markers;
FIG. 2 is a concentration optimization experiment of a surface immobilized tetrahedral DNA structure of the SERS detection chip of example 1; FIG. 2a is a SERS spectrum of different concentrations of tetrahedral DNA structure TSP-208a immobilized on a SERS detection chip for detecting miR-208 a; FIG. 2b is a plot of each line of FIG. 2a at 1331cm -1 Raman shift pairThe corresponding SERS peak intensity;
FIG. 3 is a culture (incubation) time optimization experiment of the surface immobilized tetrahedral DNA structure of the SERS detection chip of example 1; FIG. 3a is a SERS spectrum of a SERS detection chip incubated for different times with the tetrahedral DNA structure TSP-208a for detecting miR-208 a; FIG. 3b is a plot of the lines of FIG. 3a at 1331cm -1 The intensity of the SERS peak corresponding to the Raman shift;
FIG. 4 is an optimal culture (detection) time optimization experiment for detecting miR-208a by using a SERS detection kit of example 2; FIG. 4a is a SERS spectrum corresponding to different incubation (detection) times for a SERS detection kit to detect miR-208 a; FIG. 4b is a plot of the lines of FIG. 4a at 1331cm -1 The intensity of the SERS peak corresponding to the Raman shift;
FIG. 5 is an operating curve of the SERS detection kit of example 2 for detecting miR-208a at various concentrations; FIG. 5a is a SERS spectrum corresponding to different concentrations of miR-208a detected by a SERS detection kit; FIG. 5b is a plot of each line of FIG. 5a at 1331cm -1 The intensity of the SERS peak corresponding to the Raman shift;
FIG. 6 is a performance characterization of the SERS detection kit of example 3 for detecting miR-208 a; FIG. 6a is a specific characterization of SERS detection kit to detect miR-208 a; FIG. 6b is a representation of the homogeneity of a SERS detection kit for detecting miR-208 a; FIG. 6c is a reproducibility characterization of the detection of miR-208a by SERS detection kit;
FIG. 7 is a graph of the operation of the SERS detection kit of example 4 for detecting miR-499 at various concentrations; FIG. 7a is a SERS spectrum corresponding to different concentrations of miR-499 detected by a SERS detection kit; FIG. 7b is a graph of FIG. 7a showing lines at 1077cm -1 The intensity of the SERS peak corresponding to the Raman shift;
FIG. 8 is a performance characterization of the SERS detection kit of example 5 for detecting miR-499; FIG. 8a is a specific characterization of SERS detection kit for detecting miR-499; FIG. 8b is a representation of the homogeneity of a SERS detection kit for detection of miR-499; FIG. 8c is a reproducibility characterization of the detection of miR-499 by the SERS detection kit.
Detailed Description
The invention is further illustrated below in connection with specific embodiments, but the invention is not limited to the examples.
The invention provides a SERS detection kit for detecting acute myocardial infarction micro nucleic acid markers, which comprises the following components:
1. SERS detection chip
The SERS detection chip takes a silver nano rod array as a substrate, a tetrahedral DNA structure is modified on the surface of the substrate, the tetrahedral DNA structure is formed by assembling four single chains of A, B, C and D-208a (or D-499) and is fixed on the surface of the substrate through covalent bond formed by sulfydryl and silver, and the SERS detection chip can be obtained;
the working concentration of the tetrahedral DNA structure is 0.1-5 mu M, and preferably, the concentration of the tetrahedral DNA structure is 1 mu M;
2. the working concentration of hairpin DNA single-strand H2 is 5-20. Mu.M, preferably H2 concentration is 10. Mu.M;
3. SERS probe
The SERS Probe is prepared by modifying a Probe single chain and a Raman molecule on the surface of a gold nanoparticle. Wherein, the gold nanoparticles can realize strong SERS enhancement effect within a certain size and density range, and the particle size is 15-100 nm, preferably 15nm. The concentration of the SERS probe is 1-10 nM, and preferably, the concentration of the SERS probe is 2.3nM. Specifically, in the process of preparing a SERS Probe, the amount of gold nanoparticles (AuNP) is 500 mu L of 2.3nM, a single strand of Probe-208a (or Probe-499) is 10 mu L of 50 mu M, a single strand of Raman molecule 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) (or 4-mercaptobenzoic acid (4 MBA)) is 10 mu L of 100 mu M, and the SERS Probe is obtained after centrifugal purification and volume fixation to 75 mu L.
All the DNA base sequence fragments used above were obtained by artificial synthesis, and were synthesized by the division of biological engineering (Shanghai). The bolded parts in SM and DM are mismatched bases.
1. The nucleobase sequence of miR-208a to be detected in the following examples is:
5’-ATAAGA CGAGCAAAAAGC TTG T-3’
the nucleobase sequences used to detect miR-208a in the following examples are:
A(SEQ ID NO：1)：
5’-SH-(CH 2 ) 6 -TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3’
the 5' end of the A chain is connected with- (CH) 2 ) 6 -SH groups;
B(SEQ ID NO：2)：
5’-SH-(CH 2 ) 6 -TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3’
the 5' end of the B chain is connected with- (CH) 2 ) 6 -SH groups;
C(SEQ ID NO：3)：
5’-SH-(CH 2 ) 6 -TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3’
the 5' end of the C chain is connected with- (CH) 2 ) 6 -SH groups;
D-208a(SEQ ID NO：4)：
5’-ACA AGC TTT TTG CTC GTC TTA TAA GCT TGT CCA ATA AAT AAG ACG AGC AAA TTT TTT TTT TAC ATT CCT AAG TCT GAA ACA TTA CAG CTT GCT ACA CGA GAA GAG CCG CCA TAG TA-3’
H2-208a(SEQ ID NO：5)：
5’-GTC TTA TTT ATT GGA CAA GCT TAT AAG ACG AGC AGG AAG CTT GTC CAA TAA AAC CTT GCA GGA TT-3’
Probe-208a(SEQ ID NO：6)：
5’-SH-(CH 2 ) 6 -TTT TTT TTA ATC CTG CAA GGT T-3’
the 5' end of the Probe-208a is connected with- (CH) 2 ) 6 -SH groups;
2. the nucleobase sequence of miR-499 to be detected in the following examples is:
5’-TTAAGA CTT GCAGTGATG TTT-3’
the nucleobase sequences used to detect miR-499 in the following examples are:
D-499(SEQ ID NO：8)：
5’-AAA CAT CAC TGC AAG TCT TAA GAT GTT TTT AAG GAT TAA GAC TTG CAG TTT TTT TTT TTA CAT TCC TAA GTC TGA AAC ATT ACA GCT TGC TAC ACG AGA AGA GCC GCC ATA GTA-3’
H2-499(SEQ ID NO：9)：
5’-GTC TTA ATC CTT AAA AAC ATC TTA AGA CTT GCA CGG ATG TTT TTA AGG ACC ATG TTA ACC CTT-3’
Probe-499(SEQ ID NO：10)：
5’-SH-(CH 2 ) 6 -TTT TTT TTA AGG GTT AAC ATG G-3’
the 5' end of the Probe-499 is connected with- (CH) 2 ) 6 -SH groups;
3. the nucleic acid base sequences corresponding to a single base mismatch (SM), a two base mismatch (DM) and a complete mismatch (miR-106 a) of a specific experiment aiming at miR-208a and miR-499 detection of an acute myocardial infarction micro nucleic acid marker are as follows:
single base mismatch SM-208a (SEQ ID NO: 12):
5’-ATA AGA CGA GCA AAA AGC TCG T-3’
two base mismatch DM-208a (SEQ ID NO: 13):
5’-ATT AGA CGA GCA AAA AGC TCG T-3’
single base mismatch SM-499 (SEQ ID NO: 14):
5’-TTA AGA CTT GCA GTG ATG ATT-3’
two base mismatch DM-499 (SEQ ID NO: 15):
5’-TTG AGA CTT GCA GTG ATG ATT-3’
complete mismatch miR-106a (SEQ ID NO: 16):
5’-AAA AGT GCT TAC AGT GCA GGT AG-3’
referring to fig. 1, a working principle diagram of the SERS detection kit for detecting acute myocardial infarction micro nucleic acid markers according to the present invention is shown. Taking the detection of the acute myocardial infarction micro nucleic acid marker miR-208a as an example, mixing hairpin DNA single-stranded H2-208a, a SERS probe and a sample to be detected, and then dripping the mixture onto the surface of a SERS detection chip. If the sample to be detected contains the acute myocardial infarction micro nucleic acid marker miR-208a, the sample can be hybridized with hairpin DNA designed at the vertex of the tetrahedron DNA structure TSP-208a, and a Catalytic Hairpin Assembly (CHA) reaction is triggered with the aid of hairpin DNA single-chain H2-208 a. The Probe single strand on the SERS Probe is then hybridized to the exposed viscous end of H2-208a, capturing the SERS Probe to the SERS detection chip. The released miR-208a can trigger a new cycle, so that target circulation is realized, and after SERS probes are further specifically combined, raman signals from the SERS probes can be collected from an SERS detection chip, so that quick, high-sensitivity and accurate detection of the acute myocardial infarction micro nucleic acid marker miR-208a is realized.
The invention is described in further detail below with reference to specific embodiments and figures.
Preparation of SERS detection kit for detecting acute myocardial infarction micro nucleic acid marker
The SERS detection kit comprises: SERS detection chip, hairpin DNA single strand H2 and SERS probe.
1. SERS detection chip
A silver nanorod array is used as a substrate, a tetrahedral nanostructure (TSP-208 a or TSP-499) with a concentration of 1 mu M is modified on the surface of the substrate, the silver nanorod array substrate comprises 3X 10 array type pores, the pore diameter of each pore is 4mm, and the depth of each pore is 1mm.
2. Hairpin DNA single strand H2
Hairpin DNA single strand H2-208a or hairpin DNA single strand H2-499 designed and synthesized from hairpin DNA sequence extended at 5' end in D strand (D-208 a or D-499);
3. SERS probe
mu.L of 50. Mu.M Probe single strand (Probe-208 a or Probe-499) and 10. Mu.L of 100. Mu.M Raman molecule (5, 5' -dithiobis (2-nitrobenzoic acid) (DTNB) or 4-mercaptobenzoic acid (4 MBA)) were used to modify 500. Mu.L of 2.3nM gold nanoparticles having a particle size of 15nM.
The preparation method of the SERS detection kit for detecting the acute myocardial infarction micro nucleic acid marker of the embodiment comprises the following steps:
taking the detection of acute myocardial infarction micro nucleic acid marker miR-208a as an example,
1) SERS detection chip preparation:
(1) Preparing a silver nano rod array and keeping the surface clean;
(2) A, B, C and D-208a with equal molar ratio are mixed and heated to 90-95 ℃ for 5-10 min, then cooled to 25-37 ℃ in ice water bath, and assembled to form a tetrahedral DNA structure TSP-208a;
(3) Placing 10-20 mu L of 0.1-5 mu M tetrahedral DNA structure TSP-208a and silver nanorod array substrate in 25-37 ℃ and 60-80% humidity environment for co-culturing for 3-5 hours;
(4) Sequentially with reaction buffer (10 mM tris-HCl and 1mM MgCl) 2 pH 8.0) cleaning the substrate to obtain a SERS detection chip;
2) Hairpin DNA single-strand H2 preparation:
the working concentration of the hairpin DNA single-chain H2-208a designed and synthesized according to the hairpin DNA sequence extended at the 5' end in the D-208a is 10 mu M;
3) SERS probe preparation:
(1) mu.L of 50. Mu.M Probe-208a single strand was mixed with 500. Mu.L of 2.3nM AuNP solution in 0.5 XTBE solution and incubated overnight at 25℃and 300 rpm;
(2) 5, 10, 15 and 20. Mu.L of 2M NaCl solution were slowly added every 30 minutes and 4 times, and incubated overnight at 300rpm at 25℃with a final concentration of 200mM NaCl;
(3) 10. Mu.L of 100. Mu.M Raman molecule DTNB was added for reaction for 3 hours;
(4) Finally, the supernatant is removed by centrifugation, and the centrifugal sediment is dispersed by 0.5 XTBE solution and the volume is fixed to 75 mu L, thus obtaining the composite material.
Example 1 preparation of SERS detection chip for detecting acute myocardial infarction micro nucleic acid marker and investigation of reaction conditions
1) Surface-immobilized tetrahedral DNA structure concentration optimization experiment of SERS detection chip
The tetrahedral DNA structure TSP-208a is formed by four single strand assembly of A, B, C and D-208 a. In the preparation process of the SERS detection chip, the silver nanorod array is respectively co-cultured with 20 mu L of 10nM, 50nM, 100nM, 200nM, 500nM, 1 mu M, 2 mu M, 3 mu M and 5 mu M tetrahedral DNA structure TSP-208a solution, and the solution is placed in a constant temperature mixing instrument at 25 ℃ to react for 3 hours, and then the substrate is washed with reaction buffer solution and ultrapure water for multiple times in sequence, so that the SERS detection chip for detecting miR-208a is obtained. Then, 2 mu L of 10 mu M H2-208a, 15 mu L of SERS probe and 2 mu L of 100pM miR-208a are mixed in 20 mu L of reaction buffer solution, and are dripped on the surface of a SERS detection chip, and after being placed in a constant temperature mixing instrument at 25 ℃ and 300rpm for reaction for 60min, the reaction buffer solution and ultrapure water are used for cleaning the small holes and carrying out SERS detection, and SERS spectra are obtained by testing, as shown in FIG. 2, when the silver nanorod array is co-cultured with 20 mu L of 10 nM-1 mu M tetrahedral DNA structure TSP-208a solution, the SERS signal is further enhanced along with the increase of the concentration of the TSP-208a; when the TSP-208a concentration is higher than 1. Mu.M, the SERS intensity reaches a saturated state, and as the TSP-208a concentration increases, the SERS signal no longer changes with the change in the TSP-208a concentration. It can be said that 1. Mu.M TSP-208a was determined as the optimal concentration immobilized on the silver nanorod array substrate.
2) Surface-immobilized tetrahedral DNA structure culture (incubation) time optimization experiment of SERS (surface enhanced Raman scattering) detection chip
A1. Mu.M solution of tetrahedral DNA structure TSP-208a was formed by four single strand assembly of A, B, C and D-208 a. In the preparation process of the SERS detection chip, the silver nanorod array and 20 mu L of 1 mu M TSP-208a solution are respectively placed in a constant temperature mixing instrument at 25 ℃ to be cultured for 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h and 4h, and then the substrate is washed with reaction buffer solution and ultrapure water for multiple times in sequence, so that the SERS detection chip for detecting miR-208a is obtained. Then, 2 mu L of 10 mu M H-208 a, 15 mu L of SERS probe and 2 mu L of 100pM miR-208a are mixed in 20 mu L of reaction buffer solution, and are dripped on the surface of a SERS detection chip, and after being placed in a constant temperature mixing instrument at 25 ℃ and 300rpm for reaction for 60min, the reaction buffer solution and ultrapure water are used for cleaning the small holes and carrying out SERS detection, and SERS spectra are obtained by testing, as shown in figure 3, when the silver nanorod array is co-cultured with 20 mu L of 1 mu M TSP-208a solution, the SERS signal is gradually enhanced as the incubation time of the TSP-208a solution is increased to 2.5 h; when the TSP-208a solution was incubated for more than 2.5h, SERS intensity was almost saturated. It can be said that the optimal culture (incubation) time of the tetrahedral DNA structure TSP-208a in the silver nanorod array is 2.5h.
Example 2 preparation of SERS detection kit for detecting acute myocardial infarction micro nucleic acid markers
1) Optimal culture (detection) time optimization experiment for detecting miR-208a by SERS detection kit
Mixing 2 mu L of 10 mu M H-208 a, 15 mu L of SERS probe and 2 mu L of 100pM miR-208a into 20 mu L of reaction buffer, dripping the mixture onto the surface of a SERS detection chip, respectively reacting for 10, 20, 30, 40, 50, 60 and 70min in a constant temperature mixing instrument at the temperature of 25 ℃ and at the rpm, cleaning a small hole by using the reaction buffer and ultrapure water, and carrying out SERS detection, and testing to obtain SERS signals, wherein the SERS signals of the detection chip are detected after the detection is cultured for different periods of 10-40 min, the SERS intensity is gradually enhanced within 10-40 min, and the SERS signals reach the maximum saturation value when the total cultivation is carried out for 40min, so that 40min is the optimal miR-208a detection time is indicated.
2) SERS detection kit detects working curve of different concentration miR-208a
mu.L of 10 mu M H-208 a, 15 mu L of SERS probe and 2 mu L of miR-208a (100 aM-1 nM) with different concentrations are mixed into 20 mu L of reaction buffer solution, and are dripped onto the surface of a SERS detection chip, and the mixture is placed in a constant temperature mixer at 25 ℃ for reaction for 40min, and then the wells are washed by the reaction buffer solution and ultrapure water. After natural air drying, carrying out SERS test (Raman test conditions: scanning time 1s, laser power 1%, objective lens magnification 20×, cumulative times 1 time, excitation light wavelength 785 nm) on an SERS detection chip to obtain an SERS spectrum and a characteristic signal intensity value thereof, taking logarithm of target miR-208a concentration as an abscissa, taking a characteristic peak intensity value of an SERS probe as an ordinate to make a working curve, and calculating the detection limit of the SERS detection kit for detecting miR-208a according to the working curve. FIG. 5a shows SERS spectra obtained by detecting miR-208a at different concentrations, FIG. 5b shows that each line is at 1331cm -1 The corresponding SERS peak intensity. For detection of miR-208a, the working curve is I 1331 ＝354×lg C miR-208a +6688(R 2 =0.999), the limit of detection was calculated to be 12.9aM.
Example 3 characterization of Performance of SERS detection kit for detecting miR-208a
1) Specific characterization of SERS detection kit for detecting miR-208a
The target miR-208a was diluted to 100fM, single base mismatches (SM-208 a), two base mismatches (DM-208 a) and full mismatches (miR-499 and miR-106 a) were diluted to 1pM, a mixed solution (mix) of multiple miRNAs with 100fM including the target miR-208a was prepared, and a reaction buffer without any additional biomolecules added was used as a blank control. mu.L of 10 mu M H2-208a and 15 mu L of SERS probe were mixed with 2 mu L of 1pM miR-499, miR-106a, DM-208a, SM-208a, 100fM mix and target miR-208a, respectively, and blank samples (reaction buffer). And (3) dripping the mixed solution on the surface of the SERS detection chip, placing the mixed solution in a constant-temperature mixing instrument at the temperature of 25 ℃ and at the speed of 300rpm for reaction for 40min, and cleaning the small holes by using reaction buffer solution and ultrapure water. And performing SERS test on the SERS detection chip after natural air drying to obtain an SERS spectrum and a characteristic peak intensity value thereof. FIG. 6a is a graph of SERS intensity for detecting samples of different biomolecules. The prepared SERS detection kit can better distinguish the target miR-208a from a mismatched sample, which indicates that the SERS detection kit has good specificity for detecting miR-208 a.
2) Homogeneity characterization of SERS detection kit for detecting miR-208a
Mixing 2 mu L of 10 mu M H-208 a and 15 mu L of SERS probes with 2 mu L of 1fM, 1pM and 1nM miR-208a respectively, dripping the mixed solution on the surface of an SERS detection chip, placing the SERS detection chip in a constant temperature mixing instrument at the temperature of 300rpm for reaction for 40min, washing small holes by using reaction buffer solution and ultrapure water, and recording SERS signals of 30 random points on the SERS detection chip respectively to study the uniformity of the silver nanorod array substrate with the surface modified with the tetrahedral DNA structure TSP-208a. FIG. 6b is SERS intensity at 30 random spots detecting 1fM, 1pM and 1nM miR-208 a. The Relative Standard Deviation (RSD) of the SERS intensities corresponding to 30 random spots detected by miR-208a is small (RSD < 7.56%), which indicates that the SERS detection kit has good uniformity in detecting miR-208 a.
3) Repeatability characterization of SERS detection kit for detecting miR-208a
Mixing 2 mu L of 10 mu M H-208 a, 15 mu L of SERS probes and 2 mu L of 1nM miR-208a into 20 mu L of reaction buffer, respectively dripping the mixture onto the surfaces of five groups of SERS detection chips in different batches, placing the mixture into a constant-temperature mixing instrument at the temperature of 25 ℃ and at the speed of 300rpm for reaction for 40min, cleaning small holes by using the reaction buffer and ultrapure water, and respectively recording SERS signals detected by the SERS detection chips in different batches to detect the 1nM miR-208a so as to study the repeatability of the SERS detection kit. FIG. 6c is SERS intensity collected by five different sets of SERS detection chips detecting 1nM miR-208 a. The SERS intensity acquired by the SERS detection chips of five different batches corresponding to miR-208a detection is very small (RSD=3.64%), which indicates that the SERS detection kit has good repeatability in detecting miR-208 a.
Example 4 working curves of SERS detection kits for detection of different concentrations of miR-499
mu.L of 10 mu M H-499, 15 mu L of SERS probe and 2 mu L of miR-499 (100 aM-1 nM) with different concentrations are mixed into 20 mu L of reaction buffer solution, and are dripped onto the surface of a SERS detection chip, and after reaction for 40min in a constant temperature mixer at 25 ℃ and 300rpm, the wells are washed by the reaction buffer solution and ultrapure water. And performing SERS test on the SERS detection chip after natural air drying to obtain an SERS spectrum and a characteristic signal intensity value thereof, taking logarithm of the concentration of the target miR-499 as an abscissa, taking a characteristic peak intensity value of the SERS probe as an ordinate, making a working curve, and calculating the detection limit of the SERS detection kit for detecting miR-499 according to the working curve. FIG. 7a is a SERS spectrum obtained by detecting miR-499 with different concentrations, and FIG. 7b is a spectrum at 1077cm -1 The corresponding SERS peak intensity. For detection of miR-499, a working curve I was obtained 1077 ＝402×lg C miR-499 +7487(R 2 =0.994), the detection limit was 14.5aM by calculation.
Example 5 characterization of Performance of SERS detection kit to detect miR-499
1) Specific characterization of SERS detection kit for detecting miR-499
The target miR-499 was diluted to 100fM, single base mismatches (SM-499), two base mismatches (DM-499) and full mismatches (miR-208 a and miR-106 a) were diluted to 1pM, a mixed solution (mix) of multiple miRNAs with 100fM including the target miR-499 was prepared, and a reaction buffer without any additional biomolecules added was used as a blank control. mu.L of 10. Mu. M H2, 2-499, 15. Mu.L of SERS probe were mixed with 2. Mu.L of 1pM miR-208a, miR-106a, DM-499, SM-499, 100fM mix and target miR-499, respectively, as well as blank samples (reaction buffer). And (3) dripping the mixed solution on the surface of the SERS detection chip, placing the mixed solution in a constant-temperature mixing instrument at the temperature of 25 ℃ and at the speed of 300rpm for reaction for 40min, and cleaning the small holes by using reaction buffer solution and ultrapure water. And performing SERS test on the SERS detection chip after natural air drying to obtain an SERS spectrum and a characteristic peak intensity value thereof. FIG. 8a is a graph of SERS intensity for detecting samples of different biomolecules. The prepared SERS detection kit can better distinguish a target miR-499 from a mismatched sample, which indicates that the SERS detection kit has good specificity for detecting miR-499.
2) Homogeneity characterization of SERS detection kit for detecting miR-499
mu.L of 10 mu M H to 499 and 15 mu L of SERS probes are respectively mixed with 2 mu L of 1fM, 1pM and 1nM miR-499, the mixed solution is dripped on the surface of a SERS detection chip, the SERS detection chip is placed in a constant temperature mixer at 25 ℃ and 300rpm for reaction for 40min, small holes are washed by reaction buffer and ultrapure water, SERS signals of 30 random points on the SERS detection chip are respectively recorded, and the uniformity of the silver nanorod array substrate with the surface modified with the tetrahedral DNA structure TSP-499 is studied. FIG. 8b is SERS intensity at 30 random spots detected for 1fM, 1pM and 1nM miR-499. The RSD of SERS intensities corresponding to 30 random spots detected by miR-499 was small (RSD < 7.12%), indicating good homogeneity of detection of miR-499 by the SERS detection kit.
3) Repeatability characterization of SERS detection kit for detecting miR-499
Mixing 2 mu L of 10 mu M H to 499, 15 mu L of SERS probes and 2 mu L of 1nM miR-499 into 20 mu L of reaction buffer, respectively dripping the mixture onto the surfaces of five groups of SERS detection chips in different batches, placing the mixture into a constant temperature mixer at 25 ℃ and 300rpm for reaction for 40min, cleaning small holes by using the reaction buffer and ultrapure water, and respectively recording SERS signals detected by the SERS detection chips in different batches to detect 1nM miR-499 so as to study the repeatability of the SERS detection kit. FIG. 8c is SERS intensity collected by five different sets of SERS detection chips detecting 1nM miR-499. The SERS intensity collected by the SERS detection chips of five different batches corresponding to miR-499 detection had very little RSD (rsd=3.94%), which indicates that the SERS detection kit detected miR-499 with good reproducibility.
Claims (9)
1. The SERS detection kit for detecting the acute myocardial infarction micro nucleic acid marker is characterized by comprising the following parts:
(1) The surface of the silver nanorod array substrate is modified with a SERS detection chip with a tetrahedral DNA structure TSP-208a;
(2) Hairpin DNA single strand H2-208a;
(3) The SERS Probe is used in combination with a substrate, and is gold nanoparticles with surface modified Probe-208a single chain and Raman molecules of 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB);
the SERS detection chip is characterized in that the surface of the SERS detection chip is modified with a sequence shown as SEQ ID NO:1-4 and D-208a to form a tetrahedral DNA structure TSP-208a;
the sequence of the hairpin DNA single-chain H2-208a is shown as SEQ ID NO:5 is shown in the figure;
the SERS probe is modified with a sequence shown as SEQ ID NO:6 and 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB), wherein the concentration ratio of the Probe-208a single chain to the DTNB is 1:1 to 3.
2. The SERS detection kit for detecting acute myocardial infarction micronucleic acid markers as in claim 1, comprising the following:
(1) The surface of the silver nanorod array substrate is modified with a SERS detection chip with a tetrahedral DNA structure TSP-499;
(2) Hairpin DNA single strand H2-499;
(3) The SERS Probe is used in combination with a substrate, and is gold nanoparticles with surface modified Probe-499 single chains and Raman molecules of 4-mercaptobenzoic acid (4 MBA);
the SERS detection chip is characterized in that the surface of the SERS detection chip is modified with a sequence SEQ ID NO: a, B, C and the sequence shown in SEQ ID NO:8, D-499 is assembled to form a tetrahedral DNA structure silver nano rod array substrate;
the sequence of the hairpin DNA single-chain H2-499 is SEQ ID NO:9, a step of performing the process;
the SERS probe is modified with a sequence shown as SEQ ID NO:10 and 4-mercaptobenzoic acid (4 MBA), wherein the concentration ratio of the Probe-499 single strand to the 4MBA is 1:1 to 3.
3. The SERS detection kit for detecting a micro nucleic acid marker for acute myocardial infarction according to claim 1 or 2, wherein the working concentration of the tetrahedral DNA structure is 0.1 to 5 μΜ, the working concentration of the hairpin DNA single strand H2 is 5 to 20 μΜ, and the working concentration of the SERS probe is 1 to 10nM;
the silver nanorod array substrate is prepared by adopting a vacuum electron beam evaporation coating technology and comprises 3X 10 array type small holes, wherein the aperture of each small hole is 3-5 mm, and the depth is 0.8-1.2 mm; the particle size of the gold nanoparticles is 15-100 nm.
4. The method for preparing a SERS detection kit for detecting acute myocardial infarction micro nucleic acid markers according to claim 1 or 2, wherein:
a, B, C and D with equal molar ratio are mixed and assembled to form a tetrahedral DNA structure TSP, and co-cultured with a silver nanorod array substrate, so that the SERS detection chip can be prepared;
and adding the Raman molecules into the AuNP solution with single-chain functionalization of the Probe, so as to prepare the SERS Probe.
5. The method for preparing a SERS detection kit for detecting acute myocardial infarction micro nucleic acid markers according to claim 1, comprising the steps of:
preparation of SERS detection chips:
(1) Preparing a silver nano rod array and keeping the surface clean;
(2) A, B, C and D-208a with equal molar ratio are mixed and heated to 90-95 ℃ for 5-10 min, then cooled to 25-37 ℃ in ice water bath, and assembled to form a tetrahedral DNA structure TSP-208a;
(3) Placing 10-20 mu L of 0.1-5 mu M tetrahedral DNA structure TSP-208a and silver nanorod array substrate in 25-37 ℃ and 60-80% humidity environment for co-culturing for 3-5 hours;
(4) Sequentially with reaction buffer (10 mM tris-HCl and 1mM MgCl) 2 pH 8.0) cleaningA substrate to obtain a SERS detection chip;
(II) preparation of hairpin DNA single-strand H2:
designing and synthesizing hairpin DNA single-chain H2-208a with the sequence shown as SEQ ID NO.5 according to the hairpin DNA sequence extended at the 5' end in D-208 a;
(III) SERS probe preparation:
(1) Mixing 1-10 mu L of 10-100 mu M Probe-208a single strand with 100-500 mu L of 1-10 nM AuNP solution in 0.5 XTBE solution and culturing at 200-400 rpm at 25-37 ℃ overnight;
(2) The molar concentration 1 was slowly added 4 times every 30 minutes: 2:3:4, and placing the solution in 10-50 mu L of 1-3M NaCl solution at the temperature of 25-37 ℃ for culturing overnight at 200-400 rpm, wherein the final concentration of NaCl is 100-300 mM;
(3) Adding 10-100 mu L of 10-100 mu M Raman molecule DTNB for reacting for 2-4 hours;
(4) Finally centrifuging to remove the supernatant, dispersing the centrifugal sediment by using 0.5 XTBE solution and fixing the volume to 10-100 mu L to obtain the SERS probe.
6. Use of the SERS detection kit according to claim 1 or 2 for detection of acute myocardial infarction micronucleic acid markers for non-disease diagnosis purposes.
7. The use of claim 6, wherein the acute myocardial infarction micronucleic acid marker is miR-208a or miR-499.
8. The use according to claim 6, characterized by the steps of:
1) Mixing hairpin DNA single-stranded H2 and SERS probes with sample solutions containing target acute myocardial infarction micro-nucleic acid markers with different concentrations, and dripping the mixed solution onto the surface of a SERS detection chip for co-culture;
2) Washing a SERS detection chip with ultrapure water for a plurality of times, performing SERS test to obtain SERS spectrums and characteristic signal intensity values thereof corresponding to target acute myocardial infarction micro-nucleic acid markers with different concentrations, taking logarithm of the concentration of the target acute myocardial infarction micro-nucleic acid markers as an abscissa, taking the intensity value of a SERS characteristic peak as an ordinate, obtaining a working curve of a SERS detection kit, and calculating the detection limit of the SERS detection kit for detecting the acute myocardial infarction micro-nucleic acid markers according to the working curve;
3) Mixing a sample to be detected with hairpin DNA single-stranded H2 and a SERS probe, then dripping the mixture to the surface of a SERS detection chip for co-culture, cleaning the chip for a plurality of times, and then carrying out SERS test to obtain a SERS spectrum and a characteristic signal intensity value thereof, and calculating according to a working curve to obtain the concentration of the target acute myocardial infarction micro-nucleic acid marker in the sample to be detected.
9. The use according to claim 8, wherein in steps 1) and 3) the co-cultivation conditions are cultivation for 30-60 min at 200-400 rpm at 25-37 ℃.
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