CN108949906B - dsDNA-AgNCs fluorescent probe for HER2 detection and construction method and application thereof - Google Patents

dsDNA-AgNCs fluorescent probe for HER2 detection and construction method and application thereof Download PDF

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CN108949906B
CN108949906B CN201810648534.3A CN201810648534A CN108949906B CN 108949906 B CN108949906 B CN 108949906B CN 201810648534 A CN201810648534 A CN 201810648534A CN 108949906 B CN108949906 B CN 108949906B
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邓春艳
张曼曼
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Abstract

The invention discloses a dsDNA-AgNCs fluorescent probe for HER2 detection and a construction method and application thereof, wherein DNA1 and DNA2 are hybridized to form dsDNA, and the dsDNA, silver salt and a reducing agent undergo an in-situ reduction reaction to obtain the AgNCs fluorescent probe; the fluorescent probe has the advantages of strong signal, high specificity, high sensitivity and the like in the process of detecting HER2, the synthetic method is simple in steps, low in cost, mild in condition and beneficial to popularization, production and application.

Description

dsDNA-AgNCs fluorescent probe for HER2 detection and construction method and application thereof
Technical Field
The invention relates to a fluorescent probe for detecting a biomarker HER2, in particular to a dsDNA-AgNCs fluorescent probe synthesized by using a hybrid DNA strand and a method for realizing HER2 specific fluorescence detection by using the principle that a guanine (G) base sequence is close to AgNCs to enhance the fluorescent signal of AgNCs, and belongs to the technical field of biosensing.
Background
Human epidermal growth factor receptor 2(HER2, also known as Neu or ErbB2) is a protein that promotes the growth of breast cancer cells. HER2 is the most common malignancy marker (d.j.slamon, b.leyland-Jones, s.mark, h.fuchs, v.paton, v.n.engl.j.med.2001,344, 783-792.). Examples of methods for detecting HER2 that have been established to date include chromogenic in situ hybridization assay (CISH) (S.Kiyose, H.Igarashi, K.Nagura, T.Kamo, K.Kawan, H.Mori, T.Ozawa, M.Maeda, K.Konno, H.Hoshino, H.Konno, H.Ogura, K.Shinmura, N.Hattori, H.Sugimura, Pathol.Int.2012,62,728, 734.), Fluorescent In Situ Hybridization (FISH) (S.Shah, B.Chen, Pathol Res.2011, 903202-903217.), Immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA) (C.B.Moelans, R.A.D.ger, E.V.D.D.D.J.Wa.Waisn, Sa.2011, Immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA) (C.B.Moelan., R.A.D.392, E.V.D.D.J.Waisn.W.W.2011, P.2011, E.S.01, Biotech, Biol.103, Biotech, M.58, Biotech; b) C.Shen, K.Zeng, J.Luo, X.Li, M.Yang, A.Rasoly, anal.chem.2017,89, 10264-10269), etc. However, FISH and IHC require invasive biopsy specimens, specialized and dedicated instruments, and ELISA techniques are very reliable, but are generally sensitive only to the ng/mL range. The electrochemical method has the advantages of simplicity, rapidness, high selectivity, high sensitivity and the like. However, electrochemical methods have been subject to stability and reproducibility problems and are expensive. This limits the wide application of these methods. Therefore, the establishment of a simple, specific, sensitive and accurate HER2 detection method is of great significance.
In recent years, as a novel nano material, AgNCs has attracted much attention because of its great advantages over common organic dyes and quantum dots, including high photostability, sub-nanometer size, stability, low toxicity, strong fluorescence emission, and good biocompatibility. Furthermore, several reports indicate that AgNCs significantly increase their fluorescence intensity by proximity to G-rich DNA sequences (j.li, x.zhong, h.zhang, x.c.le, j.j.zhu, anal.chem.2012, 84, 5170-. This mechanism has found widespread use in a number of bioanalytical methods (a) x.liu, f.wang, r.aizen, o.yehezkeli, i.willner, j.am.chem.soc.2013,135, 11832-11839; b) l.zhang, j.zhu, s.guo, t.li, j.li, e.wang, j.am.chem.soc.2013,135, 2403-2406.).
Disclosure of Invention
Aiming at the defects of the method for detecting HER2 in the prior art, the first object of the invention is to provide a dsDNA-AgNCs fluorescent probe for HER2 specific fluorescence detection, wherein the fluorescent probe has the advantages of strong signal, high specificity, high sensitivity and the like in the process of detecting HER 2.
The second purpose of the invention is to provide a method for constructing the dsDNA-AgNCs fluorescent probe with simple steps, low cost and mild conditions.
The third purpose of the invention is to provide the application of the dsDNA-AgNCs fluorescent probe in detecting HER2, and the dsDNA-AgNCs fluorescent probe has the characteristics of strong signal, high specificity, high sensitivity and the like.
The invention provides a construction method of a dsDNA-AgNCs fluorescent probe for HER2 detection, which comprises the steps of hybridizing DNA1 and DNA2 to form dsDNA, and carrying out in-situ reduction reaction on the dsDNA, silver salt and a reducing agent to obtain the AgNCs fluorescent probe; the DNA1 is a HER2 aptamer DNA fragment; the DNA2 comprises a DNA fragment complementary to the DNA1, a template DNA fragment for synthesizing AgNCs and a DNA fragment rich in G bases, and the two ends of the DNA2 are complementary DNA fragments.
The key point of the technical scheme is that two special DNA chains are designed, DNA1 is a short chain and is an aptamer of HER2, DNA2 is a long chain and comprises a DNA fragment complementary with DNA1, a template DNA fragment for synthesizing AgNCs and a DNA fragment rich in G bases, and two ends of DNA2 are complementary DNA fragments. Firstly, hybridizing DNA2 with DNA1, and then synthesizing the silver nanoclusters in situ by using AgNCs templates. The generated hybrid double-chain part is a rigid structure, and the DNA2 is supported into a chain structure by a long chain, so that a DNA fragment rich in G basic groups is far away from the silver nanocluster and shows a weak fluorescence signal, and complementary DNA fragments at two ends of a DNA2 chain are also in a free state and cannot be specifically combined. When HER2 appears in the system, DNA1 combined with DNA2 is continuously specifically combined with HER2, so that a rigid chain segment in the middle of the DNA2 is recovered to be a free single chain, complementary DNA segments at two ends of the DNA2 are specifically combined, so that the DNA2 wraps the nano silver cluster, and the DNA segment rich in G base is close to the nano silver cluster to enhance a fluorescence signal, so that the fluorescence detection of HER2 is realized.
Preferably, the DNA1 has the sequence number: 5'-GG GGT GTG GCG ACG-3' are provided.
Preferably, the DNA2 has the sequence number: 5'-AT ATT ACCC CAA CCCC CGT CGC CAC ACCCC GGGG AAG GGGT AAT AT-3' are provided.
The DNA1 and the DNA2 of the present invention are sequences designed by themselves and synthesized by the firm Biotechnology engineering (Shanghai) Co., Ltd. The 5'-GG GGT GTG GCG ACG-3' fragment in the DNA1 sequence is a HER2 aptamer fragment, AgNCs are synthesized by utilizing a fragment rich in C base in DNA2, 5 base sequences at two ends of DNA2 are complementary DNA fragments, and DNA rich in G base in DNA2 is a fragment which generates fluorescence signal enhancement with silver nanoclusters.
In a preferable scheme, the dsDNA, silver nitrate and sodium borohydride react for 1-3 hours at room temperature in a solution system to obtain the dsDNA-AgNCs fluorescent probe. And (3) synthesizing the silver nanoclusters by in-situ reduction by using AgNCs template DNA fragments in the dsDNA.
Preferred embodiment，dsDNA、AgNO3And NaBH4The molar ratio of (A) to (B) is 1:5 to 7. In the technical scheme of the invention, dsDNA and AgNO3And NaBH4When the molar ratio of (A) to (B) is 1:6:6, relatively stable AgNCs can be synthesized. The DNA used in the experimental process is prepared by phosphate buffer solution (10mM, pH 7.4), the DNA1 and DNA2 are mixed in equal molar ratio, the mixture is uncoiled at high temperature and cooled to room temperature for hybridization complementation, and AgNO is added3The solution was shaken and mixed for 15min at room temperature, followed by the addition of freshly prepared NaBH4The method is quickly shaken and uniformly mixed for 1min, and then the mixture is placed in a constant-temperature water bath box at 25 ℃ for reaction for 2h, so that the fluorescence signal can be detected, experiments prove that the AgNCs synthesized by the method only needs 2h, and the whole DNA1-AgNCs system can be stabilized for 2 h. The system is completely stable, and the fluorescent probe for detecting the breast cancer marker is obtained.
The invention designs 4 DNA2[ DNA 2-1: 8 complements (5'-AT ATT ATT ACCC CAA CCCC CGT CGC CAC ACCCC GGGG AAG GGGT AAT AAT AT-3'); DNA 2-2: 5 complements (5'-AT ATT ACCC CAA CCCC CGT CGC CAC ACCCC GGGG AAG GGGT AAT AT-3'); DNA 2-3: 5 tails (5'-AT ATT ATT ACCC CAA CCCC CGT CGC CAC ACCCC GGGG AAG GGGT AAT AAT AT TTA TT-3'); DNA 2-4: 5G (5'-AT ATT ATT ACCC CAA CCCC CGT CGC CAC ACCCC AAGG GGGT AAT AAT AT-3') ], 4 DNA2 are changed on the basis of ACCC CAA CCCC CGT CGC CAC ACCCC GGGG AAG GGGT, and 8 complementary bases are designed at two ends of the DNA2-1 on the basis of the ACCC CAA CCCC CGT CGC CAC ACCCC GGGG AAG GGGT; on the basis of the DNA2-2, 5 complementary bases are designed at two ends; DNA2-3 has 5 additional trailing bases on the basis of 8 complementary bases; DNA2-4 was changed from 8G bases to 5G bases on the basis of 8 complements, which could enhance the fluorescence signal of silver nanoclusters. Under the same experimental conditions, a dsDNA-AgNCs fluorescent probe is synthesized by using 4 designed DNAs 2 and 1, and 85fM HER2 is detected, and the result shows that the variation of the fluorescent signals of 8 complements and 5 complements is not very large, but is obviously higher than the case of containing 5 tails and 5G bases. DNA2 containing 5 tails may affect its hybridization with DNA1 and the binding of HER2 protein to DNA1, 5G bases are less than 8G bases in number, so the change of fluorescence signal generated when dsDNA-AgNCs fluorescent probe reacts with HER2 is not as large as that generated by DNA2 containing 8G bases. Therefore, experiments are preferably carried out on DNA2-2 with 5 complementary bases at two ends, and the obtained fluorescent probe has better detection effect.
The invention provides a dsDNA-AgNCs fluorescent probe for HER2 detection, which is obtained by the construction method.
The invention also provides an application of the dsDNA-AgNCs fluorescent probe for HER2 detection, and the dsDNA-AgNCs fluorescent probe is used as a fluorescent probe for HER2 detection.
In a preferable scheme, after the dsDNA-AgNCs fluorescent probe reacts with a HER2 solution to be detected, a fluorescence signal value is detected, and the concentration of HER2 in the HER2 solution to be detected is calculated according to a standard curve of the concentration of the HER2 solution and the fluorescence signal value. The dsDNA-AgNCs fluorescent probe reacts with HER2 solution to be detected for 5-30min at 25 ℃. More preferably 20 min.
In a more preferable scheme, after the dsDNA-AgNCs fluorescent probe reacts with a series of standard HER2 solutions with different concentrations, a series of fluorescence signal values are obtained by detecting the fluorescence signal values through fluorescence, and a standard curve of the concentration of the HER2 solution and the fluorescence signal values is established.
The invention provides a method for specific fluorescence detection of HER2 based on AgNCs probe, which comprises the following steps:
1) carrying out high-temperature unwinding on the DNA1 and the DNA2, and naturally cooling to room temperature to enable the two DNA strands to be hybridized and complemented to form dsDNA;
2) sequential addition of AgNO to dsDNA3、NaBH4Synthesizing a dsDNA-AgNCs probe and detecting a signal thereof to obtain a weak fluorescent signal;
3) after the dsDNA-AgNCs fluorescent probe reacts with a series of standard HER2 solutions with different concentrations, fluorescence detection is carried out, since HER2 can be combined with aptamer chain DNA1 in a high specificity mode, DNA1 and DNA2-AgNCs are uncoiled, and a plurality of complementary base fragments are arranged at two ends of designed DNA2-AgNCs, so that a G base fragment in a DNA2-AgNCs fragment is close to AgNCs, a series of fluorescence enhanced signals can be obtained, and a standard curve of HER2 solution concentration and fluorescence signal value is established;
4) and (3) reacting the dsDNA-AgNCs fluorescent probe with a HER2 solution to be detected, performing fluorescence detection to obtain a fluorescence signal value, and calculating the concentration of the HER2 solution to be detected according to a standard curve.
The technical proposal of the invention constructs the AgNCs fluorescent probe through two DNA1 and DNA2 with complementary fragments, when DNA1 is complementary to DNA2, the G-base-rich fragment in DNA2 is far from AgNCs, the fluorescence of dsDNA-AgNCs fluorescent probe is weak, when HER2 is added, since the DNA1-HER2-aptamer specifically binds to HER2 and automatically packages HER2 to form a complex structure, and the conformation of the DNA2 without the DNA1 is changed, complementary DNA fragments at two ends of the DNA2 are specifically combined, the silver nanoclusters are wrapped, so that the fragment rich in G base is close to AgNCs, thereby leading to fluorescence enhancement, detecting the content of the breast cancer marker by fluorescence signal increase, realizing HER2 specific fluorescence detection by utilizing the principle, the detection limit can reach 1.406fM, and in the range of 4.25 fM-255 fM, the fluorescence intensity is linearly enhanced along with the increase of the concentration, and the detection range is wider than that of the traditional method.
Compared with the prior art, the technical scheme of the invention has the beneficial technical effects that:
the technical scheme of the invention realizes the construction of the dsDNA-AgNCs fluorescent probe by designing two special DNA chains, constructs the special dsDNA-AgNCs fluorescent probe, realizes the fluorescence detection of the specificity of the breast cancer marker HER2, has the advantages of strong signal, high specificity, high sensitivity, wide detection concentration range and the like, and is beneficial to popularization and application.
According to the technical scheme, the silver nano-cluster fluorescent probe stabilizes silver nano-particles through a DNA nucleic acid sequence, and has the advantages of good stability, stable signal, small size of nano-cluster and strong environmental impact resistance; and the characteristics of high selectivity and specificity of the aptamer are applied, so that the problems of low sensitivity and detection limit in the prior art can be effectively solved, the property is more stable, the operation is simpler and more convenient, and the method is favorable for popularization and application.
The dsDNA-AgNCs fluorescent probe provided by the invention is simple in construction method, low in cost, mild in condition and beneficial to popularization and application.
Drawings
Fig. 1 is a schematic diagram of an experimental method for detecting the marker HER2 by a method of fluorescence signal change.
FIG. 2 is the excitation and emission spectra of silver nanoclusters synthesized by dsDNA.
Figure 3 to design the effect of different DNA2 on HER2 detection.
FIG. 4 is a specific detection map for the target and interferents.
FIG. 5 is a fluorescence image of the detection marker HER2 content in a certain concentration range.
FIG. 6 is a linear relationship diagram of the content of the detection marker HER2 in a certain concentration range.
Detailed Description
The following examples are intended to further illustrate the present disclosure, but not to limit the scope of the claims.
Example 1
The preparation method of the silver nanocluster comprises the following steps:
mu.L of DNA1 (50. mu.M) and 40. mu.L of DNA2 (50. mu.M) were taken, and 240. mu.L of phosphate buffer (20mM, pH 7.0) was added thereto, mixed well, helicized at high temperature and then cooled to room temperature to allow hybridization complementation. Adding 12 mu L of AgNO3The aqueous solution (1mM) was shaken and mixed for 15min at room temperature, and then 12. mu.L of freshly prepared NaBH was added4(1mM) is quickly and uniformly mixed and transferred into a water bath kettle with the constant temperature of 25 ℃ for reaction for 2 hours, and then the fluorescence can be detected. DNA1 SEQ ID NO: 5'-GG GGT GTG GCG ACG-3' are provided. DNA2 SEQ ID NO: 5'-AT ATT ACCC CAA CCCC CGT CGC CAC ACCCC GGGG AAG GGGT AAT AT-3' are provided.
The detection procedure for HER2 was as follows:
HER2 was added to the reacted dsDNA-AgNCs solution at different concentrations and the fluorescence signal was detected after 20min of reaction.
The specific detection steps are as follows:
HSA, IgG and insulin, which may be present in human serum, were added to the reacted dsDNA-AgNCs solution as potential interferents (100pM) for HER2 determination under the same conditions, respectively, and the fluorescence signal was detected after 20 min.
The steps of establishing the coordinate curve are as follows:
different concentrations of HER2 solutions were prepared with phosphate buffer (10mM, pH 7.4), each solution was added to the dsDNA-AgNCs fluorescent probe solution in the same volume, and after 20min the fluorescence signal was detected, and 3 sets of experiments were repeated with a line fit graph made using origin software.
From fig. 2, it can be seen that a is the excitation spectrum of the silver nanocluster, and the excitation wavelength is at 563 nm; b is an emission spectrum with an emission wavelength of 625 nm.
As can be seen from FIG. 3, the designed DNA2 has different base sequences, and the amount of change of the generated fluorescence signal is different when dsDNA-AgNCs fluorescent probes are synthesized respectively and then target HER2 with the same concentration is added.
As can be seen from figure 4, the aptamer only has specific binding to the breast cancer marker HER2, and the experimental interference is small.
From figure 5 it can be seen that the fluorescence signal increases with increasing HER2 concentration.
From fig. 6 it can be seen that HER2 has a certain linear relationship with its corresponding response signal over a certain concentration range.

Claims (7)

1. A method for constructing a dsDNA-AgNCs fluorescent probe for HER2 detection, which is characterized by comprising the following steps: hybridizing DNA1 and DNA2 to form dsDNA, and carrying out in-situ reduction reaction on the dsDNA, silver salt and a reducing agent to obtain an AgNCs probe;
the DNA1 is a HER2 aptamer DNA fragment;
the DNA2 comprises a DNA fragment complementary to the DNA1, a template DNA fragment for synthesizing AgNCs and a DNA fragment rich in G bases, and the two ends of the DNA2 are complementary DNA fragments;
the DNA1 sequence is: 5'-GG GGT GTG GCG ACG-3', respectively;
the DNA2 sequence is: DNA 2: 5'-AT ATT ACCC CAA CCCC CGT CGC CAC ACCCC GGGG AAG GGGT AAT AT-3' are provided.
2. The method of claim 1, wherein the dsDNA-AgNCs fluorescent probe for HER2 detection comprises: and (3) reacting the dsDNA with silver nitrate and sodium borohydride in a solution system at room temperature for 1-3 h to obtain the dsDNA-AgNCs fluorescent probe.
3. The method of claim 2, wherein the dsDNA-AgNCs fluorescent probe for HER2 detection comprises: dsDNA, AgNO3 And NaBH4 The molar ratio of (a) to (b) is 1:5 to 7.
4. A dsDNA-AgNCs fluorescent probe for HER2 detection, characterized in that: the method of any one of claims 1 to 3.
5. Use of a dsDNA-AgNCs fluorescent probe for HER2 detection according to claim 4, characterized in that: as a fluorescent probe for HER2 detection.
6. Use of a dsDNA-AgNCs fluorescent probe for HER2 detection according to claim 5, characterized in that: and (3) reacting the dsDNA-AgNCs fluorescent probe with a HER2 solution to be detected, detecting a fluorescence signal value, and calculating the concentration of HER2 in the HER2 solution to be detected according to a standard curve of the concentration of the HER2 solution and the fluorescence signal value.
7. Use of a dsDNA-AgNCs fluorescent probe for HER2 detection according to claim 6, characterized in that: and (3) reacting the dsDNA-AgNCs fluorescent probe with a series of standard HER2 solutions with different concentrations, detecting a fluorescence signal value through fluorescence to obtain a series of fluorescence signal values, and establishing a standard curve of the HER2 solution concentration and the fluorescence signal value.
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