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

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

A dsDNA-AgNCs fluorescent probe for HER2 detection and its construction method and application

technical field

The present invention relates to a fluorescent probe for the detection of biological marker HER2, in particular to a dsDNA-AgNCs fluorescent probe synthesized by using hybrid DNA strands and a guanine (G) base sequence close to AgNCs to enhance the detection of AgNCs. The invention relates to a method for realizing the specific fluorescence detection of HER2 based on the principle of fluorescent signal, which belongs to the field of biosensing technology.

Background technique

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 has emerged as the most common malignancy marker (D.J.Slamon, B.Leyland-Jones, S.Shak, H.Fuchs, V.Paton, V.N.Engl.J.Med.2001, 344, 783-792.). The currently established methods for the detection of HER 2 are: chromogenic in situ hybridization assay (CISH) (S.Kiyose, H.Igarashi, K.Nagura, T.Kamo, K.Kawane, 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.), fluorescence in situ hybridization (FISH) (S.Shah, B.Chen, Patholog Res Int. 2011, 903202-903217.), immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA) (C.B.Moelans, R.A.D.Weger, E.V.D.Wall, P.J.V.Diest, Crit . Rev. Oncol. Hematol. 2011, 80, 380-392), Electrochemistry (a) M. Shamsipur, M. Emami, L. Farzin, R. Saber, Biosens. Bioelectron. 2018, 103, 54-61; b) C . Shen, K. Zeng, J. Luo, X. Li, M. Yang, A. Rasooly, Anal. Chem. 2017, 89, 10264-10269.) et al. However, FISH and IHC require invasive biopsy specimens, specialized and dedicated instruments, and ELISA techniques are very reliable, but are usually only sensitive to the ng/mL range. Electrochemical methods have the advantages of simplicity, rapidity, high selectivity and high sensitivity. However, electrochemical methods have historically faced stability and reproducibility issues and are expensive. This limits the widespread application of these methods. Therefore, it is of great significance to establish a simple, specific, sensitive and accurate HER2 detection method.

In recent years, AgNCs, as a new type of nanomaterials, due to its great advantages over common organic dyes and quantum dots, including high photostability, sub-nanometer size, stability, low toxicity, strong fluorescence emission and good The biocompatibility and other advantages have attracted widespread attention. Moreover, some reports showed that the fluorescence intensity of AgNCs was significantly enhanced by the proximity of G-rich DNA sequences (J.Li, X.Zhong, H.Zhang, X.C.Le, J.J.Zhu, Anal.Chem. 2012, 84, 5170-5174 .).) This mechanism has been widely used in many 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.).

SUMMARY OF THE INVENTION

In view of the defects in the methods for detecting HER2 in the prior art, the first object of the present invention is to provide a dsDNA-AgNCs fluorescent probe for HER2-specific fluorescent detection, which has the characteristics of detecting HER2 in the process of detecting HER2. It has the advantages of strong signal, high specificity and high sensitivity.

The second object of the present invention is to provide a method for constructing dsDNA-AgNCs fluorescent probes with simple steps, low cost and mild conditions.

The third object of the present invention is to provide the application of the dsDNA-AgNCs fluorescent probe in the detection of HER2, which exhibits 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. The method is to hybridize DNA1 and DNA2 to form dsDNA, and the dsDNA is subjected to an in-situ reduction reaction with silver salt and a reducing agent to obtain AgNCs Probe; the DNA1 is a HER2 aptamer DNA fragment; the DNA2 comprises a DNA fragment complementary to DNA1, a template DNA fragment for synthesizing AgNCs and a DNA fragment rich in G bases, and both ends of DNA2 are complementary DNAs Fragment.

The key to the technical solution of the present invention is to design two special DNA chains, DNA1 is a short chain, which is an aptamer for HER2, and DNA2 is a long chain, which includes DNA fragments complementary to DNA1, template DNA fragments for synthesizing AgNCs, and A DNA fragment rich in G bases, and the two ends of DNA2 are complementary DNA fragments. First, DNA2 was hybridized with DNA1, and then AgNCs template was used to synthesize silver nanoclusters in situ. The resulting hybrid double-stranded part is a rigid structure, which supports the DNA2 as a long chain into a chain-like structure, so that the G-base-rich DNA fragments are far away from the silver nanoclusters, showing a weaker fluorescent signal, while the complementary DNA at both ends of the DNA2 strands Fragments are also in a free state and cannot bind specifically. When HER2 appears in the system, DNA1 bound by DNA2 is continuously specifically bound by HER2, so that the rigid segment in the middle of DNA2 is restored to a free single strand, and the complementary DNA segments at both ends of DNA2 are specifically bound, so that DNA2 wraps the nano-silver cluster, The G-base-rich DNA fragment increases the fluorescence signal by approaching the nano-silver clusters, realizing the fluorescence detection of HER2.

In a preferred solution, the DNA1 sequence number is: 5'-GG GGT GTG GCG ACG-3'.

In a preferred solution, the DNA2 sequence number is: 5'-AT ATT ACCC CAA CCCC CGT CGC CAC ACCCCGGGG AAG GGGT AAT AT-3'.

The DNA1 and DNA2 of the present invention are self-designed sequences and entrusted by Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize them. The 5'-GG GGT GTG GCG ACG-3' fragment in the DNA1 sequence is a HER2 aptamer fragment, and the C base-rich fragment in DNA2 is used to synthesize AgNCs, and the 5 base sequences at both ends of DNA2 are complementary DNA fragments. The G-base-rich DNA in DNA2 is a fragment that generates fluorescence signal enhancement with silver nanoclusters.

In a more preferred solution, dsDNA reacts with silver nitrate and sodium borohydride in a solution system at room temperature for 1-3 hours to obtain dsDNA-AgNCs fluorescent probes. Silver nanoclusters were synthesized by in situ reduction using AgNCs template DNA fragments in dsDNA.

In a more preferred solution, the molar ratio of dsDNA, AgNO ₃ and NaBH ₄ is 1:5-7:5-7. In the technical scheme of the present invention, when the molar ratio of dsDNA, AgNO ₃ and NaBH ₄ is 1:6:6, more stable AgNCs can be synthesized. The DNA used in the experiment was prepared with phosphate buffer solution (10mM, pH=7.4), the DNA1 and DNA2 were mixed in an equimolar ratio, the high temperature unwinding was cooled to room temperature to make it hybridize and complement, and AgNO ₃ solution was added to mix at room temperature. 15min, then add freshly prepared NaBH ₄ , quickly shake and mix for 1min, and then place it in a constant temperature water bath at 25°C for 2h to detect its fluorescence signal. The system can reach stability in 2h. When the system reaches complete stability, a fluorescent probe for detecting breast cancer markers is obtained.

The present invention designs 4 kinds of DNA2 [DNA2-1: 8 complements (5'-AT ATT ATT ACCC CAA CCCC CGTCGC CAC ACCCC GGGG AAG GGGT AAT AAT AT-3'); DNA2-2: 5 complements (5'- AT ATT ACCC CAACCCC CGT CGC CAC ACCCC GGGG AAG GGGT AAT AT-3'); DNA2-3: 5 tails (5'-AT ATT ATTACCC CAA CCCC CGT CGC CAC ACCCC GGGG AAG GGGT AAT AAT AT TTA TT-3' ); DNA2-4: 5 G's (5'-AT ATT ATT ACCC CAA CCCC CGT CGC CAC ACCCC AAGG GGGT AAT AAT AT-3')], 4 kinds of DNA2 are in ACCC CAA CCCC CGT CGC CAC ACCCC GGGG AAG GGGT Changes are made on the basis of DNA2-1, with 8 complementary bases at both ends; DNA2-2 with 5 complementary bases at both ends; DNA2-3 at 8 complementary bases On the basis of , 5 tailing bases were added; on the basis of 8 complements, DNA2-4 was changed from 8 G bases, which can enhance the fluorescence signal of silver nanoclusters, to 5 G bases. Under the same experimental conditions, four designed DNA2 and DNA1 were used to synthesize dsDNA-AgNCs fluorescent probes, and 85fM HER2 was detected. But it is significantly higher than the case with 5 tails and 5 G bases. DNA2 containing 5 tails may affect its hybridization with DNA1 and the binding of HER2 protein to DNA1. Since the number of 5 G bases is less than 8 G bases, when dsDNA-AgNCs fluorescent probe The change in fluorescence signal produced by the reaction of needle with HER2 is not as large as that produced by DNA2 containing 8 G bases. Therefore, we prefer to carry out the experiment with DNA2-2 containing 5 complementary bases at both ends, and the obtained fluorescent probe has better detection effect.

The present invention provides a dsDNA-AgNCs fluorescent probe for HER2 detection, which is obtained by the above construction method.

The present invention also provides an application of a dsDNA-AgNCs fluorescent probe for HER2 detection, which is used as a fluorescent probe for HER2 detection.

In a preferred solution, after the dsDNA-AgNCs fluorescent probe is reacted with the HER2 solution to be tested, the fluorescence signal value is detected, and the concentration of HER2 in the HER2 solution to be tested is calculated according to the standard curve of the concentration of the HER2 solution and the fluorescence signal value. The dsDNA-AgNCs fluorescent probe of the present invention reacts with the HER2 solution to be tested at 25° C. for 5-30 minutes. More preferably, it is 20 minutes.

In a more preferred solution, after the dsDNA-AgNCs fluorescent probe is reacted with a series of standard HER2 solutions of different concentrations, the fluorescence signal values are detected by fluorescence to obtain a series of fluorescence signal values, and a standard curve between the concentration of HER2 solution and the fluorescence signal values is established.

The present invention provides a method for HER2-specific fluorescence detection based on AgNCs probe, which comprises the following steps:

1) DNA1 and DNA2 are unwound at high temperature, and naturally cooled to room temperature to make the two DNA strands hybridize and complement each other to form dsDNA;

2) Adding AgNO ₃ and NaBH ₄ to dsDNA in turn, synthesizing the dsDNA-AgNCs probe and detecting its signal, a weak fluorescent signal can be obtained;

3) After the dsDNA-AgNCs fluorescent probe is reacted with a series of standard HER2 solutions of different concentrations, fluorescence detection is performed. Since HER2 can bind to its aptamer DNA1 with high specificity, DNA1 and DNA2-AgNCs are uncoiled. The designed There are several complementary base fragments at both ends of DNA2-AgNCs, which makes the G base fragment in the DNA2-AgNCs fragment close to AgNCs, and a series of fluorescence-enhanced signals can be obtained, and a standard curve of HER2 solution concentration and fluorescence signal value can be established;

4) After reacting the dsDNA-AgNCs fluorescent probe with the HER2 solution to be tested, perform fluorescence detection to obtain the fluorescence signal value, and calculate the concentration of the HER2 solution to be tested according to the standard curve.

According to the technical scheme of the present invention, the AgNCs fluorescent probe is constructed by two DNA1 and DNA2 with complementary fragments. When DNA1 and DNA2 are complementary, the G base-rich fragment in DNA2 is far away from AgNCs, and the fluorescence of the dsDNA-AgNCs fluorescent probe is relatively low. Weak, when HER2 is added, since DNA1-HER2-aptamer specifically binds to HER2 and automatically wraps HER2 to form a complex structure, the conformation of DNA2 that loses DNA1 changes, and the complementary DNA fragments at both ends of DNA2 bind specifically to bind silver. The nanocluster encapsulates so that the G base-rich fragments will be close to the AgNCs, resulting in enhanced fluorescence, and the content of breast cancer markers can be detected by the increase of the fluorescence signal. Using this principle, HER2-specific fluorescence detection is realized, and the detection limit can reach 1.406fM, and in the range of 4.25fM～255fM, the fluorescence intensity increases linearly with the increase of concentration, and the detection range is wider than the traditional method.

Relative to the prior art, the beneficial technical effects brought by the technical solution of the present invention:

The technical scheme of the present invention realizes the construction of the dsDNA-AgNCs fluorescent probe by designing two special DNA chains, and constructs the special dsDNA-AgNCs fluorescent probe, which realizes the specific fluorescence detection of the breast cancer marker HER2, and has signal It has the advantages of strong, high specificity, high sensitivity, and wide detection concentration range, which is conducive to popularization and application.

The technical solution of the present invention is that the silver nanocluster fluorescent probe stabilizes the silver nanoparticle through DNA nucleic acid sequence, which has good stability, stable signal, small size of the nanocluster, and strong resistance to environmental influence; The characteristics of selectivity and specificity can effectively improve the problems of low sensitivity and detection limit in the prior art, and have more stable properties and simpler operation, which is beneficial to popularization and application.

The dsDNA-AgNCs fluorescent probe construction method of the invention is simple, low in cost and mild in conditions, and is favorable for popularization and application.

Description of drawings

[Fig. 1] is a schematic diagram of the experimental method for detecting the marker HER2 by the method of fluorescence signal change.

[Fig. 2] Excitation and emission patterns of dsDNA synthesized silver nanoclusters.

[Figure 3] shows the effect of different designs of DNA2 on HER2 detection.

[Figure 4] is the specific detection chart for the target and interfering substances.

[Fig. 5] is a fluorescence image of the detection marker HER2 content in a certain concentration range.

[Figure 6] is a linear relationship diagram of the detection marker HER2 content within a certain concentration range.

Detailed ways

The following examples are intended to further illustrate the content of the present invention, rather than limit the protection scope of the claims of the present invention.

Example 1

The preparation method of silver nanocluster, the steps are as follows:

Take 40 μL DNA1 (50 μM) and 40 μL DNA2 (50 μM), add 240 μL phosphate buffer (20 mM, pH 7.0), mix well, unwind at high temperature and cool to room temperature for hybridization and complementation. Add 12 μL of AgNO ₃ aqueous solution (1 mM), shake and mix for 15 min at room temperature, then add 12 μL of freshly prepared NaBH ₄ (1 mM), quickly mix and transfer to a constant temperature water bath at 25 °C for 2 h, and then the fluorescence can be detected. The sequence number of DNA1 is: 5'-GG GGT GTG GCG ACG-3'. The sequence number of DNA2 is: 5'-ATATT ACCC CAA CCCC CGT CGC CAC ACCCC GGGG AAG GGGT AAT AT-3'.

The steps to detect HER2 are as follows:

Different concentrations of HER2 were added to the already reacted dsDNA-AgNCs solution, and the fluorescence signal was detected after 20 min of reaction.

The specific detection steps are as follows:

HSA, IgG and insulin, which may exist in human serum, were added to the dsDNA-AgNCs solution that had been reacted as potential interfering substances (100 pM) for the determination of HER 2 under the same conditions, and the fluorescence signal was detected after 20 min.

The steps to create a coordinate curve are as follows:

HER2 solutions of different concentrations were prepared with phosphate buffer (10mM, pH 7.4), and the same volume of each solution was added to the dsDNA-AgNCs fluorescent probe solution. A linear fitting curve is drawn.

It can be seen from Figure 2 that a is the excitation spectrum of silver nanoclusters, and the excitation wavelength is at 563 nm; b is the emission spectrum, and the emission wavelength is at 625 nm.

It can be seen from Figure 3 that the designed DNA2 base sequences are different. Under the same experimental conditions, after synthesizing dsDNA-AgNCs fluorescent probes respectively, adding the same concentration of the target HER2, the amount of fluorescence signal changes is different.

It can be seen from Figure 4 that the aptamer only has specific binding to the breast cancer marker HER2, and the experimental interference is small.

It can be seen from Figure 5 that the fluorescence signal increases with the increase of HER2 concentration.

It can be seen from Figure 6 that there is a certain linear relationship between HER2 and its corresponding response signal within 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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