A low-abundance target protein staining reagent composition and staining method

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a low-abundance target protein staining reagent composition and a staining method. The reagent composition includes a DNA logic gate nanodevice generation reagent and a G4 nanowire / Hemin complex generation reagent. The reagent composition is conducive to the staining analysis of low-abundance targets, and its staining degree is deeper than that of traditional IHC technology.

Description

[0001] Divisional application

[0002] This application is a divisional application based on the Chinese invention patent application with application number CN202311078558.7, application date August 25, 2023, and invention name "Application of a DNA logic gate nanodevice in cell surface protein staining". Technical Field

[0003] The invention belongs to the field of biotechnology, and particularly relates to a low-abundance target protein staining reagent composition and a staining method. Background Art

[0004] Immunohistochemistry (IHC) technology, which uses the specific recognition and binding between antigens and antibodies and the deposition of color substrates catalyzed by enzyme-linked secondary antibodies, has been widely used for in situ staining analysis of proteins. IHC technology relies on the recognition of target proteins by primary antibodies, introduces peroxidase (HRP)-coupled secondary antibodies, catalyzes the oxidation of 3,3'-diaminobenzidine (DAB), and deposits it on the cell membrane surface, thereby coloring the target protein. However, immunohistochemistry can only evaluate the expression level of proteins, but cannot reflect the interaction and aggregation state between proteins, which is not conducive to the implementation of personalized treatment.

[0005] Nucleic acid aptamers are highly specific protein affinity nucleic acid sequences obtained through in vitro enrichment and screening. Because their binding ability to target substances is comparable to that of antibodies, they are called "chemical antibodies." Based on the principle of complementary pairing between DNA bases, complex two-dimensional and three-dimensional DNA structures can be designed and assembled, and then DNA logic gate nanodevices with specific functions can be developed. By integrating two or more nucleic acid aptamers into DNA logic gate nanodevices, the recognition information of nucleic acid aptamers for different protein targets can be converted into series or parallel output signals, thereby identifying protein interactions on the cell membrane surface (J. Am. Chem. Soc. 2018, 140 (31), 9793-9796; Chem. Rev. 2021, 121 (22),13797-13868). In addition, by introducing G-quadruplex / Hemin into DNA logic gate devices and utilizing its HRP enzyme-like activity to catalyze the chromogenic substrate, the recognition signal can be converted into chromogenic information (Angew. Chem., Int. Ed. 2021, 60 (40), 21673-21678.). However, the random diffusion of the chromogenic product in the solution means that its output signal can only represent the expression level of the target protein, and it is impossible to perform in situ analysis of the target.

[0006] G-quadruplex is a four-stranded helical nucleic acid structure formed by π-π stacking of the G-tetrad plane. Each layer of G-tetrad is connected by 4 guanine nucleotides through Hoogteen hydrogen bonds. The patent with application number CN2021106103891 and invention name "A new type of semiquinone free radical nanomaterial and its preparation method and application" discloses a method of annealing a nucleic acid containing a continuous G sequence to obtain an aqueous solution containing a G-quadruplex, and then adding heme to the aqueous solution to obtain a G-quadruplex / Hemin complex. The patent found that some polydopamine can be inserted between the π planes of the G-tetrad through π-π stacking, and the remaining polydopamine can adhere the G-quadruplexes together. Due to the electrostatic repulsion between G-quadruplexes, the G-quadruplexes cannot be stacked vertically, and finally arranged horizontally to form a polymer with a nanosheet morphology. If only this type of G-quadruplex / Hemin complex is introduced into a DNA logic gate device and polydopamine attached to the G-quadruplex is used as a color developer, there will be problems such as few catalytic sites and low color development, which is not conducive to the observation of the staining results.

[0007] In summary, it is necessary to propose a new method and strategy to make up for the shortcomings of existing technical solutions. Summary of the invention

[0008] In view of this, the object of the present invention is to provide a reagent composition and a staining method suitable for staining low-abundance target proteins, and the specific technical scheme is as follows.

[0009] A reagent composition for staining low-abundance target proteins on the surface of cells, the reagent composition comprising a DNA logic gate nanodevice generation reagent and a G4 nanowire / Hemin complex generation reagent; the DNA logic gate nanodevice generation reagent comprises a DNA nanotriangular prism, a second trigger sequence, a HER2 aptamer sequence, a HER3 aptamer sequence, a first complementary sequence, a second complementary sequence, a third complementary sequence and a fourth complementary sequence; the nucleotide of the second trigger sequence is shown in SEQ ID NO.5, the nucleotide sequence of the HER2 aptamer sequence is shown in SEQ ID NO.6, the nucleotide sequence of the HER3 aptamer sequence is shown in SEQ ID NO.7, the nucleotide sequence of the first complementary sequence is shown in SEQ ID NO.8, the nucleotide sequence of the second complementary sequence is shown in SEQ ID NO.9, the nucleotide sequence of the third complementary sequence is shown in SEQ ID NO.10, and the nucleotide sequence of the fourth complementary sequence is shown in SEQ ID NO.11;

[0010] The G4 nanowire / Hemin complex generating reagent includes a sequence for generating G4 nanowires and hemoglobin; the sequence for generating G4 nanowires is a first trigger sequence, a first hairpin sequence, a second hairpin sequence and a third hairpin sequence; the nucleotide sequence of the first trigger sequence is shown in SEQ ID NO.1, the nucleotide sequence of the first hairpin sequence is shown in SEQ ID NO.2, the nucleotide sequence of the second hairpin sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the third hairpin sequence is shown in SEQ ID NO.4.

[0011] Furthermore, the reagent composition also includes a self-assembly buffer, and the components of the self-assembly buffer include 20 mM Tris, 200 mM NaCl, 2 mM MgCl2 and 20 mM KCl.

[0012] Furthermore, the basic structure of the G4 nanowire is a G-quadruplex, and the molar ratio of a single G-quadruplex to the heme in the G4 nanowire is 1:1 to 1:50.

[0013] Furthermore, the molar concentration of DNA logic gate nanodevices is 10-100 nM.

[0014] Beneficial technical effects:

[0015] The present invention provides a method and application for in situ staining of cell surface proteins using DNA logic gate nanodevices. Compared with the prior art method of introducing G-quadruplex / Hemin into DNA logic gate devices and using its HRP enzyme-mimicking activity to catalyze the chromogenic substrate to convert the recognition signal into chromogenic information, the present invention truly realizes in situ staining of the target protein site. Since the G4 nanowire has multiple polydopamine oxidation deposition sites, it is equivalent to having multiple color developers, and the color development degree is better.

[0016] In order to make the staining process for protein dimers more accurate and controllable, the present invention independently designs multiple functional sequences to block the target protein nucleic acid aptamer and trigger sequence. Multiple complementary sequences block the trigger sequence and the target protein nucleic acid aptamer sequence on the DNA logic gate nanodevice, realize the input and output control process of parallel signals, prevent the binding of the aptamer with the protein monomer, and trigger the HCR reaction of the trigger sequence, thereby realizing the specific detection of protein dimers.

[0017] Finally, the DNA logic gate nanodevice synthesized independently by the present invention can form a G4 nanowire containing a repeated G-quadruplex structure at the top of the logic gate device through logic gate operation and HCR reaction, further catalyzing and inducing the oxidative deposition of polydopamine. The G-quadruplex unit not only serves as a catalytic active site of polydopamine, but also its π-π stacking effect with polydopamine can cause polydopamine to be deposited on the G4 nanowire to form a black polydopamine nanofiber. The fiber can not only realize the color development of the target protein, but also improve the color development degree of the low-abundance target protein because it has multiple polydopamine oxidative deposition sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0019] Figure 1 It is a simulation schematic diagram of the polydopamine confined oxidation deposition method of the present invention;

[0020] Figure 2 This is a gel electrophoresis image of a G-quadruplex nanowire obtained by self-assembly reaction of a guanine-rich hairpin nucleic acid in one embodiment of the present invention;

[0021] Figure 3 This is a spectrum of the HRP enzyme activity of the G nanowire / Hemin complex detected using 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a detection probe in one embodiment of the present invention;

[0022] Figure 4 This is a transmission electron microscope (TEM) photo of the polydopamine nanofibers prepared by the present invention;

[0023] Figure 5 This is an atomic force microscope (AFM) photo of the polydopamine nanofibers prepared by the present invention;

[0024] Figure 6 The schematic diagram of the synthesis and logic operation principle of the DNA logic gate nanodevice of the present invention (a is a schematic diagram of the synthesis of the DNA nanotriangular prism, and b is a schematic diagram of the assembly and identification operation of the DNA logic gate nanodevice);

[0025] Figure 7 This is a schematic diagram of the principle of staining protein dimers on the cell membrane surface using the DNA logic gate nanodevice combined with the polydopamine confined oxidation deposition method of the present invention;

[0026] Figure 8 This is a photograph of the expression results of HER2:HER3 heterodimers in different cell models detected by Western blotting in one of the embodiments of the present invention;

[0027] Fig. 9 Optical microscope photos of different cells stained by the polydopamine confined oxidation deposition method of the present invention;

[0028] Fig.10 The diagram is a principle diagram of staining protein targets on the cell membrane surface by the polydopamine confined oxidative deposition method of the present invention and the immunohistochemical method, and a comparison of staining intensities (a is a comparison of the principles of the two methods, b is the staining result of the IHC method, and c is the staining result of the confined oxidative deposition method of the present invention). DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0031] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0032] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0033] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, even more typically + / - 0.5% of the stated value.

[0034] In this specification, some embodiments may be disclosed in a format that is in a certain range. It should be understood that this description of "being in a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0035] Glossary

[0036] The "DNA logic gate nanodevice" described in the present invention refers to a biological tool that uses a chain displacement reaction to perform specific functions by connecting various functional sequences and complementary sequences on a DNA nanotriangular prism synthesized by a DNA self-assembly reaction as a basic computing component.

[0037] The "logic gate operation" described in the present invention refers to the bottom and top functional sequences on the DNA logic gate nanodevice independently synthesized by the present invention, which are combined with the complementary sequences independently synthesized by the present invention. When the bottom functional sequence binds to the target protein, the competitive release and complementary pairing between the complementary sequences promote the exposure of the top functional sequence to trigger the subsequent reaction process.

[0038] The "polydopamine nanofiber" described in the present invention refers to a G4 nanowire structure first synthesized using the method of the present invention, which is further reacted with heme to obtain a G4 nanowire / Hemin complex, which has peroxidase activity to catalyze the oxidation of dopamine into polydopamine, and finally polydopamine is specifically oxidized and deposited on a single G-quadruplex on the G4 nanowire to form a polydopamine nanofiber with a special morphology.

[0039] The G4 nanowire structure described in the present invention refers to a DNA nanofiber assembled by an HCR reaction using a hairpin nucleic acid sequence independently synthesized by the present invention, and the DNA nanofiber includes a plurality of staggered G-quadruplexes.

[0040] Example 1

[0041] This embodiment provides an example of a method for preparing polydopamine fibers by controlling the confined oxidation deposition of polydopamine.

[0042] Step 1: Hybridization chain reaction (HCR) is a cascade self-assembly reaction of several self-stable DNA hairpin structures triggered by target molecules or trigger sequences to form a long-chain DNA structure with a cut. According to the principle of hybridization chain reaction, by introducing trigger sequences and functional sequences at both ends of the hairpin sequence, a DNA nanowire with multiple G-quadruplexes, also known as a G4 nanowire, can be formed through HCR. The reaction principle is as follows Figure 1 As shown, the first trigger sequence (SEQ ID NO.1) is complementary paired with a partial sequence of the first hairpin sequence (SEQ ID NO.2), resulting in the opening of the hairpin structure of the first hairpin sequence; then the unpaired region sequence of the first hairpin sequence continues to be complementary paired with a partial assembly sequence of the second hairpin sequence (SEQ ID NO.3), resulting in the opening of the hairpin structure of the second hairpin sequence; then the partial unpaired assembly sequence of the second hairpin sequence continues to be complementary paired with the assembly sequence of the third hairpin sequence (SEQ ID NO.4), resulting in the opening of the hairpin structure of the third hairpin sequence; then the partial unpaired assembly sequence of the third hairpin sequence continues to be complementary paired with a partial assembly sequence of the second hairpin sequence, resulting in the opening of the hairpin structure of the second hairpin sequence; and then the second hairpin sequence and the third hairpin sequence are alternately paired and repeatedly extended, and finally a DNA nanowire structure is obtained. The DNA nanowire structure contains repeated second and third hairpin sequences. The poly-G sequence parts at the tail ends of every two adjacent hairpin sequences can be assembled into a G-quadruplex structure. Therefore, the DNA nanowire can contain multiple staggered G-quadruplexes, which are called G4 nanowires.

[0043] Step 2: The G-quadruplex further binds to hemin to form a G4 nanowire / Hemin complex with peroxidase activity. In the presence of H2O2, dopamine can be catalytically oxidized by the G4 nanowire / Hemin complex to form polydopamine. The oligomers formed during the polymerization of dopamine can undergo a strong π-π stacking interaction with the G-tetrad in the G-quadruplex structure, so that each G-quadruplex of the G4 nanowire serves as an oxidative deposition site for polydopamine. The confined oxidative deposition of polydopamine is regulated by the method, and finally a composite polydopamine nanofiber is obtained. The synthesis principle diagram is shown in the figure. Figure 1 shown.

[0044] Example 2

[0045] This embodiment provides an example of a specific preparation method.

[0046] 1) Add the first trigger sequence and the hairpin sequence (including the first hairpin sequence, the second hairpin sequence and the third hairpin sequence) to the self-assembly buffer and incubate at room temperature for 1-2 hours to allow all sequences to undergo HCR reaction. The molar ratio of the first trigger sequence to the hairpin sequence is 1:1~1:200. Use a nucleic acid purification kit to treat the above solution to remove the salt ions in the self-assembly buffer to obtain a G4 nanowire aqueous solution. Use gel electrophoresis to analyze the nucleic acid self-assembly process, such as Figure 2 As shown, with the increase of self-assembly steps, G4 nanowires have a stagnant pore phenomenon in gel electrophoresis due to their larger molecular weight.

[0047] 2) Add heme to the G4 nanowire aqueous solution in step 1) and incubate at room temperature for 20-40 min to obtain a G4 nanowire / Hemin complex, wherein the molar ratio of a single G-quadruplex to the heme in the G4 nanowire is 1:1-1:50. Use ABTS as a detection probe to verify the horseradish peroxidase (HRP) enzyme activity of the G4 nanowire / Hemin complex, such as Figure 3 As shown, as the molar ratio of the first trigger sequence to the hairpin sequence nucleic acid increases (1:0, 1:10, 1:20, 1:40, 1:80, 1:120, 1:200), the longer the length of the self-assembled G4 nanowires is, the faster the catalytic rate of ABTS is (the longer the G4 nanowires are, the higher the absorbance value represented by the vertical axis at 500-900nm).

[0048] 3) The G4 nanowire / Hemin complex obtained in step 2) is stirred and mixed with the dopamine aqueous solution, and H2O2 is added, wherein the mass ratio of the G4 nanowire to the dopamine is 0.1:1-1:1, and the concentration of the H2O2 is 0.1-1 M.

[0049] 4) The mixture obtained in step 3) is further stirred for 0.5-10 hours, and then centrifuged to obtain polydopamine nanofiber material; the centrifugation parameters are 10000-15000 rpm, and the centrifugation is 10-30 minutes.

[0050] The composite polydopamine nanofibers were characterized by morphology (TEM, AFM), such as Figure 4 , Figure 5 As shown, the length is 0.2~50 μm and the width is 10~200 nm.

[0051] Example 3

[0052] This embodiment provides an example of a method for staining a cell surface protein target using confined oxidative deposition of polydopamine.

[0053] Step 1: Inspired by the literature (J. Am. Chem. Soc. 2018, 140 (31), 9793-9796), DNA nanotriangular prisms were synthesized through DNA self-assembly reaction. The synthesis principle is shown in 6a. The upper and lower surfaces are single-stranded regions, which can serve as binding sites for functional sequences.

[0054] Step 2: Use the single-stranded region of the DNA nanotriangular prism to bind the second trigger sequence with the connection sequence (SEQ ID NO.5), the HER2 aptamer sequence (SEQ ID NO.6), and the HER3 aptamer sequence (SEQ ID NO.7). Afterwards, the partial sequence of the HER2 aptamer is connected to the first complementary sequence (SEQ ID NO.8), the partial sequence of the HER3 aptamer is connected to the second complementary sequence (SEQ ID NO.9), the partial sequence of the second trigger sequence is connected to the third complementary sequence (SEQ ID NO.10), and the partial sequence of the third complementary sequence is connected to the fourth complementary sequence (SEQ ID NO.11) to form a DNA logic gate nanodevice. The synthesis principle is as follows: Figure 6 As shown in b. Among them, the 3' end or 5' end of the second trigger sequence, the HER2 aptamer sequence and the HER3 aptamer sequence is a connecting sequence, which can be complementary to the single-stranded region above and below the DNA nanotriangular prism respectively; and the role of the 1st-4th complementary sequence is to competitively bind to a specific target sequence, thereby exposing the target sequence fragment to promote the logic gate operation. Among them, when there is a HER2:HER3 protein dimer on the cell membrane surface, the HER2 aptamer sequence and the HER3 aptamer sequence can bind to the protein dimer, so that the first complementary sequence and the second complementary sequence originally bound to the aptamer are released, thereby pairing with the third complementary sequence and the fourth complementary sequence, ultimately leading to the exposure of the second trigger sequence, and the exposed second trigger sequence can be triggered according to Example 2, and further catalyze the limited oxidation deposition of induced polydopamine, and finally achieve staining on the cell. The nucleic acid sequence information involved in the present invention is shown in Table 1. The sequences are all of the same molar concentration, and sequences 1-3 are tool sequences disclosed in literature.

[0055] It is to be understood that the protein dimer described in the present invention is not limited to the HER2:HER3 protein dimer exemplified in this embodiment.

[0056] Table 1

[0057]

[0058] Example 4

[0059] This embodiment provides a specific example of a dyeing method.

[0060] The process principle of this embodiment is as follows Figure 7 shown.

[0061] 1) Sequence 1, sequence 2, sequence 3, the second trigger sequence, HER2 aptamer sequence, HER3 aptamer sequence, and the first to fourth complementary sequences are added to the annealing buffer in equal proportions and incubated at room temperature to form a DNA logic gate nanodevice.

[0062] 2) In this example, SK-BR-3 cells were used as the target model, MCF-7 cells and MDA-MB-231 cells were used as the control model, and the expression and interaction of HER2:HER3 heterodimers on the cell membrane were used as the detection objects. Figure 8 As shown, SK-BR-3 cells expressed HER2 and HER3 proteins, while the control group MCF-7 cells and MDA-MB-231 cells did not express HER2 and HER3. After stimulation with hNRG-1 cytokine, the expression of phosphorylated HER2 (p-HER2) and phosphorylated HER3 (p-HER3) in SK-BR-3 cells increased, indicating the formation of HER2:HER3 heterodimers. However, the control group cells did not have the HER2:HER3 heterodimer phenomenon.

[0063] 3) Cultivate cells. SK-BR-3, MCF-7, and MDA-MB-231 cells can be cultured and seeded in 24-well plates. When the cells grow to 70-80% confluence, fix the cells with 4% paraformaldehyde and use 3% H2O2 to eliminate the HRP enzyme activity of the cells.

[0064] 4) Block all protein sites on the cell surface using bovine serum albumin and herring sperm DNA, wherein the concentration of bovine serum albumin is 1-10%, and the concentration of herring sperm DNA is 0.1-1 mg ml -1 .

[0065] 5) After washing away excess bovine serum albumin and herring sperm DNA, the DNA logic gate device is co-incubated with the cells, wherein the concentration of the DNA logic gate device is 10-100 nM.

[0066] 6) After washing away the unbound DNA logic gate nanodevice with PBS buffer, add the 1st to 3rd hairpin sequences and incubate at room temperature for 1-2 hours. The concentrations of the hairpin nucleic acid are: hairpin 1, 10-100 nM; hairpin 2, 1-10 μM; hairpin 3, 1-10 μM.

[0067] 7) After washing away the unassembled hairpin nucleic acid with PBS buffer, add hemin and incubate at room temperature for 10-60 min to form a G4 nanowire / Hemin complex. The molar ratio of each G-quadruplex to hemin is 1:1~1:50.

[0068] 8) Add dopamine and H2O2, incubate at room temperature for 0.5-10 h, and use the G4 nanowire / Hemin complex to catalyze the oxidation of dopamine, so that dopamine is oxidized to polydopamine and deposited on the G4 nanowire. The mass ratio of the G4 nanowire to the dopamine is 0.1:1-1:1, and the concentration of the H2O2 is 0.1-1 M.

[0069] The results are as follows Fig. 9 As shown in the figure, SK-BR-3 cells, because they contain HER2:HER3 heterodimers, can combine with DNA logic gate devices and deposit polydopamine on the cell membrane through a series of logic gate operations and catalytic oxidation. However, MCF-7 and MDA-MB-231 cells do not contain HER2:HER3 heterodimers and cannot trigger subsequent reactions, so there is no polydopamine staining signal.

[0070] In traditional IHC technology, each secondary antibody has only one HRP catalytic site, and the staining of low-abundance targets is often shallow, which is not conducive to result observation and analysis. In order to compare with the staining effect of the present invention, this example uses HER2 protein as the target and HER2 aptamer connected to the trigger sequence as the recognition element to perform single target recognition staining. Fig.10 As shown, the IHC method stains cells lightly, while the G4 nanowires of the present invention contain a large number of repeated G-quadruplexes that can not only catalyze the oxidation of dopamine, but also serve as deposition sites for polydopamine, so the staining degree is darker. The above results show that the method of the present invention is conducive to the staining analysis of low-abundance targets.

[0071] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A reagent composition for staining low-abundance target proteins on the cell surface, characterized in that: The reagent composition includes a DNA logic gate nanodevice generation reagent and a G4 nanowire / Hemin complex generation reagent; the DNA logic gate nanodevice generation reagent includes a DNA nanotriangular prism, a second trigger sequence, a HER2 aptamer sequence, a HER3 aptamer sequence, a first complementary sequence, a second complementary sequence, a third complementary sequence and a fourth complementary sequence; the nucleotide of the second trigger sequence is shown in SEQ ID NO.5, the nucleotide sequence of the HER2 aptamer sequence is shown in SEQ ID NO.6, the nucleotide sequence of the HER3 aptamer sequence is shown in SEQ ID NO.7, the nucleotide sequence of the first complementary sequence is shown in SEQ ID NO.8, the nucleotide sequence of the second complementary sequence is shown in SEQ ID NO.9, the nucleotide sequence of the third complementary sequence is shown in SEQ ID NO.10, and the nucleotide sequence of the fourth complementary sequence is shown in SEQ ID NO.11; The G4 nanowire / Hemin complex generating reagent includes a sequence for generating G4 nanowires and hemoglobin; the sequence for generating G4 nanowires is a first trigger sequence, a first hairpin sequence, a second hairpin sequence and a third hairpin sequence; the nucleotide sequence of the first trigger sequence is shown in SEQ ID NO.1, the nucleotide sequence of the first hairpin sequence is shown in SEQ ID NO.2, the nucleotide sequence of the second hairpin sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the third hairpin sequence is shown in SEQID NO.

4.

2. The reagent composition according to claim 1, characterized in that The reagent composition further includes a self-assembly buffer, and the components of the self-assembly buffer include 20 mM Tris, 200 mM NaCl, 2 mM MgCl2 and 20 mM KCl.

3. The reagent composition according to claim 1, characterized in that The basic structure of the G4 nanowire is a G-quadruplex, and the molar ratio of a single G-quadruplex to the heme in the G4 nanowire is 1:1-1:

50.

4. The reagent composition according to claim 1, characterized in that The molar concentration of DNA logic gate nanodevices is 10-100 nM.

Images