Analysis method for simultaneously detecting double markers by using metal ion mediated DNA (Deoxyribose Nucleic Acid) functional nano machine switch

Abstract

The invention provides an analysis method for simultaneous analysis of double markers by using a metal ion mediated DNA functional nano machine switch, and relates to the technical field of biomedical diagnos.The 'DNOP (at) One (at) X' analysis model of the analysis method comprises DNA nanosphere one-pot synthesis, and can be combined with multiple signal output modes, so that the analysis accuracy is improved. The convenient and sensitive analysis on the two trace markers is realized. Cu < 2 + > and Ag < + > mediated self-assembled DNA nanospheres are respectively pre-synthesized by a one-pot method, the aptamer of the marker is used as an initiator to trigger the conformation change of the DNA nanospheres and release a large amount of Cu < 2 + > and Ag < + >, and the Cu < 2 + > and Ag < + > are respectively recognized by fluorescence signal molecules, so that a simple, low-cost, high-sensitivity and rapid homogeneous-phase dual-marker fluorescence analysis strategy is established; time is shortened, cost is reduced, and analysis sensitivity is improved. The strategy can enrich a medical diagnosis system and provide more choices for medical diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical diagnosis, and in particular to an analysis method for using a metal ion-mediated DNA functional nanomachine switch for simultaneous analysis of dual markers. Background Art

[0002] Based on the high programmability of DNA, DNA nanomaterials with high sensitivity, high loading capacity and anti-degradation ability can be prepared by combining a variety of functional molecules, which have great application potential in the fields of biosensing and disease treatment. Among them, metal ion-mediated synthesis of DNA nanomaterials provides an effective new method for DNA modification, which gives metal nanostructures high stability and molecular recognition ability. For example, DNA is combined with metal materials or metal organic frameworks through sulfidation or thiol modification, or metal nanoparticles or nanoclusters (Au NPs, Ag NCs, CuNPs) are formed using DNA as a template, which have been applied to biomarker analysis. However, these methods usually require strict synthesis conditions and usually require the introduction of other materials or signal amplification technologies to achieve sensitive analysis of markers, which is limited in practical applications. Therefore, reducing the complexity of the synthesis process and increasing the flexibility of the strategy remain core issues in the field of DNA-metal nanomaterials.

[0003] To solve the above problems, using the base mismatch structure formed by the affinity of metal ions for specific bases as an ion fixation and release unit provides another option. + The mediated natural nucleic acid base pairing is expected to achieve sequence-defined DNA self-assembly. In addition, metal ions and phosphate coordination (such as Cu 2+ Ag) does not require the introduction of additional components and has the advantage of being fast and convenient in the preparation of composite nanomaterials. + C-Ag + -C has been used to construct recognition elements. 2+ The formation of complexes, through the conformational conversion of DNA and ion release, has achieved high-sensitivity detection of proteins and cells. Therefore, by flexibly utilizing the interaction between different metal ions and DNA, functional nanomaterials that integrate recognition, signal amplification and transduction will facilitate their clinical application. It is worth noting that the diversity of metal ions and the specificity of DNA sequences also contribute to the development of strategies for the joint analysis of multiple targets. The shortcomings of existing metal ion-mediated DNA nanomaterials are: ① The preparation process involves complex chemical reactions and delicate operation steps, which increases the difficulty and cost of preparation; ② Some materials require additional modification to achieve functionalization, which increases the complexity of application; ③ The functions are relatively single and it is difficult to meet the needs of complex biomedical applications.

[0004] In clinical disease diagnosis, the combined detection of multiple markers can accurately determine the occurrence and development of the disease, improve the detection rate, and reduce the misdiagnosis rate. In particular, for circulating tumor cells (CTCs), a type of tumor liquid biopsy marker with extremely low abundance and high heterogeneity in the human circulation, the combined detection of multiple markers is particularly important. At present, the development of efficient multiplex sensing systems has become a focus of attention in the field of diagnosis. Multiplex PCR is a commonly used method in clinical practice, but its primer design is complex, requires multiple enzymes and multiple reaction steps, and its detection sensitivity is not enough to meet the detection of important and extremely low-content disease markers in major diseases such as early infection or tumors. More importantly, its application in the analysis of other non-nucleic acid markers is limited. Aptamers, as an oligonucleotide sequence, can be used to specifically detect various types of targets, including ions, macromolecules, and cells. By combining DNA aptamers with various nucleic acid amplification technologies and testing instruments, a variety of methods have been used to achieve sensitive analysis. However, similar signal amplification technologies often require a long time for subsequent amplification because they require the presence of the target to trigger the reaction or require the assistance of multiple enzymes. At the same time, in order to achieve the detection of multiple markers, more nucleic acid designs are introduced, which causes nonspecific background leakage. Nanomaterials have great potential in signal enhancement applications because they do not require the use of enzymatic reactions or multiple signal amplification steps. Currently, the commonly used methods in clinical practice include multiple polymerase chain reaction (PCR), immunofluorescence labeling, mass spectrometry, etc. In addition, researchers have also developed many other detection methods, such as microfluidic chip technology, fluorescence in situ hybridization, multiple fluorescent labeling, etc. The shortcomings of existing multiple detection technology: ① Multiple PCR technology requires complex primer design, requires protease, and is a variable temperature process; ② Fluorescent labeling operation is complex and time-consuming, and the cost is high; ③ It depends on professional instruments and professional operators; ④ The detection sensitivity is not enough to meet the important and extremely low content (ag / mL) disease marker analysis in major diseases such as tumors. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a metal ion-mediated DNA functional nanomachine switch for a method for simultaneous analysis of dual markers to overcome the defects described in the background art.

[0006] In order to achieve the above-mentioned purpose, the present invention provides an analysis method for the simultaneous analysis of two markers using a metal ion-mediated DNA functional nanomachine switch, the method comprising the following steps: specifically binding the overexpressed mucin 1 and programmed cell death-ligand 1 on circulating tumor cells to the corresponding aptamers, and determining whether the aptamers destroy the structure of the metal ion-mediated DNA nanoballs synthesized in one pot; then effectively changing the fluorescence signal of a signal reporter based on the metal ions released by the metal ion-mediated DNA nanoballs; and then quantifying a single target based on the fluorescence signal of the signal reporter, wherein the single target is a circulating tumor cell.

[0007] As an optional embodiment, the metal ion-mediated DNA nanospheres include Y-NS@Cu 2+ and Y-NS@Ag + , the Y-NS@Cu 2+ The Y-NS@Ag + Contains the complementary strand of the programmed cell death-ligand 1 aptamer.

[0008] As an optional implementation, the Y-NS@Ag + Divided into Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag + , the Y 1-3 -NS@Ag + The Y comprises a complementary strand of the first half of the programmed cell death-ligand 1 aptamer, wherein the Y 4-6 -NS@Ag + Contains the complementary strand of the second half of the programmed cell death-ligand 1 aptamer.

[0009] As an optional embodiment, when circulating tumor cells are present, mucin 1 and programmed cell death-ligand 1 on their surface bind to the corresponding specific aptamers respectively, and the Y-NS@Cu 2+ , Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag + integrity is preserved.

[0010] As an optional embodiment, when circulating tumor cells are absent, mucin 1 and programmed cell death-ligand 1 on their surfaces are not bound to the corresponding specific aptamers, and the Y-NS@Cu 2+ , Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag +The structure of the aptamer was destroyed and a large amount of free Cu was released. 2+ and Ag + .

[0011] As an optional embodiment, the signal reporter is two of calcein, CdTe QDs, carbon dots, N-methylporphyrin dipropionate IX or CdSe QDs.

[0012] As an optional embodiment, when the signal reporter is calcein and CdTe QDs, the calcein selectively recognizes Cu 2+ To quench the fluorescence signal of calcein, the CdTe QDs and Ag + A cation exchange reaction occurs to quench the fluorescence signal of CdTe QDs.

[0013] As an optional embodiment, the mucin 1 and the programmed cell death-ligand 1 are derived from lung cancer A549 cells.

[0014] As an optional embodiment, the mucin 1 and the programmed cell death-ligand 1 can be replaced by two disease markers having aptamers among mucin 1, programmed cell death-ligand 1, cellular exosomes and bacteria.

[0015] As an optional embodiment, the lung cancer A549 cells can be expanded to other circulating tumor cells expressing the mucin 1 and the programmed cell death-ligand 1.

[0016] The metal ion-mediated DNA functional nanomachine switch provided by the present invention is used for the analysis method of simultaneous analysis of two markers, which has the following technical effects:

[0017] The metal ion-mediated DNA functional nanospheres proposed in this analytical method can be pre-synthesized in one step, and the steps are simple and fast. The aptamer of the marker is used as an initiator to trigger the conformational change of the DNA nanospheres, releasing a large amount of Cu 2+ and Ag + , and are recognized by fluorescent signal molecules respectively, establishing a simple, low-cost, highly sensitive, rapid and homogeneous dual-marker fluorescence analysis strategy; the "DNOP@One@X" analysis model proposed in the present invention is universal and can be applied to dual-marker detection of other diseases by encoding DNA sequences and replacing signal molecules; it can be combined with a variety of signal output methods, such as directly detecting the released ion concentration through an electrochemical workstation, using other dyes or fluorescent molecules that specifically recognize metal ions, other portable analytical instruments, etc., which can be applicable to a variety of medical scenarios and help promote applications. 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 description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a schematic diagram of the principle of the "DNOP@One@X" model for CTCs detection;

[0020] Figure 2 It is the synthesis and characterization of DNA nanospheres;

[0021] Figure 3 It is the feasibility verification of the “DNOP@One@X” detection system;

[0022] Figure 4 It is the analytical performance of mucin 1, PD-L1 and A549 cells. DETAILED DESCRIPTION

[0023] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0024] The principle of the analytical method of the present invention is as follows:

[0025] The "DNOP@One@X" analytical model proposed in this invention first includes the one-pot synthesis of DNA functional nanospheres mediated by metal ions (DNOP). Figure 1 As shown in Figure 1, three DNA single strands with palindromic sequences self-assemble to form a Y-shaped DNA nanosphere (Y-NS). 2+ The Y-NS@Cu 2+ .Y-NS@Ag + The formation of also follows similar conditions, except that the use of Ag + The high affinity for cytosine (C) bases uses multiple C bases to replace the palindromic sequence in the DNA single strand, cleverly utilizing C-Ag + -C structure realizes Y-NS@Ag + Self-assembly synthesis of Figure 1 II).

[0026] As a proof of concept, circulating tumor cells (CTCs), a liquid biopsy marker with extremely low levels in peripheral blood, were selected to verify the applicability of the analytical model by detecting two proteins expressed on their membrane surface: mucin 1 and programmed cell death-ligand 1 (PD-L1). 2+ The Ya chain of Y-NS@Ag was designed as the complementary chain of mucin 1 aptamer to target mucin 1. The PD-L1 aptamer is too long, which is not conducive to the stability of the nanosphere structure. + Divided into Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag + Two nanomaterials (Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag + The two chains correspond to the first and second halves of the aptamer, respectively. When CTCs are present, mucin 1 and PD-L1 on their surfaces bind to the corresponding specific aptamers, respectively, and the integrity of the nanospheres is preserved. In the absence of CTCs, the two aptamers complementarily pair with the Ya, Y1, and Y4 chains, respectively, causing changes in the structure of the nanospheres and releasing a large amount of free Cu. 2+ and Ag + The entire reaction process can be completed in one step within 75 minutes (Process 1).

[0027] Subsequently, calcein and cadmium telluride quantum dots (CdTe QDs) were selected as signal reporters. Calcein and CdTe QDs can specifically recognize Cu 2+ and Ag + , and their fluorescence emission spectra do not overlap with each other. Under specific conditions, calcein was first added to selectively recognize Cu 2+ The fluorescence signal of calcein was quenched, and then CdTeQDs and Ag were added. + A cation exchange reaction (CER) occurred to quench the fluorescence signal of QDs (Process 2 and Process 3).

[0028] Therefore, based on the significant difference in fluorescence signals between free metal ions and DNA-metal ion complexes, the co-analysis of the two proteins and the precise quantification of CTCs were achieved. It is worth mentioning that by changing the base sequence of the corresponding DNA nanospheres, the sensing system can be easily applied to the analysis of other biomarkers and disease diagnosis. In addition to fluorescence signals, the released metal ions can also use other ion detection modes, such as electrochemical direct detection, to be suitable for a variety of application scenarios.

[0029] The technical solution of the present invention is specifically verified below in conjunction with embodiments.

[0030] Example 1 Mucin 1 and PD-L1 protein co-analysis steps and A549 cell analysis steps

[0031] 1. Synthesis steps of metal ion-mediated DNA functional nanomachines

[0032] ①Y-NS@Cu 2+ : 30 μL of 50 μM Ya, Yb, and Yc DNA chains, 30 μL of 60 μM Cu(NO3)2 solution, and 180 μL of 10 mM 3-morpholinepropanesulfonic acid (MOPS) buffer (100 mM NaNO3, pH 7.6) were mixed, heated at 95 °C for 5 min, and placed at room temperature for 3 h to form Y-NS@Cu 2+ Nanospheres;

[0033] ②Y 1-3 -NS@Ag + : 30 μL of 50 μM Y1, Y2, and Y3 DNA chains, 30 μL of 600 μM AgNO3 solution, and 180 μL of 10 mM MOPS buffer (100 mM NaNO3, pH 7.6) were mixed, heated at 95°C for 5 min, and left at room temperature for 3 hours to form Y 1-3 -NS@Ag + Nanospheres;

[0034] ③Y 4-6 -NS@Ag + : 30 μL of 50 μM Y4, Y5, and Y6 DNA chains, 30 μL of 600 μM AgNO3 solution, and 180 μL of 10 mM MOPS buffer (100 mM NaNO3, pH 7.6) were mixed, heated at 95°C for 5 min, and left at room temperature for 3 hours to form Y 4-6 -NS@Ag + Nanospheres.

[0035] 2. Mucin 1 and PD-L1 protein co-analysis steps

[0036] First, 10 μL of 1nM mucin 1 aptamer, 10 μL of 100pM PD-L1 aptamer, 40 μL of mucin 1 protein with different concentrations, 40 μL of PD-L1 protein with different concentrations, and 10 μL of Y-NS@Cu 2+ , 12μL Y 1-3 -NS@Ag + , 12μL Y 4-6 -NS@Ag +Mix with 16 μL 10 mM MOPS and react at room temperature for 75 min.

[0037] Afterwards, 7 μL of 3 μM calcein was added to the reaction solution and reacted at room temperature for 1.5 min.

[0038] Finally, 9.5 μL QDs (diluted 5 times) were added, and the fluorescence signal was measured after reacting at room temperature for 2 min.

[0039] 3. Analysis steps of A549 cells

[0040] First, 10 μL of 1nM mucin 1 aptamer, 10 μL of 100pM PD-L1 aptamer, 1 mL of A549 cells of different concentrations, and 10 μL of Y-NS@Cu 2+ , 12μL Y 1-3- NS@Ag + , 12μLY 4-6 -NS@Ag + Mix with 70 μL 10 mM MOPS and react on a shaker at room temperature for 75 min.

[0041] Afterwards, 7 μL of 3 μM calcein was added to the reaction solution and reacted at room temperature for 1.5 min.

[0042] Finally, 9.5 μL QDs (diluted 5 times) were added, and the fluorescence signal was measured after reacting at room temperature for 2 min.

[0043] Example 2 Metal ion-mediated DNA functional nanomachine

[0044] like Figure 2 As shown in A, DNA chains Ya, Yb and Yc with palindromic sequences are 2+ Co-annealing to form Y-NS@Cu 2+ By adding Ag + Coordinate with the terminal C-rich Y1-Y3 and Y4-Y6 to form C-Ag + -C structure-mediated palindromic sequence, achieving Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag + Self-assembly synthesis of Figure 2 B).

[0045] In order to study the size and morphology of DNA nanospheres under different conditions, atomic force microscopy (AFM) and transmission electron microscopy (TEM) were used to characterize them. Figure 2 C, AFM image shows that the Y-DNA monomers without palindromic sequences are smaller in size and appear as evenly distributed dots. 2+It has a distinct spherical shape ( Figure 2 D).

[0046] After adding mucin 1 aptamer, Y-NS@Cu 2+ The structure is destroyed ( Figure 2 E) Similarly, Y without C base sequence at the end 4-6 -DNA monomer size is also smaller. 4-6 -NS@Ag + After adding PD-L1 aptamer, Y 4-6 -NS@Ag + The structure is destroyed ( Figure 2 FH). TEM images also confirmed the above process ( Figure 2 IN).

[0047] Afterwards, agarose gel electrophoresis was used to verify the synthesis process of the material.

[0048] In order to avoid the metal ions in the nanospheres from complexing with the thiol groups in the electrophoresis buffer, the Cu 2+ and Ag + , and the C-rich base sequence was replaced by a palindromic sequence. Since Ya, Yb, and Yc can base-pair with each other, low molecular weight complexes of Ya+Ya and Yb+Yc may be contained in lanes 5 and 6. After annealing, some new high molecular weight products appeared in lane 6, indicating that Y-NS was successfully formed ( Figure 2 O). The same method also confirmed that Y 4-6 - Successful formation of NS ( Figure 2 P).

[0049] Afterwards, dynamic light scattering (DLS) and zeta potential measurements were performed ( Figure 2 Q and R). These measurements show that Y-DNA, Y-NS@Cu 2+ and Y-NS@Cu 2+ The average diameters of the +mucin 1 aptamer complexes were 5.5 nm, 288.3 nm, and 62.3 nm, respectively.

[0050] In addition, it was found that Y-NS@Cu 2+ The UV absorption intensity of Y-NS is much lower than that of Y-DNA monomer, which may be due to the spatial structure of DNA nanospheres avoiding the exposure of conjugated double bonds, resulting in a decrease in the UV absorption peak ( Figure 2 S).

[0051] Afterwards, 4-6 -NS@Ag + The same study was conducted and similar conclusions were reached. 4-6-DNA, Y 4-6 -NS@Ag + and Y 4-6 -NS@Ag + The average diameters of the +PD-L1 aptamers were 8.3 nm, 455.2 nm, and 177.0 nm, respectively. Figure 2 TV).

[0052] In summary, the above results indicate that metal ion-mediated DNA nanospheres have been successfully synthesized and can be used for the next step of analysis.

[0053] Example 3 Selective recognition of metal ions and metal ion-mediated DNA functional nanomachines

[0054] First, calcein was selected as the 2+ CdTe QDs were selected as the fluorescent molecules to recognize Ag + Fluorescent signal molecules. Figure 3 As shown in A and 3B, the ultraviolet absorption peaks of calcein and QDs were observed respectively. When calcein was mixed with CdTeQDs, the excitation wavelength was adjusted to 486nm, and a good fluorescence double peak emission spectrum was obtained ( Figure 3 C) TEM results show that CdTe QDs are uniformly distributed in dots, while Ag + CER reaction with CdTe QDs leads to QDs clustering ( Figure 3 D).

[0055] It is worth noting that Cu 2+ and Ag + It has a quenching effect on the fluorescence of calcein and QDs, but there are differences in the response sensitivity.

[0056] Therefore, by adjusting the Cu 2+ and Ag + concentration to achieve specific recognition. Figure 3 E shows that in the same system, Cu 2+ The concentration of Cu 2+ When the concentration range is 3-6 μM, the fluorescence signal of QDs remains basically unchanged, but the fluorescence of calcein changes significantly. + At concentrations between 20 and 60 μM, the signal of calcein remained stable, while the fluorescence of QDs changed significantly ( Figure 3 F). This means that selective recognition of signal molecules can be achieved by precisely controlling the 2+ and Ag + When 60 μM Cu 2+ Formation of Y-NS@Cu with DNA 2+When Y-NS@Cu 2+ With free Cu 2+ The difference in calcein signal caused by this is the largest. 2+ The final concentration is 6 μM, which satisfies the specific recognition of Cu by calcein. 2+ conditions ( Figure 3 G) Similarly, when Ag + When the concentration reached 600 μM (final concentration was 60 μM), Y-NS@Ag + With free Ag + The QDs signal difference caused by this is the largest, which also proves that QDs selectively recognize Ag + , while calcein was not affected ( Figure 3 H).

[0057] Subsequently, to verify the feasibility of the system, the aptamer was used as a trigger to recognize and dissociate the DNA nanospheres ( Figure 3 I and J). Y-NS@Cu 2+ Binds to mucin 1 aptamer through base complementary pairing, releasing Cu 2+ The fluorescence of calcein decreased, while the QDs signal remained basically unchanged ( Figure 3 K). Similarly, after adding PD-L1 aptamer alone, Y-NS@Ag + dissociated, resulting in a decrease in the QDs signal, while calcein was not affected ( Figure 3 L). However, the simultaneous addition of these two aptamers resulted in a decrease in the signals of calcein and QDs ( Figure 3 M).

[0058] In addition, the stability of DNOP was evaluated and it was found that the dissociation effect of the aptamer on the synthesized DNA nanospheres was not affected within 48 h ( Figure 3 N). Therefore, it is believed that this analysis system can be used for the joint analysis of two markers.

[0059] Example 4 Performance evaluation of co-analysis of mucin 1 and PD-L1 and performance evaluation of A549 cell analysis

[0060] First, agarose gel electrophoresis was used for verification. Figure 4 As shown in A, a series of low molecular weight products were generated in lanes 4, 5, and 6, indicating that the addition of mucin 1 aptamer made Y-NS@Cu 2+ After the addition of mucin 1 protein, the aptamer specifically binds to mucin 1. The increase in mucin 1 concentration leads to a decrease in free aptamers, thereby preventing Y-NS@Cu 2+ The same method is also used to verify Y 4-6-NS@Ag + ( Figure 4 B).

[0061] Subsequently, the detection sensitivity of mucin 1 and PD-L1 was verified. Figure 4 As shown in C and D, the signal values ​​of calcein and QDs increased with the increase of mucin 1 and PD-L1 concentrations. The logarithmic values ​​of mucin 1 and PD-L1 concentrations (from 1ag / mL to 10fg / mL) were linearly correlated with the corresponding fluorescent molecular signal values ​​( Figure 4 E and F), the detection limits reached 4 ag / mL and 3 ag / mL, respectively. In addition, a panel of interferents including human serum albumin, immunoglobulin G, and histones were used to evaluate the selectivity of the analytical system ( Figure 4 G and H). The fluorescence signal value of the interfering substance at 10pg / mL is very close to that of the blank control group. However, low concentrations of mucin 1 or PD-L1 (10ag / mL and 10fg / mL) will cause significant changes in the signal. The above results well demonstrate the sensitivity and specificity of the system, and it has excellent detection performance for dual markers.

[0062] In order to further evaluate the analytical performance of "DNOP@One@X" on CTCs, lung cancer A549 cells were introduced as the verification object. There are multiple PD-L1 and mucin 1 expression sites on the cell surface. Figure 4 As shown in Figures I and 4J, the dual fluorescence signal increases with the increase of A549 cell concentration. In the concentration range of 1-1000 cells / mL, the logarithm of cell concentration and the fluorescence signal show a good linear relationship ( Figure 4 K and L). The linear equation determined based on QDs (related to PD-L1) is Y = 281LogC + 1577, R 2 =0.990. The linear equation determined based on calcein (related to mucin 1) is Y=75LogC+610,R 2 =0.992. Compared with existing liquid biopsy methods, the simultaneous detection of homogeneous dual markers and the non-modified labeling of "DNOP@One@X" make it valuable for detecting trace CTCs in the early stages of cancer.

[0063] In addition, this analytical strategy can also be applied to the detection of various trace markers by changing the sequence design and signal output mode according to the disease to be diagnosed.

[0064] Conclusion of Example 5

[0065] Metal ion-mediated DNA nanomaterials pre-synthesized by a one-pot method integrate signal amplification and transduction, allowing the reaction to be completed quickly in a short time. Combined with aptamer recognition and signal reporter molecules, one-pot, homogeneous and sensitive detection of dual markers was achieved. The strategy was applied to the precise analysis of two proteins and CTCs cells, proving the reliability and practicality of the strategy. Based on the adjustability of DNA nanomachine nucleic acid sequence design and the variability of nanosphere synthetic materials, for example, using other dyes as direct reporter molecules instead of metal ions, "DNOP@One@X" will be expected to achieve multiple ultra-sensitive detection of multiple disease markers, and even develop to point-of-care testing (POCT).

[0066] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A metal ion-mediated DNA functional nanomachine switch for simultaneous dual-marker analysis, characterized in that: The method includes specific binding of mucin 1 and programmed cell death-ligand 1 overexpressed on circulating tumor cells with corresponding aptamers, and whether the aptamer destroys the structure of metal ion-mediated DNA nanospheres synthesized in one pot, and then based on the metal ions released by the metal ion-mediated DNA nanospheres effectively changing the fluorescence signal of the signal reporter, and then quantifying the target based on the fluorescence signal of the signal reporter, wherein the target is mucin 1 and programmed cell death-ligand 1, or circulating tumor cells.

2. The method for simultaneous analysis of two markers using a metal ion-mediated DNA functional nanomachine switch according to claim 1, characterized in that: The metal ion-mediated DNA nanoball includes a Y-shaped DNA nanoball structure, an X-shaped DNA nanoball structure or a polyhedral DNA nanoball structure.

3. The method for simultaneous analysis of two markers using a metal ion-mediated DNA functional nanomachine switch according to claim 2, characterized in that: The Y-shaped DNA nanosphere structure includes Y-NS@Cu 2+ and Y-NS@Ag + , the Y-NS@Cu 2+ The Y-NS@Ag + Contains the complementary strand of the programmed cell death-ligand 1 aptamer.

4. The method for simultaneous analysis of two markers using a metal ion-mediated DNA functional nanomachine switch according to claim 3, characterized in that: The Y-NS@Ag + Divided into Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag + , the Y 1-3 -NS@Ag + The Y comprises a complementary strand of the first half of the programmed cell death-ligand 1 aptamer, wherein the Y 4-6 -NS@Ag + Contains the complementary strand of the second half of the programmed cell death-ligand 1 aptamer.

5. The method for simultaneous analysis of two markers using a metal ion-mediated DNA functional nanomachine switch according to claim 4, characterized in that: When circulating tumor cells are present, mucin 1 and programmed cell death-ligand 1 on their surfaces bind to the corresponding specific aptamers, respectively. 2+ , Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag + integrity is preserved.

6. The method for simultaneous analysis of two markers using a metal ion-mediated DNA functional nanomachine switch according to claim 4, characterized in that: When circulating tumor cells are absent, mucin 1 and programmed cell death-ligand 1 on their surfaces do not bind to the corresponding specific aptamers, and the Y-NS@Cu 2+ , Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag + The structure of the aptamer was destroyed and a large amount of free Cu was released. 2+ and Ag + .

7. The method for simultaneous analysis of two markers using a metal ion-mediated DNA functional nanomachine switch according to claim 6, characterized in that: The signal reporter is two of calcein, CdTe QDs, carbon dots, N-methylporphyrin dipropionate IX or CdSe QDs.

8. The method for simultaneous analysis of two markers using a metal ion-mediated DNA functional nanomachine switch according to claim 7, characterized in that: When the signal reporter is calcein and CdTe QDs, the calcein selectively recognizes Cu 2+ To quench the fluorescence signal of calcein, the CdTe QDs and Ag + A cation exchange reaction occurs to quench the fluorescence signal of CdTe QDs.

9. The method for simultaneous analysis of dual markers using a metal ion-mediated DNA functional nanomachine switch according to claim 1, characterized in that: The mucin 1 and the programmed cell death-ligand 1 are derived from lung cancer A549 cells.

10. The method for simultaneous analysis of two markers using a metal ion-mediated DNA functional nanomachine switch according to claim 1, characterized in that: The mucin 1 and the programmed cell death-ligand 1 can be replaced by two disease markers having aptamers among mucin 1, programmed cell death-ligand 1, cellular exosomes and bacteria.

11. The method for simultaneous analysis of two markers using a metal ion-mediated DNA functional nanomachine switch according to claim 9, characterized in that: The lung cancer A549 cells can be expanded to other circulating tumor cells expressing the mucin 1 and the programmed cell death-ligand 1.

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