Metal ion-mediated DNA functional nanomachine switch for simultaneous detection of two markers

Abstract

The present invention provides an analytical method for simultaneous analysis of two markers using a metal ion-mediated DNA functional nanomachine switch, which relates to the field of biomedical diagnostic technology. The "DNOP@One@X" analytical model of this analytical method includes a one-pot synthesis of DNA nanospheres and can be combined with multiple signal output methods to achieve convenient and sensitive analysis of two trace markers. 2+ and Ag + The aptamer of the marker is used as an initiator to trigger the conformational change of the DNA nanosphere and release a large amount of Cu 2+ and Ag + , and are recognized by fluorescent signal molecules, respectively. This establishes a simple, low-cost, highly sensitive, rapid, and homogeneous dual-marker fluorescence analysis strategy, shortening time, reducing costs, and improving analytical sensitivity. This strategy can enrich the medical diagnostic system and provide more options for medical diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical diagnosis, and particularly relates to an analysis method for double-marker simultaneous analysis by using a metal ion-mediated DNA functional nanomachine switch. BACKGROUND

[0002] Based on the high programmability of DNA, by combining various functional molecules, DNA nanomaterials with high sensitivity, high loading capacity and anti-degradation capacity can be prepared, which have great application potential in the fields of biosensing and disease treatment. Among them, the synthesis of metal ion-mediated DNA nanomaterials provides an effective new method for DNA modification, which endows metal nanostructures with high stability and molecular recognition ability. For example, DNA is modified by sulfuration or mercapto to combine with metal materials or metal organic frameworks, or to form metal nanoparticles or nanoclusters (Au NPs, Ag NCs, Cu NPs), which have been applied to biomarker analysis. However, these methods usually require strict synthesis conditions, and usually require the additional introduction of other materials or signal amplification technology to achieve sensitive analysis of markers, which is limited in practical application. Therefore, reducing the complexity of the synthesis process and increasing the flexibility of the strategy are still the core problems in the field of DNA-metal nanomaterials.

[0003] To solve the above problems, the base mismatch structure formed by using the affinity of metal ions for specific bases as an ion fixation and release unit provides another option. For example, Ag + mediated natural nucleic acid base pairing is expected to realize sequence-defined DNA self-assembly. In addition, metal ion and phosphate coordination (such as Cu 2+ and DNA) does not require the introduction of additional components, and has the advantages of being fast and convenient when preparing composite nanomaterials. Ag + mediated base pairs C-Ag + -C have been used to construct recognition elements. By using self-assembled DNA nanomachines and Cu 2+ forming complexes, high-sensitivity detection of proteins and cells has been achieved through DNA conformational conversion and ion release. Therefore, by flexibly utilizing the interaction of different metal ions with 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 multiple target co-analysis strategies. The existing metal ion-mediated DNA nanomaterials have the following deficiencies: ① The preparation process involves complex chemical reactions and delicate operation steps, which increases the difficulty and cost of preparation; ② Some materials need additional modification to realize functionalization, which increases the complexity of application; ③ The function is relatively single, which 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 development process of the disease, improve the detection rate, and reduce the misdiagnosis rate. Especially for circulating tumor cells (CTCs), this kind 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 system has become the focus in the field of diagnosis. Multiplex PCR is a commonly used method in clinic, but its primer design is complex, multiple enzymes and multiple reaction steps are needed, and its detection sensitivity is not enough to meet the detection of important and extremely low content disease markers in early infection or tumor and other major diseases. More importantly, its application in the analysis of other non-nucleic acid markers is limited. As an oligonucleotide sequence, aptamer can be used for specific detection of multiple types of targets, including ions, macromolecules and cells, etc. By combining DNA aptamer with multiple nucleic acid amplification techniques and detection instruments, a variety of methods have realized sensitive analysis. However, similar signal amplification techniques need to trigger the reaction when the target exists, or need the assistance of multiple enzymes, and the subsequent amplification often needs a long time. At the same time, in order to realize the detection of multiple markers, more nucleic acid designs are introduced, thereby causing non-specific 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. The methods commonly used in clinic are multiplex 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 fluorescence labeling, etc. The shortcomings of the existing multiplex detection technology are: ① multiplex PCR technology needs complex primer design, needs protease, and is a variable temperature process; ② the operation of fluorescence labeling is complex and time-consuming, and the cost is high; ③ it depends on professional instruments and equipment and professional operators; ④ the detection sensitivity is not enough to meet the analysis of important and extremely low (ag / mL) disease markers in tumor and other major diseases. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a metal ion-mediated DNA functional nanomachine switch for simultaneous analysis of double markers, in order to overcome the defects described in the background art.

[0006] In order to achieve the above-mentioned purpose, the present application provides an analysis method of metal ion-mediated DNA functional nanomachine switch for double-marker simultaneous analysis, which comprises specific binding of overexpressed mucin 1 and programmed cell death-ligand 1 on circulating tumor cells with corresponding aptamers, simultaneous destruction of the structure of one-pot synthesized metal ion-mediated DNA nanoballs based on the aptamers, then effective change of the fluorescence signal of the signal reporter based on the metal ion released by the metal ion-mediated DNA nanoballs, and quantification of single targets, i.e. circulating tumor cells, based on the fluorescence signal of the signal reporter.

[0007] As an optional embodiment, the metal ion-mediated DNA nanoballs comprise Y-NS@Cu 2+ and Y-NS@Ag + , the Y-NS@Cu 2+ contains complementary strands of mucin 1 aptamer, and the Y-NS@Ag + contains complementary strands of programmed cell death-ligand 1 aptamer.

[0008] As an optional embodiment, the Y-NS@Ag + is divided into Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag + , the Y 1-3 -NS@Ag + contains complementary strands of the first half of programmed cell death-ligand 1 aptamer, and the Y 4-6 -NS@Ag + contains complementary strands of the second half of programmed cell death-ligand 1 aptamer.

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

[0010] As an optional embodiment, when circulating tumor cells do not exist, mucin 1 and programmed cell death-ligand 1 on the surface of the circulating tumor cells are not combined with corresponding specific aptamers, respectively, and the integrity of the Y-NS@Cu 2+ , Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag +The structure of the metal ion is destroyed by the aptamer, and a large amount of free Cu 2+ and Ag + .

[0011] As an optional implementation, the signal reporter is two of calcein, CdTe QDs, carbon dots, N-methyl porphyrin dipropionic acid IX or CdSe QDs.

[0012] As an optional implementation, when the signal reporter is calcein and CdTe QDs, the calcein selectively recognizes Cu 2+ and quenches the fluorescence signal of calcein, and the CdTe QDs undergoes a cation exchange reaction with Ag + to quench the fluorescence signal of CdTe QDs.

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

[0014] As an optional implementation, the mucin 1 and the cell programmed death-ligand 1 can be replaced by two of mucin 1, cell programmed death-ligand 1, cell exosome and bacteria with aptamer disease markers.

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

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

[0017] The metal ion-mediated DNA functional nanosphere proposed in the analysis method can be synthesized in advance, and the steps are simple and fast; the aptamer of the marker is used as an initiator to trigger the conformation change of the DNA nanosphere, release a large amount of Cu 2+ and Ag + , and is respectively recognized by a fluorescence signal molecule, establishing a simple, low-cost, high-sensitivity, rapid and homogeneous double-marker fluorescence analysis strategy; the "DNOP@One@X" analysis model proposed in the application has universality, and can be applied to the detection of double markers of other diseases by encoding DNA sequences and replacing signal molecules; a variety of signal output means can be combined, such as directly detecting the concentration of released ions by an electrochemical workstation, using other specific recognition metal ion dyes or fluorescent molecules, other portable analyzers, etc., which can be applied to various medical scenes and help popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any 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 The analytical performance of mucin 1, PD-L1 and A549 cells. DETAILED DESCRIPTION

[0023] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within 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 Y-shaped DNA nanospheres (Y-NS). 2+ Y-NS@Cu is formed by binding to DNA through coordination 2+ .Y-NS@Ag + The formation of also follows similar conditions, except that, using Ag + It has a high affinity for cytosine (C) bases and uses multiple C bases to replace the palindromic sequence in the DNA single strand, making clever use of C-Ag + -C structure implementation of Y-NS@Ag + Self-assembly synthesis of Figure 1 II).

[0026] As a proof of concept, the liquid biopsy marker circulating tumor cells (CTCs) with extremely low content in peripheral blood were chosen to verify the applicability of the analysis model by detecting two proteins expressed on the membrane surface of CTCs: mucin 1 and programmed cell death-ligand 1 (PD-L1). Y-NS@Cu 2+ The Ya strand of Y-NS@Cu + was designed as the complementary strand of the mucin 1 aptamer for targeting mucin 1. The PD-L1 aptamer was too long to be beneficial to the stability of the nanosphere structure, so Y-NS@Ag 1-3 was divided into Y + -NS@Ag 4-6 and Y + -NS@Ag 1-3 Two nanomaterials (Y + -NS@Ag 4-6 and Y 4-6 -NS@Ag + The strands correspond to the first half and the second half of the aptamer, respectively). When CTCs exist, mucin 1 and PD-L1 on the surface of CTCs bind to the corresponding specific aptamer, respectively, and the integrity of the nanosphere is maintained. In the absence of CTCs, the two aptamers respectively pair with the Ya, Y1 and Y4 strands by base complementation, causing the nanosphere structure to change and releasing a large amount of free Cu 2+ and Ag + . The whole reaction process can be completed in one step in 75 minutes (Process 1).

[0027] Subsequently, Calcein and cadmium telluride quantum dots (CdTe QDs) were chosen 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 certain conditions, Calcein is first added to selectively recognize Cu 2+ and quench the fluorescence signal of Calcein, and then CdTe QDs are added to undergo a cation exchange reaction (CER) with Ag + 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 two proteins and the accurate quantification of CTCs are achieved. It is worth mentioning that by changing the base sequence of the corresponding DNA nanosphere, this sensing system can be easily applied to the analysis of other biomarkers and disease diagnosis. The released metal ions can also be detected using other ion detection modes, such as direct electrochemical detection, in addition to fluorescence signals, to be suitable for a variety of application scenarios.

[0029] The technical solutions of the application are verified in detail below in combination with examples.

[0030] Example 1: Steps of co-analysis of mucin 1 and PD-L1 proteins and steps of A549 cell analysis

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

[0032] ① Y-NS@Cu 2+ : 30 μL of 50 μM Ya, Yb, Yc DNA strands, 30 μL of 60 μM Cu(NO3)2 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-NS@Cu 2+ nanospheres;

[0033] ② Y 1-3 -NS@Ag + : 30 μL of 50 μM Y1, Y2, Y3 DNA strands, 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] ③ Y 4-6 -NS@Ag + : 30 μL of 50 μM Y4, Y5, Y6 DNA strands, 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. Steps of co-analysis of mucin 1 and PD-L1 proteins

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

[0037] After that, 7 μL 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 1 nM mucin 1 aptamer, 10 μL 100 pM PD-L1 aptamer, 1 mL A549 cells with different concentrations, 10 μL Y-NS@Cu 2+ , 12 μL Y 1-3- NS@Ag + , 12 μL Y 4-6 -NS@Ag + were mixed with 70 μL 10 mM MOPS, and reacted at room temperature on a shaker for 75 min.

[0041] After that, 7 μL 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 Two Metal ion-mediated DNA functional nanomachine

[0044] As shown in Figure 2 A, DNA strands Ya, Yb and Yc with palindromic sequences were co-annealed with Cu 2+ to form Y-NS@Cu 2+ . By coordinating Ag + with C-rich bases at the ends of Y1-Y3 and Y4-Y6, a C-Ag + -C structure-mediated palindromic sequence was achieved, realizing the self-assembly synthesis of Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag + ( 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 for characterization. As shown in Figure 2 C, the AFM image shows that the Y-DNA monomer without palindromic sequence is small in size and uniformly distributed in point shape. Y-NS@Cu 2+ ​showed obvious spherical shape Figure 2 D).

[0046] After adding mucin 1 aptamer, the structure of Y-NS@Cu 2+ was destroyed Figure 2 E). Similarly, Y 4-6 -DNA monomer size without C-base sequence at the end was also small. Y 4-6 -NS@Ag + showed obvious spherical shape. After adding PD-L1 aptamer, the structure of Y 4-6 -NS@Ag + was destroyed Figure 2 F-H). TEM image results also confirmed the above process Figure 2 I-N).

[0047] After that, the synthesis process of the material was verified using agarose gel electrophoresis.

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

[0049] After that, dynamic light scattering (DLS) and Zeta potential measurements were performed Figure 2 Q and R). These measurement results showed that the average diameters of Y-DNA, Y-NS@Cu 2+ and Y-NS@Cu 2+ +mucin 1 aptamer complex were 5.5 nm, 288.3 nm and 62.3 nm, respectively.

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

[0051] After that, similar studies were conducted on Y 4-6 -NS@Ag + , and similar conclusions were drawn that Y 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 identify Ag + Fluorescent signal molecules. Figure 3 As shown in A and 3B, the UV absorption peaks of calcein and QDs were observed respectively. When calcein was mixed with CdTeQDs, the excitation wavelength was adjusted to 486 nm, and a good fluorescence double peak emission spectrum was obtained ( Figure 3 C) TEM results show that CdTe QDs are uniformly distributed in the form of 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 both calcein and QDs, but there are differences in 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 effect of Cu concentration on calcein and QDs. 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 60μM Cu 2+ Forming 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 difference in QDs signal 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, aptamers were used as initiators to recognize and dissociate DNA nanospheres ( Figure 3 I and J). Y-NS@Cu 2+ Binds to the 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 + was dissociated, resulting in a decrease in the QDs signal, while calcein was unaffected ( 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 hours ( Figure 4 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, use agarose gel electrophoresis to verify. Figure 4 As shown in A, lanes 4, 5, and 6 produced a series of low molecular weight products, 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 the degradation of 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 1 ag / mL to 10 fg / mL) were linearly correlated with the corresponding fluorescent molecule signal values ​​( Figure 4 E and F), the detection limits reached 4 ag / mL and 3 ag / mL, respectively. In addition, a panel of interfering substances 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 10 pg / mL was very close to that of the blank control. However, low concentrations of mucin 1 or PD-L1 (10 ag / mL and 10 fg / mL) caused significant signal changes. These results well demonstrate the sensitivity and specificity of this system, demonstrating its excellent dual-marker detection performance.

[0062] In order to further evaluate the analytical performance of “DNOP@One@X” for CTCs, lung cancer A549 cells were introduced as the validation object. There are multiple PD-L1 and mucin 1 expression sites on the cell surface. Figure 4 As shown in 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 ( ​ K and L). The linear equation determined based on QDs (associated with 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 "DNOP@One@X" method's label-free and homogeneous dual-marker simultaneous detection makes 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] The metal ion-mediated DNA nanomaterials pre-synthesized by one-pot method integrate signal amplification and transduction, and make the reaction complete quickly in a short time. Combined with aptamer recognition and signal reporter molecules, the two markers are realized one-pot, homogeneous and sensitive detection. The strategy is applied to the precise analysis of two proteins and CTC cells, which proves the reliability and practicability of the strategy. Based on the adjustability of DNA nanomachine nucleic acid sequence design and the variability of nanosphere synthesis material, for example, other dyes are used as direct reporter molecules instead of metal ions, "DNOP@One@X" will be expected to realize the multiplex ultra-sensitive detection of various disease markers, and even develop to point-of-care testing (POCT).

[0066] The above merely describes a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for simultaneous dual-marker analysis using a metal ion-mediated DNA functional nanomachine switch, characterized in that: The method includes determining whether the overexpressed mucin 1 and programmed cell death-ligand 1 on circulating tumor cells specifically bind to corresponding aptamers, and whether the aptamers destroy the structure of metal ion-mediated DNA nanospheres synthesized in one pot. Then, based on the metal ions released by the metal ion-mediated DNA nanospheres, the fluorescence signal of the signal reporter is effectively changed. The target is quantified based on the fluorescence signal of the signal reporter. The target is mucin 1 and programmed cell death-ligand 1, or circulating tumor cells. The metal ion-mediated DNA nanospheres include a Y-shaped DNA nanosphere structure; The Y-shaped DNA nanosphere structure includes Y-NS@Cu 2+ and Y-NS@Ag + , the Y-NS@Cu 2+ Containing the complementary chain of the mucin 1 aptamer, the Y-NS@Ag + comprising a complementary strand of a programmed cell death-ligand 1 aptamer; The Y-NS@Ag + Divided into Y 1-3 -NS@Ag + and Y 4-6 -NS@Ag + , the Y 1-3 -NS@Ag + Contains the complementary chain of the first half of the programmed cell death-ligand 1 aptamer, the Y 4-6 -NS@Ag + A complementary strand comprising the second half of the programmed cell death-ligand 1 aptamer; The Y-NS@Cu 2+ It is composed of DNA chains Ya, Yb and Yc with palindromic sequences and Cu 2+ Co-annealing formation; The Y 1-3 -NS@Ag + and the Y 4-6 -NS@Ag + By + Coordinate with the terminal C-rich Y1-Y3 and Y4-Y6 to form C-Ag + -C structure-mediated palindromic sequence and self-assembly synthesis; Wherein, C is cytosine; The signal reporters are calcein and CdTe QDs; The calcein and the CdTe QDs specifically recognize Cu 2+ and Ag + , and their fluorescence emission spectra do not overlap with each other. Under specific conditions, calcein is first added for selective recognition of Cu 2+ The fluorescence signal of calcein was quenched, and then CdTe QDs and Ag were added. + A cation exchange reaction occurs to quench the fluorescence signal of QDs.

2. The method for simultaneous dual-marker analysis using a metal ion-mediated DNA functional nanomachine switch according to claim 1, 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.

3. The method for simultaneous dual-marker analysis using a metal ion-mediated DNA functional nanomachine switch according to claim 1, wherein: 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 is destroyed and a large amount of free Cu is released. 2+ and Ag + .

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

5. The method for simultaneous dual-marker analysis using a metal ion-mediated DNA functional nanomachine switch according to claim 1, wherein: 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.

6. The method for simultaneous dual-marker analysis using a metal ion-mediated DNA functional nanomachine switch according to claim 4, 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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