Analytical method based on functional dna nanomachines and selective coordination recognition and application

Abstract

The application provides an analysis method and application based on a functional DNA nanomachine and selective coordination recognition, relates to the technical field of biomedical diagnosis analysis methods, and systematically discusses the interaction between Cu 2+ and DNA, and improves the application mode of the material; self-assembled DNA nanospheres and calcium chlorin which can be selectively complexed and adjusted are used as signal molecules, and a mucin 1 aptamer is used as a recognition probe to establish a simple, accurate, low-cost, high-sensitivity, rapid and homogeneous lung cancer CTC fluorescence analysis strategy, which can simplify the existing tumor diagnosis process and operation, shorten the time, reduce the cost and improve the analysis sensitivity.

Description

Technical Field

[0001] This invention relates to the field of biomedical diagnostic analysis methods, and in particular to an analytical method and its application based on functional DNA nanomachines and selective coordination recognition. Background Technology

[0002] Metal ions are widely distributed in the biological world, and many applications currently revolve around metal ions (especially metal cations), covering fields ranging from bioanalysis to medical treatment. For example, metal ions (Cu...) 2+ Ag + Hg 2+ Cd 2+ Pd 2 + Metal ions exhibit strong coordination interactions with the thiol, carboxylic acid, and amino groups of cysteine ​​(Cys), the imidazole group of histidine (His), and the phosphate ion of pyrophosphate (PPi), leading to the development of various sensing platforms. Metal ions can also bind to biomolecules such as proteins and nucleic acids, showing great potential in the biomedical field. However, DNA strands are particularly unique due to their programmability, addressability, freely adjustable sequence length, and ease of modification. Based on the coordination interactions between metal ions and bases, nanoscale DNA-template metal nanoclusters have been synthesized using DNA templates of different configurations (such as single strands and hairpin structures).

[0003] Representative examples include the directed synthesis of nanomaterials with controllable morphology and different physical and chemical properties using DNA as a template, such as gold and silver nanoclusters (AuNCs, AgNCs), silver nanoparticles (AgNPs), and copper nanoparticles (CuNPs); Hg 2+ Ag + Mediated base pairing, for example: T-Hg 2+ -T、C-Ag + -C; i-motif structures and G-quadruplexes, etc. In addition, Hg 2+ Ag + Mediated base pairing, especially T-Hg 2+ -T、C-Ag + -C has been used to construct recognition elements. Cytosine-rich i-motif structures and guanine-rich G-tetramers have both been used in disease detection and cancer treatment.

[0004] The shortcomings of existing DNA-templated metal nanomaterials include: ① Difficulty in synthesizing existing DNA-templated metal nanoclusters; ② Ag + Needs to be kept away from light, Hg 2+ It has biological toxicity; ③ The stability and conformation of the G-quadruplex are affected by cations present in the solution (such as K+).+ and Na + ) Impact; ④ Contains Cu 2+ DNA template nanomaterials have poor photostability, weak emission intensity, and are difficult to apply.

[0005] Contains Cu 2+ DNA template nanomaterials possess advantages such as low cost, non-toxicity, and high copper abundance. However, they exhibit poor photostability and weak emission intensity. Researchers have attempted to improve their fluorescence stability by incorporating high concentrations of histones and fructose. However, little research has been conducted on Cu... 2+ The study of the interaction forces between Cu and DNA. If Cu can be properly utilized... 2+ Coordination with DNA and combination with luminescent materials will effectively avoid photostability issues, while simplifying and broadening the application of Cu-based technologies. 2+ Applications of nanomaterials.

[0006] Liquid biopsy for tumors is a technique that can replace tissue biopsy, allowing for direct and effective detection through non-invasive sampling, thereby enabling early diagnosis and dynamic monitoring of tumors. Circulating tumor cells (CTCs) have become promising biomarkers in liquid biopsy of malignant tumors due to their ability to carry molecular and biological information about primary and metastatic tumors. However, the number of peripheral blood CTCs is scarce (a few to tens per milliliter), and peripheral blood contains a large number of red blood cells and white blood cells (103). 6 -10 8 With extremely high background noise, the efficient enrichment, separation, and sensitive detection of trace amounts of CTCs from peripheral blood, and the accurate quantification of these CTCs, is a major challenge in clinical practice and a research hotspot in recent years.

[0007] Precise quantification of CTCs mainly involves two core strategies: (1) capture and enrichment, and (2) identification and detection. Capture and enrichment usually involve the physical interaction between CTCs and materials or antibody-antigen interaction, such as immunoaffinity enrichment, immunomagnetic separation, ultrafine membrane filtration, density gradient centrifugation, and new technologies such as microfluidic chips; detection and identification include various methods and technologies, such as fluorescence microscopy, fluorescence spectroscopy, flow cytometry, surface-enhanced Raman scattering, or electrochemical methods. Currently, in clinical practice, enrichment methods such as immunomagnetic beads and microfluidics are often used in combination with fluorescence microscopy and PCR for CTC detection, but these methods are usually cumbersome, time-consuming, and require professional technicians, which limits their widespread application.

[0008] The shortcomings of existing CTC detection methods are as follows: ① The extraction of CTCs uses strategies such as heterogeneous immunomagnetic bead enrichment, which are complex, time-consuming, have low sample throughput, and are costly due to the use of antibodies as recognition probes, posing certain biosafety risks. ② PCR technology requires proteases and is a temperature-dependent process; fluorescence microscopy requires sophisticated instruments and professional operators. ③ Point-of-care testing (POCT) is difficult to achieve. ④ The cost is high, exceeding 2000 RMB per test (taking West China Hospital as an example), and it can only be performed in large hospitals, making it difficult to promote in primary hospitals and for patient self-monitoring.

[0009] Researchers have already designed T-Hg 2+ -T、C-Ag + -C hairpin structure triggered enzyme-free cascade amplification, enabling homogeneous analysis of CTCs. However, as mentioned earlier, Ag... + It needs to be shaded, and Hg 2+ Harmful to the human body. In recent years, the development of DNA nanosphere structures may offer new options. These are obtained through predictable and programmable self-assembly of DNA molecules, possessing multivalent and anisotropic properties. Their multidimensional structure can attach multiple signal molecules, thereby achieving rapid signal amplification. If Cu can be well utilized... 2+ By leveraging the interaction between DNA and enzyme-free nanospheres and aptamers, a novel, rapid, convenient, and highly sensitive precise quantitative detection technology for CTCs is expected to be developed. Summary of the Invention

[0010] In view of this, one of the objectives of this invention is to provide an analytical method based on functional DNA nanomachines and selective coordination recognition, in order to address the lack of existing methods regarding Cu. 2+ The study of the interaction forces between Cu and DNA, if properly utilized... 2+ Coordination with DNA and combination with luminescent materials will effectively avoid photostability issues, while simplifying and broadening the application of Cu-based technologies. 2+ Technical issues related to the application of nanomaterials.

[0011] The second objective of this invention is to provide an application of an analytical method based on functional DNA nanomachines and selective coordination recognition.

[0012] To achieve one of the above objectives, the present invention provides an analytical method based on functional DNA nanomachines and selective coordination recognition, the method comprising bases and Cu based on DNA nanospheres. 2+ Combined to form DNA nanospheres-Cu 2+ Complex, the DNA nanospheres-Cu 2+ Complex and Cu 2+The fluorescence signal of calcein can be effectively altered, and a single target analyte, namely mucin 1 or circulating tumor cells, can be quantified based on the fluorescence signal of calcein.

[0013] Preferably, the DNA nanospheres are formed by the self-assembly of single-stranded DNA carrying palindromic sequences through complementary base pairing.

[0014] Preferably, the single-stranded DNA includes a first sequence, a second sequence, and a third sequence, wherein the palindromic sequence of the first sequence binds to itself, and the palindromic sequences of the second and third sequences both bind to themselves and pair with each other.

[0015] Preferably, the palindromic sequence of the first sequence is longer than the palindromic sequences of the second and third sequences.

[0016] Preferably, Cu 2+ The purine N7 and N1 atoms, the O atom on the ring, the N3 atom and the phosphate oxygen atom of the DNA nanospheres combine to form an intrasphere complex.

[0017] Preferably, when the mucin 1 or the circulating tumor cells are absent, the aptamer pairs with the first sequence, disrupting the conformation of the DNA nanospheres and releasing Cu. 2+ Increases and inhibits the fluorescence signal of calcein.

[0018] Preferably, when the mucin 1 or the circulating tumor cells are present, the aptamer specifically recognizes and binds to them, thus preserving the DNA nanospheres.

[0019] Preferably, the excitation wavelength of the calcein is 486 nm.

[0020] Preferably, the fluorescence signal of the calcein is obtained using a fluorometer.

[0021] To achieve the second objective mentioned above, the present invention provides an application of an analytical method based on functional DNA nanomachines and selective coordination recognition, wherein the application includes applying any of the aforementioned analytical methods based on functional DNA nanomachines and selective coordination recognition to the analysis of circulating tumor cells in lung cancer.

[0022] The analytical method and application based on functional DNA nanomachines and selective coordination recognition provided by this invention have the following technical effects:

[0023] This analytical method and its application, based on functional DNA nanomachines and selective coordination recognition, systematically explores the role of Cu in DNA template metal nanomaterials. 2+The interaction with DNA improves the application of materials. By employing self-assembled DNA nanospheres and selectively complexable calcein as signaling molecules, combined with mucin-1 aptamers as recognition probes, a simple, accurate, low-cost, highly sensitive, rapid, and homogeneous fluorescence analysis strategy for lung cancer CTCs is established. This strategy will simplify existing tumor diagnostic procedures and operations, shorten time, reduce costs, and improve analytical sensitivity. This strategy can enrich the medical diagnostic system, provide more options for medical diagnosis, lay the foundation for POCT analysis of lung cancer CTCs, and provide theoretical guidance for the construction of portable home analysis devices. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic diagram illustrating the extraction and detection principles of CTCs;

[0026] Figure 2 It is Cu 2+ A schematic diagram illustrating the application of coordination with DNA or small molecules in signal modulation of luminescent materials;

[0027] Figure 3 It is Cu 2+ Coordination with DNA or small molecules for signal modulation of luminescent materials;

[0028] Figure 4 The characterization of DNA nanospheres and their feasibility for use in mucin 1 protein analysis;

[0029] Figure 5 This refers to the performance of mucin 1 protein analysis;

[0030] Figure 6 This refers to the performance analysis of A549 lung cancer cells. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0032] The principle of the analytical method of this invention is as follows:

[0033] like Figure 1 As shown, this invention is based on DNA nanospheres combined with Cu 2+ Significant regulation of calcein signaling, using mucin 1 as a marker and its aptamer as a recognition probe, was demonstrated in lung cancer A549 cells, enabling homogeneous, rapid, and portable analysis of CTCs.

[0034] The analytical principle of CTCs consists of two parts: separation of CTCs and homogeneous detection.

[0035] A three-step centrifugation method was used to isolate CTCs. First, an equal volume of phosphate-buffered saline (PBS) was added to 4 mL of collected blood to reduce blood viscosity. Lymphocyte separation medium was added and centrifuged. The second lymphocyte layer and the third separation medium layer were collected and mixed with an equal volume of PBS. The mixture was centrifuged again, the precipitate was retained, and erythrocyte lysis buffer was added to dissolve any remaining erythrocytes. After a third centrifugation, the precipitate in the tube contained the desired free CTCs. Finally, 10% fetal bovine serum (FBS) was added for cell storage.

[0036] Homogeneous portable quantification of CTCs includes using mucin 1, which is highly expressed on the surface of tumor cells, as a marker and its aptamer as a recognition probe, self-assembled DNA nanospheres, and Cu. 2+ Selective complexation recognition of calcein and handheld instrument for detection.

[0037] First, the first (Y1), second (Y2), and third (Y3) sequences of single-stranded DNA (ssDNA) carrying palindromic sequences self-assemble into DNA nanospheres through complementary base pairing.

[0038] It is worth noting that the palindromic sequence of Y1 can bind to itself, while the palindromic sequences of Y2 and Y3 can bind to themselves or pair with each other. The palindromic sequence of Y1 is longer than that of Y2 and Y3, which makes the space of Y1 relatively larger, which is beneficial to the binding of aptamers to Y1 in subsequent experiments.

[0039] Cu 2+ It can combine with the N7 and N1 atoms of purines (A and G) in DNA, the O atom on the ring, the N3 atom of pyrimidines (T and C), and the phosphate oxygen atom to form an intrasphere complex.

[0040] In the absence of mucin 1 or CTCs, the aptamer pairs with Y1, disrupting the conformation of the DNA nanospheres and leading to the release of free Cu. 2+ Increased levels suppress the fluorescence signal of calcein. When mucin 1 or CTCs are present, they can be specifically recognized and bound by aptamers, thus retaining a large number of DNA nanospheres. 2+ The bases of the DNA nanospheres bind to form DNA nanospheres-Cu2+ Complex. Due to Cu 2+ The interaction force with DNA is similar to that of calcein, therefore the fluorescence of calcein cannot be quenched, thus exhibiting Cu... 2+ Selective coordination recognition. Therefore, based on free Cu 2+ and DNA nanospheres-Cu 2+ The significant difference in fluorescence signal caused by the complex enabled highly sensitive homogeneous analysis of mucin 1 and CTCs.

[0041] Regarding signal output, both a traditional fluorometer and a self-developed handheld fluorescence spectrometer were employed, both yielding quantitative results. Particularly for the handheld instrument, simply drop the reaction solution into the circular area of ​​the test strip, place the strip in the detection slot, and measure the fluorescence at the 510nm emission wavelength to obtain a digital fluorescence intensity value for mucin 1 or CTCs analysis. This method is simple, efficient, and enables rapid, portable quantification of CTCs within 3 hours. Furthermore, based on Cu... 2+ This method, which is independent of DNA sequence and involves altering the sequence design of DNA self-assembly machinery, will have wider applications in disease diagnosis.

[0042] The technical solution of the present invention will be specifically verified below with reference to the embodiments.

[0043] 1. Steps for analyzing mucin 1 protein and A549 cells

[0044] 1.1 Synthesis steps of DNA nanospheres

[0045] 30 μL of 50 μM Y1, Y2, and Y3 DNA strands were mixed with 210 μL of 10 mM 3-morpholinopropanesulfonic acid (MOPS) buffer (100 mM NaNO3, pH 7.6), heated at 95 °C for 5 min, and then placed at room temperature for 3 h to form DNA nanospheres.

[0046] 1.2 Analysis steps for mucin 1 protein

[0047] First, 10 μL of 12 μM mucin 1-aptamer, 40 μL of mucin 1 protein at different concentrations, and 40 μL of DNA nanospheres were mixed with 70 μL of 10 mM MOPS and reacted at room temperature for 60 min. Then, 10 μL of 5 μM CuSO4 was added to the above reaction solution, and the reaction was carried out at room temperature for 50 min. Finally, 6 μL of 10 μM calcein was added, and the fluorescence signal was measured after reacting for 2 min.

[0048] 1.3 Analytical Procedures for A549 Cells

[0049] First, 10 μL of 12 μM mucin 1-aptamer, 40 μL of A549 cells at different concentrations, and 40 μL of DNA nanospheres were mixed with 70 μL of 10 mM MOPS and reacted on a shaker at room temperature for 60 min. Then, 10 μL of 5 μM CuSO4 was added to the above reaction solution, and the mixture was reacted on a shaker at room temperature for 50 min. Finally, 6 μL of 10 μM calcein was added, and the fluorescence signal was measured after reacting for 2 min.

[0050] 2.Cu 2+ Coordination between DNA and luminescent materials is used to regulate their signal.

[0051] Due to Cu 2+ It exhibits a coordination relationship with calcein similar to that of DNA, while cadmium telluride quantum dots (CdTe QDs) can interact with Cu. 2+ A chemical reaction occurs; therefore, this invention uses calcein and QDs as representative luminescent materials, and studies the effects of Cu on their respective luminescent properties. 2+ Coordination forces with DNA and biomolecules (PPi and His) modulate the luminescent material ( Figure 2 A).

[0052] Calcein's ultraviolet absorption peak is located at 480 nm. Figure 3 A). When Cu 2+ In its presence, the fluorescence of calcein increases with increasing concentration of double-stranded DNA (dsDNA). Figure 3 B). Similar phenomena (3C and 3D) were also observed in PPi and His. This indicates that Cu 2+ After forming a complex with dsDNA and PPi or His, the remaining Cu 2+ The fluorescence of calcein was quenched, while the coordinated Cu... 2+ It hardly reacts with calcein ( Figure 2 G).

[0053] Therefore, the signaling of calcein can be modulated by controlling the amount of the generated coordination complex. It is worth noting that dsDNA-Cu 2+ The regulation of calcein was more sensitive than that of PPi (5 mM) and His (100 μM). Subsequently, the signaling molecules were replaced with QDs and characterized. Figure 3 E). QDs and Cu 2+ A cation exchange reaction (CER) occurs, resulting in aggregation. Figure 3 F). dsDNA-Cu 2+ The complex cannot modulate the signal of QDs. Figure 3 G), but PPi-Cu 2+ -PPi and His-Cu 2+-His can modulate the fluorescence of QDs ( Figure 3 H and 3I).

[0054] It can be used with more Cu 2+ Coordinated DNA nanospheres to replace dsDNA ( Figure 2 B) It was found that a small number of nanospheres could cause a significant change in calcein signaling, while QDs remained unaffected. Figure 3 J). These experimental results indicate that Cu 2+ The interaction force with DNA is weaker than that between ionic bonds and Cu. 2+ Coordination forces between the molecule and small molecules.

[0055] Then, an attempt was made to assemble a four-armed DNA nanostructure using streptavidin (SA) and four biotin-labeled hairpin DNA probes to form a hydrogel network with high steric hindrance, enabling signal modulation via cross-linked hybridization chain reaction (cHCR). Figure 2 F).

[0056] First, the effects of SA and biotin on Cu were tested. 2+ The impact. For example... Figure 3 As shown in K and 3L, the fluorescence signals of both calcein and QDs increased after the addition of SA, indicating that SA has a positive effect on Cu. 2+ It has a significant adsorption effect, while biotin has almost no interference.

[0057] Subsequently, SA and Cu were carefully adjusted. 2+ The amount of [amount] was used to exclude the influence of SA in the system, and ordinary HCR was compared with cHCR. The results showed that only the successful formation of a network polymer via cHCR could modulate the signaling of calcein and QDs. Figure 3 P), and neither HCR nor the addition of SA or biotin alone could alter the signaling of QDs (P). Figure 2 C, D, E and Figure 3 M,N,O).

[0058] In conclusion, it is believed that DNA and Cu 2+ The synergistic effects between Cu and Cu can be used to modulate luminescent materials based on coordination, and similarly, chemical reaction-based luminescent materials can be modulated by increasing steric hindrance and local concentration, thus expanding the application of Cu in luminescent materials. 2+ New applications of DNA nanomaterials.

[0059] 3. Characterization of nanomaterials and feasibility verification of mucin 1 analysis

[0060] Based on DNA nanospheres combined with Cu 2+Significant modulation of calcein signaling was achieved by utilizing the programmability and self-assembly of DNA nanospheres, with mucin 1 as a marker and its aptamer as a recognition probe for CTC detection. The DNA nanomachines, composed of Y-type DNA monomers, consist of three single-stranded DNAs (ssDNA) (Y1, Y2, and Y3), all possessing palindromic sequences (black portion) and capable of hybridizing to form Y-DNA. Figure 4 A), which then self-assembles to form DNA nanospheres.

[0061] Next, the size and morphology of the DNA nanospheres under different conditions were determined using atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), respectively. Figure 4 As shown in B-4E, in the AFM image, the Y-DNA monomers without palindromic sequences are small in size and appear as uniform dots, while the DNA nanospheres are clearly spherical. The structure of the DNA nanospheres is disrupted upon the addition of nucleic acid aptamers; however, with further addition of mucin 1, the aptamers bind to proteins, and the spherical structure is preserved. (SEM) Figure 4 F-4I) and TEM Figure 4 Consistent results were also obtained for J-4M. Dynamic light scattering (DLS) and Zeta potential results ( Figure 4 The above process was verified by N and 4O. The average diameters of Y-DNA monomer, DNA nanosphere, DNA nanosphere + aptamer and DNA nanosphere + aptamer + mucin 1 were 3.20 nm, 273.10 nm, 48.37 nm and 145.10 nm, respectively.

[0062] And when Cu 2+ After the addition of DNA nanospheres, the nanosphere diameter significantly increased to 2885.42 nm, and the Zeta potential increased, indicating the formation of Cu. 2+ -DNA nanosphere complex.

[0063] Furthermore, agarose gel electrophoresis clearly demonstrated the reaction process between the aptamer and mucin 1 with the DNA nanospheres. Figure 4 P) proves that the system can be used for the analysis of mucin 1. Figure 4 Q shows the electrophoresis results of cHCR. When SA and initiator chain P2 were added (lane 10), a high molecular weight product was successfully generated by cHCR, thus allowing for the regulation of Cu. 2+ Signals from nanomaterials with stronger interactions.

[0064] 4. Performance evaluation of mucin 1 analysis

[0065] The analytical sensitivity of mucin 1 was investigated, such as... Figure 5 As shown, within the protein concentration range of 10 ag / mL to 10 fg / mL, the fluorescence signal of calcein showed a strong linear relationship with the logarithm of the protein concentration. Figure 5 The limit of detection (LOD) calculated based on a signal-to-noise ratio of 3 (A, B, C) is 3 ag / mL.

[0066] To achieve rapid and accurate quantification, the inventors independently developed a handheld fluorescence instrument. Figure 5 D). The detection principle is similar to that of traditional fluorometers, using a light-emitting diode (LED) as the excitation source. The fluorescence of calcein is obtained by adjusting the light source and rapidly reading the value at a specific point within the emission wavelength range (e.g., 510 nm). After the reaction is complete, the solution is dropped into the corresponding circular area of ​​the test paper for detection. The signal of calcein shows a good linear relationship with 10 ag / mL - 10 fg / mL of mucin1. Figure 5 With E and 5F, and a LOD of 4 ag / mL, portable detection was achieved.

[0067] The selectivity of mucin 1 analysis was investigated within this system, such as... Figure 5 As shown in G, the change in calcein fluorescence signal caused by a higher concentration of the potential interfering protein (10 fg / mL) was similar to that of the blank solution and was almost negligible. However, low concentrations and the same concentrations of mucin 1 (0.1 and 10 fg / mL) caused a significant increase in fluorescence signal. Therefore, this system exhibits good target selectivity.

[0068] 5. Performance evaluation of A549 cell analysis

[0069] Inspired by the superior performance of mucin 1 analysis described above, this strategy was extended to the cellular level. Given the high expression of mucin 1 on the surface of lung cancer CTCs, A549 cells were used as a model to indirectly assess cell concentration by detecting mucin 1 on their surface. The fluorescence signal correlated with cell concentration in the range of 1–10. 3 A good linear relationship was observed within the range of cells / mL. Figure 6 (A, B, C), with a LOD of 1 cell / mL. Similarly, the cell concentration standard curve was measured using a handheld fluorometer, with a linear range of 1-100 cells / mL and a LOD of 2 cells / mL, which is comparable to the results obtained using a traditional fluorometer. Figure 6 E and 6F). To assess cell specificity, various mucin 1-negative cells were measured (E and 6F). Figure 6 (G) Negative cells at a high concentration of 100 cells / mL showed a signal comparable to the blank solution. In contrast, the presence of A549 cells resulted in a significant increase in fluorescence signal, indicating that this method has good cell specificity.

[0070] In summary, this strategy offers a simple enrichment process and a precise, sensitive dual-signal output mode. Firstly, a three-step centrifugation pretreatment step achieves complete and gentle enrichment of CTCs. The test strips used only require printing and cutting on chromatographic paper, making them both economical and offering long-term storage. More importantly, the independently developed handheld fluorescence instrument is ideal for on-site detection. Therefore, this strategy can be applied to the detection of CTCs in clinical samples.

[0071] 6. Conclusion

[0072] Based on Cu 2+ The coordination interaction between the luminescent material and DNA modulates the signal of the luminescent material. Through self-assembling DNA machinery and selective complexation recognition with calcein, homogeneous and portable detection of lung cancer CTCs was achieved within 3 hours. For signal output, both a fluorometer and a self-designed handheld fluorescence spectrometer can obtain quantitative results. Therefore, a highly sensitive, accurate, homogeneous, low-cost, and simple fluorescent lung cancer A549 cell analysis strategy has been successfully constructed, and its significant advantages lay the foundation for future clinical translation. In addition, based on Cu... 2+ This method, which is independent of DNA sequence and can be used more widely in disease diagnosis by changing the sequence design of DNA self-assembly machinery, will also enable convenient point-of-care testing (POCT) analysis of diseases using handheld fluorescence spectrometers.

[0073] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An analytical method for non-disease diagnostic purposes based on functional DNA nanomachines and selective coordination recognition, characterized in that, The method includes bases and Cu based on DNA nanospheres. 2+ Combined to form DNA nanospheres-Cu 2+ Complex, the DNA nanospheres-Cu 2+ Complexes and free Cu 2+ The fluorescence signal of calcein can be effectively altered, and a single target analyte, namely mucin 1 or circulating tumor cells, can be quantified based on the fluorescence signal of calcein. The DNA nanospheres are formed by the self-assembly of single-stranded DNA carrying palindromic sequences through complementary base pairing. The single-stranded DNA includes a first sequence, a second sequence, and a third sequence. The palindromic sequence of the first sequence binds to itself, and the palindromic sequences of the second and third sequences both bind to themselves and pair with each other. The palindrome of the first sequence is longer than the palindromes of the second and third sequences. In the absence of mucin 1 or circulating tumor cells, the aptamer pairs with the first sequence, disrupting the conformation of the DNA nanospheres and releasing Cu. 2+ Increases and inhibits the fluorescence signal of calcein; When the mucin 1 or the circulating tumor cells are present, they are specifically recognized and bound by the aptamer, thus preserving the DNA nanospheres.

2. The analytical method for non-disease diagnostic purposes based on functional DNA nanomachines and selective coordination recognition according to claim 1, characterized in that, Cu 2+ The purine N7 and N1 atoms, the O atom on the ring, the N3 atom and the phosphate oxygen atom of the DNA nanospheres combine to form an intrasphere complex.

3. The analytical method for non-disease diagnostic purposes based on functional DNA nanomachines and selective coordination recognition according to claim 1, characterized in that, The excitation wavelength of the calcein is 486 nm.

4. The analytical method for non-disease diagnostic purposes based on functional DNA nanomachines and selective coordination recognition according to claim 1, characterized in that, The fluorescence signal of the calcein was obtained using a fluorometer.

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