Homogeneous rapid immunoassay method for circulating tumor cells by using metal ion mediated self-assembly functional nucleic acid nano machine

Abstract

The invention provides a homogeneous rapid immunoassay method for circulating tumor cells by using a metal ion mediated self-assembly functional nucleic acid nano-machine, relates to the technical field of biomedical diagnosis, and is used for establishing a simple, rapid, sensitive and high-sensitivity immune sensor system from the aspects of construction of a hypersensitive homogeneous immunosensing system, application of the metal ion functional nucleic acid nano-machine and the like. On the basis of a circulating tumor cell analysis strategy of various surface markers, expensive instruments or reagents are not needed, all materials are prepared in advance, the detection time is within 1 hour, multi-target simultaneous high-sensitivity detection under the room temperature condition is realized, and compared with a clinical imaging and pathology'gold standard 'detection result, the consistency reaches 90% or above. According to the invention, a medical diagnosis system can be enriched, and technical support is provided for rapid, non-invasive and accurate tumor diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical diagnosis technologies, and particularly to a homogeneous rapid immunoassay method for circulating tumor cells using a metal ion-mediated self-assembled functional nucleic acid nanomachine. Background Art

[0002] Circulating tumor cells (CTCs) are cancer cells shed from the primary tumor site and can spread through the circulatory system, leading to the formation of secondary tumors at distant sites. Due to carrying complete tumor cell information and the change in concentration level being parallel to the tumor situation, CTCs have great potential and broad prospects in disease diagnosis, treatment, and prognosis evaluation.

[0003] In the research of biomarkers such as CTCs, many applications take metal ions (especially metal cations) as the core and cover various fields from bioanalysis to medical treatment. For example, the strong coordination of metal ions (such as Cu 2+ , Ag + , Hg 2+ , Cd 2+ , Pd 2+ etc.) with the thiol group, carboxylic acid group, and amine group of cysteine; the imidazole group of histidine; and the phosphate group of pyrophosphate. Metal ions can also bind to biological macromolecules such as proteins and nucleic acids and have great potential in the biomedical field. Among them, functional nucleic acid (FNA), a nucleic acid molecule with a special structure or performing specific biological functions, has the characteristics of being easy to cut and modify, strong programmability, and good biocompatibility. Through design and modification, it can possess various biological functions such as catalysis, recognition, and transmission, and has been widely applied in many fields such as biosensing, bioimaging, biomedicine, nanotechnology, and data storage. By using the self-assembly characteristics of some functional nucleic acids and combining molecular biology and nanotechnology, the constructed nanomaterials, also known as functional nucleic acid nanomachines (FNA nanomachines), not only have the biological activity of nucleic acids but also possess the unique physical and chemical properties of nanomaterials. Previous studies have shown that metal ion-base mismatch secondary structures mediated by Ag + , Hg 2+ (C-Ag + -C, T-Hg 2+Functional nucleic acid nanomachines based on ( -T) have been used to construct a variety of recognition and signal transduction elements. By changing the number of nucleotides and the content of purine or pyrimidine bases therein, etc., a precisely controllable spatial structure is formed, and it is expected to achieve sequence-defined DNA self-assembly at room temperature. Further, when different types of target molecules (antigens) are combined with the above functional nucleic acid nanomachines through specific recognition ligands (antibodies), the reaction equilibrium of the coordination of metal ions with functional nucleic acid bases in the nanomachines can be changed, forcing them to undergo conformational changes and release metal ions. Thus, by detecting the concentration of the corresponding metal ions, different targets can be effectively quantitatively detected, realizing hypersensitive and rapid analysis of multiple markers in liquid biopsy.

[0004] Most biomarker detection technologies rely on the immune affinity of antigen-antibody to identify specific targets (except nucleic acid biomarkers), and can be divided into two types: homogeneous detection and heterogeneous detection. Homogeneous immunoassay means that the label used in the reaction can only produce a signal after binding to the antigen or antibody, and the unbound label present in the reaction system has no effect on the measurement result. Therefore, there is no need to wash and separate it, that is, it has the advantages of separation-free or even label-free, rapid detection and simple sample processing. In the detection of tumor markers, homogeneous immunoassay can be used as a rapid and simple screening method.

[0005] Some researchers have completed the homogeneous analysis of CTCs by designing hairpin nucleic acid structures such as C-Ag + -C to trigger multiple cascade enzyme-free amplifications. However, additional steps such as catalytic hairpin self-assembly will extend the detection time to several hours and increase the system complexity, limiting the popularization and application of the technology. At the same time, the existing CTC detection methods still have the following deficiencies: ① The number of CTCs in peripheral blood is extremely scarce (about several to dozens per milliliter), and the sensitivity needs to reach the single-cell level to achieve accurate analysis; ② The high heterogeneity of CTCs leads to easy missed detection in single-target detection, and multi-target combination is required to improve the reliability of the results; ③ The system highly depends on enzymatic reactions and heterogeneous separation, with a long time-consuming and strict operation requirements, and new rapid technologies that are easy to be clinically popularized and applied are needed. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a method for homogeneous and rapid immunoanalysis of circulating tumor cells using a metal ion-mediated self-assembled functional nucleic acid nanomachine, so as to solve the technical problems that the existing homogeneous immunoassay technology has low sensitivity and is easily interfered by the sample matrix, and thus it is difficult to meet the detection requirements of trace markers (ag / mL or single-cell level).

[0007] In order to achieve the above-mentioned purpose, the present invention provides a metal ion-mediated self-assembled functional nucleic acid nanomachine for a homogeneous rapid immunoassay method of circulating tumor cells, the method is based on the immune recognition reaction between circulating tumor cells with multiple surface markers and metal ion-mediated self-assembled functional nucleic acid nanomachines to regulate the conformation of double-stranded nucleic acids, release corresponding metal ions, and quantify the circulating tumor cells with the multiple surface markers based on the electrochemical signal intensity of the metal ions.

[0008] Optionally, the metal ion-mediated self-assembly functional nucleic acid nanomachine is a C-Ag + -C, T-Hg 2+ -T double-stranded nucleic acid, based on Ag + and Hg 2+ The electrochemical signal intensity can achieve sensitive analysis of the markers at the pg / mL level.

[0009] Optionally, the metal ion-mediated self-assembled functional nucleic acid nanomachine is a four-membered circular DNA molecule mediated by multiple metal ions, and sensitive analysis of the marker to be detected at the fg / mL level is achieved based on the electrochemical signal intensity of the metal ions.

[0010] The homogeneous immune system of the four-membered circular DNA molecular switch mediated by multiple metal ions proposed in the present invention can be pre-synthesized with simple steps, laying the foundation for the rapid identification of subsequent targets; the intermolecular tensile force between the target and the antibody is used to regulate the "C-Ag + -C", "T-Hg 2+ -T" and other nucleic acid conformations, releasing corresponding metal ions, and constructing a simple, rapid, and highly sensitive circulating tumor cell analysis strategy based on multiple surface markers; the detection system proposed in the present invention is universal and can be applied to multi-marker detection of other diseases by replacing corresponding antibodies and DNA sequences; there is no need to use expensive instruments or reagents, all materials are prepared in advance, the detection time is within 1 hour, and multi-target simultaneous high-sensitivity detection under room temperature is achieved; the concentration of released ions is directly detected by the electrochemical workstation, and there is no need to introduce additional signal reporter molecules, which simplifies the operation steps. The electrochemical workstation can be further miniaturized and is suitable for a variety of medical scenarios, which is conducive to promotion and application.

[0011] Optionally, the metal ion-mediated self-assembled functional nucleic acid nanomachine is a self-assembled functionalized three-dimensional nucleic acid spherical nanomachine that can load a large amount of metal ions, and can achieve sensitive analysis of the marker to be tested at the ag / mL level based on the electrochemical signal intensity of the metal ions.

[0012] The three-dimensional nucleic acid spherical nanomachines proposed in the present invention can be pre-synthesized with simple steps, laying the foundation for the rapid identification of subsequent targets; the intermolecular tensile force between the target and the antibody is used to regulate the "C-Ag+ -C”, “T-Hg 2+ -T” and other nucleic acid conformations to release corresponding metal ions, and a simple, rapid, and highly sensitive analysis strategy for circulating tumor cells based on multiple surface markers was constructed; the detection system proposed by the present invention has universality and can be applied to the multi-marker detection of other diseases by replacing the corresponding antibodies and DNA sequences; expensive instruments or reagents are not required, all materials are prepared in advance, the detection time is within 1 hour, and highly sensitive simultaneous multi-target detection under room temperature conditions is achieved; the concentration of the released ions is directly detected by an electrochemical workstation without introducing additional signal reporting molecules, which simplifies the operation steps, and the electrochemical workstation can be further miniaturized and is applicable to various medical scenarios, which helps to promote the application.

[0013] Optionally, the metal ion-mediated self-assembled functional nucleic acid nanomachine is a Y-shaped DNA nanosphere containing C-Ag + -C, T-Hg 2+ -T structure;

[0014] The Y-shaped DNA nanosphere includes three single-stranded DNAs, and the single-stranded DNAs have continuous C bases or T bases and can hybridize with each other to form Y-DNA, and then self-assemble into a DNA nanosphere.

[0015] Optionally, the circulating tumor cell is an A549 cell, and the markers of the circulating tumor cell are programmed death ligand-1 and / or mucin 1.

[0016] Optionally, the electrochemical signal intensity of the metal ion uses a pre-treated indium tin oxide electrode as the working electrode, a saturated Ag / AgCl as the reference electrode, and a platinum wire electrode as the auxiliary electrode, and differential pulse voltammetry is selected for measurement in the range of 0.2 - 0.7V.

[0017] The metal ion-mediated self-assembled functional nucleic acid nanomachine provided by the present invention for the homogeneous rapid immunoassay method of circulating tumor cells has the following technical effects:

[0018] The present invention starts from aspects such as the construction of a hypersensitive homogeneous immunosensing system and the application of metal ion-functional nucleic acid nanomachines, establishes a simple, rapid, and sensitive analysis strategy for circulating tumor cells based on multiple surface markers, does not require expensive instruments or reagents, all materials are prepared in advance, the detection time is within 1 hour, highly sensitive simultaneous multi-target detection under room temperature conditions is achieved, and after comparison with the inspection results of clinical imaging and pathology “gold standards” and other results, the consistency reaches more than 90%. The invention can enrich the medical diagnosis system and provide technical support for fast, non-invasive, and accurate tumor diagnosis. Description of the Drawings

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0020] Figure 1 is a schematic diagram of homogeneous immunodetection of CTCs based on metal ion-functional nucleic acid;

[0021] Figure 2 is the characterization related to the formation and dissociation of functional nucleic acid nanomachines;

[0022] Figure 3 is the analysis performance and clinical sample verification; Detailed implementation manners

[0023] To make the objectives, technical solutions and advantages of the present invention clearer, the following will describe the technical solutions of the present invention in detail. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts belong to the scope protected by the present invention.

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

[0025] Based on metal ion-base coordination functional nucleic acid, the present invention uses a biotin-streptavidin connection system. The complex of the CTCs sample to be detected and its surface marker antibody can be used as a bridge to initiate homogeneous immune reaction. By regulating the C-Ag + -C, T-Hg 2+ -T and other double-stranded nucleic acid conformations through molecular weight change and intermolecular stretching force, the corresponding metal ions are released, and the electrochemical signal intensity is changed. It has been verified that by detecting this signal change, sensitive analysis of biomarkers in the pg / mL level can be achieved.

[0026] For target molecules in the fg / mL level, certain signal amplification means need to be combined. To avoid increasing the complexity of the system, a multi-metal ion-mediated four-way circular DNA molecular switch can be introduced, so that lower concentrations of analytes can achieve the same or even stronger signal response without additional modification or amplification.

[0027] Finally, to further meet the detection requirements of CTCs at ultra-trace levels, this system also integrates pre-synthesized self-assembled functionalized three-dimensional nucleic acid spherical nanomachines that can load a large amount of metal ions, realizing ultra-sensitive dynamic biosensing at the ag / mL level and single-cell, single-molecule levels.

[0028] Finally, combined with the existing rapid CTCs separation method, taking two surface markers, programmed death ligand-1 (PD-L1) and mucin 1 (MUC1) in non-small cell lung cancer as examples, the constructed sensing system and workflow are as Figure 1 shown.

[0029] Example 1 Synthesis of functional nucleic acid nanomachines

[0030] The DNA strands used for synthesizing functional nucleic acid nanomachines are shown in Table 1.

[0031] Table 1. Sequence information of functional nucleic acid nanomachines

[0032]

[0033] Synthesis steps of antibody-conjugated metal ion-functional nucleic acid nanomachines:

[0034] Add 4 μL of C1 (10 μM), HP1 (10 μM), 15 μL of Ag + (200 μM) and 100 μL of 3-morpholinopropanesulfonic acid (MOPS) buffer (100 mM NaNO 3 , pH 7.4) into an EP tube protected from light. At the same time, add 4 μL of C2 (10 μM), HP2 (10 μM), 20 μL of Hg 2+ (200 μM) and 100 μL of MOPS buffer (100 mM NaNO 3 , pH 7.4) into another EP tube, and react for 30 minutes to form quaternary cyclic molecular switches containing C-Ag + -C and T-Hg 2+ -T structures respectively.

[0035] Subsequently, add 10 μL of biotin-labeled PD-L1 and MUC1 antibodies, and 5 μL of streptavidin (1 mg / mL), and react for 30 minutes to couple them with DNA.

[0036] Synthesis steps of metal ion-mediated functionalized self-assembled three-dimensional nucleic acid nanospheres:

[0037] Add 2 μL of Y1, Y2, Y3 (50 μM), 20 μL of Ag + (200 μM) and 200 μL of MOPS buffer (100 mM NaNO 3 , pH 7.4) into an EP tube protected from light. At the same time, add 2 μL of Y4, Y5, Y6 (50 μM), 20 μL of Hg 2+ (200 μM) and 200 μL of MOPS buffer (100 mM NaNO 3, pH 7.4) was added to another EP tube and heated in a 95 °C water bath for 5 minutes, followed by a reaction at room temperature for 3 hours to form Y-shaped DNA nanospheres containing C-Ag + -C, T-Hg 2+ -T structures.

[0038] Example 2 Nanomaterial Characterization and Feasibility Verification

[0039] The DNA nanomachine composed of Y-shaped DNA monomers includes three single-stranded DNAs (ssDNAs), all of which have continuous C or T bases (black parts), and can hybridize with each other in the presence of Ag + or Hg 2+ to form Y-DNA ( Figure 2 A), and then self-assemble into DNA nanospheres.

[0040] Next, atomic force microscopy (AFM) was used to measure the size and morphology of DNA nanospheres under different conditions.

[0041] Y-DNA@Ag + nanospheres are shown in Figure 2 B-2D. In the AFM image, the size of Y-DNA monomers without continuous C sequences is smaller, showing uniform dots, while the DNA nanospheres are clearly spherical; after adding the HP1 strand complementary to Y1, the structure of the DNA nanospheres is disrupted.

[0042] Y-DNA@Hg 2+ nanospheres are shown in Figure 2 E-2G, and similar results are obtained.

[0043] Zeta potential and dynamic light scattering (DLS) analyses were performed on the above three states of DNA nanospheres, and the results are shown in Figure 2 H and 2I. The changes in their average potential and particle size indirectly verified the occurrence of the above process.

[0044] Figure 2 J shows the results of agarose gel electrophoresis. When three ssDNAs are added, Y-shaped DNA monomers and nanospheres are successfully formed (lanes 8 and 9); when HP1 / HP2 are added (lanes 10-12), the bands become lighter and show tailing, and products dissociated from small molecular weight nanospheres are successfully generated, proving that the reaction is successfully triggered.

[0045] Example 3 Electrochemical Analysis Steps for PD-L1, MUC1 Proteins and Cells

[0046] Mix the two groups of functional nucleic acid nanomachines synthesized in Example 1, add 20 μL of PD-L1 / MUC1 proteins or PD-L1 / MUC1-positive cells (such as A549 cells) at different concentrations, and after reacting at room temperature for 30 min, detect the electrochemical signals.

[0047] The electrochemical signal detection was carried out on an electrochemical workstation equipped with a three-electrode system, using a pretreated indium tin oxide (ITO) electrode as the working electrode, a saturated Ag / AgCl as the reference electrode, and a platinum wire electrode as the auxiliary electrode. Differential pulse voltammetry (DPV) was selected for measurement in the range of 0.2 - 0.7 V.

[0048] Example 4 Analysis of performance and clinical verification

[0049] Taking lung cancer CTCs as an example, through simple and rapid separation and enrichment, and detecting the levels of PD-L1 and MUC1 on their surfaces, a standard curve was established to indirectly evaluate the cell concentration ( Figure 3 A).

[0050] The electrochemical signal and the protein concentration showed a good linear relationship in the range of 10 - 10 5 ag / mL ( Figure 3 B and 3C), with LODs of 3 ag / mL and 4 ag / mL respectively; and showed a good linear relationship with the A549 cell concentration in the range of 10 - 10 4 cells / mL ( Figure 3 D and 3E), with an LOD of 2 cells / mL.

[0051] Apply it to the detection of 30 clinical samples (10 healthy volunteers and 20 patients with non-small cell lung cancer diagnosed by intraoperative pathology), and the results are as Figure 3 F and Figure 3 G shows that there are significant differences in the detection results of PD-L1 and MUC1-positive CTCs between the two groups of patients.

[0052] The results of the receiver operating characteristic (ROC) curve analysis ( Figure 3 H and Figure 3 I) show that the areas under the curve (AUC) reach 0.93 and 0.95 respectively, proving that the present invention has good diagnostic efficacy in this cohort. By collecting the preoperative imaging examination and intraoperative pathological examination results of some patients ( Figure 3 J and Figure 3 K), it can be seen that the results are in good agreement with the CTCs levels obtained by testing, further verifying the reliability of this method.

[0053] Example 5 Conclusion

[0054] The present invention utilizes a pre-synthesized functional nucleic acid nanomachine with a metal ion-base mismatch secondary structure as the core to improve the homogeneous immunosensing system and develop a new method for detecting CTCs that is simple, rapid, accurate, and highly accessible. The detection can be completed within only 1 hour at room temperature. Through preliminary clinical verification, accurate analysis of CTCs in lung cancer patients can be achieved, and its prominent advantages can lay a foundation for future translational applications.

[0055] In addition, by changing the sequence design of the DNA self-assembly machine and the types of conjugated antibodies, the present invention will find wider applications in various medical scenarios, providing reliable technical support for disease diagnosis and treatment.

[0056] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A metal ion-mediated self-assembled functional nucleic acid nanomachine for a homogeneous rapid immunoassay method for circulating tumor cells, characterized in that: The method is based on the immune recognition reaction between circulating tumor cells with multiple surface markers and self-assembled functional nucleic acid nanomachines mediated by metal ions, regulates the self-assembled functional nucleic acid nanomachines mediated by metal ions, releases corresponding metal ions, and quantifies the circulating tumor cells with multiple surface markers based on the electrochemical signal intensity of the metal ions.

2. The method for homogeneous rapid immunoassay of circulating tumor cells using a metal ion-mediated self-assembled functional nucleic acid nanomachine according to claim 1, characterized in that: The metal ion-mediated self-assembly functional nucleic acid nanomachine comprises C-Ag + -C, T-Hg 2+ -T double-stranded nucleic acid, based on Ag + and Hg 2+ The electrochemical signal intensity can achieve sensitive analysis of the markers at the pg / mL level.

3. The method for homogeneous rapid immunoassay of circulating tumor cells using metal ion-mediated self-assembled functional nucleic acid nanomachines according to claim 1, characterized in that: The metal ion-mediated self-assembly functional nucleic acid nanomachine is a four-membered circular DNA molecule mediated by multiple metal ions, and can achieve sensitive analysis of the marker to be tested at the fg / mL level based on the electrochemical signal intensity of the metal ions.

4. The method for homogeneous rapid immunoassay of circulating tumor cells using metal ion-mediated self-assembled functional nucleic acid nanomachines according to claim 1, characterized in that: The metal ion-mediated self-assembled functional nucleic acid nanomachine is a self-assembled functionalized three-dimensional nucleic acid spherical nanomachine that can load a large amount of metal ions, and can achieve sensitive analysis of the marker to be tested at the ag / mL level based on the electrochemical signal intensity of the metal ions.

5. The method for homogeneous rapid immunoassay of circulating tumor cells using metal ion-mediated self-assembled functional nucleic acid nanomachines according to claim 4, characterized in that: The metal ion-mediated self-assembly functional nucleic acid nanomachine comprises C-Ag + -C, T-Hg 2+ -T structured Y-shaped DNA nanoball; the Y-shaped DNA nanoball comprises three single-stranded DNAs, the single-stranded DNAs have continuous C bases or T bases, and can hybridize with each other to form Y-DNA monomers, and then self-assemble to form DNA nanoballs.

6. The method for homogeneous rapid immunoassay of circulating tumor cells using metal ion-mediated self-assembled functional nucleic acid nanomachines according to claim 1, characterized in that: The circulating tumor cells are A549 cells, and the markers of the circulating tumor cells are programmed death ligand-1 and / or mucin 1.

7. The method for homogeneous rapid immunoassay of circulating tumor cells using metal ion-mediated self-assembled functional nucleic acid nanomachines according to claim 1, characterized in that: The electrochemical signal intensity of the metal ions is proportional to the concentration of the cells to be measured. The electrochemical workstation used uses a pretreated indium tin oxide electrode as a working electrode, a saturated Ag / AgCl electrode as a reference electrode and a platinum wire electrode as an auxiliary electrode, and selects differential pulse voltammetry to perform measurements in the range of 0.2-0.7V.

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