Disease marker fluorescence instant detection method based on protein scaffold DNA-Cu2 + functional nanospheres

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a disease marker fluorescence instant detection method based on protein scaffold DNA-Cu2 + functional nanospheres, which comprises the following steps: combining an insulin-like growth factor 1 (IGF-1) aptamer or cortisol aptamer, protein-DNA and DNA-metal ions; the protein scaffold DNA-Cu < 2 + > multifunctional integrated nanospheres are synthesized by a one-step method at room temperature. Based on the function integration of the nanosphere, one-step detection of IGF-1 or cortisol is realized, a plurality of analysis modes including fluorescence detection and smartphone-RGB are provided by combining a test strip and smartphone red, green and blue (RGB) analysis technologies, and a rapid and sensitive disease marker instant detection method is constructed. Finally, rapid and convenient detection of multiple markers in hundreds of clinical samples of multiple diseases is realized, and a new solution is provided for paper-based fluorescence POCT.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a method based on a protein scaffold DNA-Cu 2+ Functional nanospheres for real-time fluorescence detection of disease markers. Background Art

[0002] In recent years, a large number of biomarkers for disease diagnosis and treatment have been discovered and studied. Detecting different types of biomarkers can obtain comprehensive information about the disease from different levels, helping clinicians to make accurate clinical assessments and develop personalized treatment plans. At present, a variety of methods such as polymerase chain reaction, colorimetry and immunoelectrochemiluminescence have been developed for clinical detection. The versatility and sensitivity of polymerase chain reaction make it widely used in the field of nucleic acid detection, especially in the diagnosis and treatment of new crown and other infectious diseases. However, the dependence of polymerase chain reaction on precision instruments and equipment and specific operating environments limits its wide application in resource-limited areas. In addition, various immunoassay methods based on antigen-antibody in clinical practice can detect a variety of biomarkers from hormones to cells. However, the high cost and instability of antibody preparation limit its applicability in the detection of low immunogenic biomarkers and resource-poor areas. Therefore, in the field of biosensing, how to accurately detect disease biomarkers in a sensitive, direct, universal and easily accessible way to accelerate the development of precision medicine is a challenging task.

[0003] The current detection process usually involves multi-step reactions, which limits the simplicity and practicality of the method. The development of a one-step reaction strategy can reduce the complexity of the operation, shorten the detection time, reduce the dependence on professionals, and improve accessibility. According to the clinical demand for rapid and accurate detection, the one-step method has great application potential in integrating various detection instruments and analytical equipment in different application scenarios. For example, integrating a one-step reaction on a paper substrate can enable the sensing strategy to have multiple advantages, such as portability, user-friendliness, and easy handling, which is expected to promote the development of point-of-care testing (POCT). Due to the limited sensitivity of some colorimetric paper-based POCT, it cannot meet the needs of different types of biomarker detection in clinical practice. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a protein scaffold DNA-Cu 2+ Functional nanospheres for real-time fluorescence detection of disease markers.

[0005] The detection method of the present invention is a universal fluorescence instant detection strategy (named "Ⅰ@Ⅰ@X"), which can synthesize protein scaffold DNA-Cu in one step at room temperature. 2+Functional nanospheres can sense disease biomarkers in one step and provide multiple detection modes including red, green and blue (RGB) on smartphones. By using streptavidin (SA) to regulate the spatial distribution of biotinylated DNA hairpins, a cross-shaped protein scaffold DNA tetramer is formed. The aptamer is used as a target recognition element and trigger chain to initiate a bidirectional cross-linking hybridization chain reaction (bi-cHCR) between the four protein scaffold tetramers. Then, Cu 2+ Self-assembly of protein scaffold DNA-Cu by interaction with DNA base pairs 2+ Nanospheres. The composite nanospheres integrate the functions of target recognition, signal amplification and conduction. From target recognition and binding, nanospheres depolymerize to Cu 2+ Release, one-step reaction can be completed within 45 minutes. Further using quantum dots (QDs) with excellent optical properties as signal molecules, combined with test strips and smartphone-RGB analysis technology, a paper-based fluorescence instant detection method was constructed. Ultimately, rapid and convenient detection of multiple markers such as insulin-like growth factor 1 (IGF-1) in hundreds of clinical samples of multiple diseases was achieved, providing a new solution for paper-based fluorescence POCT.

[0006] The technical solution adopted by the present invention is:

[0007] A protein scaffold based DNA-Cu 2+ The method for real-time fluorescence detection of disease markers of functional nanospheres comprises the following steps:

[0008] (1) Biotinylated DNA hairpin binds to SA to form a cross-shaped protein scaffold-DNA tetramer;

[0009] Add IGF-1 aptamer or cortisol aptamer, Cu 2+ and buffer, and after mixed reaction, DNA functionalized nanospheres are obtained;

[0010] (2) Add IGF-1 or cortisol to the DNA functionalized nanospheres obtained in step (1), react at room temperature, and add the recognition Cu 2+ The fluorescent probe is used to detect fluorescence immediately after the reaction.

[0011] In step (1), when the IGF-1 aptamer is added to the obtained DNA functionalized nanosphere, the biotinylated DNA hairpin includes biotin-H1, biotin-H2, biotin-H3 and biotin-H4;

[0012] The nucleotide sequences of the four hairpins H1, H2, H3, and H4 in the biotinylated DNA hairpin are shown as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively;

[0013] The nucleotide sequence of the IGF-1 aptamer is shown in SEQ ID No.9.

[0014] In step (1), when the cortisol aptamer is added to the obtained DNA functionalized nanosphere, the biotinylated DNA hairpin includes biotin-H5, biotin-H6, biotin-H7 and biotin-H8.

[0015] In step (1), the nucleotide sequences of the four hairpins H5, H6, H7, and H8 in the biotinylated DNA hairpin are shown as SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively;

[0016] The nucleotide sequence of the cortisol aptamer is shown in SEQ ID No.10.

[0017] In step (1), the buffer is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.

[0018] In step (1), the mixing reaction is carried out at 20-28° C. for 2-6 hours.

[0019] In step (2), the reaction time at room temperature is 30-60 minutes.

[0020] In step (2), the identification Cu 2+ The fluorescent probes are QDs, calcein and Cu 2+ -Any of the probes.

[0021] Based on previous research work, the inventors of this application prepared a protein scaffold DNA metal mineralized nanosphere inspired by functional nucleic acids, confinement theory and metal-DNA coordination effects, which can realize the multifunctional integration of target recognition, signal transduction and amplification. Based on this functional composite nanosphere, we combined with the independently designed test strips to develop a fast and universal paper-based fluorescence sensing strategy that is easy to operate. The corresponding biotin-coupled DNA hairpin is designed according to the aptamer sequence. SA binds to biotin with high affinity, and the DNA hairpin is anchored to SA to form a cross-shaped protein scaffold-DNA tetramer. In addition, the aptamer acts as a trigger chain to trigger bi-cHCR between DNA tetramers. In Cu 2+ Self-assembly of protein scaffold DNA-Cu under interaction with DNA base pairs 2+Nanospheres. The inventors of this application have further found that after adding the target to be tested, the target induces the conformational change of the aptamer in the nanosphere, and the aptamer folds into a specific secondary structure and binds to the target with high affinity. As the target concentration increases, a large number of aptamers in the nanosphere bind to the target, causing the nanosphere to gradually disaggregate and release a large amount of Cu 2+ The released Cu 2+ Can be recognized by various fluorescent probes, including QDs, calcein, and Cu 2+ -probes, etc. QDs were selected as signal molecules for subsequent research because they have excellent optical properties and are suitable for the development of fluorescent instant detection platforms. In order to visualize the test results, we used inkjet printing technology to make test strips. 10μL of reaction solution was dropped on the circular detection area with a diameter of 1cm on the test strip, and the color change corresponding to the concentration of the target could be observed by the naked eye under ultraviolet light. In addition, taking pictures with a smartphone and converting the colors into RGB values ​​can be performed for more accurate quantitative analysis.

[0022] It should be noted that the protein scaffold DNA-Cu 2+ The nanospheres can be replaced with other nucleic acid structures.

[0023] The RGB analysis mode can also be replaced by other color digital conversion technologies such as HSV.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a protein scaffold DNA-Cu 2+ The functional nanospheres are used for real-time detection of disease markers by fluorescence. First, the aptamers, protein scaffolds, hybridization chain reactions, and DNA and Cu are integrated to detect disease markers such as IGF-1 or cortisol. 2+ Interaction, construction of protein scaffold DNA-Cu 2+ Composite nanospheres; specifically, by using SA to regulate the spatial distribution of biotinylated DNA hairpins, a cross-shaped protein scaffold DNA tetramer was formed. The aptamer was used as a target recognition element and trigger chain to initiate bi-cHCR between the four protein scaffold tetramers; then, Cu 2+ Self-assembly of protein scaffold DNA-Cu by interaction with DNA base pairs 2+ Then, the DNA-Cu 2+IGF-1 or cortisol is added to the composite nanospheres. After the reaction, CdTe QDs with excellent optical properties are selected as signal molecules. Combined with test strips and smartphone-RGB analysis technology, multiple analysis modes including fluorescence detection and smartphone-RGB are provided to construct a rapid and sensitive disease marker instant detection method. The composite nanospheres integrate target recognition, signal amplification and conduction functions. From target recognition binding, nanosphere depolymerization to Cu 2+ Release, one-step reaction can be completed within 45 minutes. In addition, by collecting hundreds of peripheral blood samples from patients with sarcopenia, Cushing's syndrome and healthy donors, the clinical practicality of the detection method of the present invention was verified. It only takes 1 hour from sample collection to data acquisition, and it has good consistency with the corresponding clinical indicators and diagnostic results. In summary, the rapid one-step universal fluorescence instant detection strategy of the present invention will provide a valuable option for bedside detection of disease markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 The invention shows that the protein scaffold DNA-Cu 2+ Schematic diagram of the rapid one-step universal detection strategy of disease markers using functional nanospheres;

[0028] Figure 2 The feasibility and analytical performance of IGF-1 detection are shown; (a) is a schematic diagram of the process of IGF-1 fluorescence instant detection; (b) is a DNA agarose gel electrophoresis diagram under different conditions; (c) is a fluorescence change diagram under different conditions; (d)-(e) are molecular fluorescence change trend diagrams and analytical linear ranges dependent on IGF-1 concentration; (f) is a schematic diagram of result visualization and smartphone-RGB analysis; (g) is the linear range in smartphone-RGB analysis mode; (h) is the selectivity and anti-interference verification of the detection strategy;

[0029] Figure 3Shows the fluorescence detection of clinical blood samples of sarcopenia patients and the analysis of the results; wherein, (a) is a schematic diagram of the clinical sample detection process; (b) is the fluorescence detection results of 63 clinical samples; (c)-(d) are the comparisons of IGF-1 detection results between the healthy control group and the disease group, and between different stages of the disease; (e) is the operating characteristic curve (ROC) analysis of the healthy control group and the disease group; (f) is the operating characteristic curve (ROC) analysis of sarcopenia and severe sarcopenia; (g)-(i) are the correlation analysis between the clinical indicators used to diagnose sarcopenia and the IGF-1 detection results; (j) is the IGF-1 detection results under the development and changes of sarcopenia;

[0030] Figure 4 The performance of cortisol analysis and fluorescence detection of blood samples from patients with Cushing's syndrome and the analysis of the results are shown; wherein (a) is a schematic diagram of cortisol detection in clinical samples; (b)-(d) are molecular fluorescence change trend diagrams and analysis linear ranges dependent on cortisol concentration; (e) is a visualization of cortisol detection results and a flowchart of smartphone-RGB analysis; (f) is the linear range in smartphone-RGB analysis mode; (g) is the selectivity and anti-interference verification of the cortisol detection strategy; (h) is the fluorescence detection results of cortisol in 34 clinical samples; (i) ROC curve analysis; (j) is the consistency evaluation between the method of the present invention and the clinical cortisol detection results;

[0031] Figure 5 A strip-smartphone RGB test and result analysis showing blood samples from patients with Cushing's syndrome; (a) is a strip-smartphone visualization of cortisol levels in serum samples; (b) is a smartphone-RGB quantitative analysis. DETAILED DESCRIPTION

[0032] 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.

[0033] The protein scaffold DNA-Cu 2+ Schematic diagram of a rapid one-step universal detection strategy for disease biomarkers using functional nanospheres Figure 1 First, by integrating aptamers of multiple disease markers such as IGF-1 and cortisol, streptavidin-biotin-coupled DNA hairpins and Cu 2+ , a one-step synthesis of protein scaffold DNA-Cu at room temperature 2+Functional nanospheres. The corresponding biotin-coupled DNA hairpins were designed according to the aptamer sequence (Table 1). SA binds to biotin with high affinity, and the DNA hairpin is anchored on SA to form a cross-shaped protein scaffold-DNA tetramer. The aptamer is used as a trigger chain to trigger a bidirectional cross-linking hybridization chain reaction between multiple DNA tetramers. 2+ Self-assembly of protein scaffold DNA-Cu under coordination with DNA base pairs 2+ Nanospheres.

[0034] After adding the target to be tested, the target induces conformational changes in the aptamer in the nanosphere. The aptamer folds into a specific secondary structure and binds to the target with high affinity. As the target concentration increases, a large number of aptamers in the nanosphere bind to the target, causing the nanosphere to gradually disaggregate and release a large amount of Cu 2+ The released Cu 2+ It can be recognized by various fluorescent probes, including QDs, calcein, and organic dyes. QDs have excellent optical properties and are suitable for the development of fluorescent instant detection platforms, so they were selected as signal molecules for subsequent research. In order to visualize the test results, we used inkjet printing technology to make test strips. 10μL of reaction solution was dropped on the circular detection area with a diameter of 1cm on the test strip, and the color change corresponding to the concentration of the target can be observed by the naked eye under ultraviolet light. In addition, taking pictures with a smartphone can convert the colors into RGB values ​​for more accurate quantitative analysis.

[0035] Materials and Reagents

[0036] Oligonucleotides with different sequences were synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China), and the sequences are shown in Table 1. Copper sulfate pentahydrate (CuSO4·5H2O), sodium tellurite (Na2TeO3), and 3-mercaptopropionic acid (MPA) were ordered from Aladdin Reagent Co., Ltd. (Shanghai, China). Sodium chloride (NaCl), trisodium citrate dihydrate (Na3C6H5O7·2H2O), cadmium chloride (CdCl2), potassium borohydride (KBH4), hydrochloric acid (HCl), sodium hydroxide (NaOH), and magnesium chloride (MgCl2) were purchased from Cologne Chemical Reagent Co., Ltd. (Chengdu, China). Recombinant human programmed cell death ligand 1 (PD-L1) protein, streptavidin (SA), cortisol, and insulin-like growth factor-1 (IGF-1) were purchased from Shanghai Sangon Biotechnology Co., Ltd. Human serum albumin (HSA), trypsin, glucose oxidase (GOD), immunoglobulin G (IgG), pepsin, prostate-specific antigen (PSA), transferrin, interferon-γ (IFN-γ), papain, glypican 3 (GPC3), lysozyme, thrombin, and human immunodeficiency virus (HIV) p24 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human mucin 1 (MUC1) protein was ordered from Sino Biological (Beijing, China). DNA Marker (50-1031 bp) and 4SGelred were purchased from BBI Co., Ltd. (Shanghai, China). 3-(N-morpholino)propanesulfonic acid (MOPS) was purchased from Solarbio Technology Co., Ltd. (Beijing, China). All working solutions were prepared with MOPS buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4). Phosphate buffered saline (PBS) was ordered from Corning (New York, USA). All reagents used in the present invention are of analytical grade or higher and do not require further purification when used. All experimental water uses a water purification system from Chengdu Ultrapure Technology Co., Ltd. (Chengdu, China) with a resistivity of 18.25 MΩ·cm. Serum samples from patients with sarcopenia (Chengdu, China, approval number: 2023836), patients with Cushing's syndrome (Chengdu, China, approval number: 20221833), and healthy people were donated by West China Hospital of Sichuan University. All solutions were stored in a refrigerator at 4°C before use.

[0037] Table 1 - Oligonucleotide sequences of aptamers and DNA hairpins

[0038]

[0039]

[0040] Synthesis of CdTe QDs

[0041] The common synthesis process of CdTe QDs has been reported previously. The main operation is as follows: First, prepare a 50mL solution containing CdCl2 (0.5mM) and trisodium citrate (0.2g). Then immediately add mercaptopropionic acid (MPA, 52μL) to the above solution, and adjust the pH of the solution to 10.5 with NaOH. Subsequently, Na2TeO3 (0.1mM) and KBH4 (50mg) were added to the above solution, and refluxed in batches to obtain CdTe QDs. Subsequently, high-purity CdTe QDs were obtained by precipitation with n-propanol and centrifugation (11000rpm). The purified red CdTe QDs were redispersed in ultrapure water before use.

[0042] Pretreatment of clinical samples

[0043] Peripheral blood samples were collected from patients with sarcopenia (approval number: 2023836), Cushing syndrome (approval number: 20221833) and healthy donors at West China Hospital, Sichuan University, Chengdu, China. Serum was separated by centrifugation at room temperature (3000 rpm, 10 min) and stored at -80°C until use. Samples were diluted 10,000 times with ultrapure water before testing for subsequent analysis.

[0044] Example 1

[0045] This embodiment provides a protein scaffold DNA-Cu 2+ The IGF-1 fluorescence instant detection method of functionalized nanospheres is as follows:

[0046] 10 μM IGF-1 aptamer (5 μL), 10 μM streptavidin-biotin-H1 to H4 (5 μL) and 50 μM Cu 2+ (10 μL) was mixed with 70 μL 3-(N-morpholine) propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4) and incubated at room temperature for 2 hours. Then 40 μL of different concentrations of IGF-1 were added and reacted at room temperature for 45 minutes. Subsequently, 5 μL QDs solution (concentration was 1 / 10 of the original solution) was added and reacted for 1 minute. Fluorescence detection was performed at an excitation wavelength of 365 nm.

[0047] The pattern and size of the test strips were designed using Adobe Illustrator 2021, and the Whatman chromatography paper was prepared into the corresponding test strips using inkjet printing technology, which were sealed in a plastic bag and placed in a dry, dark environment at room temperature. According to the above experimental process, 10 μL of the reaction solution was dropped on the test strip and placed under ultraviolet (UV) irradiation. The color changes corresponding to different marker concentrations were observed with the naked eye. Photos were taken using a smartphone, and the RGB values ​​were analyzed using the colorpicker application (version 0.0.20).

[0048] Example 2

[0049] This embodiment provides a protein scaffold DNA-Cu 2+ The method for real-time detection of cortisol fluorescence using functionalized nanospheres is as follows:

[0050] 10 μM cortisol aptamer (5 μL), 10 μM streptavidin-biotin-H5 to H8 (5 μL) and 55 μM Cu 2+ (10 μL) was mixed with 70 μL 3-(N-morpholine) propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4) and incubated at room temperature for 2 hours. Then 40 μL of cortisol at different concentrations was added and reacted at room temperature for 45 minutes. Subsequently, 4 μL QDs solution (concentration was 1 / 10 of the original solution) was added and reacted for 1 minute. Fluorescence detection was performed at an excitation wavelength of 365 nm.

[0051] The pattern and size of the test strips were designed using Adobe Illustrator 2021, and the chromatographic paper was prepared into the corresponding test strips using inkjet printing technology, which were sealed in a plastic bag and placed in a dry, dark environment at room temperature. According to the above experimental process, 10 μL of the reaction solution was dropped on the test strip and placed under ultraviolet (UV) irradiation. The color changes corresponding to different marker concentrations were observed with the naked eye. Photos were taken using a smartphone, and the RGB values ​​were analyzed using the colorpicker application (version 0.0.20).

[0052] Example 3

[0053] This embodiment provides a protein scaffold DNA-Cu 2+ The IGF-1 fluorescence instant detection method of functionalized nanospheres is as follows:

[0054] 15 μM IGF-1 aptamer (5 μL), 15 μM streptavidin-biotin-H1 to H4 (5 μL) and 40 μM Cu 2+(10 μL) was mixed with 70 μL 3-(N-morpholine) propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4) and incubated at room temperature for 4 hours. Then 40 μL of IGF-1 at different concentrations was added and reacted at room temperature for 30 minutes. Subsequently, 7 μL QDs solution (concentration was 1 / 10 of the original solution) was added and reacted for 1 minute. Fluorescence detection was performed at an excitation wavelength of 365 nm.

[0055] The pattern and size of the test strips were designed using Adobe Illustrator 2021, and the Whatman chromatography paper was prepared into the corresponding test strips using inkjet printing technology, which were sealed in a plastic bag and placed in a dry, dark environment at room temperature. According to the above experimental process, 12 μL of the reaction solution was dropped on the test strip and placed under ultraviolet (UV) irradiation. The color changes corresponding to different marker concentrations were observed with the naked eye. Photos were taken using a smartphone, and the RGB values ​​were analyzed using the colorpicker application (version 0.0.20).

[0056] Example 4

[0057] This embodiment provides a protein scaffold DNA-Cu 2+ The method for real-time detection of cortisol fluorescence using functionalized nanospheres is as follows:

[0058] 20 μM cortisol aptamer (5 μL), 15 μM streptavidin-biotin-H5 to H8 (5 μL) and 65 μM Cu 2+ (10 μL) was mixed with 70 μL 3-(N-morpholine) propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4) and incubated at room temperature for 6 hours. Then 40 μL of cortisol at different concentrations was added and reacted at room temperature for 60 minutes. Subsequently, 10 μL QDs solution (concentration was 1 / 10 of the original solution) was added and reacted for 75 seconds. Fluorescence detection was performed at an excitation wavelength of 365 nm.

[0059] The pattern and size of the test strips were designed using Adobe Illustrator 2021, and the Whatman chromatography paper was prepared into the corresponding test strips using inkjet printing technology, which were sealed in a plastic bag and placed in a dry, dark environment at room temperature. According to the above experimental process, 12 μL of the reaction solution was dropped on the test strip and placed under ultraviolet (UV) irradiation. The color changes corresponding to different marker concentrations were observed with the naked eye. Photos were taken using a smartphone, and the RGB values ​​were analyzed using the colorpicker application (version 0.0.20).

[0060] Experimental example

[0061] 1. IGF-1 detection feasibility and analytical performance evaluation

[0062] The feasibility and analytical performance of this sensing strategy were first evaluated using IGF-1 ( Figure 2 a). Using IGF-1 aptamer as trigger chain, triggering 5'-end biotin-modified DNA hairpin hybridization chain reaction ( Figure 2 b, lanes 2-6). Compared with lane 3, lane 7 showed a band with a higher molecular weight, indicating that DNA tetramers were successfully formed after the addition of SA ( Figure 2 b, lane 3 compared with lane 7). The introduction of SA-biotin greatly improved the amplification efficiency of the chain hybridization reaction ( Figure 2 b, lane 6 compared with lane 10).

[0063] In addition, as the concentration of IGF-1 increased, the low molecular weight bands gradually increased and the high molecular weight bands gradually decreased ( Figure 2 b, lanes 10-12), indicating that IGF-1 binding to the aptamer modulates the protein-scaffold DNA network. 2+ The DNA-Cu complex was complexed by the sulfhydryl reagent in the gel electrophoresis loading buffer. 2+ Feasibility of Nanospheres for Detecting IGF-1 ( Figure 2 c) Aptamers, DNA hairpins (in monomeric or tetrameric form) or conventional hybridization chain reaction products to Cu 2+ Quenching QDs fluorescence has no significant effect ( Figure 2 c, Groups 3-6 compared with Group 2). However, DNA nanospheres could significantly inhibit the 2+ Cation exchange reaction between QDs ( Figure 2 c, comparison between group 6 and group 7). As IGF-1 increases, it induces DNA nanospheres to disassemble and release Cu 2+ , the fluorescence intensity gradually decreased ( Figure 2 c, groups 7–9).

[0064] In the optimized protein scaffold DNA-Cu 2+ After nanosphere preparation and IGF-1 detection conditions, the analytical performance of IGF-1 detection was evaluated. As the concentration of IGF-1 increased, the fluorescence signal gradually weakened. 5 fg / mL concentration range, the fluorescence signal and the logarithmic value of IGF-1 concentration showed a good linear relationship ( Figure 2de). Compared with the most recent IGF-1 detection method, this detection method achieved a detection limit of 5 fg / mL in 46 minutes (based on the three-fold signal-to-noise ratio calculation method). Subsequently, the test strips were prepared according to the pre-designed pattern using inkjet printing technology to further improve the portability and economy of the method. 10 μL of the reaction solution was dropped on the circular detection area of ​​the test strip, and the IGF-1 concentration-dependent color change was observed by naked eye under a UV light box ( Figure 2 f). Use a smartphone to take a photo and convert its color into RGB value to visualize and digitize the test results. The red-to-blue ratio (R / B) and IGF-1 concentration (100-10 5 fg / mL) have a good linear relationship between the logarithmic values ​​( Figure 2 g). Although the test sensitivity is reduced in the test strip-smartphone detection mode, it is more economical, simple and practical. To evaluate the specificity, we tested the effects of 16 potential interfering proteins on the detection system. Compared with the blank solution, low concentrations of IGF-1 (100fg / mL and 10pg / mL) caused significant signal changes, while the high concentration of 16 interfering substances (10ng / mL) was comparable to the blank group, showing excellent selectivity ( Figure 2 h). In general, the good analytical performance of this method laid a foundation for subsequent clinical sample testing.

[0065] 2. Clinical applicability of IGF-1 analysis method

[0066] Clinical peripheral blood samples from healthy elderly people and patients with sarcopenia were collected and tested for IGF-1 ( Figure 3 a). Among the 43 patients, the fluorescence results of most patients were significantly higher than those of the negative control group ( Figure 3 b). This indicates that IGF-1 levels are lower in patients with sarcopenia, which is consistent with previous research results. In addition, IGF-1 levels were significantly different not only between the healthy control group and the disease group (P<0.001), but also between different stages of the disease (P<0.01), which means that IGF-1 plays an important role in screening and monitoring the progression of sarcopenia ( Figure 3 cd). Receiver operating characteristic curve (ROC) analysis showed that IGF-1 had excellent diagnostic performance in distinguishing controls from patients, with an area under the curve (AUC) of 0.894 ( Figure 3 e). However, in terms of distinguishing sarcopenia from severe sarcopenia, the diagnostic performance of IGF-1 was slightly lower, with an AUC of 0.84 ( Figure 3 f). This result needs to be validated in a larger clinical cohort to provide better clinical guidance.

[0067] Subsequently, the clinical indicators used to diagnose sarcopenia were correlated with the IGF-1 test results. Compared with grip strength and gait speed, IGF-1 had the best correlation with skeletal muscle index ( Figure 3 gi). As the disease progresses, the IGF-1 level gradually decreases, and the fluorescence results are negatively correlated and gradually increase ( Figure 3 j). Although the diagnosis of two patients (P9 and P10) changed from 2021 to 2023, their IGF-1 levels remained low, which is consistent with the diagnosis of severe sarcopenia in the second year. This suggests that IGF-1 may be valuable in monitoring disease progression and prognosis. In addition, some clinical samples were analyzed using smartphone RGB mode, which can distinguish sarcopenic patients from healthy people by color changes. More obvious differences were observed when quantitative analysis was performed using the R / B ratio. In summary, the clinical practicality and reliability of this strategy were confirmed by testing samples at different time points and disease stages.

[0068] 3. Analytical performance and clinical applicability of cortisol testing

[0069] Cortisol, as a small molecule representative, was used to evaluate the versatility of the proposed sensing strategy for the detection of different types of disease markers ( Figure 4 a). Under the optimal experimental conditions, the fluorescence intensity of QDs decreased with the increase of cortisol concentration ( Figure 4 bc). There is a good linear relationship between the fluorescence intensity and the logarithmic value of cortisol concentration (10fg / mL-100pg / mL) ( Figure 4 d). Drop the reaction solution on the test strip and place it under a UV light box. As the cortisol concentration increases, the color of the test strip detection area changes from purple to blue. Use a smartphone to take a photo, and the color software converts the color into RGB values ​​( Figure 4 e). There is a clear linear correlation between the R / B ratio and the logarithm of cortisol concentration ( Figure 4 f). When evaluating the specificity of the method, when the cortisol concentration was 100 fg / mL, the fluorescence signal changed significantly, while the high concentration of potential interference (10 ng / mL) did not cause a significant signal change and was close to the blank control ( Figure 4 g), which indicates that the method has good specificity. Compared with the cortisol detection methods in recent years, this method has comparable sensitivity and is simpler and faster.

[0070] The clinical applicability of this method has been preliminarily verified in 34 clinical samples. The fluorescence signal of the Cushing syndrome patient group was significantly lower than that of the healthy control group, and the heat map more directly showed the difference between the groups ( Figure 4 h). In addition, the ROC curve AUC was 0.94, indicating good diagnostic performance ( Figure 4i). In addition, the good correlation with clinical cortisol test results indicates the good clinical applicability and reliability of this method ( Figure 4 j).

[0071] In addition, the potential of this method in practical POCT application was demonstrated by analyzing the cortisol level in serum samples using smartphone RGB mode. The test strip can distinguish patients with Cushing syndrome from healthy people based on the color change ( Figure 5 a). When using R / B for quantitative analysis, the difference is more obvious ( Figure 5 b) Based on the good performance in detecting samples of cortisol-related endocrine diseases, the applicability and reliability of this strategy as a general method were preliminarily verified.

[0072] The detection method of the present invention uses aptamers, protein scaffolds, hybridization chain reactions and DNA and Cu to detect multiple disease markers such as insulin-like growth factor 1 (IGF-1) and cortisol. 2+ Interaction, construction of protein scaffold DNA-Cu 2+ Composite nanospheres. The nanospheres achieve multifunctional integration, thereby completing the construction of a one-step fluorescence sensing system. Combined with test strips and smartphone RGB analysis technology, the test results are visualized and digitized, which can meet the application needs of semi-quantitative and quantitative analysis in different application scenarios. The versatility of this strategy has been verified in two different types of disease markers. In addition, the clinical practicality of this strategy has been confirmed by testing hundreds of clinical samples. It only takes 1 hour from sample collection to obtaining results, and it has good consistency with clinical results.

[0073] 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 DNA-Cu based on protein scaffold 2+ The method for real-time fluorescence detection of disease markers using functional nanospheres is characterized in that: The steps include: (1) Biotinylated DNA hairpin binds to streptavidin to form a cross-shaped protein scaffold-DNA tetramer; Add IGF-1 aptamer or cortisol aptamer, Cu 2+ and buffer, and after mixed reaction, DNA functionalized nanospheres are obtained; (2) Add IGF-1 or cortisol to the DNA functionalized nanospheres obtained in step (1), react at room temperature, and add the recognition Cu 2+ The fluorescent probe is used and fluorescence analysis is performed after the reaction.

2. The fluorescence instant detection method according to claim 1, characterized in that: In step (1), when the IGF-1 aptamer is added to the obtained DNA functionalized nanosphere, the biotinylated DNA hairpin includes biotin-H1, biotin-H2, biotin-H3 and biotin-H4.

3. The fluorescence instant detection method according to claim 2, characterized in that: The nucleotide sequences of the four hairpins H1, H2, H3, and H4 in the biotinylated DNA hairpin are shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively; The nucleotide sequence of the IGF-1 aptamer is shown in SEQ ID No.

9.

4. The fluorescence instant detection method according to claim 1, characterized in that: In step (1), when the cortisol aptamer is added to the obtained DNA functionalized nanosphere, the biotinylated DNA hairpin includes biotin-H5, biotin-H6, biotin-H7 and biotin-H8.

5. The fluorescence instant detection method according to claim 4, characterized in that: In step (1), the nucleotide sequences of the four hairpins H5, H6, H7, and H8 in the biotinylated DNA hairpin are shown as SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively; The nucleotide sequence of the cortisol aptamer is shown in SEQ ID No.

10.

6. The fluorescence instant detection method according to claim 1, characterized in that: In step (1), the buffer is 3-(N-morpholino)propanesulfonic acid buffer.

7. The fluorescence instant detection method according to claim 1, characterized in that: In step (1), the mixing reaction is carried out at 20-28° C. for 2-6 hours.

8. The fluorescence instant detection method according to claim 1, characterized in that: In step (2), the reaction time at room temperature is 30-60 minutes.

9. The fluorescence instant detection method according to claim 1, characterized in that: In step (2), the identification Cu 2+ The fluorescent probes are QDs, calcein and Cu 2+ -Any of the probes.

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