Tumor small cell extracellular vesicle electrochemical detection platform based on OR logic gate as well as preparation method and application of tumor small cell extracellular vesicle electrochemical detection platform

Abstract

The invention discloses a tumor small cell extracellular vesicle electrochemical detection platform based on an OR logic gate as well as a preparation method and application thereof. The electrochemical detection platform comprises a DNA probe containing an aptamer, a substrate probe linker, a probe PAP and a gold electrode, the DNA probe comprises three types of aptamers, namely AP1, AP2 and AP3. According to the invention, various aptamers are adopted to specifically recognize different proteins, so that high-sensitivity detection of tumor small extracellular vesicles is realized, and the problems of insufficient signal intensity and difficulty in detection of low-abundance vesicles in a single aptamer recognition mode are solved; on the other hand, compared with a traditional single protein detection scheme, the existence states of multiple target proteins on the surfaces of the tumor small cell extracellular vesicles can be synchronously captured, differential recognition and characterization of different phenotypes of tumor small cell extracellular vesicles are achieved, and more comprehensive molecular marker information is provided for precise diagnosis of tumors.

Description

Technical Field

[0001] This invention relates to the field of tumor small cell extracellular vesicle detection technology, specifically to an electrochemical detection platform for tumor small cell extracellular vesicles based on "OR" logic gates, its preparation method, and its application. Background Technology

[0002] Small extracellular vesicles (sEVs) are double-membrane vesicles, 30-150 nm in diameter, secreted by parental cells. They contain lipids, proteins, nucleic acids, and other substances, carrying the genetic material of the parent cell and playing a crucial role in intercellular communication and physiological and pathological processes. Compared to sEVs from normal cells, sEVs secreted by diseased cells contain more biomarkers, providing strong support for early diagnosis and treatment of diseases.

[0003] Tumor sevus cells (sEVs) not only carry transmembrane proteins such as CD9, CD63, and CD81, but also abundant characteristic proteins, such as carcinoembryonic antigen (CEA), mucin-1 (MUC1), epithelial cell adhesion molecule (EpCAM), and protein tyrosine kinase 7 (PTK7). These characteristic proteins can specifically bind to antibodies and aptamers. Based on this, tumor sEVs can be combined with various detection methods, including fluorescence, electrochemistry, and electrochemiluminescence, providing a highly sensitive and specific detection platform for tumor sEVs. The concentration of tumor sEVs as biomarkers is very low in the early stages of disease. Most existing tumor sEV detection methods detect single proteins, but the signal changes from single proteins are small, which is not conducive to obtaining low detection limits and makes it difficult to detect low concentrations of sEVs. Therefore, there is an urgent need for a method with low detection limits for early disease diagnosis. Summary of the Invention

[0004] Objective of the Invention: To address the aforementioned technical problems, this invention aims to provide an electrochemical detection platform for tumor microcellular extracellular vesicles based on an "OR" logic gate. This platform constructs a "multi-aptamer-multi-protein" recognition system by designing DNA probes containing multiple specific aptamers. The signal from the "OR" logic gate serves as the input signal, and the RuHeX electrochemical signal serves as the output signal. The degree of signal change (ΔI) reflects the content of biomarkers on the surface of tumor microcellular extracellular vesicles. This invention utilizes multiple aptamers to specifically recognize different proteins, achieving highly sensitive detection of tumor microcellular extracellular vesicles and solving the problems of insufficient signal intensity and difficulty in detecting low-abundance vesicles under single-aptamer recognition mode. Furthermore, compared to traditional single-protein detection schemes, this invention can simultaneously capture the presence of multiple target proteins on the surface of tumor microcellular extracellular vesicles, enabling differentiated identification and characterization of tumor microcellular extracellular vesicles with different phenotypes, providing more comprehensive molecular marker information for the precise diagnosis of tumors.

[0005] The present invention also provides a method for preparing and applying an electrochemical detection platform for extracellular vesicles of tumor small cells based on "OR" logic gates.

[0006] Technical Solution: To achieve the above objectives, the present invention provides an electrochemical detection platform for extracellular vesicles of tumor small cells based on an "OR" logic gate. The electrochemical detection platform comprises a DNA probe containing aptamers, a substrate probe linker, a probe PAP, and a gold electrode. The DNA probe includes three aptamers, AP1, AP2, and AP3, which specifically recognize PTK7, CD63, and EpCAM proteins, respectively. All three aptamers, AP1, AP2, and AP3, contain the universal sequence L1, which can complementarily pair with the substrate probe linker to construct a specific recognition and cleavage site for the Nb.BbvCI enzyme. After action by the Nb.BbvCI enzyme, a DNA1 fragment is generated. The PAP probe is pre-modified onto the surface of the gold electrode, which can hybridize with the enzyme-digested product DNA1 to form a double-stranded complex.

[0007] The sequences of the aptamers AP1, AP2, and AP3 are as follows: AP1: 5'-ATC TAA CTG CTG CGC CGCCGG GAA AAT ACT GTA CGG TTA GAA AAA AAA AAA AAT CCT CAG CAG TTA-3', AP2: 5'-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TAA CT AAA AAA AAT CCT CAG CAG TTAGCA TT-3', AP3: 5'-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGCCTG AAAAAAA AT CCT CAG CAG TTA ACC TCT GTA GTG-3'.

[0008] The sequence of the substrate probe linker is 5'-TCA GCA GGG AGG AAG ACA ATA TTA ACTGCT GAG GAT AAA CG-3'.

[0009] The L1 sequence is 5'-AT CCT CAG CAG TTA-3', and the DNA1 sequence formed after Nb.BbvCI enzyme cleavage is 5'-TCA GCA GGG AGG AAG ACA ATA TTA ACT GC-3'.

[0010] The sequence of the probe PAP is 5'-CCT CCC TGC TGA A CAC ACA CAC AAA AAA AAAAAA AAA AAA AAA AAA CAC ACA CAC A GC AGT TAA TAT TG-3'.

[0011] The preparation method of the electrochemical detection platform for extracellular vesicles of tumor small cells based on "OR" logic gates according to the present invention includes the following steps:

[0012] (1) Tumor small cell extracellular vesicles are co-incubated with AP1, AP2 and AP3 to form sEVs-aptamer complexes, while exposing the same sequence L1, which contains restriction enzyme sites.

[0013] (2) Take the sEVs-aptamer complex prepared in step (1) and incubate it with the substrate probe linker to construct the complete Nb.BbvCI enzyme cleavage site; then add Nb.BbvCI enzyme to the system to specifically cleave the enzyme site and release the DNA1 fragment.

[0014] (3) The probe PAP is incubated with the pretreated gold electrode overnight and then immersed in 6-mercaptoethanol (MCH) to block non-specific sites, thus obtaining the modified electrode.

[0015] (4) Incubate the solution obtained in step (2) with the modified electrode obtained in step (3). The detection electrode obtained after incubation can be used as an electrochemical detection platform.

[0016] In step (1), the tumor microcellular extracellular vesicles are microcellular extracellular vesicles secreted by CCRF-CEM cells, MCF-7 cells, HeLa cells or Ramos cells.

[0017] Further, the tumor small extracellular vesicles described in step (1) are obtained by culturing cells in RPMI-1640 medium or DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, collecting the culture medium, and operating according to the instructions of the cell exosome kit to obtain the corresponding sEVs precipitate. The precipitate is then resuspended in sterile PBS buffer (10 mM K2HPO4 and 0.1 M NaCl, pH=7.4) to form an sEVs suspension and stored at -20 ℃ for use.

[0018] The concentration of extracellular vesicles in the tumor small cells was 2.75 × 10⁻⁶. 2 -5.5×10 8 The concentration of particles / mL, the concentration of substrate probe linker is 1.5-3.5 μM, the concentration of Nb.BbvCI enzyme is 0.2-0.7 U / μl, and the concentration of probe PAP is 0.01-2 μM.

[0019] The specific method for pretreating the electrode in step (3) is as follows: first, polish the gold electrode with Al2O3 powder of 0.3 μm and 0.05 μm in sequence, then sonicate it with ethanol and water, immerse the electrode in piranha (concentrated sulfuric acid: H2O2=3:1) after sonication, then scan the cyclic voltammetry curve in 0.5 M sulfuric acid solution to remove oxides on the electrode surface, soak it in ethanol and blow the surface dry with nitrogen to obtain the treated electrode.

[0020] The application of the electrochemical detection platform for tumor small cell extracellular vesicles based on "OR" logic gates described in this invention in the detection of small cell extracellular vesicles.

[0021] The application includes the following steps:

[0022] (1) An electrochemical detection platform was used as the working electrode, a platinum wire electrode as the counter electrode, and a saturated calomel electrode as the reference electrode. Electrochemical analysis and detection were performed using a three-electrode system. The electrochemical detection parameters were 0.1 V to -0.6 V, amplitude 0.05 V, and pulse width 0.05 s.

[0023] (2) The extracellular vesicles of tumor cells are quantitatively analyzed and detected by detecting the signal difference obtained by different concentrations of tumor microcell extracellular vesicles.

[0024] Furthermore, to verify the feasibility of detecting small extracellular vesicles based on "OR" logic gates, polyacrylamide gel electrophoresis (PAGE) experiments were performed on them.

[0025] Furthermore, differential pulse voltammetry (DPV) was used to verify the feasibility of the biosensor. This involved the following steps:

[0026] In the presence of 5 μM hexaammineruthenium ([Ru(NH3)6) 3+ In Tris-HCl of RuHex, a three-electrode system was used for electrochemical analysis and detection, with a functionalized electrode as the working electrode, a platinum wire electrode as the counter electrode, and a saturated calomel electrode as the reference electrode. The electrochemical detection parameters were 0.1 V to -0.6 V, amplitude 0.05 V, and pulse width 0.05 s.

[0027] Furthermore, the extracellular vesicles of tumor microcells were quantitatively analyzed and detected by detecting the signal difference obtained from different concentrations of tumor microcell extracellular vesicles.

[0028] This invention differs from detecting a single protein by detecting three proteins, achieving higher sensitivity and a wider detection range. By adjusting the sequence length of polyadenine (polyA), the density of the gold surface probe can be controlled. Furthermore, polyA exhibits high affinity comparable to Au-S bonds when assembling oligonucleotides on the gold surface, avoiding costly oligonucleotide modifications and complex pretreatment processes. This invention indirectly reflects the content of biomarkers on tumor sEVs by monitoring signal changes at different concentrations of tumor sEVs, significantly improving the sensitivity of tumor sEV detection. Based on the DNA OR logic gate, CD63, PTK7, and EpCAM are selected as sEV targets to achieve the detection and analysis of various tumor sEVs.

[0029] The principle of this invention is as follows: For signal input, to obtain comprehensive biomarker information, a DNA "OR" logic gate is used to recognize PTK7, CD63, and EpCAM proteins on the surface of extracellular vesicles of tumor microcells. The aptamer sequences of probes AP1, AP2, and AP3 specifically recognize PTK7, CD63, and EpCAM proteins on the surface of extracellular vesicles, respectively, exposing the same enzyme sequence L1. The linker, as a substrate, has a sequence complementary to L1 and can form a complete restriction site with L1. After the Nb.BbvCI enzyme cleaves the site, the linker breaks into two segments, one of which is the target DNA1 binding to the probe PAP on the electrode. For signal output, the probe PAP is immobilized on a gold electrode. The probe PAP consists of three parts: one part is a polyadenine sequence, which exhibits a high affinity comparable to the Au-S bond when assembled on the gold surface; another part is a sequence complementary to the target DNA1, used to capture the target DNA1; and the middle part is a spacer sequence, so that one end of the probe connects to the gold electrode and the other end captures the target DNA1. RuHex, as an electroactive substance, is adsorbed onto DNA strands. Double-stranded DNA adsorbs more RuHex than single-stranded DNA, resulting in a significant signal change. The degree of signal change (ΔI) reflects the content of biomarkers on the surface of small extracellular vesicles. Signal changes occur in the presence of one or more proteins; no signal change occurs only when any protein is absent.

[0030] This invention, based on the "OR" logic gate design, enables multiple signal inputs and the same signal output. The designed three DNA sequences contain different aptamer regions and the same hidden universal sequence. When the aptamer specifically binds to the target protein, the change in spatial conformation exposes the hidden sequence. This sequence hybridizes with a complementary linker to form a double-stranded structure, which is recognized and cleaved by the Nb.BbvCI enzyme, producing free DNA1. The capture probe PAP modified on the gold electrode can hybridize with DNA1. Combined with the electrochemical signaling molecule RuHex, this achieves highly sensitive detection of sEVs and enables the analysis of the types and amounts of proteins on the surface of sEVs.

[0031] This invention indirectly reflects the content of surface proteins on small extracellular vesicles (sEVs) through changes in electrical signals. Multi-aptamer recognition generates a larger electrochemical signal than single-aptamer recognition, lowering the detection limit, increasing sensitivity, and enabling more sensitive detection of low concentrations of small extracellular vesicles.

[0032] In this invention, multiple signal inputs can generate a larger electrochemical signal, resulting in high sensitivity, low detection limit, and a wider detection range for detecting surface proteins of sEVs. Regarding probe immobilization, PolyA replaces thiol groups, avoiding the thiol reduction process before immobilization and saving on DNA modification costs.

[0033] The core of this invention is the use of multiple aptamers to recognize various proteins on the surface of sEVs, forming an "OR" logic gate. When each aptamer binds to its corresponding surface protein, the aptamer conformation changes, and the binding of substrate and enzyme can form an enzyme cleavage cycle, generating free DNA1. The probe on the gold electrode can capture DNA1, thereby amplifying the electrochemical signal. Multiple aptamers can form multiple enzyme cleavage cycles, generating more free DNA1, and the probe on the gold electrode can capture more DNA1, thus generating a stronger electrochemical signal than with only one aptamer. The high signal lowers the detection limit of sEVs, making the detection of sEVs more sensitive, and even low concentrations of sEVs can be detected, which provides strong support for the early diagnosis and treatment of cancer. Existing technologies mostly design detection schemes based on single proteins, which can only obtain information about one protein on the surface of sEVs. However, multiple aptamers recognize multiple proteins on the surface of sEVs, obtaining information about multiple proteins on the surface of sEVs. This makes this invention applicable to the detection of other sEVs besides MCF-7 sEVs, and has universality. This invention can simultaneously detect three surface proteins: EpCAM, PTK7, and CD63, reducing the detection limit, increasing sensitivity, and enabling more sensitive detection of low concentrations of small extracellular vesicles.

[0034] In this invention, based on the properties of aptamers, one aptamer corresponds to one protein, exhibiting specificity. Compared to existing technologies, multi-aptamer recognition provides more comprehensive biological information than single-aptamer recognition. It also demonstrates excellent high sensitivity and low detection limit. This invention eliminates the need for pretreatment steps such as PAP reduction, shortening experimental time. Furthermore, it eliminates the need for thiol modification of DNA, as the synthetic cost of modifying DNA ends with thiol groups is higher than the cost of synthesizing only the DNA strand.

[0035] This invention introduces an enzyme cyclic cleavage mechanism to amplify the detection signal. After the Nb.BbvCI enzyme completes the cleavage of a single linker, it can continue to act on new cleavage sites and repeat the cleavage process. The mechanism is as follows: the aptamer binding to the target surface protein exposes a shared sequence L1, which can bind complementaryly to the linker to form a complete cleavage site. After cleavage by the Nb.BbvCI enzyme, the linker is cleaved into two single strands without complementary ability and can no longer bind to L1. At this point, the L1 sequence is re-exposed and can bind to new free linkers in the system to form new cleavage sites, thus initiating the next round of cleavage reaction. Without this cyclic mechanism, a single L1 sequence can only bind to and cleave one linker, releasing only one DNA1 fragment, resulting in a weak detection signal, far less effective than the signal amplification effect of the enzyme cyclic cleavage strategy.

[0036] The electrochemical detection platform of this invention includes a DNA probe containing an aptamer, a substrate probe linker, a probe PAP, and a gold electrode. The DNA probe includes a universal sequence capable of complementary hybridization with the linker DNA and an aptamer sequence. Different aptamer sequences (AP1, AP2, and AP3) target and recognize PTK7, CD63, and EpCAM proteins, respectively. When the aptamer binds to the corresponding surface protein, the probe conformation changes, the hidden universal sequence is exposed, and hybridizes with the linker DNA. The resulting double-stranded DNA is recognized and cleaved by Nb.BbvCI enzyme. The resulting free DNA fragment can hybridize with the PAP fixed on the surface of the gold electrode. After binding with the signal molecule RuHex, a signal can be output. This invention employs multiple aptamers to specifically recognize different proteins, achieving highly sensitive detection of extracellular vesicles in tumor microcells. This solves the problems of insufficient signal intensity and difficulty in detecting low-abundance vesicles under single aptamer recognition mode. On the other hand, compared with traditional single-protein detection schemes, this invention can simultaneously capture the presence of multiple target proteins on the surface of extracellular vesicles in tumor microcells, enabling differentiated recognition and characterization of extracellular vesicles in tumor microcells with different phenotypes, and providing more comprehensive molecular marker information for the accurate diagnosis of tumors.

[0037] Beneficial effects: Compared with existing methods, the present invention has the following advantages:

[0038] (1) Specificity and comprehensiveness of the detection:

[0039] This invention employs a DNA OR logic gate to specifically recognize PTK7, CD63, and EpCAM proteins on the surface of extracellular vesicles of tumor microcells, achieving highly sensitive detection of these vesicles. Compared to the detection of single proteins, this invention can obtain information on the types and amounts of multiple proteins, improving the reliability of the detection.

[0040] (2) High sensitivity and low detection limit:

[0041] The detection platform provided by this invention exhibits high sensitivity and a low detection limit. Using an OR gate, the detection limit for MCF-7 sEVs via three aptamers is 223 particles / mL. Without using an OR gate, the detection limit for MCF-7 sEVs via only the EpCAM aptamer is 462 particles / mL. The use of the OR gate demonstrates high sensitivity for sEVs, enabling more sensitive detection of low concentrations of sEVs, which is helpful for identification and diagnosis in the early stages of disease.

[0042] (3) Wide detection linear range:

[0043] The detection method provided by this invention is at 2.75 × 10⁻⁶. 2Up to 5.5×10 8 Within the concentration range of particles / mL sEVs, a good linear relationship was observed, with the linear equation being ΔI = 0.31857lgCsEVs - 0.63298, and the correlation coefficient being R. 2 =0.99889. This detection method has a wide linear response range, which can effectively capture sEVs at different concentration levels, and provides a guarantee for the quantitative analysis of sEVs samples with various concentration gradients.

[0044] (4) Low cost and simple operation process:

[0045] This invention utilizes polyA to replace the terminal thiol group of DNA, omitting the step of reducing the thiol group before DNA fixation and significantly shortening the experimental time. Simultaneously, the synthesis cost of polyA is lower than that of the terminal thiol group of DNA, greatly saving experimental costs. Furthermore, the experimental operation does not rely on expensive equipment and requires no complex operations or sample processing, making it suitable for diagnosing early-stage diseases in a relatively short time. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the principle of the electrochemical detection method for small extracellular vesicles based on the "OR" logic gate (A: schematic diagram of three protease digestion (I), schematic diagram of one protease digestion (II); B: PAP probe on the electrode captures target DNA1 for electrochemical detection).

[0047] Figure 2 Characterization of small extracellular vesicles (A: Transmission electron microscopy image (TEM); B: Particle size distribution map).

[0048] Figure 3 The construction and feasibility analysis of biosensors (A: Polyacrylamide gel electrophoresis (PAGE), where "+" indicates the addition of DNA strands, Nb.BbvCI or sEVs; B: Differential pulse voltammetry (DPV) curves including bare electrode + PAP + MCH (a), bare electrode + PAP + MCH + AP1 + AP2 + AP3 + linker + Nb.BbvCI (b), bare electrode + PAP + MCH + AP1 + AP2 + AP3 + linker + Nb.BbvCI + sEVs (c)).

[0049] Figure 4 The study explored the optimal experimental conditions for biosensors (A: exploration of different concentrations of PAP on electrodes; B: exploration of different concentrations of Nb.BbvCI enzyme; C: exploration of different concentrations of linker; D: exploration of the incubation time of DNA1 with PAP).

[0050] Figure 5The electrochemical performance of biosensors (A: detection of three proteins in sEVs at different concentrations (a) and standard curves (b); B: detection and analysis of surface proteins of sEVs from different cell sources; C: electrochemical signals in complex systems). Detailed Implementation

[0051] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0052] The abbreviations of technical terms used in this invention are as follows: FBS: Fetal bovine serum; UC: Ultracentrifugation.

[0053] MCF-7 cells: American type culture collection; (ATCC number HTB-22)

[0054] HeLa cells: American type culture collection; (ATCC number CCL-2)

[0055] CCRF-CEM cells: American type culture collection; (ATCC number CCL-119)

[0056] Ramos cells: American type culture collection; (ATCC number CRL-1596)

[0057] Nb.BbvCI enzyme: purchased from New England Biolabs (catalog number: R0631S)

[0058] The electrochemically active substance was RuHex, the detection solution was 10 mM tris-HCl (pH=7.4) buffer, and the electrochemical signal was recorded by differential pulse voltammetry (DPV).

[0059] In this embodiment of the invention, the working electrode is gold, the reference electrode is saturated calomel, and the auxiliary electrode is platinum.

[0060] All sequences in this invention were synthesized directly by a biotechnology company.

[0061] The aptamer chain sequences are as follows: AP1 5'-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACTGTA CGG TTA GAA AAA AAA AAA AAT CCT CAG CAG TTA-3', AP2 5'-CAC CCC ACC TCG CTCCCG TGA CAC TAA TGC TAA CT AAA AAA AAT CCT CAG CAG TTA GCA TT-3', and AP3 5'-CACTAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG AAAAAAA AT CCTCAG CAG TTA ACC TCT GTA GTG-3'.

[0062] L1 sequence 5'-AT CCT CAG CAG TTA-3'.

[0063] Linker sequence 5'-TCA GCA GGG AGG AAG ACA ATA TTA ACT GCT GAG GAT AAA CG-3'.

[0064] PAP sequence 5'-CCT CCC TGC TGA A CAC ACA CAC AAA AAA AAA AAA AAA AAA AAAAAA CAC ACA CAC A GC AGT TAA TAT TG-3'.

[0065] The DNA 1 sequence formed after Nb.BbvCI enzyme cleavage is 5'-TCA GCA GGG AGG AAG ACA ATA TTA ACTGC-3'.

[0066] Example 1

[0067] Isolation and characterization of small extracellular vesicles

[0068] (1) Isolation of small extracellular vesicles: CCRF-CEM and Ramos cells were cultured in RPMI-1640 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum, while MCF-7 and HeLa cells were cultured in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum. All cells were grown in a humid atmosphere of 37°C and 5% CO2. When the cell density reached 70%-80%, the medium was changed to serum-free RPMI-1640 medium or serum-free DMEM medium, and the cell supernatant was collected after starvation culture for 48 hours. Then, small extracellular vesicles secreted by MCF-7, HeLa, CCRF-CEM and Ramos cancer cell lines were obtained using a cell supernatant exosome extraction kit (Beyotime, C3620S), resuspended in PBS, and stored at -20°C.

[0069] (2) Characterization of small extracellular vesicles: The obtained MCF-7 sEVs were imaged using transmission electron microscopy (TEM) on an H-7500 (Hitachi), and nanoparticle tracking analysis (NTA) was performed on ZetaView (Particle Metrix, Germany) to obtain the particle size distribution and concentration of MCF-7 sEVs. Figure 2 TEM images of cell A show small extracellular vesicles resembling tea saucers, with a diameter of approximately 130 nm, which is largely consistent with the NTA results. Figure 2 The particle size distribution map of B showed that the peak concentration of small extracellular vesicles was around 130 nm, and the original concentration measured by NTA was 5.5 × 10⁻⁶. 8 particles / mL.

[0070] Example 2

[0071] Preparation and electrochemical feasibility of gold electrodes

[0072] (1) Preparation of gold electrode: Bare gold electrodes (GE) with a diameter of 2 mm were manually polished with alumina slurries of 0.3 μm and 0.05 μm, respectively, and then ultrasonically cleaned in anhydrous ethanol and ultrapure water for 30 s. The bare GE was immersed in freshly prepared piranha solution (98% H2SO4:30% H2O2 = 3:1) for 30 minutes to further clean residual impurities. After rinsing with ultrapure water, the electrode was scanned by cyclic voltammetry (CV) in 0.5 M H2SO4 until a stable CV curve was obtained. The electrode was then immersed in anhydrous ethanol for 30 minutes to remove residual sulfuric acid. After rinsing with ultrapure water, the electrode surface was dried with nitrogen. To functionalize the gold electrode, PAP was diluted to 10 μM with 10 mM Tris-HCl buffer and treated at 95 °C for 5 minutes. The annealed PAP was then diluted to 1 μM with 10 mM Tris-HCl containing 1 M NaCl and 1 mM EDTA. 10 μL of the diluted PAP was then dropped onto the pretreated electrode and incubated overnight at room temperature. The next day, unoccupied binding sites on the electrode surface were blocked with 1 mM MCH for 1 hour at room temperature, followed by washing before use in subsequent experiments.

[0073] (2) Verify the feasibility of the biosensor:

[0074] The feasibility of the method was verified using 12% polyacrylamide gel electrophoresis (PAGE). The DNA strands were hybridized, and the results were as follows: Figure 3 As shown in Figure A, lanes 1, 2, 3, and 4 represent AP1, AP2, AP3, and the linker, respectively. In the absence of MCF-7 sEVs (lanes 5, 7, 9, and 11), AP1, AP2, AP3, and the linker can coexist stably in solution without being cleaved by the Nb.BbvCI enzyme, indicating that AP1 and the linker, AP2 and the linker, and AP3 and the linker can all exist stably without hybridization, and the recognition sequence of the Nb.BbvCI enzyme is not exposed, thus preventing cleavage. In the presence of MCF-7 sEVs (lanes 6, 8, 10, and 12), the aptamers in the hairpin structures of AP1, AP2, and AP3 bind to PTK7, CD63, and EpCAM on the sEV membrane, causing structural changes that trigger hybridization between AP1 and the linker, AP2 and the linker, and AP3 and the linker. After forming a double strand, the Nb.BbvCI enzyme can recognize specific nucleotide sequences and cleave the linker, producing the new band, which is DNA1. This initially confirms the feasibility of the experiment.

[0075] 1 μM AP1, AP2, and AP3 and 3.0 μM linker were combined with 5.5 × 10⁻⁶ 8MCF-7sEVs particles / mL were first incubated at 37°C for 1 hour, followed by incubation with 0.5 U / μl Nb.BbvCI enzyme for another 1 hour to obtain a reaction mixture containing the cleaved target DNA1. 10 μl of the reaction mixture was added to the electrode modified in step (1), and incubated for 1.5 hours to allow PAP to capture the target DNA1. After the reaction, unbound DNA strands were washed with PBS, resulting in a functionalized electrode, which can then be used as an electrochemical detection platform. Finally, the DPV signal was tested in Tris-HCl containing 5 μM RuHex (ruthenium hexaammonium trichloride). The electrochemical detection parameters were 0.1 V to -0.6 V, amplitude 0.05 V, and pulse width 0.05 s. A three-electrode system was used for electrochemical analysis and detection, with the functionalized electrode as the working electrode, a platinum wire electrode as the counter electrode, and a saturated calomel electrode as the reference electrode. All data were obtained in triplicate.

[0076] Figure 3 As shown in curve B, when MCF-7 sEVs are present (curve c), the DPV signal is significantly increased, which is attributed to the adsorption of a large amount of RuHex on the DNA double strand; in the absence of MCF-7 sEVs (curve b), although DNA strands and Nb.BbvCI enzyme are added, the DPV signal is almost indistinguishable from the background signal (curve a).

[0077] Example 3

[0078] Optimization of experimental conditions

[0079] Following the method of Example 2, with other conditions kept constant, the probe density immobilized on the gold electrode was adjusted by changing the concentration of the probe PAP, and electrochemical analysis and detection were performed. Figure 4 As shown in Figure A, the concentrations ranged from 0.01 μM to 2 μM, and the signal was the DPV signal in 5 μM RuHex after PAP bound to target DNA1. The results showed that the DPV signal was the largest at 1 μM PAP. Since the DNA strand is negatively charged, excessively high concentrations would cause a repulsive reaction between probes, resulting in a slight decrease in the DPV signal. Therefore, 1 μM PAP was chosen as the experimental condition for subsequent experiments.

[0080] Furthermore, keeping other conditions constant, the enzyme concentration determines whether substrate cleavage is complete, such as... Figure 4 As shown in B, the enzyme was diluted with 1×rCutSmart to 0.2 U / μl-0.7 U / μl. The signal reached its peak when the enzyme concentration was 0.5 U / μl, so 0.5 U / μl was chosen as the experimental condition for subsequent experiments.

[0081] The substrate concentration affected the concentration of target DNA1. To determine the optimal substrate concentration, keeping other conditions constant, concentration gradients of 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, and 3.5 μM were selected. The results are as follows: Figure 4 As shown in C, the substrate concentration reached saturation at 3.0 μM, and further increasing the substrate concentration had no effect. Therefore, 3.0 μM was chosen as the experimental condition for subsequent experiments.

[0082] Figure 4 D represents the incubation time between the target DNA1 and the probe PAP, which determines whether the reaction between the target DNA1 and the probe PAP is complete. Keeping other conditions constant, the incubation time was adjusted. The results showed that 1.5 hours was the optimal reaction time, at which point the target DNA1 and the probe PAP reacted completely. Increasing the reaction time further did not increase the signal, so 1.5 hours was chosen as the condition for subsequent experiments.

[0083] Based on the above optimizations, the final selected combination of parameters is as follows: PAP concentration: 1 μM; Nb.BbvCI enzyme concentration: 0.5 U / μl; substrate linker concentration: 3.0 μM; incubation time with electrode surface: 1.5 hours. The optimized experimental conditions increase signal sensitivity while avoiding unnecessary waste, ensuring experimental stability and reproducibility.

[0084] Example 4

[0085] Electrochemical performance of biosensors

[0086] (1) Construction of the standard curve: The specific operation is the same as in Example 2. Under the above-mentioned optimal experimental conditions, MCF-7 sEVs were diluted to different concentrations for DPV measurement, and a standard curve was constructed as a function of the logarithm of small extracellular vesicle concentration and the change in electrochemical signal. The results are as follows: Figure 5 As shown in Figure A, the DPV signal increases with increasing sEVs concentration, and the linear equation is ΔI = 0.31857lgCsEVs - 0.63298, with a correlation coefficient of R. 2 =0.99889, and the detection limit calculated according to the 3σ rule is 223 particles / mL. Using the same experimental procedure, only AP3 was added to recognize the single protein EpCAM on the surface of MCF-7 sEVs (the method in Example 2 does not include AP1 and AP2), and the detection limit was 462 particles / mL. Compared with the single aptamer recognizing a single surface protein, the multi-aptamer recognition method has a lower detection limit and is more sensitive to low concentrations of sEVs. This indicates that the present invention has certain advantages in detecting low concentrations of sEVs, achieving comprehensive and highly sensitive detection of sEV surface proteins.

[0087] (2) Universality of the biosensor: To verify the universal feasibility of the biosensor, the same preparation and detection methods as in Example 2 were used, except that MCF-7 sEVs were replaced with CCRF-CEM sEVs, Ramos sEVs, and HeLa sEVs, while other procedures remained unchanged. Small extracellular vesicles secreted by PBS, MCF-7, HeLa, CCRF-CEM, and Ramos cancer cell lines (containing no small extracellular vesicles) were subjected to DPV testing. Each group underwent at least three parallel experiments. Figure 5 As shown in Figure B, all small extracellular vesicles exhibited a current response, with MCF-7 showing a higher current signal intensity than the other small extracellular vesicles. This indicates that MCF-7 contains a higher total content of CD63, PTK7, and EpCAM proteins. These results demonstrate that this detection strategy is suitable for the general detection of small extracellular vesicles and has the potential for clinical diagnostic applications.

[0088] (3) Application of biosensors in complex systems: First, fetal bovine serum (FBS) was added to the cell culture medium at concentrations of 30% and 60%, respectively. SEVs were removed by ultracentrifugation, following these steps: initial centrifugation at 300g for 10 minutes at 4°C, followed by centrifugation at 2000g for 20 minutes to remove cells and debris. The supernatant was centrifuged at 11000g for 30 minutes and filtered through a 0.22μm filter membrane to further remove large particulate impurities. SEV precipitation was removed by ultracentrifugation at 110000g RCF for 120 minutes, yielding the supernatant, which was named ultracentrifuged FBS (UC FBS). The MCF-7 sEVs prepared in Example 1 were added to UCFBS at volume ratios of 30% and 60%, respectively, following the same preparation and detection methods as in Example 2. The results are as follows: Figure 5 As shown in Figure C, the signal is essentially the same as when PBS is added when different concentrations of UC FBS are incorporated. This demonstrates the good applicability of this detection platform in complex systems, showcasing its potential for detecting untreated clinical samples.

Claims

1. An electrochemical detection platform for extracellular vesicles of small tumor cells based on "OR" logic gates, characterized in that, The electrochemical detection platform includes a DNA probe containing aptamers, a substrate probe linker, a probe PAP, and a gold electrode. The DNA probe includes three aptamers, AP1, AP2, and AP3, which specifically recognize PTK7, CD63, and EpCAM proteins, respectively. All three aptamers, AP1, AP2, and AP3, contain a universal L1 sequence, which is complementary to the substrate probe linker and constructs a specific recognition and cleavage site for the Nb.BbvCI enzyme. After being acted upon by the Nb.BbvCI enzyme, a DNA1 fragment is generated. The PAP probe is pre-modified on the surface of the gold electrode, and it hybridizes with the above-mentioned enzyme digestion product DNA1 to form a double-stranded complex.

2. The electrochemical detection platform for extracellular vesicles of tumor small cells based on "OR" logic gates according to claim 1, characterized in that, The preferred sequences of the aptamers AP1, AP2, and AP3 are as follows: AP1: 5'-ATC TAA CTG CTG CGC CGCCGG GAA AAT ACT GTA CGG TTA GAA AAA AAA AAA AAT CCT CAG CAG TTA-3', AP2: 5'-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TAA CT AAA AAA AAT CCT CAG CAG TTAGCA TT-3', AP3: 5'-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGCCTG AAAAAAA AT CCT CAG CAG TTA ACC TCT GTA GTG-3'.

3. The electrochemical detection platform for extracellular vesicles of tumor small cells based on "OR" logic gates according to claim 1, characterized in that, The sequence of the substrate probe linker is 5'-TCA GCA GGG AGG AAG ACA ATA TTA ACT GCTGAG GAT AAA CG-3'.

4. The electrochemical detection platform for extracellular vesicles of tumor small cells based on "OR" logic gates according to claim 1, characterized in that, The L1 sequence is 5'-AT CCT CAG CAG TTA-3', and the DNA1 sequence formed after Nb.BbvCI enzyme cleavage is 5'-TCA GCA GGG AGG AAG ACA ATA TTA ACT GC-3'.

5. The electrochemical detection platform for extracellular vesicles of tumor small cells based on "OR" logic gates according to claim 1, characterized in that, The sequence of the probe PAP is 5'-CCT CCC TGC TGA A CAC ACA CAC AAA AAA AAA AAAAAA AAA AAA AAA AAA CAC ACA CAC A GC AGT TAA TAT TG-3'.

6. A method for preparing the electrochemical detection platform for extracellular vesicles of tumor small cells based on "OR" logic gates as described in claim 1, characterized in that, Includes the following steps: (1) Tumor small cell extracellular vesicles are co-incubated with AP1, AP2 and AP3 to form sEVs-aptamer complexes, exposing the same sequence L1, which contains an enzyme cleavage site; (2) Take the sEVs-aptamer complex prepared in step (1) and incubate it with the substrate probe linker to construct the complete Nb.BbvCI enzyme cleavage site; then add Nb.BbvCI enzyme to the system to specifically cleave the enzyme site, thereby releasing the DNA1 fragment and forming a solution. (3) The probe PAP is incubated with the pretreated gold electrode overnight and then immersed in 6-mercaptoethanol (MCH) to block non-specific sites, thus obtaining the modified electrode. (4) Incubate the solution obtained in step (2) with the modified electrode obtained in step (3). The detection electrode obtained after incubation can be used as an electrochemical detection platform.

7. The preparation method of the electrochemical detection platform for extracellular vesicles of tumor small cells based on "OR" logic gates according to claim 6, characterized in that, The tumor microcellular extracellular vesicles mentioned in step (1) are microcellular extracellular vesicles secreted by CCRF-CEM cells, MCF-7 cells, HeLa cells or Ramos cells.

8. The preparation method of the electrochemical detection platform for extracellular vesicles of tumor small cells based on "OR" logic gates according to claim 6, characterized in that, The concentration of extracellular vesicles in the tumor small cells was 2.75 × 10⁻⁶. 2 -5.5×10 8 The concentration of particles / mL, the concentration of substrate probe linker is 1.5-3.5 μM, the concentration of Nb.BbvCI enzyme is 0.2-0.7 U / μl, and the concentration of probe PAP is 0.01-2 μM.

9. The application of the electrochemical detection platform for tumor small cell extracellular vesicles based on "OR" logic gates as described in claim 1 in the detection of small cell extracellular vesicles.

10. The application according to claim 9, characterized in that, The application includes the following steps: (1) Electrochemical analysis and detection were performed using a three-electrode system with an electrochemical detection platform as the working electrode, a platinum wire electrode as the counter electrode, and a saturated calomel electrode as the reference electrode. (2) The extracellular vesicles of tumor cells are quantitatively analyzed and detected by detecting the signal difference obtained by different concentrations of tumor microcell extracellular vesicles.

Images