A biosensor and a method for preparing the same

Abstract

The present application provides a biosensor and a preparation method thereof, wherein the biosensor comprises: an electrode substrate, and a composite nano-conductive base material, cDNA and a competitive probe connected in sequence on the electrode substrate; wherein the composite nano-conductive base material is made of NH2-UiO-66@AQ, AuNPs and IL-rGO; the competitive probe is made by pairing Apt-Bio and DSN-Probe; in the presence of HER-2, the two current signals generated by the biosensor in the present application change, and the HER-2 content is determined by the two current signals. Compared with traditional single signal detection, the external or system detection error is greatly reduced, thereby improving the accuracy of HER-2 concentration detection.

Description

Technical Field

[0001] The present application relates to the field of biosensor technology, and in particular to a biosensor and a preparation method thereof. Background Art

[0002] Changes in HER-2 levels are important early warning signs of breast cancer recurrence and metastasis. HER-2 levels can also be measured in serum. However, existing biosensors for detecting HER-2 in serum often fail to achieve the required level of accuracy, resulting in ambiguous diagnostic results near critical thresholds. Therefore, the development of an accurate and sensitive biosensor is crucial for early diagnosis, efficacy monitoring, and prognostic assessment of breast cancer. Summary of the Invention

[0003] In view of this, the purpose of this application is to propose a biosensor and a preparation method thereof to solve the problem that current biosensors are not accurate enough in detecting HER-2.

[0004] Based on the above objectives, the present application provides a biosensor comprising: an electrode substrate, and a composite nano-conductive substrate material, cDNA, and a competitive probe sequentially connected on the electrode substrate; wherein the composite nano-conductive substrate material is made of NH2-UiO-66@AQ, AuNPs, and IL-rGO; the competitive probe is made by pairing Apt-Bio and DSN-Probe; and the DSN-Probe is made of MOF@FeTCPP.

[0005] The sequence of the Apt-Bio is shown as 5'-Biotin-SEQ ID NO: 1-3', specifically 5'-Biotin-GGGCCG TCG AAC ACG AGC ATG GTG CGT GGA CCT AGG ATG ACC TGA GTA CTG TCC-3'; the sequence of the cDNA is shown as 5'-SH-(CH2)6-SEQ ID NO: 2-3', specifically 5'-SH-(CH2)6-TTT TTG GACAGT ACT-3'.

[0006] Optionally, the NH2-UiO-66@AQ is prepared by the following method:

[0007] Dissolving zirconium tetrachloride, benzoic acid, and aminoterephthalic acid in DMF and reacting to obtain a crude NH2-UiO-66 product; washing and drying the crude NH2-UiO-66 product to obtain NH2-UiO-66;

[0008] Dissolving the NH2-UiO-66, AQ and carboxyl activator in a methanol solution and reacting the solution to obtain an intermediate reaction solution.

[0009] N-hydroxysuccinimide was added to the intermediate reaction solution to continue the reaction to obtain NH2-UiO-66@AQ.

[0010] Optionally, the IL-rGO is prepared by the following method:

[0011] IL-NH2 and KOH are placed in a graphene homogeneous solution for reaction to obtain IL-rGO.

[0012] Optionally, the AuNPs are prepared by the following method:

[0013] dissolving trisodium citrate dihydrate in deionized water and heating to boiling to obtain a trisodium citrate dihydrate solution;

[0014] adding HAuCl4 solution to the trisodium citrate dihydrate solution to obtain a reaction solution;

[0015] After the reaction solution changes color, it is cooled, and trisodium citrate dihydrate and HAuCl4 solution are alternately added to the cooled reaction solution to obtain AuNPs.

[0016] Optionally, the competitive probe is prepared by pairing Apt-Bio and DSN-Probe; wherein the MOF@FeTCPP is coupled to the Apt-Bio via a coupling agent.

[0017] Optionally, the coupling agent is a streptavidin coupling agent.

[0018] Optionally, the MOF@FeTCPP is prepared by the following method:

[0019] Dissolving benzene trimecchymic acid and FeTCPP in a mixture of ethanol and DMF to obtain a benzene trimecchymic acid-FeTCPP mixture;

[0020] Then, a Cu(NO3)2·3H2O aqueous solution is added to the benzenetricarboxylic acid-FeTCPP mixed solution, and the mixture is ultrasonically dispersed and then heated for reaction to obtain the MOF@FeTCPP.

[0021] Optionally, the MOF@FeTCPP is prepared by the following method:

[0022] Dissolving Cu(NO3)2·3H2O and polyvinyl pyrrolidone in methanol and stirring uniformly to obtain a first methanol solution;

[0023] Adding benzenetricarboxylic acid to methanol and stirring evenly to obtain a second methanol solution;

[0024] adding FeTCPP to methanol to obtain a third methanol solution;

[0025] adding the second methanol solution to the first methanol solution until the solution becomes turbid to obtain a turbid solution;

[0026] The third methanol solution is added to the turbid liquid to react to obtain the MOF@FeTCPP.

[0027] Based on the same inventive concept, the present disclosure also provides a method for preparing a biosensor, comprising the following steps:

[0028] Adding the composite nano-conductive base material solution dropwise onto the pretreated surface of the electrode substrate and drying the resulting solution to obtain a first electrode substrate;

[0029] adding the cDNA solution dropwise onto the surface of the first electrode substrate, incubating and then washing to obtain a second electrode substrate;

[0030] Adding MCH blocking solution dropwise to the surface of the second electrode substrate, reacting and washing to obtain a third electrode substrate;

[0031] adding an Apt-Bio solution dropwise onto the surface of the third electrode substrate, incubating and then washing to obtain a fourth electrode substrate;

[0032] The DSN-Probe solution is dropped onto the surface of the fourth electrode substrate, and the biosensor is obtained after incubation.

[0033] As can be seen from the above description, the biosensor provided in this application includes: an electrode substrate, and a composite nanoconductive substrate material, cDNA, and competitive probe sequentially connected to the electrode substrate. The electrode substrate serves as the physical support and electron conduction foundation of the entire biosensor. The composite nanoconductive substrate material supported on its surface is synthesized from NH2-UiO-66@AQ, AuNPs, and IL-rGO. The interlayered IL-rGO supported on the electrode substrate serves as both an electron transport platform and a binding site for AuNPs and NH2-UiO-66@AQ. AuNPs have excellent electron conductivity, enabling the construction of a continuous electron transport channel within the composite material. They also provide binding sites for cDNA, facilitating the subsequent connection of the competitive probe. NH2-UiO-66@AQ is fabricated from the metal-organic framework NH2-UiO-66 and the electroactive molecule AQ. The biosensor is capable of generating a first current signal based on AQ. The competitive probe is made by pairing Apt-Bio and DSN-Probe. The competitive probe binds complementary to the cDNA through Apt-Bio, and the biosensor generates a second current signal based on the DSN-Probe. When HER-2 is present in solution, the binding affinity of HER-2 to the cDNA is greater than that of Apt-Bio. As a result, some of the competitive probe dissociates from the cDNA and enters the solution, reducing the amount of competitive probe remaining on the cDNA. Consequently, the second current signal generated by the DSN-Probe in the biosensor decreases. Furthermore, because the competitive probe is non-conductive, the first current signal generated by the biosensor based on AQ increases after some of the competitive probe dissociates. The ratio of the first and second current signals accurately determines the HER-2 concentration. Compared to traditional single-signal detection, this significantly reduces external or systemic detection errors, thereby improving the accuracy of HER-2 concentration detection.

[0034] In addition, a colorimetric test is performed on the competitive probe dissociated from the cDNA to obtain a colorimetric signal. The HER-2 content is determined based on the colorimetric signal, and the HER-2 content determined by the first current signal and the second current signal is calibrated against the HER-2 content determined by the first current signal, thereby reducing errors caused by interfering substances in the sample or accidental factors in the detection process, further improving the reliability of the detection results, and providing more accurate data support for clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in this application or related technologies, the following briefly introduces the drawings required for use in the embodiments or related technical descriptions. Obviously, the drawings described below are merely embodiments of this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0036] Figure 1 A flow chart showing a method for detecting HER-2 using a biosensor according to an embodiment of the present application;

[0037] Figure 2 This is a characterization diagram of MFN-Substrate in the embodiment of this application;

[0038] Figure 2 A in the middle is a scanning electron microscope (SEM) image of NH2-UiO-66 in the embodiment of the present application;

[0039] Figure 2 Figure B is a transmission electron microscope (TEM) image of NH2-UiO-66 in the examples of the present application;

[0040] Figure 2 C is a scanning electron microscope image of NH2-UiO-666@AQ in the embodiment of the present application;

[0041] Figure 2 D in the middle is a transmission electron microscope image of NH2-UiO-66@AQ in the embodiment of the present application;

[0042] Figure 2 E in the middle is a scanning electron microscope image of MFN-Substrate in the embodiment of the present application;

[0043] Figure 2 Figure F is a transmission electron microscope image of the MFN-Substrate in the embodiment of the present application;

[0044] Figure 2 Figure G is a particle size analysis diagram of NH2-UiO-66 in the examples of this application;

[0045] Figure 2 H in the figure is the N2 adsorption-desorption isotherm of NH2-UiO-66 and NH2-UiO-66@AQ in the examples of the present application;

[0046] Figure 2 1 is a pore size distribution diagram of NH2-UiO-66 and NH2-UiO-66@AQ in the examples of this application;

[0047] Figure 2In the examples of this application, the X-ray diffraction (XRD) patterns of AQ, NH2-UiO-66 and NH2-UiO-66@AQ are shown;

[0048] Figure 2 In the examples of this application, K is the Fourier transform infrared spectrum (FT-IR) of AQ, NH2-UiO-66 and NH2-UiO-66@AQ;

[0049] Figure 2 Middle L in the examples of this application is the ultraviolet-visible (UV-Vis) spectra of AQ, NH2-UiO-66 and NH2-UiO-66@AQ;

[0050] Figure 3 This is a characterization diagram of DSN-Probe in the embodiment of this application;

[0051] Figure 3 A in the middle is a scanning electron microscope (SEM) image of HKUST-1 in the examples of this application;

[0052] Figure 3 B is a transmission electron microscope (TEM) image of HKUST-1 in the examples of this application;

[0053] Figure 3 C in the middle is the SEM image of MOF@FeTCPP in the embodiment of this application;

[0054] Figure 3 D in the middle is a TEM image of MOF@FeTCPP in the embodiment of the present application;

[0055] Figure 3 Figure E is a SEM image of the DSN-Probe in the embodiment of the present application;

[0056] Figure 3 F is a TEM image of DSN-Probe in the embodiment of the present application;

[0057] Figure 3 G in the middle is the particle size analysis of HKUST-1 and MOF@FeTCPP in the examples of this application;

[0058] Figure 3 H in the figure is the N2 adsorption-desorption isotherm of HKUST-1 and MOF@FeTCPP in the examples of this application;

[0059] Figure 3 Figure 1 is the pore size distribution diagram of HKUST-1 and MOF@FeTCPP in the examples of this application;

[0060] Figure 3 J in the figure is the X-ray diffraction (XRD) pattern of FeTCPP, HKUST-1, MOF@FeTCPP and DSN-Probe in the examples of this application;

[0061] Figure 3 K in the figure is the Fourier transform infrared spectra (FT-IR) of FeTCPP, HKUST-1, MOF@FeTCPP and DSN-Probe in the examples of this application;

[0062] Figure 3 L in the figure is the ultraviolet-visible (UV-Vis) spectra of FeTCPP, HKUST-1, MOF@FeTCPP and DSN-Probe in the examples of this application;

[0063] Figure 3 M is the absorbance variation curve of MOF@FeTCPP synthesized by three different methods in the examples of this application at 650 nm over time;

[0064] Figure 3 N is the Lineweaver-Burk diagram of MOF@FeTCPP synthesized by three different methods in the examples of this application;

[0065] Figure 3 O is a curve of the absorbance change at 650 nm of MOF@FeTCPP synthesized with different FeTCPP concentrations in the examples of this application over time;

[0066] Figure 3 P in the figure is the Lineweaver-Burk plot of MOF@FeTCPP synthesized at different FeTCPP concentrations in the examples of this application;

[0067] Figure 4 This is a graph showing the performance of the biosensor in the examples of this application;

[0068] Figure 4 A in the figure is the cyclic voltammetry (CV) response diagram of GCE (a), IL-rGO / GCE (b), IL-rGO-NH2-UiO-66@AQ / GCE (c), and MFN-Substrate / GCE (d) in the examples of the present application;

[0069] Figure 4 Figure B is a CV response diagram of MFN-Substrate / GCE at different scan rates from 10 mV・s⁻¹ to 100 mV・s⁻¹ in an embodiment of the present application;

[0070] Figure 4Figure C is a graph showing the relationship between the redox peak current and the square root of the scan rate in the electrochemical reaction of MFN-Substrate / GCE in an embodiment of the present application;

[0071] Figure 4 D in the figure is the electrochemical impedance spectroscopy of GCE (a), MFN-Substrate / GCE (b), cDNA / MFN-Substrate / GCE (c), MCH / cDNA / MFN-Substrate / GCE (d), Apt-Bio / MCH / cDNA / MFN-Substrate / GCE (e), DSN-Probe / Apt-Bio / MCH / cDNA / MFN-Substrate / GCE (f), and HER-2 / DSN-Probe / Apt-Bio / MCH / cDNA / MFN-Substrate / GCE (g) in the examples of the present application;

[0072] Figure 4 Figure E is the differential pulse voltammetry (DPV) response diagram of GCE (a), MFN-Substrate / GCE (b), cDNA / MFN-Substrate / GCE (c), MCH / cDNA / MFN-Substrate / GCE (d), Apt-Bio / MCH / cDNA / MFN-Substrate / GCE (e), DSN-Probe / Apt-Bio / MCH / cDNA / MFN-Substrate / GCE (f), and HER-2 / DSN-Probe / Apt-Bio / MCH / cDNA / MFN-Substrate / GCE (g) in the examples of the present application;

[0073] Figure 4 Figure F is the DPV response graph of GCE (a), IL-rGO-NH2-UiO-66 / GCE (b), IL-rGO-NH2-UiO-66@AQ / GCE (c), MFN-Substrate / GCE (d), DSN-Probe / GCE (e), and DSN-Probe / Apt-Bio / MCH / cDNA / MFN-Substrate / GCE (f) in the examples of the present application;

[0074] Figure 4 Figure G in the middle is a 10-repeat DPV response graph of MFN-Substrate / GCE in an embodiment of the present application;

[0075] Figure 4 H in the figure is a feasibility verification diagram of the electrochemical model in the embodiment of the present application;

[0076] Figure 4 Figure 1 is a feasibility verification diagram of the colorimetric mode in the embodiment of the present application;

[0077] Figure 5 This is the detection diagram of the biosensor for HER-2 standard;

[0078] Figure 5 A in the middle is the electrochemical detection graph of the biosensor for HER-2 at different concentrations from 0 fg / mL to 150 ng / mL;

[0079] Figure 5 Figure B is the calibration curve of different HER-2 concentrations from 100 fg / mL to 100 ng / mL in electrochemical mode;

[0080] Figure 5 Center C is the colorimetric detection graph of the biosensor for HER-2 at different concentrations from 0 fg / mL to 150 ng / mL;

[0081] Figure 5 Center D is the calibration curve of different HER-2 concentrations from 100 fg / mL to 150 ng / mL in colorimetric mode;

[0082] Figure 5 Middle E is the image of the test solution with different HER-2 concentrations under sunlight;

[0083] Figure 5 Middle F is a calibration curve based on ImageJ grayscale values ​​and different HER-2 concentrations from 100 fg / mL to 150 ng / mL;

[0084] Figure 5 G in the middle is the specific analysis diagram in electrochemical mode;

[0085] Figure 5 Middle H is the specificity analysis diagram in colorimetric mode;

[0086] Figure 5 Figure I is the reproducibility analysis graph of five groups of biosensors independently constructed under the same conditions;

[0087] Figure 5 Middle J is the storage stability analysis chart of the biosensor (tested once a month and every week for 1 month);

[0088] Figure 6 The figure shows the analysis of HER-2 levels in cell lines and clinical human serum by the biosensor;

[0089] Figure 6 A in the figure shows 1×10 6 HER-2 detection signal intensity diagram of each cell;

[0090] Figure 6 B shows 1×10 6 HER-2 detection signal intensity diagram of each cell;

[0091] Figure 6 C shows the biosensor quantification of 1×10 6 HER-2 concentration in each cell;

[0092] Figure 6 Figures DE are HER-2 analysis of SK-BR-3 (D) and BT-474 (E) cell lines: electrochemical response (a) and colorimetric response (b);

[0093] Figure 6 Figures F and G are the signal intensity diagrams of HER-2 detection in human serum samples under electrochemical mode (F) and colorimetric mode (G);

[0094] Figure 6 ZhongHI is the difference in HER-2 concentration between healthy controls (n=10) and breast cancer patients (n=10);

[0095] Figure 6 J in the middle is the receiver operating characteristic curve (ROC curve). DETAILED DESCRIPTION

[0096] In order to make the objectives, technical solutions and advantages of this application more clear, this application is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0097] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present application should have the usual meanings understood by people with ordinary skills in the field to which this application belongs. The "first", "second" and similar words used in the embodiments of the present application do not indicate any order, quantity or importance, but are only used to distinguish different components. "Include" or "comprise" and similar words mean that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Connect" or "connected" and similar words are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0098] As mentioned in the background, human epidermal growth factor receptor 2 (HER-2) is a transmembrane receptor tyrosine kinase that plays a key role in cell proliferation and differentiation. HER-2 overexpression or amplification occurs in approximately 25–30% of primary breast cancers and is closely associated with aggressive tumor growth, high recurrence rates, and poor prognosis. Furthermore, changes in HER-2 levels are important early warning signs of breast cancer recurrence and metastasis, particularly in HER-2-positive patients. Brain metastases, which show a progressively increasing trend, pose a life-threatening threat. Currently, HER-2 levels are primarily assessed clinically using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). However, these assays typically rely on tumor biopsy or surgery, which are associated with high invasiveness, complex procedures, expensive equipment, the need for specialized personnel, poor patient compliance, unsuitability for dynamic disease monitoring, poor concordance between IHC and ISH results, and the risk of false-negative results. Studies have shown that the HER-2 extracellular domain (ECD) can be enzymatically cleaved and released into the blood. The optimal cutoff value for distinguishing HER-2 in the serum of healthy individuals (2-15 ng / mL) from that of breast cancer patients (15-75 ng / mL) is 15 ng / mL. Therefore, HER-2 levels can also be measured in serum, and the development of accurate serum HER-2 quantification methods is crucial to meet clinical needs.

[0099] Biosensors are universal chemical sensors that utilize biologically derived molecular recognition mechanisms. Specifically, they are devices that convert substrate-specific signals, such as enzymes or ion channels, into electrical signals that are accompanied by changes in the movement of substances, chemical potential, heat, or light. When biosensors are applied to the detection of HER-2, HER-2 can cause changes in the biosensor's electrical signal, which can be used to qualitatively analyze HER-2 levels. Existing biosensors for detecting HER-2 concentrations often fail to achieve the required level of accuracy, resulting in ambiguous diagnostic results near critical thresholds. Therefore, the development of an accurate and sensitive biosensor is crucial for early diagnosis, therapeutic efficacy monitoring, and prognostic assessment of breast cancer.

[0100] The following is attached Figure 1-6 The embodiments of the present application will be described in detail.

[0101] In some embodiments, the biosensor includes: an electrode substrate, and a composite nano-conductive substrate material, cDNA, and a competitive probe sequentially connected to the electrode substrate; wherein the composite nano-conductive substrate material is made of NH2-UiO-66@AQ (amino-functionalized metal-organic framework material loaded with anthraquinone-2-carboxylic acid, Amino-functionalized UiO-66@Anthraquinone-2-Carboxylic Acid), AuNPs (gold nanoparticles), and IL-rGO (Ionic Liquid Functionalized Reduced Graphene Oxide); the competitive probe is made by pairing Apt-Bio (Aptamer-Biotin Conjugate) and DSN-Probe (Duplex-Specific Nuclease Probe);

[0102] The sequence of the Apt-Bio is shown as 5'-Biotin-SEQ ID NO: 1-3', and the sequence of the cDNA is shown as 5'-SH-(CH2)6-SEQ ID NO: 2-3'.

[0103] like Figure 1 As shown, a method for detecting HER-2 with a biosensor comprises:

[0104] S100: placing the biosensor in a solution containing HER-2 to dissociate a portion of the competitive probe from the cDNA;

[0105] Specifically, the prepared biosensor is placed in a solution containing HER-2. Because the binding affinity of HER-2 to cDNA is greater than the binding affinity of the competing probe to cDNA, when HER-2 molecules are present, they compete with the competing probe bound to the cDNA. Consequently, some of the competing probe dissociates from the cDNA and enters the solution.

[0106] S200: generating, by the biosensor, a first current signal based on the composite nano-conductive base material and a second current signal based on the competitive probe remaining on the cDNA;

[0107] Specifically, after interacting with HER-2, the biosensor was removed from the HER-2 solution, washed, and then a first current signal and a second current signal were simultaneously collected using an electrochemical workstation. Because the competitive probe (non-conductive substance) detached from the cDNA in the presence of HER-2, the first current signal generated by the composite nano-conductive substrate on the biosensor increased. Simultaneously, because the competitive probe detached from the cDNA in the presence of HER-2, the amount of competitive probe remaining on the cDNA decreased, resulting in a decrease in the second current signal generated by the biosensor based on the competitive probe.

[0108] S300: Determine the HER-2 content based on the first current signal and the second current signal.

[0109] Specifically, the biosensor accurately converts the ratio of the first and second current signals into a numerical HER-2 concentration value by combining it with a pre-established standard curve. This dual-signal collaborative detection mechanism significantly reduces detection error compared to traditional single-signal detection, enabling highly sensitive quantitative analysis of HER-2.

[0110] In this example, the electrode substrate serves as the physical support and electron conduction foundation for the entire biosensor. The composite nanoconductive substrate material supported on its surface is composed of NH2-UiO-66@AQ, AuNPs, and IL-rGO. The interlayered IL-rGO, supported on the electrode substrate, serves as both an electron transport platform and provides binding sites for the AuNPs and NH2-UiO-66@AQ. The AuNPs, with their excellent electron conductivity, enable the formation of a continuous electron transport channel within the composite material. They also provide binding sites for cDNA, facilitating the subsequent attachment of a competitive probe. The NH2-UiO-66@AQ is fabricated from the metal-organic framework NH2-UiO-66 and the electroactive molecule AQ. The biosensor generates a primary current signal based on the AQ. The competitive probe is prepared by pairing Apt-Bio with DSN-Probe. The competitive probe binds complementary to the cDNA through Apt-Bio, resulting in a secondary current signal based on the DSN-Probe. When HER-2 is present in the solution, since the binding affinity of HER-2 to cDNA is greater than the binding affinity of Apt-Bio to cDNA, some of the competitive probes will dissociate from the cDNA and enter the solution, reducing the competitive probes remaining on the cDNA. Therefore, the second current signal generated by the biosensor based on the DSN-Probe in the competitive probe is reduced. In addition, since the competitive probe is a non-conductive substance, after some of the competitive probes dissociate, the first current signal generated by the biosensor based on AQ is enhanced. The ratio of the first current signal to the second current signal can accurately obtain the HER-2 concentration value. Compared with traditional single signal detection, this greatly reduces external or system detection errors, thereby improving the accuracy of HER-2 concentration detection.

[0111] In some embodiments, a method for detecting HER-2 with a biosensor further comprises:

[0112] S400: performing a color development test on the competitive probe dissociated from the cDNA, and obtaining a color development signal;

[0113] Specifically, a colorimetric test is performed on the competitor probe that has dissociated from the cDNA, and a colorimetric signal is obtained. The principle of this colorimetric test is based on the specific reaction of the colorimetric reagent with the competitor probe. After the colorimetric reagent reacts with the competitor probe to develop color, the optical density corresponding to the color change can be accurately measured using a spectrophotometer or other equipment to obtain the colorimetric signal. The intensity of the colorimetric signal can also be determined by color identification using a smartphone. The intensity of the colorimetric signal is related to the amount of dissociated competitor probe, and the amount of dissociated competitor probe is correlated with the HER-2 content in the sample. Therefore, the colorimetric signal can reflect the HER-2 content.

[0114] S500: determining the HER-2 content based on the color development signal;

[0115] Specifically, determining HER-2 levels based on colorimetric signals requires the establishment of an accurate standard curve. When testing actual samples, the measured colorimetric signal intensity is substituted into the standard curve, and through curve fitting, the HER-2 content in the sample can be accurately calculated.

[0116] In the step S300, after determining the HER-2 content based on the first current signal and the second current signal, the method further includes:

[0117] S600: Calibrate the HER-2 content determined by the first current signal and the second current signal using the HER-2 content determined by the color development signal.

[0118] Specifically, compared to single-detection methods, the colorimetric mode effectively covers a wider range of HER-2 concentrations. When testing low-concentration samples, the colorimetric signal changes correlate with the amount of competitive probe dissociation, accurately capturing signal differences caused by low HER-2 levels. For high-concentration samples, the colorimetric mode avoids the problem of "signal saturation," ensuring that the detection signal continues to change with increasing HER-2 concentration, ensuring the validity and accuracy of the test results and significantly improving the detection system's adaptability to samples of varying concentrations. The colorimetric mode verifies HER-2 content from a different detection principle, reducing errors caused by interfering substances in the sample or accidental factors during the detection process, further improving the reliability of test results and providing more accurate data support for clinical diagnosis and treatment.

[0119] The technical solutions in this application are described below with reference to specific embodiments.

[0120] Example 1

[0121] In some embodiments, the composite nano-conductive substrate material is prepared by the following method:

[0122] NH2-UiO-66@AQ (amino-functionalized metal-organic framework material loaded with anthraquinone-2-carboxylic acid, Amino-functionalized UiO-66@Anthraquinone-2-Carboxylic Acid), AuNPs (gold nanoparticles) and IL-rGO (Ionic Liquid Functionalized Reduced Graphene Oxide, ionic liquid functionalized reduced graphene oxide) are placed in deionized water and mixed and ultrasonicated to obtain the composite nano-conductive substrate material.

[0123] Specifically, 1 mL of the NH2-UiO-66@AQ aqueous solution (from Example 4), 1 mL of the AuNPs aqueous solution (from Example 3), and 1 mL of the IL-rGO aqueous solution (from Example 2) were mixed and sonicated for 2 hours to obtain the MFN-substrate (Metal-Functionalized Nanocomposite Substrate), which serves as the composite nanoconductive substrate. The MFN-substrate was then redissolved in 1 mL of deionized water to obtain the MFN-substrate aqueous solution, which was stored at 4°C until use.

[0124] In this embodiment, a composite nanoconductive substrate material is synthesized from NH2-UiO-66@AQ, AuNPs, and IL-rGO. IL-rGO serves as an electron transport platform. IL-rGO forms an interlayer structure, is loaded on an electrode substrate, and provides binding sites for AuNPs and NH2-UiO-66@AQ. AuNPs have excellent electron conductivity and can construct a continuous electron transport channel in the composite material. At the same time, AuNPs provide binding sites for cDNA for subsequent connection to competitive probes. NH2-UiO-66@AQ is NH2-UiO-66 (amino-functionalized UiO-66, an amino-functionalized metal-organic framework material) loaded with AQ (anthraquinone-2-carboxylic acid). As a metal-organic framework material, NH2-UiO-66 has an ultra-high specific surface area and a regular pore structure, which is a suitable material for loading AQ (anthraquinone-2-carboxylic acid). Providing an ideal nanoscale "reservoir," AQ, an electroactive molecule, is loaded into the pores of NH2-UiO-66 via π-π stacking or coordination. In electrochemical detection, AQ acts as an electron mediator, rapidly transferring electrons and significantly enhancing the charge transfer efficiency between the electrode and biomolecules, thereby improving detection sensitivity. The porous structure of NH2-UiO-66 not only protects AQ from environmental interference but also further amplifies the signal by enriching target molecules (such as HER-2), enabling highly sensitive detection of low-concentration biomarkers. This composite nanoconductive substrate exhibits excellent conductivity and provides binding sites for cDNA to attach to competing probes. Upon dissociation of the competing probe, an enhanced primary current signal is generated, providing accurate evidence for HER-2 detection.

[0125] Example 2

[0126] The IL-rGO was prepared by the following method:

[0127] IL-NH2 (amino-functionalized ionic liquid) and KOH are placed in a graphene homogeneous solution for reaction to obtain IL-rGO.

[0128] Specifically, 5 g of IL-NH2 viscous liquid was dispersed in 25 mL of a graphene homogeneous solution with a concentration of 0.5 mg / mL, and the mixture was continuously stirred after ultrasonic treatment for 30 minutes to obtain a mixed solution; 25 mgKOH was added to the above mixed solution, and the mixture was ultrasonically treated again for 50 minutes, at which time the mixed solution was converted into a homogeneous solution; the above homogeneous solution was refluxed and vigorously stirred at 80°C for 24 hours, and then the product was collected by centrifugation and washed with ethanol and deionized water in sequence to obtain IL-rGO; IL-rGO was dispersed in deionized water and the concentration was adjusted to 1.5 mg / mL to obtain an IL-rGO aqueous solution, which was stored at 4°C for later use.

[0129] Example 3

[0130] The AuNPs were prepared by the following method:

[0131] dissolving trisodium citrate dihydrate in deionized water and heating to boiling to obtain a trisodium citrate dihydrate solution;

[0132] adding HAuCl4 solution to the trisodium citrate dihydrate solution to obtain a reaction solution;

[0133] After the reaction solution changes color, it is cooled, and trisodium citrate dihydrate and HAuCl4 solution are alternately added to the cooled reaction solution to obtain AuNPs.

[0134] Specifically, 51.76 mg of trisodium citrate dihydrate was added to 80 mL of deionized water to obtain a mixed solution; the mixed solution was placed in a three-necked flask, stirred vigorously and heated to boiling for 15 minutes to obtain a trisodium citrate dihydrate solution; 533 μL of HAuCl4 solution was slowly added to the trisodium citrate dihydrate solution to obtain a reaction solution; the color of the reaction solution changed from light yellow to bright red within 10 minutes, indicating that gold nanoparticles were successfully synthesized, and then the reaction solution was cooled to 90°C, and 553 μL of trisodium citrate solution and 533 μL of HAuCl4 solution were alternately added to the reaction solution in sequence, with an interval of 2 minutes between the two, and this operation was repeated seven times to obtain AuNPs; the obtained AuNPs were naturally cooled to room temperature, centrifuged at 8000 rpm for 20 minutes, and then resuspended in 10 mL of deionized water to obtain an AuNPs aqueous solution, which was stored at 4°C for use.

[0135] Example 4

[0136] The NH2-UiO-66@AQ was prepared by the following method:

[0137] Dissolving zirconium tetrachloride, benzoic acid, and aminoterephthalic acid in DMF and reacting to obtain a crude NH2-UiO-66 product. Washing and drying the crude NH2-UiO-66 product to obtain NH2-UiO-66 (amino-functionalized UiO-66, an amino-functionalized metal-organic framework material, MOF).

[0138] Dissolving the NH2-UiO-66, AQ and carboxyl activator in a methanol solution and reacting the solution to obtain an intermediate reaction solution.

[0139] N-hydroxysuccinimide was added to the intermediate reaction solution to continue the reaction to obtain NH2-UiO-66@AQ.

[0140] Specifically, 120 mg of ZrCl₄, 1.9 g of benzoic acid, and 110 mg of aminoterephthalic acid were completely dissolved in 10 mL of N,N-dimethylformamide (DMF) and sonicated to obtain a mixture. The mixture was then transferred to a polytetrafluoroethylene-lined stainless steel autoclave and reacted at 120°C for 24 h to obtain a crude NH₂-UiO-66 product. The crude NH₂-UiO-66 product was washed three times with DMF and three times with methanol, respectively, and dried in vacuo at 60°C overnight to obtain NH₂-UiO-66.

[0141] 2 mg of NH2-UiO-66 and 2 mg of AQ were added to 2 mL of ethanol and sonicated to fully dissolve. Then, 1.5 mg of EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) was added and shaken for 30 min to obtain an intermediate reaction solution.

[0142] 1.5 mg of NHS (N-Hydroxysuccinimide) was added to the intermediate reaction solution, and the mixture was ultrasonicated for 2 h and shaken for another 3 h to obtain a crude NH2-UiO-66@AQ product. The crude NH2-UiO-66@AQ product was collected by centrifugation at 8000 rpm for 10 min and washed twice with ethanol and deionized water to obtain NH2-UiO-66@AQ. NH2-UiO-66@AQ was dissolved in 2 mL of deionized water to obtain an NH2-UiO-66@AQ aqueous solution for later use.

[0143] Example 5

[0144] In order to construct a high-performance biosensor, a stable substrate material is crucial. Therefore, the performance of NH2-UiO-66, NH2-UiO-66@AQ obtained in Example 4 and MFN-Substrate obtained in Example 1 were characterized ( Figure 2 ), the specific analysis process is as follows:

[0145] like Figure 2 As shown in the SEM and TEM images of A and B, NH2-UiO-66 exhibits a regular octahedral structure with good dispersion and an average particle size of approximately 162.51 nm ( Figure 2 Middle G), after encapsulating AQ, its morphology remains basically unchanged ( Figure 2 Middle C and Figure 2 (D). Figure 2 As shown in Figure 1, the N2 adsorption-desorption isotherms of NH2-UiO-66 and NH2-UiO-66@AQ both exhibit type I isotherms, indicating that they have a microporous structure. The specific surface area of ​​NH2-UiO-66 is 1504.07 m 2 / g, while the specific surface area of ​​NH2-UiO-66@AQ is relatively small, only 857.54 m 2 / g, which may be due to the entry of AQ molecules into the NH2-UiO-66 framework. Figure 2 As shown in Figure 1, the pore volumes of NH2-UiO-66 and NH2-UiO-66@AQ are 0.87 cm 3 / g and 0.56 cm 3 / g, and the average pore sizes measured by density functional theory (DFT) model are 1.96 nm and 1.81 nm, respectively. These decreases in pore volume and pore size further indicate that AQ is mainly located in the nanochannels of NH2-UiO-66.

[0146] In addition, the X-ray diffraction (XRD) pattern of NH2-UiO-66 showed characteristic diffraction peaks at 7.34° and 8.48°, indicating that its crystal structure is good ( Figure 2 J). The XRD pattern of NH2-UiO-66@AQ is consistent with that of NH2-UiO-66, indicating that the introduction of AQ did not change the lattice structure of NH2-UiO-66. To further verify the formation of NH2-UiO-66@AQ, Fourier transform infrared spectroscopy (FT-IR) and ultraviolet-visible absorption spectroscopy (UV-Vis) tests were carried out. Figure 2 As shown in K and its illustration, compared with NH2-UiO-66, the FT-IR spectrum of NH2-UiO-66@AQ is at 1675 cm -1A new stretching vibration peak appears at 3376 cm, which corresponds to the carbonyl group on the central ring structure of AQ. In addition, the amino group (-NH2) peak (3376 cm -1 and 3485 cm -1 ) disappears in NH2-UiO-66@AQ, which may be due to the fact that a certain amount of AQ is connected to NH2-UiO-66 through amide bonds, covering some amino sites. Figure 2 As shown in Figure 1, the UV-Vis spectrum of NH2-UiO-66@AQ shows absorption peaks at 272 nm and 390 nm, corresponding to the π→ In addition, characteristic absorption peaks of AQ were observed at 256 nm and 331 nm in the UV-visible absorption spectrum, further confirming the successful integration of AQ into NH2-UiO-66. These results demonstrate that NH2-UiO-66@AQ was successfully prepared in Example 4 and can be used to construct biosensor substrates with strong electrochemical signals.

[0147] Subsequently, NH2-UiO-66@AQ (via π-π stacking) and AuNPs (via Au-N bonds) were co-loaded onto IL-rGO to form an integrated MFN-Substrate. As shown in the SEM and TEM images ( Figure 2 In Figures E and F, NH2-UiO-66@AQ and AuNPs are evenly distributed on the IL-rGO surface, and the three components are well mixed. In summary, the MFN-Substrate obtained in Example 1 has excellent electrical conductivity, generating a stable electrochemical signal while effectively immobilizing cDNA.

[0148] Example 6

[0149] The competitive probe is prepared by pairing Apt-Bio (Aptamer-Biotin Conjugate) and DSN-Probe (Duplex-Specific Nuclease Probe); wherein the DSN-Probe is prepared by MOF@FeTCPP and a coupling agent, and the MOF@FeTCPP is coupled to the Apt-Bio via the coupling agent.

[0150] Specifically, the DSN-Probe is prepared by MOF@FeTCPP and a coupling agent, specifically comprising:

[0151] 3 mg of MOF@FeTCPP (from Synthesis Method 3 in Example 7) was dissolved in 1 mL of deionized water. 1.2 mg of EDC and 1.2 mg of N-hydroxysuccinimide (NHS) were added, mixed thoroughly, and reacted at room temperature for 20 min. After the reaction, the intermediate product was collected by centrifugation and washed multiple times with deionized water to remove excess EDC and NHS. The intermediate product was then redispersed in 500 μL of deionized water, 50 μL of a 1 mg / mL SA (Streptavidin) solution was added, and the mixture was sonicated for 4 h to obtain the DSN-Probe. The DSN-Probe was dissolved in 1 mL of deionized water to obtain the DSN-Probe aqueous solution, which was stored at 4°C for later use.

[0152] In this embodiment, Apt-Bio is a pairing partner of cDNA, and the competitive probe is connected to the biosensor via Apt-Bio. When HER-2 is present, the affinity between HER-2 and cDNA is greater than that between Apt-Bio and cDNA, causing Apt-Bio to dissociate from cDNA, thereby dissociating the competitive probe from cDNA.

[0153] Example 7

[0154] In this application, there are three methods for synthesizing MOF@FeTCPP, namely, synthesis method 1, synthesis method 2 and synthesis method 3.

[0155] The first method for synthesizing MOF@FeTCPP includes the following steps:

[0156] Dissolving benzene trimecchymic acid and FeTCPP in a mixture of ethanol and DMF to obtain a benzene trimecchymic acid-FeTCPP mixture;

[0157] Then, a Cu(NO3)2·3H2O aqueous solution is added to the benzenetricarboxylic acid-FeTCPP mixed solution, and the mixture is ultrasonically dispersed and then heated for reaction to obtain the MOF@FeTCPP.

[0158] Specifically, 0.5 g of BTC (1,3,5-Benzenetricarboxylic acid) and 30 mg of iron (III)-tetrakis(4-carboxyphenyl)chlorinated porphyrin (FeTCPP) were dissolved in 15 mL of a 1:1 volume ratio mixture of ethanol and DMF to obtain a benzenetricarboxylic acid-FeTCPP mixture; the above benzenetricarboxylic acid-FeTCPP mixture was mixed with 7.5 mL of an aqueous solution containing 1.04 g of Cu(NO3)2·3H2O and then ultrasonically treated. The mixture was heated at 60°C for 7 h, cooled to room temperature, and then centrifuged at 8000 rpm for 15 min to obtain a crystalline material; the crystalline material was thoroughly washed with ethanol and dried in vacuo at 60°C overnight to obtain MOF@FeTCPP.

[0159] The difference between the second synthesis method of MOF@FeTCPP and the first synthesis method is that the amount of ethanol and DMF mixture used is 50 mL, and the amount of Cu(NO3)2·3H2O aqueous solution used is 40 mL.

[0160] The third method for synthesizing MOF@FeTCPP includes the following steps:

[0161] Dissolving Cu(NO3)2·3H2O and polyvinyl pyrrolidone in methanol and stirring uniformly to obtain a first methanol solution;

[0162] Adding benzenetricarboxylic acid to methanol and stirring evenly to obtain a second methanol solution;

[0163] adding FeTCPP to methanol to obtain a third methanol solution;

[0164] adding the second methanol solution to the first methanol solution until the solution becomes turbid to obtain a turbid solution;

[0165] The third methanol solution is added to the turbid liquid to react to obtain the MOF@FeTCPP.

[0166] Specifically, 0.9 g Cu(NO3)2·3H2O and 0.4 g PVP (polyvinyl pyrrolidone) were dissolved in 50 mL of methanol and gently stirred for 5 min to obtain a first methanol solution; 0.43 g BTC was dissolved in 48 mL of methanol to obtain a second methanol solution; 30 mg FeTCPP was dissolved in 2 mL of methanol (15 mg / mL) to obtain a third methanol solution; the second methanol solution was slowly added dropwise to the first methanol solution until the solution changed from transparent blue to cobalt blue and became turbid, obtaining a turbid solution; the third methanol solution was slowly added dropwise to the turbid solution, mixed thoroughly, and aged in a constant temperature water bath at 27°C for 24 h, and then the MOF@FeTCPP crude product was collected by centrifugation; the MOF@FeTCPP crude product was thoroughly washed with methanol and dried at 60°C overnight to obtain MOF@FeTCPP, i.e., HKUST-1 (MOF) loaded with FeTCPP.

[0167] In the third synthesis method of MOF@FeTCPP, HKUST-1 loaded with FeTCPP was prepared by a one-pot method. In order to compare the effect of FeTCPP on the performance of HKUST-1, HKUST-1 was prepared separately using the third synthesis method.

[0168] The preparation steps of HKUST-1 include:

[0169] 0.9 g Cu(NO3)2·3H2O and 0.4 g PVP (polyvinyl pyrrolidone) were dissolved in 50 mL of methanol and gently stirred for 5 min to obtain a first methanol solution; 0.43 g BTC was dissolved in 48 mL of methanol to obtain a second methanol solution; the second methanol solution was slowly added dropwise to the first methanol solution until the solution changed from transparent blue to cobalt blue and became turbid, obtaining a turbid solution; the turbid solution was aged in a constant temperature water bath at 27°C for 24 h, and the crude HKUST-1 product was collected by centrifugation; the crude HKUST-1 product was thoroughly washed with methanol and dried at 60°C overnight to obtain HKUST-1.

[0170] Example 8

[0171] The HKUST-1, MOF@FeTCPP obtained in Example 7 and the DSN-Probe obtained in Example 6 were characterized ( Figure 3 ).

[0172] The SEM and TEM images of HKUST-1 and MOF@FeTCPP show that they both present a typical regular octahedral structure with uniform size ( Figure 3After encapsulating FeTCPP, the prepared MOF@FeTCPP retains its octahedral structure due to the hydrophobic interaction between the porphyrin on FeTCPP and the organic ligands in the HKUST-1 framework. Notably, the average particle size of MOF@FeTCPP is 300.12 nm, smaller than the 336.52 nm of HKUST-1, making it more suitable for use as a signal probe ( Figure 3 Middle G).

[0173] like Figure 3 As shown in Figure 1, the N2 adsorption-desorption isotherms of HKUST-1 and MOF@FeTCPP are both type IV isotherms. The obvious adsorption phenomenon in the low-pressure region and the hysteresis loop in the medium- and high-pressure regions indicate the coexistence of micropores and mesopores in the materials. Since the porphyrin molecules are stored in the pores of HKUST-1, the BET specific surface area of ​​MOF@FeTCPP (509.28 m 2 / g) is less than HKUST-1 (810.20 m 2 / g). In addition, the pore volume of MOF@FeTCPP (0.31 cm 3 / g) and the average pore size (2.41 nm) are also smaller than those of HKUST-1 (0.53 cm 3 / g, 2.64 nm), indicating that the FeTCPP molecules are effectively confined in the nanopores of HKUST-1 ( Figure 3 Middle I).

[0174] To obtain the best-performing MOF@FeTCPP, the synthesis method and FeTCPP concentration were optimized. In the presence of a constant concentration of H2O2, the catalytic performance of MOF@FeTCPP synthesized by different methods was evaluated using TMB (3,3′,5,5′-Tetramethylbenzidine) as a substrate. Figure 3 As shown in M, the MOF@FeTCPP synthesized by method 3 has the highest catalytic activity. In addition, the Michaelis constants (Km) calculated based on the Lineweaver-Burk double reciprocal plot are 0.689 mM (method 1), 0.562 mM (method 2), and 0.358 mM (method 3), respectively. Figure 3 The MOF@FeTCPP synthesized by method 3 has the smallest Km value, indicating that it has the strongest affinity for the substrate TMB.

[0175] Subsequently, a series of MOF@FeTCPP were synthesized by using method 3 and varying the FeTCPP concentration. The catalytic activity of MOF@FeTCPP synthesized at different FeTCPP concentrations was evaluated in the same manner as before ( Figure 3The results showed that at a FeTCPP concentration of 15 mg / mL, MOF@FeTCPP exhibited optimal enzyme activity, with the lowest Km (0.261 mM), surpassing those of MOF@FeTCPP synthesized at 5 mg / mL (0.420 mM), 10 mg / mL (0.358 mM), 20 mg / mL (0.335 mM), and 25 mg / mL (0.397 mM). High FeTCPP concentrations led to decreased enzyme activity, likely due to poor morphology and low FeTCPP encapsulation efficiency. Therefore, a green, room-temperature, one-step method (synthesis method three) was selected for the subsequent preparation of MOF@FeTCPP nanozymes, using 15 mg / mL of FeTCPP to obtain uniform particle size.

[0176] In order to improve the recognition efficiency, streptavidin (SA) was modified on the surface of MOF@FeTCPP to construct MOF@FeTCPP@SA (DSN-Probe). Figure 3 As shown in Figures E and F, after SA modification, aggregates appeared on the surface of MOF@FeTCPP, indicating that SA protein was successfully loaded on its surface. Figure 3 As shown in Figure 1, the XRD diffraction peaks of MOF@FeTCPP and DSN-Probe are completely consistent with those of HKUST-1, indicating that the introduction of FeTCPP and SA does not affect the fine crystal structure of HKUST-1. The most prominent characteristic peak in the XRD pattern is located at 11.70°.

[0177] Subsequently, the formation of DSN-Probe was further verified by FT-IR and UV-Vis spectroscopy. Figure 3 As shown in K, compared with HKUST-1, FeTCPP, MOF@FeTCPP and DSN-Probe all observed 1002 cm -1 The characteristic peaks of α, which correspond to the C–H bending vibration of the pyrrole ring, further confirm that FeTCPP is successfully encapsulated in the pores of HKUST-1. Figure 3 As shown in Figure 1, the maximum Soret absorption peak of FeTCPP in ethanol is located at 413 nm, while the maximum absorption peaks of MOF@FeTCPP and DSN-Probe are red-shifted to approximately 425 nm. This red-shift is likely due to the hydrophobicity of the octahedral cavity and the sensitivity of the Soret absorption band to the dielectric constant of the solvent. Taken together, these results confirm the successful construction of the DSN-Probe, which can output enhanced dual signals in both electrochemical and colorimetric modes.

[0178] Example 9

[0179] A method for preparing a biosensor comprises the following steps:

[0180] Adding the composite nano-conductive base material solution dropwise onto the pretreated surface of the electrode substrate and drying the resulting solution to obtain a first electrode substrate;

[0181] adding the cDNA solution dropwise onto the surface of the first electrode substrate, incubating and then washing to obtain a second electrode substrate;

[0182] Adding MCH blocking solution dropwise to the surface of the second electrode substrate, reacting and washing to obtain a third electrode substrate;

[0183] adding an Apt-Bio solution dropwise onto the surface of the third electrode substrate, incubating and then washing to obtain a fourth electrode substrate;

[0184] The DSN-Probe solution is dropped onto the surface of the fourth electrode substrate, and the biosensor is obtained after incubation.

[0185] Specifically, a glassy carbon electrode (GCE, 3 mm in diameter) was polished for 2 min by dipping alumina powder (particle size 0.1 and 0.05 μm) on the surface of suede, and then washed with deionized water and anhydrous ethanol for 3 min each, and finally dried under nitrogen to obtain pretreated glassy carbon; 6 μL of MFN-Substrate aqueous solution (from Example 1) was dropped on the clean GCE surface and dried at 37°C to form a uniform film to obtain a first electrode matrix; cDNA was placed in TE buffer (a buffer solution prepared by Tris (trishydroxymethylaminomethane) and EDTA (ethylenediaminetetraacetic acid)) to prepare a cDNA solution, 6 μL of 10 μM cDNA solution was dropped on the surface of the first electrode matrix, and placed at 4°C overnight to allow the cDNA to connect to the AuNPs in the MFN-Substrate on the electrode surface to obtain a second electrode matrix; in order to block nonspecific binding sites, 6 μL of 1 μM cDNA solution was dropped on the surface of the second electrode matrix at room temperature. mM 6-mercaptohexanol (MCH, 6-Mercaptohexanol) solution was reacted for 30 minutes to obtain the third electrode substrate; Apt-Bio was placed in TE buffer to prepare an Apt-Bio solution, and 6 μL of a 5 μM Apt-Bio solution was dropwise added to the electrode surface and incubated at 37°C for 2 hours to allow Apt-Bio to couple with cDNA; 6 μL of a 3 mg / mL DSN-Probe aqueous solution (from Example 6) was dropwise added to the electrode surface and incubated at 37°C for 1 hour to finally form a DSN-Probe / Apt-Bio / MCH / cDNA / MFN-Substrate / GCE biosensor.

[0186] The sequence of the Apt-Bio is shown as 5'-Biotin-SEQ ID NO:1-3', and the sequence of the cDNA is shown as 5'-SH-(CH2)6-SEQ ID NO:2-3'. During each modification step, the electrode was washed with 10 mM PBS buffer (pH 7.4) to remove unbound molecules from the electrode surface. For HER-2 detection, the assembled biosensor was immersed in a HER-2 standard solution or actual sample for testing.

[0187] Example 10

[0188] The performance of the biosensor (from Example 9) was evaluated ( Figure 4 ), the specific evaluation process is as follows:

[0189] The conductivity of the DSN-Probe / Apt-Bio / MCH / cDNA / MFN-Substrate / GCE biosensor was evaluated by cyclic voltammetry (CV) in a solution containing 0.1 M KCl at a scan rate of 50 mV / s using 10 mM K3[Fe(CN)6] as a probe. Figure 4 (A). Compared to the bare GCE electrode (a), the IL-rGO-modified GCE (b) exhibits significantly enhanced redox peak current, validating the ability of IL-rGO to promote electron transport at the electrode interface. After NH2-UiO-66@AQ was modified on IL-rGO, the current signal of IL-rGO-NH2-UiO-66@AQ / GCE (c) decreased slightly. However, due to the incorporation of highly electroactive AuNPs, the MFN-substrate / GCE (d) produced the largest current response, demonstrating that MFN-substrate is an excellent conductive nanocomposite material suitable as a biosensor substrate.

[0190] In addition, the Randles-Sevcik equation was used to calculate the effective electrochemical active surface area of ​​the interface modified by different materials:

[0191]

[0192] Where Ipa is the anodic peak current (A), n is the number of electron transfers, and Aeff is the effective electrode surface area (cm 2 ), D is the diffusion coefficient of K3[Fe(CN)6] (6.73 × 10 -6 cm 2 / s), ν is the scan rate (V / s), C0 is the K3[Fe(CN)6] concentration (mol / cm 3). The calculated effective surface area of ​​MFN-Substrate / GCE is 0.0632 cm 2 , which are 2.01, 1.10, and 1.29 times those of bare GCE, IL-rGO / GCE, and IL-rGO-NH2-UiO-66@AQ / GCE, respectively. MFN-Substrate / GCE shows the largest effective surface area and excellent electrochemical performance, which is attributed to the excellent conductivity of IL-rGO and AuNPs.

[0193] The CV curves of MFN-Substrate / GCE at scan rates of 10-100 mV / s were further measured. Figure 4 As shown in Figure B, as the scan rate increases, the redox current response also increases. The peak current has a good linear relationship with the square root of the scan rate, and the linear equation is: pa = 13.518v 1 / 2 + 2.034 ( = 0.998) and Ipc = -12.010v 1 / 2 –4.657 (R2 2 = 0.997)( Figure 4 These results indicate that the electrochemical reaction on MFN-Substrate / GCE is a typical diffusion-controlled process.

[0194] In order to characterize the construction process of the electrode interface, electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) tests were performed in 0.1 M KCl solution containing 10 mM K3[Fe(CN)6]. Figure 4As shown in D, the diameter of the semicircle in the Nyquist plot of EIS is related to the charge transfer resistances (Rct) of the different modified electrodes. Compared with bare GCE (a), the Rct of MFN-Substrate / GCE (b) is significantly reduced, indicating that the excellent conductivity of the MFN-Substrate material is conducive to electron transport. However, as the electrode is modified with cDNA (c), MCH (d), Apt-Bio (e) and DSN-Probe (f) in sequence, Rct gradually increases. This is because non-conductive biological molecules hinder electron conduction, verifying the success of the layer-by-layer self-assembly process of the HER-2 biosensor. After incubating HER-2, the Rct of HER-2 / DSN-Probe / Apt-Bio / MCH / cDNA / MFN-Substrate / GCE (g) ct This is because HER-2 competes with Apt-Bio-DSN-Probe, causing some non-conductive molecules to dissociate from the electrode surface. As expected, the current response trend of DPV is consistent with R ct The opposite change ( Figure 4 The above results indicate that the HER-2 biosensor has been successfully constructed.

[0195] To study the electrochemical response of the substrate material and its probe, DPV tests were performed in 10 mM PBS buffer at pH = 7.4 after adding 10 mM H2O2. Figure 4 As shown in Figure (F), no significant peak current signals were observed for bare GCE (a) and IL-rGO-NH2-UiO-66 / GCE (b). However, after the introduction of the electroactive molecule AQ, a significant peak current signal was observed for IL-rGO-NH2-UiO-66@AQ / GCE (c) at -660 mV. In contrast, the current signal for MFN-Substrate / GCE (d) was further enhanced due to the high conductivity of AuNPs. Furthermore, the electrocatalytic current response of MOF@FeTCPP to H2O2 was observed at -304 mV for DSN-Probe / GCE (e). As the addition of more non-conductive molecules gradually blocked the electron transport channel, the current signal generated by AQ gradually weakened in DSN-Probe / Apt-Bio / MCH / cDNA / MFN-Substrate / GCE (f). The electrochemical signals generated by AQ and MOF@FeTCPP were observed simultaneously, verifying their feasibility as electroactive probes for ratiometric sensors.

[0196] In addition, the stability of MFN-Substrate is crucial to the performance of biosensors. Figure 4As shown in Figure G, 10 consecutive DPV measurements were performed under the same conditions, and a stable and consistent signal response was obtained, indicating that MFN-Substrate has excellent stability as a substrate material for HER-2 biosensors.

[0197] To verify the feasibility of the biosensor for HER-2 detection, its electrochemical and colorimetric signal responses were analyzed. Figure 4 As shown in Figure H, in the absence of a target, the MOF@FeTCPP nanozyme can catalyze H2O2 to produce a strong electrochemical signal, while the current signal of AQ is weak. However, after the addition of 1 ng / mL HER-2, the current response of the MOF@FeTCPP nanozyme decreased significantly, while the AQ signal was significantly enhanced. This is because HER-2 has a high affinity for Apt-Bio-DSN-Probe, which causes some non-conductive biomolecules to dissociate from the electrode surface. In the colorimetric mode, compared with the absence of a target, the presence of 1 ng / mL HER-2 triggers the competitive dissociation of the MOF@FeTCPP nanozyme, thereby significantly enhancing the absorbance response of the TMB colorimetric solution, which is attributed to the excellent peroxidase-like activity of the MOF@FeTCPP nanozyme ( Figure 4 The significant signal change indicates that the designed biosensor has good feasibility in HER-2 detection.

[0198] Example 11

[0199] Detection of HER-2 standards by biosensor

[0200] For HER-2 detection, the assembled biosensor was immersed in a HER-2 standard solution or actual sample for testing. In electrochemical mode, the electrode was washed with PBS buffer and air-dried at room temperature before DPV measurement was performed in 10 mM PBS buffer (pH = 7.4) containing 10 mM H2O2. In colorimetric mode, 100 μL of a single-component TMB colorimetric solution was added to a 96-well plate containing the competitively released nanozymes. The reaction was incubated in the dark at 37°C for 15 minutes, followed by the addition of 100 μL of a stop solution. Subsequently, the ultraviolet-visible (UV-Vis) absorption spectrum was monitored using a microplate reader, and images were taken with a smartphone within 15 minutes and analyzed using ImageJ software.

[0201] The constructed biosensor (Example 9) was used to detect the HER-2 standard ( Figure 5 In electrochemical mode, DPV responses were recorded in 10 mM PBS buffer, pH = 7.4, containing 10 mM H2O2 ( Figure 5The results show that as the HER-2 concentration increases, the current signal (I1) of AQ gradually increases, while the current signal (I2) generated by MOF@FeTCPP nanozyme gradually decreases. This trend indicates that the change in electrochemical signal can effectively reflect the change in HER-2 content in the system. Figure 5 As shown in Figure B, the signal intensity ratio of AQ to MOF@FeTCPP (I1 / I2) showed a good linear relationship with the logarithm of HER-2 concentration in the range of 100 fg / mL to 100 ng / mL, and the regression equation was: y = 0.194 × lgC HER-2 + 0.398 (R 2 = 0.994). Based on 3S D / m (SD is the standard deviation of 10 blank sample measurements, and m is the slope of the calibration curve) and the limit of detection (LOD) was 28.509 fg / mL.

[0202] In the colorimetric mode, as the HER-2 concentration increases, the absorption peak of the TMB colorimetric solution in the UV-Vis spectrum is significantly enhanced ( Figure 5 (C), which is attributed to the excellent peroxidase-like activity of the competitively released MOF@FeTCPP nanozyme. Figure 5 As shown in Figure D, in the range of 100 fg / mL to 150 ng / mL, the absorbance at 450 nm showed a good linear relationship with the logarithm of the HER-2 concentration, and the regression equation was: y = 0.113 × lg C HER-2 + 0.058 (R 2 = 0.995), and its detection limit was 30.226 fg / mL.

[0203] In addition, HER-2 detection can be achieved through color recognition on smartphones ( Figure 5 (E). Figure 5 As shown in Figure F, the grayscale value obtained from ImageJ and the logarithm of HER-2 concentration showed good linearity in the range of 100 fg / mL to 150 ng / mL, and the regression equation was: y = -11.326 × lg C HER-2 + 214.013 (R 2 = 0.993), and its detection limit was 34.170 fg / mL.

[0204] It is worth noting that the electrochemical detection mode has a lower detection limit, while the colorimetric detection mode provides a wider detection range. In the detection of HER-2, the performance of the two detection modes complement each other: the electrochemical mode effectively reduces the detection limit and improves detection sensitivity; the colorimetric mode expands the detection range and effectively avoids the "signal saturation" phenomenon in high-concentration samples. In addition, the colorimetric mode has good visual readability, which improves user convenience and expands its potential scenarios in practical applications.

[0205] Subsequently, the key performance indicators of the biosensor, including specificity, reproducibility and stability, were evaluated. To verify the specificity, the HER-2 detection method was applied to the testing of multiple non-target analytes, including human epidermal growth factor receptor-1 (HER-1), human epidermal growth factor receptor-3 (HER-3), human epidermal growth factor receptor-4 (HER-4), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) and prostate specific antigen (PSA) (all at a concentration of 10 ng / mL). Figure 5 As shown in Figures G and H, compared with the strong signal exhibited by 1 ng / mL HER-2, the signals of these interferents were close to those of the blank control and had almost no effect on the current response and absorbance values ​​in the mixture, indicating that the biosensor has good specificity in both electrochemical and colorimetric modes.

[0206] To evaluate the reproducibility, five independent batches of biosensors were prepared for HER-2 detection. The electrochemical and fluorescence responses showed only minor variations, with relative standard deviations (RSDs) of 1.98% and 2.25%, respectively, indicating that the biosensor had good reproducibility ( Figure 5 I).

[0207] To investigate the stability of the biosensor, it was stored at 4°C and tested regularly over four consecutive weeks. Figure 5As shown in Figure J, the signal response decreased slightly after four weeks, while the electrochemical and fluorescence signals remained at 93.17% and 91.62% of their initial values, respectively, indicating that the biosensor has good long-term stability. The excellent performance of this biosensor makes it a promising tool for HER-2 quantitative analysis in clinical medicine.

[0208] Real sample analysis in cell lines and human serum

[0209] To evaluate the applicability and reliability of the constructed biosensor, HER-2 levels in cell lines and clinical human serum were analyzed ( Figure 6 A variety of cell lines were selected as research models, including HER-2-positive breast cancer cell lines (human breast adenocarcinoma cells, SK-BR-3 and human breast carcinoma cells, BT-474), HER-2-negative breast cancer cell lines (Michigan Cancer Foundation-7 breast cancer cells, MCF-7), HER-2-negative lung cancer cell lines (human lung adenocarcinoma cells, A-549), and non-cancerous cell lines (human embryonic kidney cells, HEK-293T). All cells were cultured in modified basal medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin / streptomycin at 37°C in a humidified atmosphere with 5% CO2. After cell counting, the cells were lysed using lysis buffer according to the reagent instructions. The supernatant was collected by centrifugation and stored at -80°C. Subsequently, the HER-2 content in the cell lysate was detected using the established biosensor platform.

[0210] The relative HER-2 expression levels in each cell line were evaluated and compared by recording signal changes. Figure 6 As shown in Figures A and B, the electrochemical and colorimetric signal responses of HER-2 positive cell lines were significantly enhanced and significantly higher than those of HER-2 negative cell lines. This trend is consistent with the actual expression levels of HER-2 in each cell line ( Figure 6 Among them, SK-BR-3 showed the highest HER-2 concentration, followed by BT-474, which had slightly lower but still high expression levels. HER-2 expression was low in MCF-7, very low in A-549, and almost undetectable in HEK-293T.

[0211] In addition, the biosensor was applied to cell lysates of SK-BR-3 and BT-474 cells, and the number of cells detected was 10 3 to 106 HER-2 levels within the range. Figure 6 As shown in D and E, there is a good linear relationship between the measured signal and the logarithm of the HER-2 concentration measured by ELISA (R 2 The results further validated the broad applicability of this biosensor for quantitative detection of HER-2 at the cellular level.

[0212] To evaluate the clinical diagnostic capability of this method, the constructed biosensor was used to quantitatively detect the HER-2 levels in the serum of healthy donors (n = 10) and HER-2 positive breast cancer patients (n = 10). Figure 6 As shown in Figures F and G, there are significant differences in electrochemical and colorimetric signal responses between healthy individuals and breast cancer patients, with patients showing stronger signal responses. Similarly, HER-2 concentrations in patient serum are significantly higher than those in healthy individuals ( Figure 6 (H, P < 0.0001). Figure 6 As shown in Figure 1, the results obtained by this method are highly consistent with those of commercially available ELISA kits, indicating that the biosensor has excellent detection accuracy. At the same time, the receiver operating characteristic (ROC) curve shows that the biosensor has extremely high accuracy in cancer diagnosis (area under the curve AUC = 1), and can effectively distinguish healthy people from breast cancer patients ( Figure 6 These results indicate that the biosensor can achieve accurate clinical diagnosis of breast cancer without complex operations and has great application potential.

[0213] During actual sample analysis, the biosensor simultaneously outputs electrochemical and colorimetric signals, with the concentration values ​​calculated by the two modes highly consistent, enabling cross-validation and ensuring the accuracy of HER-2 quantification, while avoiding false positive or false negative results that may arise from a single signal. Furthermore, the ratiometric electrochemical method, based on a built-in correction factor, effectively eliminates external or systematic errors, further improving the accuracy of HER-2 concentration detection. This collaborative self-calibration strategy, by integrating the built-in correction of ratiometric electrochemistry with the cross-validation of dual-modality detection, significantly enhances the accuracy and reliability of the biosensor for HER-2 analysis in real samples.

[0214] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present application (including the claims) is limited to these examples. Within the scope of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0215] While the present application has been described in conjunction with specific embodiments thereof, many alternatives, modifications and variations of these embodiments will be apparent to those skilled in the art in light of the foregoing description.

[0216] The embodiments of the present application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present application should be included in the scope of protection of this application.

Claims

1. A biosensor, characterized in that: include: An electrode substrate, and a composite nano-conductive base material, cDNA, and a competitive probe sequentially connected on the electrode substrate; wherein the composite nano-conductive base material is prepared by NH2-UiO-66@AQ, AuNPs, and IL-rGO; the competitive probe is prepared by pairing Apt-Bio and DSN-Probe; the DSN-Probe is prepared by MOF@FeTCPP, and AQ in the NH2-UiO-66@AQ is anthraquinone-2-carboxylic acid; The sequence of the Apt-Bio is shown as 5'-Biotin-SEQ ID NO: 1-3', and the sequence of the cDNA is shown as 5'-SH-(CH2)6-SEQ ID NO: 2-3'.

2. A biosensor according to claim 1, characterized in that: The NH2-UiO-66@AQ was prepared by the following method: Dissolving zirconium tetrachloride, benzoic acid, and aminoterephthalic acid in DMF and reacting to obtain a crude NH2-UiO-66 product; washing and drying the crude NH2-UiO-66 product to obtain NH2-UiO-66; Dissolving the NH2-UiO-66, AQ and carboxyl activator in a methanol solution and reacting the solution to obtain an intermediate reaction solution. N-hydroxysuccinimide was added to the intermediate reaction solution to continue the reaction to obtain NH2-UiO-66@AQ.

3. The biosensor according to claim 1, wherein: The IL-rGO was prepared by the following method: IL-NH2 and KOH are placed in a graphene homogeneous solution for reaction to obtain IL-rGO.

4. The biosensor according to claim 1, wherein: The AuNPs were prepared by the following method: dissolving trisodium citrate dihydrate in deionized water and heating to boiling to obtain a trisodium citrate dihydrate solution; adding HAuCl4 solution to the trisodium citrate dihydrate solution to obtain a reaction solution; After the reaction solution changes color, it is cooled, and trisodium citrate dihydrate and HAuCl4 solution are alternately added to the cooled reaction solution to obtain AuNPs.

5. The biosensor according to claim 1, wherein: The MOF@FeTCPP is coupled to the Apt-Bio via a coupling agent.

6. The biosensor according to claim 5, characterized in that: The coupling agent is a streptavidin coupling agent.

7. The biosensor according to claim 5, characterized in that: The MOF@FeTCPP was prepared by the following method: Dissolving benzene trimecchymic acid and FeTCPP in a mixture of ethanol and DMF to obtain a benzene trimecchymic acid-FeTCPP mixture; Then, a Cu(NO3)2·3H2O aqueous solution is added to the benzenetricarboxylic acid-FeTCPP mixed solution, and the mixture is ultrasonically dispersed and then heated for reaction to obtain the MOF@FeTCPP.

8. The biosensor according to claim 5, characterized in that: The MOF@FeTCPP was prepared by the following method: Dissolving Cu(NO3)2·3H2O and polyvinyl pyrrolidone in methanol and stirring uniformly to obtain a first methanol solution; Adding benzenetricarboxylic acid to methanol and stirring evenly to obtain a second methanol solution; adding FeTCPP to methanol to obtain a third methanol solution; adding the second methanol solution to the first methanol solution until the solution becomes turbid to obtain a turbid solution; The third methanol solution is added to the turbid liquid to react to obtain the MOF@FeTCPP.

9. A method for preparing the biosensor according to any one of claims 1 to 8, characterized in that: The following steps are involved: Adding the composite nano-conductive base material solution dropwise onto the pretreated surface of the electrode substrate and drying the resulting solution to obtain a first electrode substrate; adding the cDNA solution dropwise onto the surface of the first electrode substrate, incubating and then washing to obtain a second electrode substrate; Adding MCH blocking solution dropwise to the surface of the second electrode substrate, reacting and washing to obtain a third electrode substrate; adding an Apt-Bio solution dropwise onto the surface of the third electrode substrate, incubating and then washing to obtain a fourth electrode substrate; The DSN-Probe solution is dropped onto the surface of the fourth electrode substrate, and the biosensor is obtained after incubation.

Images