DR1 ratio type electrochemical-colorimetric dual-mode detection kit based on Cu-MOF and AuPt-coated PBA

Abstract

The invention discloses a DR1 ratio type electrochemical-colorimetric dual-mode detection kit based on Cu-MOF and AuPt-coated PBA. The kit mainly comprises a glassy carbon electrode, a magnetic bead, an antibody 1, bovine serum albumin, an AuPt-coated PBA-Ab2 biological conjugate and a copper-based metal organic framework Cu-MOF. Firstly, Ab1 is added into a centrifugal tube, Ab1 is coupled with MBs activated by EDC / NHS so as to be modified to the surfaces of the MBs through amido bonds, and then BSA is added to block unreacted binding sites; then adding a target object DR1, finally adding an AuPt-coated PBA-Ab2 conjugate, and carrying out magnetic separation to obtain a precipitate and a supernatant; the separated precipitate is used for colorimetric detection, and the supernatant is dropwise added to the surface of the Cu-MOF modified GCE through electrostatic adsorption for ratio-type electrochemical detection. The invention has the advantages of high sensitivity, strong selectivity, good stability, excellent anti-interference capability and the like, can realize ultra-sensitive and accurate detection of DR1, and has good applicability in human serum detection.

Description

Technical Field

[0001] This invention relates to a highly sensitive ratiometric electrochemical-colorimetric dual-mode detection kit based on copper-based metal-organic frameworks (Cu-MOF) and gold-platinum@Prussian blue analogs (AuPt@PBA MPs), for detecting transcription downregulatory factor 1 (DR1), a key biomarker of Hashimoto's thyroiditis (HT), and belongs to the field of bioanalytical technology. Background Technology

[0002] Hashimoto's thyroiditis (HT) is a prevalent autoimmune disease that continues to pose an increasing threat to human health and quality of life. Down-regulator of transcription 1 (DR1) is a key biomarker for HT, therefore, developing efficient DR1 detection methods is crucial for monitoring disease progression.

[0003] Currently, various sensing detection methods for disease biomarkers have been developed, including electrochemical, colorimetric, fluorescence, and photoelectrochemical methods. However, these single-mode, single-signal detection methods rely on a single output signal, leading to unreliable results. Changes in the detection environment or the presence of interfering substances can easily result in false positives, and the accuracy and reliability of the detection cannot be verified independently. Furthermore, they suffer from low sensitivity and poor selectivity.

[0004] In recent years, ratiometric sensors and dual-mode sensors have attracted widespread attention as two advanced sensing strategies due to their unique self-calibration and cross-verification capabilities. However, the success of this design still depends on two key components: first, a stable internal reference nanomaterial anchored to the electrode surface; and second, nanomaterials that can synergistically generate both electrochemical and colorimetric signals. This invention aims to construct a ratiometric electrochemical-colorimetric dual-mode detection kit based on Cu-MOF and AuPt@PBA nanomaterials to achieve ultrasensitive and accurate detection of DR1. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a ratiometric electrochemical-colorimetric dual-mode detection kit (biosensor) for DR1 based on Cu-MOF and AuPt@PBA and its application. This kit overcomes the drawbacks of traditional single-mode DR1 detection methods, such as unreliable results, susceptibility to interference, and low sensitivity. It offers advantages such as high selectivity, high sensitivity, and cross-verification of detection results, and has good applicability in human serum detection.

[0006] To achieve the above objectives, one aspect of the technical solution of the present invention is to provide a ratiometric electrochemical-colorimetric dual-mode detection kit for detecting transcription downregulation factor 1 (DR1), comprising the following core materials: glassy carbon electrode (GCE), magnetic beads (MBs), antibody 1 (Ab1), bovine serum albumin (BSA), AuPt@PBA-Ab2 bioconjugate, and copper-based metal-organic framework (Cu-MOF).

[0007] Furthermore, the detection kit also includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), hydrogen peroxide (H2O2), tetramethylbenzidine (TMB), sodium acetate (NaOAc) buffer, and phosphate buffer (PBS).

[0008] Furthermore, the preparation method of Cu-MOF is as follows: First, Cu(NO3)2·3H2O and terephthalic acid (PTA) were dissolved in DMF and reacted. After the reaction was completed, blue crystals were collected by centrifugation, washed, and Cu-MOF was obtained. The reaction temperature was 150℃ and the reaction time was 24 h.

[0009] Furthermore, the preparation method of AuPt@PBA-Ab2 bioconjugate is as follows: (1) Preparation of PBA powder K3[Fe(CN)6] was dissolved in deionized water; NiCl2·6H2O and Na3C6H5O7·2H2O were separately dissolved in deionized water; the two solutions were mixed, stirred for a period of time, centrifuged to collect the precipitate, washed, and dried to obtain PBA powder; (2) Preparation of AuPt@PBA The PBA powder obtained above was dispersed in deionized water, sonicated, and then PVP was added and stirred; then a mixture of HAuCl4·3H2O and K2PtCl4 was added, and sodium borohydride (NaBH4) solution was added under ice bath stirring and the reaction was carried out; the precipitate was collected by centrifugation, washed, and vacuum dried to obtain AuPt@PBA. (3) Preparation of AuPt@PBA-Ab2 bioconjugate The AuPt@PBA powder obtained above was added to deionized water to obtain an AuPt@PBA suspension. An Ab2 solution was added to the AuPt@PBA suspension and the mixture was shaken and incubated. Subsequently, the mixture was centrifuged, washed, and unbound Ab2 was removed to obtain the AuPt@PBA-Ab2 bioconjugate.

[0010] Furthermore, in step (1), the stirring time is 24 h; in step (2), the sonication time is 30 min, the stirring time is 1 h, and the reaction time is 30 min; in step (3), the incubation temperature is 37℃ and the time is overnight.

[0011] On the other hand, the technical solution of the present invention provides a DR1 detection method based on the detection kit, comprising the following steps: (1) Preparation of the sensing system ① Add the Ab1 solution dropwise into a centrifuge tube, where it couples with the activated MBs from EDC / NHS in the centrifuge tube via amide bonds; ② Add BSA solution and incubate to block unreacted active sites; ③ Add the solution to be tested and incubate; ④ Add AuPt@PBA-Ab2 bioconjugate solution and incubate; ⑤ After incubation, the mixed system is subjected to magnetic separation treatment, and the precipitate and supernatant are collected separately; (2) Dual-mode detection ① Colorimetric detection: Take the precipitate obtained by magnetic separation, add H2O2 solution, TMB solution and NaOAc buffer, and then transfer it to a 96-well plate; react in the dark, and finally use an ELISA reader to measure the absorbance; ② Ratio-dependent electrochemical detection: A Cu-MOF suspension was drop-coated onto the surface of a glassy carbon electrode, dried, washed, and dried with N2. The supernatant obtained from magnetic adsorption was then drop-coated onto the Cu-MOF-modified electrode surface, dried, washed, and dried with N2. The modified electrode was placed in PBS buffer for electrochemical testing, and the characteristic peak currents of Cu-MOF and AuPt@PBA were recorded. The peak current ratio (Ir) was calculated. Cu 2 + / I AuPt@PBA This enables the quantitative detection of DR1.

[0012] Furthermore, before use, the glassy carbon electrode (GCE) is pretreated by polishing it to a mirror finish with 0.3 μm alumina powder, ultrasonically cleaning it with ultrapure water and anhydrous ethanol, and then drying it with N2.

[0013] Furthermore, electrochemical detection includes, but is not limited to, square wave voltammetry (SWV), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS); the SWV scanning parameters are: scan range -0.6 V to 0.8 V, scan rate 1.0 V / s, and potential increment 4 mV; CV measurements are performed in the voltage range of -0.3 V to 0.65 V, with a scan rate of 0.2 V / s. -1The number of scan segments is 40; the EIS test frequency range is 0.1 Hz to 0.1 MHz, the applied AC voltage is 5 mV, the amplitude is 0.005 Hz, and the supporting electrolyte is a 5 mM potassium ferricyanide solution containing 0.1 M potassium nitrate.

[0014] Furthermore, in step (1), the reaction temperature is 36℃-38℃ and the reaction time is 1 h; the reaction temperature is 36℃-38℃ and the reaction time is 20-40 min; the reaction temperature is 36℃-38℃ and the reaction time is 1 h; and the reaction temperature is 36℃-38℃ and the reaction time is 50-70 min.

[0015] On the other hand, the technical solution of the present invention is to provide an application of the detection kit in the preparation of DR1 detection products.

[0016] A schematic diagram of the construction process of the reagent kit of the present invention is shown below. Figure 1 As shown in the diagram, antibody 1 (Ab1) was first coupled to the surface of activated MBs using an amide. Unreacted active sites were then blocked with bovine serum albumin (BSA), followed by the addition of DR1. Ab1 specifically recognizes DR1, and finally, AuPt@PBA-Ab2 was added, which specifically recognizes DR1. After the reaction, magnetic separation was performed. The precipitate obtained was the MBs / Ab1 / BSA / DR1 / AuPt@PBA-Ab2 system formed in the reaction. The supernatant contained free AuPt@PBA-Ab2. Because AuPt@PBA in the precipitate has peroxidase activity, it can oxidize TMB to oxTMB in a mixture of H2O2, TMB, and NaOAc buffer. The absorbance value was then read at 652 nm using a microplate reader. The free AuPt@PBA-Ab2 in the supernatant, due to the redox activity of AuPt@PBA, can generate a signal peak in electrochemistry. Finally, DR1 was quantitatively analyzed by measuring the ratio of the electrochemical signals of Cu-MOF and AuPt@PBA and the absorbance of oxTMB.

[0017] Beneficial effects of the present invention 1. This invention uses Cu-MOF as an electrochemical internal reference signal material, which significantly improves the accuracy of the detection signal and effectively avoids errors caused by environmental changes or interfering substances in single signal detection. AuPt@PBA nanozyme has stable peroxidase-like activity. In the presence of hydrogen peroxide (H2O2), it can oxidize TMB to generate a colorimetric signal. It also has redox activity, which brings good electrochemical performance to the sensing system and realizes the synergistic generation of dual-mode signals.

[0018] 2. A ratiometric electrochemical-colorimetric dual-mode design is adopted, allowing for mutual verification of detection results between the two modes, further reducing experimental errors. Under optimal conditions, the detection limit in electrochemical mode is as low as 13.60 fg / mL, with a linear range of 1×10⁻⁶. -1 ~ 1×10 5 pg / mL; the detection limit in colorimetric mode is as low as 4.49 fg / mL, and the linear range is 1×10 pg / mL. -2 ~ 1×10 4 pg / mL, possessing ultrasensitive detection performance.

[0019] 3. The biosensor system constructed in this invention possesses excellent selectivity, stability, and anti-interference capabilities. Both electrochemical and colorimetric modes exhibit extremely low responses to interfering substances such as human epidermal growth factor receptor 2 (HER2), progastrin-releasing peptide (proGRP), and estrogen receptor α (ERα). After storage at 4°C for 4 weeks, it retains more than 92% of the signal response. The signal in 5% and 10% human serum is close to that in PBS buffer, enabling reliable detection of DR1 in complex biological samples. The system is simple to operate and low in cost, and is expected to be extended to the clinical monitoring of Hashimoto's thyroiditis and the detection of other biomarkers. Attached Figure Description

[0020] Figure 1 Schematic diagrams of the material synthesis (A and B) and kit construction (C and D) processes.

[0021] Figure 2 (A) Electrochemical sensing signals without Ab1 (curve a), without DR1 (curve b), without AuPt@PBA-Ab2 (curve c), without Cu-MOF (curve d), and with complete modification (curve e). (B) Ultraviolet (UV) absorption peaks without Ab1 (curve a), without DR1 (curve b), without AuPt@PBA-Ab2 (curve c), and with complete modification (curve d).

[0022] Figure 3 It contains 0.1 M KNO3 and 5 mM [Fe(CN)6] 3- / 4- EIS curves (A) measured by electrodes modified at different stages in a solution containing 0.1 M KNO3 and 5 mM [Fe(CN)6] 3- / 4- In the solution, the CV curves (B) were measured by electrodes modified at different stages.

[0023] Figure 4SEM images of Cu-MOF (A) and AuPt@PBA (E), TEM images of Cu-MOF (C) and AuPt@PBA (G), mapping (B) and EDX (D) images of Cu-MOF, and mapping (F) and EDX (H) images of AuPt@PBA.

[0024] Figure 5 The UV absorption spectra of PBA+TMB + H2O2 and AuPt@PBA+TMB + H2O2 solutions are shown.

[0025] Figure 6 Hysteresis loop (VSM) test for magnetic beads and AuPt@PBA.

[0026] Figure 7 To optimize experimental conditions: the effect of reaction time of Ab1 on the electrochemical ratio (A) and the colorimetric sensing system (C). The effect of reaction time of DR1 on the electrochemical ratio (B) and the colorimetric sensing system (D). The effect of the added H2O2 volume on the colorimetric signal was further analyzed (E).

[0027] Figure 8 shows the current signals corresponding to different concentrations of DR1 protein (A). The linear relationship between the logarithm of DR1 concentration and the peak ratio of the electrochemical signal (B). The linear relationship between the logarithm of DR1 concentration and the colorimetric signal (C). (Curves ag in Figure A correspond to DR1 concentrations of 1×10⁻⁶ and ag, respectively.) -1 1×10 0 1×10 1 1×10 2 1×10 3 1×10 4 and 1×10 5 (pg / mL).

[0028] Figure 9 The responses of the ratiometric electrochemical sensing system (A) and the colorimetric sensing system (B) to different analytes. The stability of the ratiometric electrochemical sensing system (C) and the colorimetric sensing system (D). The reproducibility of the ratiometric electrochemical sensing system (E) and the colorimetric sensing system (F).

[0029] Figure 10 The signal responses of the ratio electrochemical sensing system (A) and the colorimetric sensing system (B) to different concentrations of DR1 in PBS buffer, 5% human serum and 10% human serum are shown. Detailed Implementation

[0030] The specific embodiments of the present invention will be further described in detail below with reference to examples. Unless otherwise specified, the reaction temperature of the present invention is room temperature.

[0031] Antibody 1 (Ab1) is a DR1 rabbit polyclonal antibody, purchased from Beijing Bio-Sen Biotechnology Co., Ltd., catalog number: bs-9055R; Antibody 2 (Ab2) is a DR1 mouse polyclonal antibody, purchased from Abogen (Shanghai) Co., Ltd., catalog number: ab88597; Transcription downregulation factor 1 (DR1) is a DR1 antigen, purchased from Guangzhou Aikenman Biotechnology Co., Ltd., catalog number: sc-515083.

[0032] Example 1: Material Preparation (1) Preparation of Cu-MOF (e.g.) Figure 1 (As shown in A) First, 0.97 g of copper nitrate trihydrate (Cu(NO3)2·3H2O) and 0.67 g of terephthalic acid (PTA) were dissolved in 20 mL of N,N-dimethylformamide (DMF). The resulting solution was then transferred to a 50 mL polytetrafluoroethylene-lined stainless steel autoclave and reacted at 150 °C for 24 h. After the reaction, blue crystals were collected by centrifugation and washed alternately with sufficient anhydrous ethanol and deionized water to obtain Cu-MOF. Next, Cu-MOF was dispersed in deionized water to prepare a Cu-MOF suspension (1 mg / mL) for later use.

[0033] (2) Preparation of AuPt@PBA-Ab2 (e.g.) Figure 1 (as shown in B) ①Preparation of PBA powder Dissolve 0.65 g potassium ferricyanide (K3[Fe(CN)6]) in 80 mL of deionized water; separately dissolve 0.47 g nickel chloride hexahydrate (NiCl2·6H2O) and 1.18 g sodium citrate dihydrate (Na3C6H5O7·2H2O) in 80 mL of deionized water; mix the two solutions, stir at room temperature for 24 h, centrifuge to collect the precipitate, wash repeatedly with deionized water, and dry at 60 °C overnight to obtain PBA powder.

[0034] ②Preparation of AuPt@PBA The PBA powder obtained above was dispersed in 10 mL of deionized water and sonicated for 30 min. 5 mg of polyvinylpyrrolidone (PVP) was added and stirred for 1 h. A mixture of 1 mL of tetrachloroauric acid trihydrate (HAuCl4·3H2O) (5 mmol / L) and potassium tetrachloroplatinate (K2PtCl4) (2.5 mmol / L) was added. 0.5 mL of sodium borohydride (NaBH4) solution (0.1 mol / L) was rapidly added under vigorous stirring in an ice bath and reacted for 30 min. The precipitate was collected by centrifugation, washed twice with 0.1 mM sodium hydroxide (NaOH) solution, and dried under vacuum at 60 ℃ to obtain AuPt@PBA.

[0035] ③Preparation of AuPt@PBA-Ab2 bioconjugates The AuPt@PBA powder obtained above was added to 1 mL of deionized water to obtain an AuPt@PBA suspension. 20 μL of antibody 2 (Ab2) solution (50 μg / mL) was added to the AuPt@PBA suspension, and the mixture was gently incubated overnight at 37°C with shaking. Subsequently, the mixture was centrifuged and washed twice with phosphate-buffered saline (PBS) to remove unbound Ab2, yielding the AuPt@PBA-Ab2 bioconjugate, which was stored at 4°C for later use.

[0036] Example 2: Reagent Kit A DR1 ratiometric electrochemical-colorimetric dual-mode detection kit based on Cu-MOF and AuPt@PBA comprises the following components: glassy carbon electrode (GCE), magnetic beads (MBs), antibody 1 (Ab1), bovine serum albumin (BSA), AuPt@PBA-Ab2 bioconjugate, copper-based metal-organic framework (Cu-MOF), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), hydrogen peroxide (H2O2), tetramethylbenzidine (TMB), sodium acetate (NaOAc) buffer, phosphate buffered saline (PBS), deionized water, and anhydrous ethanol. Some of the above components can be prepared into solutions as needed.

[0037] Example 3: Detection method of the kit (1) Electrode pretreatment The glassy carbon electrode (GCE) was polished with 0.3 μm alumina powder until the electrode surface was mirror-like; then the electrode was ultrasonically cleaned three times each with ultrapure water and anhydrous ethanol, and finally dried with N2.

[0038] (2) Preparation of the sensing system ① Take 10 μL of Ab1 solution (100 ng / mL) and add it dropwise to a centrifuge tube. Couple it with the EDC / NHS activated magnetic beads (MBs) (10 mg / mL, 10 μL) in the centrifuge tube through amide bonds in a shaker at 37℃ for 1 h. Magnetic separation is performed and unreacted substances are washed with PBS buffer. EDC / NHS activation method for MBs: The mixture of magnetic beads (10 mg / mL) and EDC / NHS (20 mM / 10 mM) is reacted in a shaker at 37℃ for 2 h to activate the carboxyl groups on the magnetic beads. The mixture is then washed with PBS buffer to obtain the activated MBs. ② Add 10 μL of bovine serum albumin (BSA) solution (1% by mass) to a centrifuge tube, incubate in a shaker at 37°C for 30 min to block unreacted active sites, magnetically separate and wash unreacted material with PBS buffer; ③ Add 10 μL of the test solution to the centrifuge tube, incubate in a shaker at 37°C for 1 h, perform magnetic separation, and wash with PBS buffer to remove unreacted substances; ④ Add 10 μL of AuPt@PBA-Ab2 bioconjugate solution (1 mg / mL) to a centrifuge tube and incubate in a shaker at 37°C for 1 h; ⑤ After incubation, the mixed system is subjected to magnetic separation treatment, and the precipitate and supernatant are collected separately.

[0039] The above is the preferred scheme for the preparation of the sensing system in step (2). The reaction time in step ② can be 20-40 min; the reaction time in step ④ can be 50-70 min.

[0040] (3) Dual-mode detection ① Colorimetric detection: Take the precipitate obtained by magnetic separation, add 20 μL of hydrogen peroxide (H2O2) solution (10 mM), 10 μL of tetramethylbenzidine (TMB) solution (5 mM) and 170 μL of sodium acetate (NaOAc) buffer (0.5 M), and then transfer it to a 96-well plate; react at 37℃ in the dark for 10 min, and finally use an ELISA reader to measure the absorbance by colorimetric signal.

[0041] ② Ratio-dependent electrochemical detection: 2.5 μL of Cu-MOF suspension (1 mg / mL) was drop-coated onto the surface of a glassy carbon electrode. After drying at room temperature, it was rinsed with PBS buffer and dried with N2. Then, 5 μL of the supernatant obtained from magnetic adsorption was drop-coated onto the surface of the Cu-MOF-modified electrode. After drying at room temperature, it was rinsed with PBS buffer and dried with N2. The modified electrode was placed in 0.1 M, pH 7.4 phosphate-buffered saline (PBS) for square wave voltammetry (SWV) testing. The characteristic peak currents of Cu-MOF and AuPt@PBA were recorded, and the peak current ratio (Ir) was calculated. Cu 2+ / I AuPt@PBA This enables the quantitative detection of DR1.

[0042] In step (3), the colorimetric detection was performed using an ELISA reader (SpectraMax 190, USA) with a detection wavelength of 652 nm. Electrochemical detection employed a conventional three-electrode system, consisting of: a modified glassy carbon electrode (3 mm diameter, working electrode), a saturated calomel electrode (SCE, reference electrode), and a platinum wire counter electrode. A CHI760E electrochemical workstation was used, and electrochemical signals were detected using square wave voltammetry (SWV). The detection range was -0.6 V to 0.8 V, the scan rate was 1.0 V / s, the potential increment was 4 mV, and PBS buffer was used as the electrolyte.

[0043] Example 4: Feasibility Verification To evaluate the feasibility of using the kit of this invention to determine DR1, the detection system was validated using SWV and UV-Vis spectroscopy, respectively. The SWV test results are as follows: Figure 2 As shown in Figure A, when Ab1 (curve a) and DR1 (curve b) are not added, the characteristic peak of AuPt@PBA is very large because AuPt@PBA is not adsorbed as a precipitate. When AuPt@PBA-Ab2 is not added (curve c), no signal peak of AuPt@PBA is observed. When Cu-MOF is not added (curve d), no characteristic peak of Cu-MOF is observed. In contrast, when all the above components are added simultaneously, a bimodal signal is produced (attributed to Cu-MOF and AuPt@PBA respectively), and the peak value of AuPt@PBA is lower than when the components are absent (curve e). The UV-Vis spectroscopy results are as follows... Figure 2 As shown in Figure B, no significant absorption peaks were observed without the addition of Ab1 (curve a), DR1 (curve b), and AuPt@PBA-Ab2 (curve c). Notably, a distinct UV absorption peak (curve d) only appeared after all components were fully assembled. These results preliminarily confirm that the proposed ratiometric electrochemical-colorimetric dual-mode biosensor system can detect DR1.

[0044] Example 5: Characterization of the electrode modification process The stepwise modification process of the electrode surface was characterized using electrochemical impedance spectroscopy (EIS): such as... Figure 3 As shown in the Nyquist plot of A, the charge transfer resistance (Rct) of the bare glassy carbon electrode (GCE) is approximately 214 Ω (curve a); after modification with Cu-MOF, Rct increases to approximately 791 Ω (curve b), which is due to the relatively low conductivity of the Cu-MOF layer and the presence of steric hindrance; after adding the supernatant containing AuPt@PBA-Ab2, Rct further increases to approximately 1100 Ω (curve c), confirming that the immune complex has been successfully bound to the electrode surface.

[0045] Electrochemical characterization of the progressively modified electrode was performed using cyclic voltammetry (CV). Figure 3 B): Compared to the bare glassy carbon electrode (GCE) (curve a), the low conductivity of Cu-MOF after modification hinders electron transfer, leading to a decrease in current intensity (curve b). After the supernatant is dropped onto the electrode surface, AuPt@PBA-Ab2 is anchored to the electrode surface, and charge transfer at the electrode interface is gradually suppressed, resulting in a gradual decrease in current intensity (curve c). These results are consistent with the characterization results of electrochemical impedance spectroscopy (EIS), confirming the successful fabrication of the sensor.

[0046] To verify the successful synthesis of Cu-MOF and AuPt@PBA nanomaterials, their morphology and elemental composition were comprehensively analyzed using techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), and elemental mapping.

[0047] like Figure 4 As shown in Figure A, the SEM image of Cu-MOF reveals a regular rod-like structure with a relatively uniform surface. The corresponding TEM image (…) Figure 4 C) further reveals its distinct rod-like morphology. These observations collectively confirm the successful synthesis of Cu-MOF, a material possessing the typical structural characteristics of a metal-organic framework. Furthermore, in mapping ( Figure 4 In (B), the signals of carbon, oxygen, and copper elements completely coincide with the spatial distribution of the nanorods. Combined with EDX ( Figure 4 The carbon, oxygen, and copper characteristic peaks detected in D) not only confirm the core elemental composition of Cu-MOF (where copper acts as a metal node and the organic ligands contain carbon and oxygen), but also reflect the structural characteristics of uniform dispersion of elements in the material—which is completely consistent with the design expectations of metal-organic frameworks.

[0048] For the AuPt@PBA complex, SEM ( Figure 4 E) clearly shows its cubic structure, indicating that the gold-platinum nanoparticles have been successfully anchored on the Prussian blue analogue (PBA) matrix. TEM ( Figure 4 G) further confirmed this result, with the uniformly dispersed cubic particles in the image showing good dispersibility. Furthermore, mapping ( Figure 4 F) shows that Fe, Ni, Pt, and Au are uniformly distributed in the material, confirming the uniform loading of AuPt nanoparticles in the PBA matrix. EDX ( Figure 4 H) confirmed the presence of Ni, Fe, Pt and Au elements, consistent with the expected elemental composition of AuPt@PBA.

[0049] In summary, these comprehensive characterization results strongly demonstrate that Cu-MOF and AuPt@PBA nanomaterials have been successfully synthesized with the expected morphology and elemental composition, laying a solid foundation for their subsequent application in dual-mode sensors.

[0050] Example 6: Peroxidase Activity Analysis of Materials The verification results of the peroxidase catalytic activity of PBA and AuPt@PBA are as follows: Figure 5As shown, when PBA nanozymes were added to a solution containing TMB and H2O2, an absorption peak was observed at 652 nm. When AuPt@PBA was added to the solution containing TMB and H2O2, a stronger absorption peak was observed at 652 nm compared to PBA nanozymes. This phenomenon indicates that the catalytic activity of the composite nanozyme obtained by combining PBA and AuPt was significantly enhanced.

[0051] Example 7: Magnetic Analysis of Magnetic Beads and AuPt@PBA To verify that AuPt@PBA nanomaterials will not be magnetically adsorbed and precipitated if they are not attached to the target material DR1, we performed hysteresis loop (VSM) tests on both purchased magnetic beads and our self-synthesized AuPt@PBA. Figure 6 As shown in Figure A, MBs exhibit typical superparamagnetic material response characteristics. Under the action of an applied magnetic field, the magnetization increases rapidly with increasing magnetic field, reaching saturation at approximately 5 kOe, with a relatively high saturation magnetization. The curve shows no significant remanence or coercivity near zero magnetic field, indicating that MBs can achieve rapid magnetic separation under an applied magnetic field, and there is no hysteresis residue after the magnetic field is removed, demonstrating excellent magnetic response speed and cyclic recovery capability. AuPt@PBA ( Figure 6 The magnetization in A and 6B is close to zero and the curves are approximately horizontal, indicating that the AuPt@PBA composite material itself has no significant intrinsic magnetic response. Its magnetic separation into precipitation mainly depends on the specific binding of AuPt@PBA-Ab2 with DR1. This also confirms that the modification of AuPt@PBA did not introduce additional magnetic interference and did not damage the core magnetic properties of the magnetic beads.

[0052] Example 8: Optimization of Detection Conditions To further improve the analytical performance of the sensing system, key parameters in the sensor fabrication process were systematically optimized for two different detection modes: (1) Optimization of Ab1 reaction time: In electrochemical systems, such as Figure 7 As shown in A, I Cu²⁺ / I AuPt@PBA The signal ratio gradually increases with the extension of Ab1 reaction time, reaching a peak at 60 min. After this time, the signal ratio remains stable; in colorimetric systems, such as... Figure 7 As shown in Figure C, the colorimetric signal gradually increased with the increase of Ab1 reaction time, reaching a peak at 60 min. After this time, the colorimetric signal remained essentially unchanged. This indicates that the binding of Ab1 to MBs is essentially saturated after 60 min. Therefore, 60 min was chosen as the optimal reaction time for Ab1.

[0053] (2) Optimization of DR1 incubation time: such as Figure 7As shown in B and 7D, with the extension of DR1 reaction time, I Cu² ⁺ / I AuPt@PBA The signal ratio and colorimetric signal gradually increased, reaching a peak at 60 min. Thereafter, the signal ratio and colorimetric signal remained relatively stable in the electrochemical mode, indicating that the binding of DR1 to Ab1 was essentially complete after 60 min. Therefore, 60 min was determined to be the optimal reaction time for DR1.

[0054] (3) Optimization of H2O2 volume: To further enhance the signal, this study investigated the effect of H2O2 volume on the contrast color signal. For example... Figure 7 As shown in E, the colorimetric signal intensity gradually increased with the volume of H2O2, reaching a peak at 20 μL. No further signal enhancement was observed when the reaction volume exceeded 20 μL. Therefore, 20 μL was selected as the optimal volume of H2O2 for subsequent detection.

[0055] Example 9: Performance Analysis Under optimal conditions, the analytical performance of the dual-mode sensing system is evaluated by detecting the signal response of the sensing system to a series of different concentrations of DR1: Electrochemical mode: at 1 × 10 -1 pg / mL ~ 1 × 10 5 Within the DR1 concentration range of pg / mL, I Cu 2+ / I AuPt@PBA The peak ratio steadily increased with increasing DR1 concentration. Figure 8A The fitting yielded the calibration curve as y = 0.05498 × lg( C DR1 ) +0.21298 (R² = 0.998, Figure 8B Based on this, the limit of detection (LOD) was calculated to be 13.60 fg / mL.

[0056] Colorimetric mode: in 1 × 10 -2 pg / mL ~ 1 × 10 4 Within the DR1 concentration range of pg / mL, the colorimetric signal intensity showed a good linear correlation with the logarithm of the DR1 concentration. Figure 8C The corresponding linear regression equation is y = 0.05147×lg( C DR1 ) + 0.55306 ( R (²=0.997); based on this, the limit of detection (LOD) is calculated to be 4.49 fg / mL.

[0057] Compared with reported biosensors (Table 1), the ratiometric electrochemical-colorimetric dual-mode sensor constructed in this invention has a wider detection range and higher sensitivity, enabling ultrasensitive detection of DR1.

[0058] Table 1 Example 10: Selectivity, stability, and reproducibility of the kit To investigate the selectivity of this invention for DR1, the same concentrations of other biomarkers (human epidermal growth factor receptor 2 (HER2), progastrin-releasing peptide (proGRP), estrogen receptor α (ERα), and a blank control group) were measured using this method. Figure 9 As shown in Figure A, the electrochemical response values ​​of HER2, proGRP, ERα, and the blank group were significantly lower than those of DR1, with peak current ratios only 16.75%, 15.10%, 26.29%, and 8.70% of that of DR1, respectively. Figure 9 As shown in Figure B, the colorimetric signal intensity of the aforementioned interfering substances is significantly weaker than that of DR1, at 19.24%, 16.16%, 19.75%, and 10.61% of the DR1 signal, respectively. This indicates that the present invention has high selectivity for DR1.

[0059] To demonstrate the stability of this invention, two sets of ratiometric electrochemical and colorimetric biosensors with identical modification conditions were prepared: one set was immediately detected using square wave voltammetry (SWV) and an ELISA reader, while the other set was stored at 4°C and saturated humidity for 4 weeks before detection. Figure 9 As shown in C and 9D, after 4 weeks of storage, the biosensor retained 92.27% of the electrochemical signal and 93.59% of the colorimetric signal, respectively.

[0060] To assess reproducibility, intra-batch and inter-batch experiments (n=3) were conducted for both the electrochemical biosensor and the colorimetric biosensor, such as... Figure 9 As shown in E and 9F, the intra-batch and inter-batch RSDs of the electrochemical biosensor are 2.22% and 2.25%, respectively; the intra-batch and inter-batch RSDs of the colorimetric biosensor are 1.951% and 2.004%, respectively, indicating that the present invention has good reproducibility.

[0061] Example 11: Practical Application Value To verify the detection performance of the sensing system in complex media, the SWV response signal ratio and colorimetric signal intensity of 1 pg / mL, 100 pg / mL, and 10 ng / mL DR1 were detected using the system in different environments (PBS buffer, 5% normal human serum v / v, and 10% normal human serum v / v). Figure 10As shown in Figure A, when the DR1 concentration was 1 pg / mL, the electrochemical signal intensities after dilution with 5% and 10% human serum were 97.64% and 98.39% of the PBS control group, respectively; at a concentration of 100 pg / mL, the corresponding values ​​were 92.71% and 92.31%, respectively; and at a concentration of 10 ng / mL, the signal intensity reached 96.41% and 97.61% of the control group, respectively. The colorimetric detection results are as follows... Figure 10 As shown in Figure B, when the DR1 concentration is 1 pg / mL, the colorimetric signal intensities after dilution with 5% and 10% human serum are 96.90% and 104.78% of the signal in PBS buffer, respectively; when the concentration is 100 pg / mL, the signal intensities at the two serum concentrations are 100.54% and 101.59% of the signal in PBS buffer, respectively; and when the concentration is 10 ng / mL, the corresponding values ​​are 99.17% and 100.66%, respectively. This indicates that the biosensor system has excellent anti-interference performance and strong potential for practical applications.

[0062] Furthermore, to evaluate the reliability of this biosensor system in practical clinical applications, this invention used the method to test six clinical human serum samples provided by the Third Affiliated Hospital of Henan University of Traditional Chinese Medicine, and compared the results with those of an ELISA kit (the serum was diluted 5-fold in electrochemical, colorimetric, and ELISA detection methods). As shown in Table 2, the detection results of this method are basically consistent with those of the kit, with a relative error of <5%. This further demonstrates that the biosensor system has good reliability in clinical sample detection and high practical value.

[0063] Table 2

Claims

1. A ratiometric electrochemical-colorimetric dual-mode detection kit for detecting transcription downregulation factor 1 (DR1), characterized in that, The core materials include: glassy carbon electrode (GCE), magnetic beads (MBs), antibody 1 (Ab1), bovine serum albumin (BSA), AuPt@PBA-Ab2 bioconjugate, and copper-based metal-organic framework (Cu-MOF).

2. The detection kit according to claim 1, characterized in that, It also includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), hydrogen peroxide (H2O2), tetramethylbenzidine (TMB), sodium acetate (NaOAc) buffer, and phosphate buffer (PBS).

3. The detection kit according to claim 1, characterized in that, The preparation method of Cu-MOF is as follows: First, Cu(NO3)2·3H2O and terephthalic acid (PTA) were dissolved in DMF and reacted. After the reaction was completed, blue crystals were collected by centrifugation, washed, and Cu-MOF was obtained. The reaction temperature was 150℃ and the reaction time was 24 h.

4. The detection kit according to claim 1, characterized in that, The preparation method of AuPt@PBA-Ab2 bioconjugate is as follows: (1) Preparation of PBA powder Dissolve K3[Fe(CN)6] in deionized water; separately dissolve NiCl2·6H2O and Na3C6H5O7·2H2O in deionized water; The two solutions were mixed and stirred for a period of time. The precipitate was collected by centrifugation, washed, and dried to obtain PBA powder. (2) Preparation of AuPt@PBA The PBA powder obtained above was dispersed in deionized water, sonicated, and then PVP was added and stirred; then a mixture of HAuCl4·3H2O and K2PtCl4 was added, and sodium borohydride (NaBH4) solution was added under ice bath stirring and the reaction was carried out; the precipitate was collected by centrifugation, washed, and vacuum dried to obtain AuPt@PBA. (3) Preparation of AuPt@PBA-Ab2 bioconjugate The AuPt@PBA powder obtained above was added to deionized water to obtain an AuPt@PBA suspension. An Ab2 solution was added to the AuPt@PBA suspension and the mixture was shaken and incubated. Subsequently, the mixture was centrifuged, washed, and unbound Ab2 was removed to obtain the AuPt@PBA-Ab2 bioconjugate.

5. The detection kit according to claim 4, characterized in that, In step (1), the stirring time is 24 h; in step (2), the sonication time is 30 min, the stirring time is 1 h, and the reaction time is 30 min; in step (3), the incubation temperature is 37℃ and the time is overnight.

6. A method for detecting DR1 based on the detection kit according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Preparation of the sensing system ① Add the Ab1 solution dropwise into a centrifuge tube, where it couples with the activated MBs from EDC / NHS in the centrifuge tube via amide bonds; ② Add BSA solution and incubate to block unreacted active sites; ③ Add the solution to be tested and incubate; ④ Add AuPt@PBA-Ab2 bioconjugate solution and incubate; ⑤ After incubation, the mixed system is subjected to magnetic separation treatment, and the precipitate and supernatant are collected separately; (2) Dual-mode detection ① Colorimetric detection: Take the precipitate obtained by magnetic separation, add H2O2 solution, TMB solution and NaOAc buffer, and then transfer it to a 96-well plate; react in the dark, and finally use an ELISA reader to measure the absorbance; ② Ratio-modified electrochemical detection: A Cu-MOF suspension was drop-coated onto the surface of a glassy carbon electrode, and the supernatant obtained from magnetic adsorption was then drop-added onto the Cu-MOF-modified electrode surface. The modified electrode was placed in PBS buffer for electrochemical testing, and the characteristic peak currents of Cu-MOF and AuPt@PBA were recorded. The peak current ratio (Ir) was calculated. Cu 2+ / I AuPt@PBA This enables the quantitative detection of DR1.

7. The detection method according to claim 6, characterized in that, Before use, the glassy carbon electrode (GCE) is pretreated by polishing it to a mirror finish with 0.3 μm alumina powder, ultrasonically cleaning it with ultrapure water and anhydrous ethanol, and then drying it with N2.

8. The detection method according to claim 6, characterized in that, Electrochemical detection includes, but is not limited to, square wave voltammetry (SWV), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The SWV scanning parameters are: scan range -0.6 V to 0.8 V, scan rate 1.0 V / s, and potential increment 4 mV. CV measurements are performed within a voltage range of -0.3 V to 0.65 V, with a scan rate of 0.2 V / s. -1 The number of scan segments is 40; the EIS test frequency range is 0.1 Hz to 0.1 MHz, the applied AC voltage is 5 mV, the amplitude is 0.005 Hz, and the supporting electrolyte is a 5 mM potassium ferricyanide solution containing 0.1 M potassium nitrate.

9. The detection method according to claim 6, characterized in that, In step (1), the reaction temperature is 36℃-38℃ and the reaction time is 1 h; the reaction temperature is 36℃-38℃ and the reaction time is 20-40 min; the reaction temperature is 36℃-38℃ and the reaction time is 1 h; the reaction temperature is 36℃-38℃ and the reaction time is 50-70 min.

10. The use of the detection kit as described in any one of claims 1-5 in the preparation of a product for detecting DR1.

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