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CN-122016972-A - DR1 ratio type electrochemical-colorimetric dual-mode detection kit based on Cu-MOF and AuPt@PBA

CN122016972ACN 122016972 ACN122016972 ACN 122016972ACN-122016972-A

Abstract

The invention discloses a DR1 ratio type electrochemical-colorimetric dual-mode detection kit based on Cu-MOF and AuPt@PBA, which mainly comprises a glassy carbon electrode, magnetic beads, an antibody 1, bovine serum albumin, an AuPt@PBa-ab2 bioconjugate and a copper-based metal organic framework Cu-MOF. Firstly adding Ab1 into a centrifuge tube, modifying the Ab1 to the surface of MBs through an amide bond by coupling with EDC/NHS activated MBs, then adding BSA to block unreacted binding sites, then adding target DR1, finally adding AuPt@PBa-Ab2 conjugate, performing magnetic separation to obtain a precipitate and a supernatant, and using the separated precipitate for colorimetric detection, wherein the supernatant is dripped on the surface of GCE modified with Cu-MOF through electrostatic adsorption for ratio electrochemical detection. The invention has the advantages of high sensitivity, strong selectivity, good stability, excellent anti-interference capability and the like, can realize the ultra-sensitive and accurate detection of DR1, and has good applicability in human serum detection.

Inventors

  • ZHANG YAPING
  • CAI QIYONG
  • LIU YANJU
  • ZHAI XIAOHUI
  • LI JIYANG
  • YANG HUAIXIA

Assignees

  • 河南中医药大学

Dates

Publication Date
20260512
Application Date
20260226

Claims (10)

  1. 1. A ratio-type electrochemical-colorimetric dual-mode detection kit for detecting transcription downregulating factor 1 (DR 1) is characterized by comprising the following core raw materials of a Glassy Carbon Electrode (GCE), magnetic Beads (MBs), an antibody 1 (Ab 1), bovine Serum Albumin (BSA), an AuPt@PBa-Ab2 bioconjugate and a copper-based metal organic framework (Cu-MOF).
  2. 2. The test kit of claim 1, further comprising 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), hydrogen peroxide (H 2 O 2 ), tetramethylbenzidine (TMB), sodium acetate (NaOAc) buffer, phosphate Buffer (PBS).
  3. 3. The detection kit according to claim 1, wherein the Cu-MOF is prepared by: firstly, cu (NO 3 ) 2 ·3H 2 O and terephthalic acid (PTA) are dissolved in DMF to react, after the reaction is finished, blue crystals are collected through centrifugation and washed to obtain Cu-MOF; The reaction temperature was 150℃and the time was 24 h.
  4. 4. The detection kit according to claim 1, wherein the preparation method of the aupt@pbA-Ab2 bioconjugate comprises: (1) Preparation of PBA powder Dissolving K 3 [Fe(CN) 6 in deionized water, dissolving NiCl 2 ·6H 2 O and Na 3 C 6 H 5 O 7 ·2H 2 O in deionized water, mixing the two solutions, stirring for a period of time, centrifuging, collecting precipitate, washing, and drying to obtain PBA powder; (2) Preparation of AuPt@PBA Dispersing the obtained PBA powder in deionized water, performing ultrasonic treatment, adding PVP, stirring, adding mixed solution of HAuCl 4 ·3H 2 O and K 2 PtCl 4 , adding sodium borohydride (NaBH 4 ) solution under ice bath stirring, reacting, centrifuging, collecting precipitate, washing, and vacuum drying to obtain AuPt@PBA; (3) Preparation of AuPt@PBA-Ab2 bioconjugate Adding the AuPt@PBA powder obtained above into deionized water to obtain AuPt@PBA suspension, adding Ab2 solution into the AuPt@PBA suspension, shaking for incubation, centrifuging, washing, and removing unbound Ab2 to obtain the AuPt@PBa-Ab2 bioconjugate.
  5. 5. The test kit according to claim 4, wherein in the step (1), the stirring time is 24 h, in the step (2), the ultrasonic time is 30min, the stirring time is 1h, the reaction time is 30min, and in the step (3), the incubation temperature is 37 ℃ and the incubation time is overnight.
  6. 6. A DR1 detection method based on the detection kit of any one of claims 1-5, comprising the steps of: (1) Preparation of a sensing System ① Dripping Ab1 solution into a centrifuge tube, and performing coupling reaction on the Ab1 solution and the EDC/NHS activated MBs in the centrifuge tube through an amide bond; ② Adding BSA solution, and incubating to block unreacted active sites; ③ Adding a solution to be detected, and incubating; ④ Adding an AuPt@PBA-Ab2 bioconjugate solution, and incubating; ⑤ After incubation, carrying out magnetic separation treatment on the mixed system, and respectively collecting sediment and supernatant; (2) Dual mode detection ① Colorimetric detection, namely taking a precipitate obtained by magnetic separation, adding an H 2 O 2 solution, a TMB solution and a NaOAc buffer solution, transferring into a 96-well plate, carrying out light-proof reaction, and finally measuring absorbance by using an enzyme-labeled instrument; ② The ratio type electrochemical detection comprises the steps of dripping Cu-MOF suspension on the surface of a glassy carbon electrode, dripping supernatant obtained by magnetic adsorption on the surface of an electrode for modifying Cu-MOF, placing the modified electrode in PBS buffer solution for electrochemical test, recording characteristic peak currents of Cu-MOF and AuPt@PBA, and calculating a peak current ratio (I Cu 2+ /I AuPt@PBA ) to realize quantitative detection of DR 1.
  7. 7. The method according to claim 6, wherein the Glass Carbon Electrode (GCE) is pretreated before use by polishing with 0.3 μm alumina powder to a mirror state, ultrasonic cleaning with ultrapure water and absolute ethanol, and drying with N 2 .
  8. 8. The method of claim 6, wherein the electrochemical test includes, but is not limited to, square Wave Voltammetry (SWV), cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS), wherein the SWV has a scan parameter ranging from-0.6V to 0.8V, a scan rate of 1.0V/s, a potential increase of 4 mV, a CV measurement ranging from-0.3V to 0.65V, a scan rate of 0.2V s -1 , a scan number of 40 segments, an EIS test frequency ranging from 0.1 Hz to 0.1 MHz, an applied AC voltage of 5 mV, an amplitude of 0.005 Hz, and a supporting electrolyte of 5 mM potassium ferricyanide solution containing 0.1M potassium nitrate.
  9. 9. The method according to claim 6, wherein the reaction temperature of ① in the step (1) is 36 to 38 ℃ and the reaction time is 1 h, the reaction temperature of the step ② is 36 to 38 ℃ and the reaction time is 20 to 40 min, the reaction temperature of the step ③ is 36 to 38 ℃ and the reaction time is 1 h, the reaction temperature of the step ④ is 36 to 38 ℃ and the reaction time is 50 to 70 min.
  10. 10. Use of the detection kit of any one of claims 1-5 for the preparation of a product for detecting DR 1.

Description

DR1 ratio type electrochemical-colorimetric dual-mode detection kit based on Cu-MOF and AuPt@PBA Technical Field The invention relates to a high-sensitivity ratio type electrochemical-colorimetric dual-mode detection kit constructed based on a copper-based metal organic framework (Cu-MOF) and a gold platinum@Prussian blue analogue (AuPt@PBA MPs), which is used for detecting a thyroiditis (HT) key biomarker transcription downregulation factor 1 (DR 1), and belongs to the technical field of biological analysis. Background Hashimoto's Thyroiditis (HT) is a highly autoimmune disease that continues to exacerbate the threat to human health and quality of life. Transcription down-regulating factor 1 (down-regulator of transcription, DR 1) is a key biomarker for HT, so developing an efficient DR1 detection method is essential for monitoring disease progression. Currently, various sensing methods for disease biomarkers have been developed, including electrochemical, colorimetric, fluorescent, and photoelectrochemical methods. However, such single-mode and single-signal detection methods rely on a single output signal, which results in unreliable detection results. Once the detection environment changes or interference substances exist, false positive results are easy to appear in a single signal, the detection accuracy and reliability cannot be verified by itself, and meanwhile, the problems of low sensitivity and poor selectivity exist. In recent years, ratio-based sensors and dual-mode sensors have received widespread attention as two advanced sensing strategies, by virtue of their unique self-calibration and mutual authentication capabilities. However, the success of the design still depends on two key components, namely, a stable internal reference nanomaterial anchored on the surface of an electrode and a nanomaterial capable of cooperatively generating electrochemical and colorimetric signals. The invention aims to construct a ratio type electrochemical-colorimetric dual-mode detection kit based on Cu-MOF and AuPt@PBA nano materials, so as to realize ultrasensitive and accurate detection of DR 1. Disclosure of Invention Aiming at the defects of the prior art, the invention aims to provide a DR1 ratio type electrochemical-colorimetric dual-mode detection kit (biosensor) based on Cu-MOF and AuPt@PBA and application thereof, overcomes the defects of unreliable results, easiness in interference, low sensitivity and the like in the traditional single-mode detection DR1 method, has the advantages of strong selectivity, high sensitivity, mutual verification of detection results and the like, and has good applicability in human serum detection. In order to achieve the aim, the technical scheme of the invention provides a ratio type electrochemical-colorimetric dual-mode detection kit for detecting transcription downregulating factor 1 (DR 1), which comprises the following core raw materials of a Glassy Carbon Electrode (GCE), magnetic Beads (MBs), an antibody 1 (Ab 1), bovine Serum Albumin (BSA), auPt@PBa-Ab2 bioconjugate and a copper-based metal organic framework (Cu-MOF). Further, the detection kit also comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), hydrogen peroxide (H 2O2), tetramethyl benzidine (TMB), sodium acetate (NaOAc) buffer solution and Phosphate Buffer Solution (PBS). Further, the preparation method of the Cu-MOF comprises the following steps: First, cu (NO 3)2·3H2 O) and terephthalic acid (PTA) were dissolved in DMF and reacted, after the reaction was completed, blue crystals were collected by centrifugation and washed to obtain Cu-MOF wherein the reaction temperature was 150℃and the time was 24 h. Further, the preparation method of the AuPt@PBA-Ab2 bioconjugate comprises the following steps: (1) Preparation of PBA powder Dissolving K 3[Fe(CN)6 in deionized water, dissolving NiCl 2·6H2 O and Na 3C6H5O7·2H2 O in deionized water, mixing the two solutions, stirring for a period of time, centrifuging, collecting precipitate, washing, and drying to obtain PBA powder; (2) Preparation of AuPt@PBA Dispersing the obtained PBA powder in deionized water, performing ultrasonic treatment, adding PVP, stirring, adding mixed solution of HAuCl 4·3H2 O and K 2PtCl4, adding sodium borohydride (NaBH 4) solution under ice bath stirring, reacting, centrifuging, collecting precipitate, washing, and vacuum drying to obtain AuPt@PBA; (3) Preparation of AuPt@PBA-Ab2 bioconjugate Adding the AuPt@PBA powder obtained above into deionized water to obtain AuPt@PBA suspension, adding Ab2 solution into the AuPt@PBA suspension, shaking for incubation, centrifuging, washing, and removing unbound Ab2 to obtain the AuPt@PBa-Ab2 bioconjugate. Further, in step (1), the stirring time was 24 h, in step (2), the ultrasonic time was 30 min, the stirring time was 1h, the reaction time was 30 min, and in step (3), the incubation temperature was 37 ℃ and the time was overnight. On the othe