WO2021109384A1 - Preparation method for nano electrochemical aptamer sensor for detecting stress-induced phosphorus protein - Google Patents

Abstract

Disclosed in the present invention is a preparation method for a nano electrochemical aptamer sensor for detecting stress-induced phosphoprotein: using a solvothermal method to prepare a manganese-doped nickel-based metal framework compound Mn-MOF and drip-coating same onto the surface of a glassy carbon electrode GCE, then drip-coating a single-stranded nucleic acid aptamer with a methylene blue labelled tail end MB-DNA to construct an MB-DNA/Mn-MOF/GCE sensing interface. Adding stress-induced phosphoprotein STIP1, the MB-DNA aptamer specifically binding with the STIP1 to form an MB-DNA-STIP1 complex, making the MB-DNA detach from the surface of the GCE and causing the MB oxidation current peak intensity to weaken. Fitting the linear relationship between the MB current peak intensity and the STIP1 concentration to construct a nano electrochemical aptamer sensor for detecting STIP1. The method of the present invention has convenient operation, high sensitivity, and good specificity, and can serve as a new method for quantitative detection of STIP1.

Description

Preparation method of nano electrochemical aptamer sensor for detecting stress-induced phosphoprotein Technical field

The invention belongs to the technical field of preparation of functional nanocomposite materials and electrochemical aptamer sensors, and specifically relates to a preparation method of a nano electrochemical aptamer sensor for detecting stress-induced phosphoproteins. The prepared sensor can be used for application in biomedical samples. Highly sensitive and selective detection of phosphoprotein induced by stimulation.

Background technique

Stress-induced phosphoprotein (STIP1) is a protein secreted by ovarian cancer tissue into the peripheral blood of patients. The detection of STIP1 can help improve the level of early diagnosis of ovarian cancer. In recent years, studies on STIP1 as a potential tumor marker for detecting human ovarian cancer have been reported successively. Huang et al. prepared a hybrid nanocomposite-based fluorescence on-off control based on gold nanoparticles for the detection of human STIP1 (Yue Huang, Hao Li, Lei Wang, Xiaoxia Mao, Genxi Li, Highly sensitive protein detection based on smart hybrid nanocomposite-controlled switch of DNA polymerase activity, ACS Applied Materials & Interfaces, 2016, 8, 28202-28207); Chen et al. prepared a dual-function sensing interface based on reduced graphene oxide for fluorescence and Raman scattered light detection of STIP1 (Feng Chen, Yi Liu, Chunyan Chen, Hang Gong, Changqun Cai, Xiaoming Chen, Respective and Simultaneous detection tumor markers CA125 and STIP1 using aptamer-based fluorescent, 470 and RLS sensors, Sensors, 2017: 245 ; Judy Wanderwater and others combined biological samples from the mother with the biomarker STIP1 antibody to develop a method for preventing or reducing the risk of autism spectrum disorders in fetuses or children (Judy Wander Watt; Daniel Braunschweig, Methods of Diagnosing and Treating Autism, Patent Publication No. CN105911275A). At present, there are few literature reports on the quantitative detection of STIP1, and the reported detection methods still have some challenges. It is difficult to achieve the goal of simple, rapid, highly sensitive and highly selective detection of STIP1 in actual samples.

Based on this, the present invention discloses a preparation method of a nano electrochemical aptamer sensor for detecting stress-induced phosphoprotein. First, prepare manganese-doped nickel-based metal framework compound (Mn-MOF), drop its ethanol dispersion onto the surface of a clean glassy carbon electrode (GCE), incubate for a period of time and then dry naturally; then label the methylene blue end with a single-stranded nucleic acid suitable The aqueous solution of MB-DNA is dripped onto the surface of Mn-MOF/GCE, incubated for a period of time and then naturally dried; finally the surface-modified GCE is inserted into the electrolytic cell, phosphate water is used as the electrolyte solution, and a certain amount of STIP1 is added to it , Incubate for a period of time. Due to the specific binding between MB-DNA aptamer and STIP1 to form a MB-DNA/STIP1 complex, MB-DNA is separated from the surface of GCE, causing the redox effect of MB to decrease, that is, the current peak intensity is weakened. By fitting the linear relationship between the MB current peak intensity and the added concentration of STIP1, a nanoelectrochemical aptamer sensor for detecting STIP1 can be constructed. Up to now, no domestic and foreign literature and patent reports about the MB-DNA/Mn-MOF nanocomposite and the construction of a nanoelectrochemical aptamer sensor based on the composite for the quantitative detection of STIP1 have not been retrieved.

Summary of the invention

The purpose of the present invention is to overcome the above-mentioned problems in the prior art and design a new method for stress-induced phosphoprotein detection with convenient operation, high sensitivity and good specificity.

In order to achieve the above objective, the present invention relates to a method for preparing a nano-electrochemical aptamer sensor for detecting stress-induced phosphoproteins. The specific preparation method includes the following steps:

1. A preparation method of a nano electrochemical aptamer sensor for detecting stress-induced phosphoproteins, characterized in that the method specifically includes the following steps:

(1) Preparation of manganese-doped nickel-based metal framework compound (Mn-MOF): 0.465g Ni(NO ₃ ) ₂ ·6H ₂ O and 0.01g Mn(NO ₃ ) ₂ ·4H ₂ O were dissolved in 25mL dimethyl In DMF, solution A was formed after stirring for 30 min. 0.133 g of 1,4-phthalic acid was dissolved in 25 mL of DMF, and solution B was formed after stirring for 30 min. Under magnetic stirring, solution B was slowly added to solution A, and then dilute hydrochloric acid was added dropwise to obtain a green solution, which was transferred to an autoclave and stirred at 180°C for 24h. The reaction product was centrifuged to obtain a precipitate, which was washed 3 times with DMF and ethanol, and the product was naturally dried.

(2) Methylene blue end-labeled single-stranded nucleic acid aptamer/manganese-doped nickel-based metal framework compound/glassy carbon electrode (MB-DNA/Mn-MOF/GCE) sensing interface construction: in a clean glassy carbon electrode (GCE) 5μL of Nafion solution (5% w/w) was dripped on the surface, and Mn-MOF aqueous dispersion was dripped after 5min incubation, the concentration of Mn-MOF was 0.1-1mg/mL, and the amount of dripping was 5-50μL. Incubate MB-DNA (2μM) at 37°C for 2h, measure 5～25μL and apply it to the surface of Mn-MOF/GCE. After incubating for 12h, _{dry it with N 2} and rinse with double distilled water and ethanol for 3 times. The product is naturally dried to obtain the surface modified GCE.

(3) Insert MB-DNA/Mn-MOF/GCE as the working electrode into the electrolytic cell, add 0.1M phosphate water buffer as the electrolyte (pH 7.4), add STIP1 to it, and the concentration of STIP1 in the electrolyte is 0～500ng/mL. The CHI 660E electrochemical workstation containing a three-electrode system was used to determine the electrochemical square wave voltammetry curve in the presence of _{different STIP1 concentrations (C STIP1} ), and the linearity between the _{oxidation current peak intensity (I MB} ) of _{methylene blue and C STIP1 was fitted by} To construct a nano-electrochemical aptamer sensor for quantitative detection of STIP1. The linear detection range of STIP1 concentration is 1～500ng/mL, and the detection limit is 1～10ng/mL.

The effect of the present invention is: a preparation method of a nano electrochemical aptamer sensor for detecting stress-induced phosphoprotein is reported. The manganese-doped nickel-based metal framework compound (Mn-MOF) was prepared in one pot by solvothermal method. A small amount of Nafion was dripped on the surface of the glassy carbon electrode (GCE) as a cross-linking agent, and then an aqueous dispersion of Mn-MOF was dripped. , To build Mn-MOF/GCE. The aqueous solution of methylene blue end-labeled single-stranded nucleic acid aptamer (MB-DNA) was dripped onto the surface of Mn-MOF/GCE to construct the MB-DNA/Mn-MOF/GCE sensing interface. Insert the surface-modified GCE into the electrolytic cell, and use phosphate water as the electrolyte solution. When a certain amount of STIP1 is added, the MB-DNA aptamer specifically binds to STIP1 to form a MB-DNA/STIP1 complex, which causes MB-DNA to detach from the GCE surface, causing the redox effect of MB to decrease, that is, the current peak intensity decreases. By fitting the linear relationship between the MB current peak intensity and the added concentration of STIP1, a nanoelectrochemical aptamer sensor for detecting STIP1 can be constructed. Compared with the prior art, the method of the present invention is convenient to operate, has high sensitivity and good specificity, and can be used as a new STIP1 detection method for the quantitative detection of STIP1 in biomedical samples.

Description of the drawings

Figure 1 is a schematic diagram of the preparation method of a nano-electrochemical aptamer sensor based on the MB-DNA/Mn-MOF/GCE sensing interface and the principle of quantitative detection of STIP1.

Figure 2 shows the electrochemical square wave voltammetry curves measured with MB-DNA/Mn-MOF/GCE as the working electrode in the presence of different STIP1 concentrations.

Figure 3 shows the methylene blue redox current peak intensity (I _MB ) corresponding to different STIP1 concentrations, fitting the linear relationship between _{different I MB and exosome} concentration (C STIP1 ).

Detailed ways

The present invention will be described in detail below with reference to the drawings and specific embodiments.

Example 1

The preparation method of the nano-electrochemical aptamer sensor based on the MB-DNA/Mn-MOF/GCE sensing interface and the principle schematic diagram of the quantitative detection STIP1 involved in this embodiment are shown in FIG. 1, and the specific steps are as follows:

Preparation of manganese-doped nickel-based metal framework compound (Mn-MOF): 0.465g Ni(NO ₃ ) ₂ ·6H ₂ O and 0.01g Mn(NO ₃ ) ₂ ·4H ₂ O were dissolved in 25mL dimethylformamide (DMF), after stirring for 30 minutes, solution A is formed; 0.133g 1,4-phthalic acid is dissolved in 25mL DMF, and after stirring for 30 minutes, solution B is formed; under magnetic stirring, solution B is slowly added to solution A, and then gradually Add dilute hydrochloric acid dropwise to obtain a green solution, transfer it to an autoclave, and stir for 24 hours at 180°C; the reaction product is centrifuged to obtain a precipitate, which is washed with DMF and ethanol for 3 times, and the product is naturally dried;

Methylene blue end-labeled single-stranded nucleic acid aptamer/manganese-doped nickel-based metal framework compound/glassy carbon electrode (MB-DNA/Mn-MOF/GCE) sensing interface construction: drip coating on the surface of clean glassy carbon electrode (GCE) 5μL Nafion solution (5%w/w), after incubating for 5min, drip Mn-MOF aqueous dispersion, where the Mn-MOF concentration is 0.2mg/mL, and the dripping dosage is 10μL; put MB-DNA (2μM) at 37℃ After incubating for 2 hours, 10μL was dripped on the surface of Mn-MOF/GCE, after incubating for 12 hours, dried with N ₂ and then rinsed with double distilled water and ethanol for 3 times, the product was naturally dried to obtain the surface modified GCE;

Insert MB-DNA/Mn-MOF/GCE as working electrode into the electrolytic cell, add 0.1M phosphate water buffer as electrolyte (pH 7.4), add STIP1 to it, the concentration of STIP1 in this electrolyte is 0～275ng /mL; CHI 660E electrochemical workstation containing a three-electrode system was used to measure _{the electrochemical square wave voltammetry curve in the presence of different STIP1 concentrations (C STIP1} ) (as shown in Figure 2), and by fitting the methylene blue oxidation current peak intensity ( The linear relationship between I _MB ) and C _STIP1 (as shown in Figure 3), the construction of a nanoelectrochemical aptamer sensor for quantitative detection of STIP1; the linear detection range of STIP1 concentration is 5～275 ng/mL, and the detection limit is 2ng/ mL.

Example 2

The preparation method of the nano-electrochemical aptamer sensor based on the MB-DNA/Mn-MOF/GCE sensing interface and the principle schematic diagram of quantitative detection of STIP1 involved in this embodiment, and the preparation steps of Mn-MOF are the same as in embodiment 1, other specifics The preparation steps are as follows:

Methylene blue end-labeled single-stranded nucleic acid aptamer/manganese-doped nickel-based metal framework compound/glassy carbon electrode (MB-DNA/Mn-MOF/GCE) sensing interface construction: drip coating on the surface of clean glassy carbon electrode (GCE) 5μL Nafion solution (5%w/w), after incubating for 5min, drip Mn-MOF aqueous dispersion, where the Mn-MOF concentration is 0.4mg/mL, and the dripping dosage is 20μL; put MB-DNA (2μM) at 37℃ After incubating for 2h, weigh 15μL and apply it to the surface of Mn-MOF/GCE. After incubating for 12h, _{dry it with N 2} and rinse with double distilled water and ethanol for 3 times. The product is naturally dried to obtain surface-modified GCE;

Insert MB-DNA/Mn-MOF/GCE as working electrode into the electrolytic cell, add 0.1M phosphate water buffer as electrolyte (pH 7.4), add STIP1 to it, the concentration of STIP1 in this electrolyte is 1-500ng /mL; CHI 660E electrochemical workstation containing a three-electrode system was used to determine _{the electrochemical square wave voltammetry curve in the presence of different STIP1 concentrations (C STIP1} ), and by fitting the methylene blue oxidation current peak intensity (I _MB ) and C _STIP1 To construct a nanoelectrochemical aptamer sensor for quantitative detection of STIP1; the linear detection range of STIP1 concentration is 2～250ng/mL, and the detection limit is 1ng/mL.

Example 3

The preparation method of the nano-electrochemical aptamer sensor based on the MB-DNA/Mn-MOF/GCE sensing interface and the principle schematic diagram of quantitative detection of STIP1 involved in this embodiment, and the preparation steps of Mn-MOF are the same as in embodiment 1, other specifics The preparation steps are as follows:

Methylene blue end-labeled single-stranded nucleic acid aptamer/manganese-doped nickel-based metal framework compound/glassy carbon electrode (MB-DNA/Mn-MOF/GCE) sensing interface construction: drip coating on the surface of clean glassy carbon electrode (GCE) 5μL Nafion solution (5%w/w), after 5min incubation, drip Mn-MOF aqueous dispersion, where the Mn-MOF concentration is 0.8mg/mL, and the dripping dosage is 30μL; put MB-DNA (2μM) at 37℃ After incubating for 2h, measure 20μL and apply it to the surface of Mn-MOF/GCE. After incubating for 12h, _{dry it with N 2} and rinse with double distilled water and ethanol for 3 times. The product is naturally dried to obtain surface-modified GCE;

Insert MB-DNA/Mn-MOF/GCE as working electrode into the electrolytic cell, add 0.1M phosphate water buffer as electrolyte (pH 7.4), add STIP1 to it, the concentration of STIP1 in this electrolyte is 1-500ng /mL; CHI 660E electrochemical workstation containing a three-electrode system was used to determine _{the electrochemical square wave voltammetry curve in the presence of different STIP1 concentrations (C STIP1} ), and by fitting the methylene blue oxidation current peak intensity (I _MB ) and C _STIP1 To construct a nanoelectrochemical aptamer sensor for quantitative detection of STIP1; the linear detection range of STIP1 concentration is 2～500ng/mL, and the detection limit is 2ng/mL.

Claims (1)

1. A preparation method of a nano electrochemical aptamer sensor for detecting stress-induced phosphoproteins, characterized in that the method specifically includes the following steps:

   (1) Preparation of manganese-doped nickel-based metal framework compound Mn-MOF: 0.465g Ni(NO ₃ ) ₂ ·6H ₂ O and 0.01g Mn(NO ₃ ) ₂ ·4H ₂ O were dissolved in 25mL dimethylformaldehyde In amide DMF, solution A is formed after stirring for 30 minutes; 0.133g 1,4-phthalic acid is dissolved in 25mL DMF, and solution B is formed after stirring for 30 minutes; under magnetic stirring, solution B is slowly added to solution A, and then gradually Add dilute hydrochloric acid dropwise to obtain a green solution, transfer it to an autoclave, and stir for 24 hours at 180°C; the reaction product is centrifuged to obtain a precipitate, which is washed with DMF and ethanol for 3 times, and the product is naturally dried;

   (2) Methylene blue end-labeled single-stranded nucleic acid aptamer/manganese-doped nickel-based metal framework compound/glassy carbon electrode MB-DNA/Mn-MOF/GCE sensing interface construction: drop 5μL on the clean glassy carbon electrode GCE surface Nafion solution with a mass concentration of 5%, after incubating for 5 minutes, drip Mn-MOF aqueous dispersion, where the Mn-MOF concentration is 0.1-1mg/mL, and the dripping dosage is 5-50μL; the molar concentration of MB-DNA is 2μM incubation at 37 ℃ 2h, weighed 5 ~ 25μL drop coating the Mn-MOF / GCE surface, after 12h incubation with dry N _2, and then rinsed 3 times with double distilled water and ethanol was naturally dried, to give surface-modified GCE;

   (3) Insert MB-DNA/Mn-MOF/GCE as the working electrode into the electrolytic cell, add 0.1M phosphate water buffer with pH 7.4 as the electrolyte, and add the stress-inducing phosphoprotein STIP1 to it, where STIP1 is electrolyzed The concentration in the solution is 0～500ng/mL; CHI 660E electrochemical workstation with three-electrode system is used to measure _{the electrochemical square wave voltammetry curve in the presence of different STIP1 concentrations C STIP1} , and by fitting the methylene blue oxidation current peak intensity I _MB The linear relationship with C _STIP1 is used to construct a nano electrochemical aptamer sensor for quantitative detection of STIP1; the linear detection range of STIP1 concentration is 1～500ng/mL, and the detection limit is 1～10ng/mL.

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