Method for preparing ratiometric electrochemical miR3123 aptasensor based on metal-organic framework composite

Patent metadata

Field	Value
Publication number	US-11099150-B1
Application number	US-201917051781-A
Country	US
Kind code	B1
Filing date	Aug 30, 2019
Priority date	Aug 27, 2019
Publication date	Aug 24, 2021
Grant date	Aug 24, 2021

Abstract

A method for preparing a ratiometric electrochemical miR3123 aptasensor based on a copper-based metal-organic framework (Cu-MOF) composite doped with black phosphorus nanosheets (BPNSs) and thionine (TH) is provided. TH/Cu-MOF is prepared by reacting TH with Cu-MOF precursor, and BPNSs/TH/Cu-MOF is prepared by drop coating the BPNSs and drop coated onto an electrode. A ferrocene (Fc)-labeled single-stranded DNA aptamer is adsorbed on the BPNSs to prepare aptamer-BPNSs/TH/Cu-MOF. Target molecule miR3123 is bonded with the single-stranded DNA aptamer Fc-DNA, Fc-DNA is forced to escape from the BPNSs. Electrochemical signals of Fc are, therefore, weakened while TH signals are slightly affected. The ratiometric electrochemical miR3123 aptasensor is constructed by fitting a linear relationship between peak current intensity ratios IFc/ITH and concentrations of the miR3123.

First claim

What is claimed is:

1. A method for preparing a ratiometric electrochemical miR3123 aptasensor based on a metal-organic framework (MOF) composite comprising the following steps:
(1) preparation of a thionine (TH)/copper-based metal-organic framework (Cu-MOF) composite: separately weighing 4 mg of copper nitrate trihydrate, 10 μL of trifluoroacetic acid and 10 mg of polyvinylpyrrolidone (PVP) and adding the copper nitrate trihydrate, the trifluoroacetic acid and the PVP into 12 mL of a first mixed solvent containing 9 mL of N,N-dimethylformamide and 3 mL of ethanol for a uniform stirring to form a first mixture; separately weighing 4 mg of 4,4′,4″,4″-(porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid) and 4 mg of TH and adding the 4,4′,4″,4′″-(porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid) and the TH to a second mixed solvent containing 3 mL of N,N-dimethylformamide and 1 mL of ethanol for a uniform stirring to form a second mixture; adding the first mixture dropwise to the second mixture to obtain a third mixture for stirring and sonicating for 10 min; heating the third mixture to 80° C. and reacting for 3 h to obtain a first resulting product solution; centrifuging the first resulting product solution for 10 min at 8,000 rpm to obtain a second resulting product solution, washing the second resulting product solution with ethanol and distilled water to obtain a third resulting product solution, and drying the third resulting product solution to obtain the TH/Cu-MOF composite, and the TH/Cu-MOF composite is dispersed in ethanol to obtain a TH/Cu-MOF composite dispersion for later use;
(2) preparation of a black phosphorus nanosheets (BPNSs)/TH/Cu-MOF composite: weighing 15 mg of black phosphorus bulk crystals and adding the black phosphorus bulk crystals into 30 mL of 1-methyl-2-pyrrolidone to obtain a fourth mixture, sonicating the fourth mixture in an ultrasonic cleaner for 6 h, and then transferring the fourth mixture to a probe-type ultrasonic generator and sonicating for 4 h; centrifuging the fourth mixture for 20 min at 10,000 rpm to remove larger-size products from the fourth mixture to obtain an upper dispersion, and centrifuging the upper dispersion for 20 min at 3,500 rpm to obtain a BPNSs dispersion; adding the BPNSs dispersion dropwise to the TH/Cu-MOF composite dispersion to obtain a fifth mixture, sonicating the fifth mixture for 1 h under a stirring, and then centrifuging the fifth mixture for 15 min at 8,000 rpm to prepare a BPNSs/TH/Cu-MOF composite dispersion for later use;
(3) preparation of an aptamer-BPNSs/TH/Cu-MOF nanocomposite: adding a cross-linking agent Nafion dropwise on a surface of a polished bare glassy carbon electrode to obtain a first resulting carbon electrode, and then drop coating the BPNSs/TH/Cu-MOF composite dispersion on the first resulting carbon electrode to form a second resulting glassy carbon electrode, wherein the second resulting glassy carbon electrode is modified by the BPNSs/TH/Cu-MOF composite; immersing the second resulting glassy carbon electrode in phosphate buffered saline (PBS) containing 1-10 μM of miR3123-corresponding single-stranded DNA aptamer Fc-DNA to obtain a sixth mixture, and incubating the sixth mixture for 2 h at 37° C. to obtain a third resulting glassy carbon electrode; then taking out the third resulting glassy carbon electrode, and drying the third resulting glassy carbon electrode naturally to prepare the aptamer-BPNSs/TH/Cu-MOF nanocomposite on a surface of the third resulting glassy carbon electrode; and
(4) using an aptamer-BPNSs/TH/Cu-MOF nanocomposite modified glassy carbon electrode as a working electrode and placing the aptamer-BPNSs/TH/Cu-MOF nanocomposite modified glassy carbon electrode in a three-electrode system of an electrochemical workstation; using PBS as an electrolyte, adding a predetermined amount of miR3123 to determine electrochemical square wave voltammetry curves at different concentrations of the miR3123; using a redox peak current intensity of the TH as a reference signal, and using a redox peak current intensity of Fc as a response signal, respectively, fitting a linear relationship between peak current intensity ratios I Fc /I TH and the concentrations of the miR3123 to construct a ratiometric electrochemical aptasensor for a quantitative detection of the miR3123, wherein a linear detection concentration of the miR3123 is 1 nM-10 μM, and a detection limit is 1-5 nM.

Assignees

• Univ Qingdao

Inventors

• Gui Rijun
• Sun Yujiao
• Jin Hui
• Jiang Xiaowen

Classifications

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q2600/178

  miRNA, siRNA or ncRNA · CPC title

• G01N27/3278 Primary

  involving nanosized elements, e.g. nanogaps or nanoparticles (nanopores G01N33/48721; magnetic beads G01N27/745) · CPC title

• G01N27/3276

  being a hybridisation with immobilised receptors (using a FET type sensor G01N27/4145; concerning the hybridisation C12Q1/68) · CPC title

• B05D1/30

  performed by gravity only, i.e. flow coating · CPC title