METHOD FOR PREPARING DUAL-FUNCTIONAL HYBRID THIN-FILM FOR SELF-CALIBRATION DETECTION OF TUMOR-DERIVED EXOSOMES

Abstract

A method for preparing a dual-functional hybrid thin-film for self-calibration detection of tumor-derived exosomes is disclosed. A dual-functional hybrid thin-film, aptamer-BPNSs/Fc/ZIF-67/ITO, is constructed by facile self-assembly of a cobalt-based metal-organic framework (ZIF-67) composite doped with black phosphorus nanosheets (BPNSs), an aptamer and ferrocene (Fc) on an indium tin oxide (ITO) electrode. Methylene blue (MB) labeled aptamer specifically binds to CD63 protein to precisely capture protein. The protein is a specific biomolecule carried by breast cancer MCF-7 cell exosome, and realizes the detection of the tumor cell exosome. A self-calibration sensor for quantitative detection of the tumor exosome is constructed by using MB as a response signal and Fc as a reference. Compared with the prior art, the present invention features convenient operation, high sensitivity, low cost and excellent specificity, and can be used as a novel exosome self-calibration detection method for quantitative detection of the exosomes in biomedical samples.

Claims

1. A method for preparing a dual-functional hybrid thin-film for a self-calibration detection of tumor-derived exosomes, comprising: (1) preparation of a ferrocene-doped cobalt-based metal-organic framework (Fc/ZIF-67) composite: weighing a predetermined amount of Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole and adding the predetermined amount of Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole into a mixed solvent containing 47 mL of ethanol and 3 mL of deionized water to obtain a first mixture, magnetically stirring the first mixture to form an uniform first mixture, and adjusting a concentration of the Co(NO.sub.3).sub.2.6H.sub.2O to 0.1-0.5 mol/L and adjusting a concentration of the 2-methylimidazole to 0.8-1.5 mol/L, respectively; adding the uniform first mixture mixed solution to an electrolytic cell; with Ag/AgCl as a reference electrode, platinum wire as a counter electrode, and indium tin oxide (ITO) as a first working electrode, electrodepositing −the Fc/ZIF-67 composite on a surface of the first working electrode through a cyclic voltammetry at a constant voltage of −5 to −10 V for 100 to 500 s; (2) preparation of a black phosphorus nanosheets and ferrocene-doped cobalt-based metal-organic framework (BPNSs/Fc/ZIF-67) composite: weighing 10-30 mg of black phosphorus crystals and adding the black phosphorus crystals into 50 mL of 1-methyl-2-pyrrolidone to obtain a second mixture; sonicating the second mixture in an ultrasonic cleaner for 1-6 h, transferring the second mixture to a probe-type ultrasonic generator and sonicating the second mixture for 1-4 h to obtain a product dispersion; centrifuging the product dispersion for 15 min at 12,000 rpm to obtain an upper dispersion, and centrifuging the upper dispersion for 15 min at 5,000 rpm to obtain a black phosphorus nanosheets (BPNSs) dispersion; adding the black phosphorus nanosheets (BPNSs) dispersion dropwise to a surface of the Fc/ZIF-67 composite to obtain a third mixture, drying the third mixture naturally, to obtain the BPNSs/Fc/ZIF-67 composite on the surface of the first working electrode as a BPNSs/Fc/ZIF-67/ITO film electrode; (3) preparation of an aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film: immersing the BPNSs/Fc/ZIF-67/ITO film electrode in phosphate buffered saline (PBS) containing 1-10 μM of CD63 transmembrane protein corresponding to a single-stranded DNA aptamer to obtain a fourth mixture, incubating the fourth mixture at 37° C. for 30-120 min to obtain a resulting film electrode, taking out the resulting film electrode, drying the resulting film electrode naturally, to obtain an aptamer-BPNSs/Fc/ZIF-67 composite on the surface of the first working electrode, wherein the aptamer-BPNSs/Fc/ZIF-67 composite on the surface of the first working electrode is the aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film; and (4) preparation of a hybrid thin-film sensor: placing-a the aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film in a three-electrode system of an electrochemical workstation, wherein the aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film serves as a second working electrode, adding exosomes in PBS, wherein the exosomes are extracted from breast cancer MCF-7 cells and the PBS serves as an electrolyte, and measuring electrochemical square wave voltammetry curves at different concentrations of the exosomes; with methylene blue (MB) modified on an aptamer strand as a response signal, Fc doped in the ZIF-67 metal-organic framework as a reference signal, and ratios of peak current intensities I.sub.Fc/I.sub.MB as a self-calibrated signal output, fitting a linear relationship between the ratios of the I.sub.Fc/I.sub.MB and the concentrations of the exosomes to construct a self-calibrating sensor for a quantitative detection of the exosomes, wherein a linear detection range of the concentrations of the exosomes is 1×10.sup.2−1×10.sup.6 particles μL.sup.−1, and a detection limit is 50-100 particles μL.sup.−1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is a schematic diagram showing a preparation of a self-assembled dual-functional hybrid thin-film material based on a cobalt-based metal-organic framework composite doped with BPNSs and Fc, and a principle of application thereof in precise capture of CD63 transmembrane protein and self-calibration detection of breast cancer exosomes;

[0014] FIG. 2A is a diagram showing electrochemical square wave voltammetry curves determined with the hybrid thin-film material as a working electrode at different concentrations of the exosomes; and

[0015] FIG. 2B is a diagram showing a linear relationship between different ratios of I.sub.Fc/I.sub.MB and concentrations of the exosomes fitted by ratios of redox peak current intensities I.sub.Fc/I.sub.MB of ferrocene versus methylene blue corresponding to different concentrations of the exosomes.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] The present invention will be described in detail below with reference to the drawings.

Embodiment 1

[0017] A schematic diagram of the preparation method and detection principle of a self-assembled dual-functional hybrid thin-film based on a cobalt-based metal-organic framework composite doped with BPNSs and Fc according to embodiment 1 is shown in FIG. 1, and the specific preparation steps are as follows:

[0018] Preparation of a Fc/ZIF-67 composite: A predetermined amount of Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole were weighed in a mixed solvent containing 47 mL of ethanol and 3 mL of deionized water, and magnetically stirred to form an uniform mixed solution. Concentrations of Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole were adjusted to 0.1 and 0.8 mol/L, respectively. The mixed solution was added to an electrolytic cell; with Ag/AgCl as a reference electrode, platinum wire as a counter electrode, and ITO as a working electrode. An Fc/ZIF-67 composite was electrodeposited on the surface of the ITO electrode through cyclic voltammetry at a constant voltage of −5 V for 100 s.

[0019] Preparation of a BPNSs/Fc/ZIF-67 composite: 10 mg of black phosphorus crystals were weighed and added into 50 mL of 1-methyl-2-pyrrolidone, sonicated in an ultrasonic cleaner for 1 h, and then transferred to a probe-type ultrasonic generator and sonicated for 1 h. A product dispersion was centrifuged for 15 min at 12,000 rpm, and an upper dispersion was centrifuged for 15 min at 5,000 rpm. A prepared BPNSs dispersion was added dropwise to the surface of the Fc/ZIF-67 composite, followed by natural drying, and the BPNSs/Fc/ZIF-67 composite was prepared on the surface of the ITO electrode.

[0020] Preparation of an aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film: A BPNSs/Fc/ZIF-67/ITO film electrode was immersed in phosphate buffered saline (PBS) containing 1 μM of CD63 transmembrane protein corresponding to single-stranded DNA aptamer, and incubated at 37° C. for 30 min. The film electrode was taken out and dried naturally, and an aptamer-BPNSs/Fc/ZIF-67 composite was prepared on the surface of the ITO electrode, i.e., the aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film was prepared.

[0021] Preparation of a hybrid thin-film sensor: A hybrid thin-film as a working electrode was placed in a three-electrode system of an electrochemical workstation, exosomes extracted from breast cancer MCF-7 cells were added in PBS as an electrolyte, and electrochemical square wave voltammetry curves were measured at different concentrations of the exosomes (as shown in FIG. 2A). With MB modified on an aptamer strand as a response signal, Fc doped in the ZIF-67 metal-organic framework as a reference signal, and ratios of peak current intensities I.sub.Fc/I.sub.MB as self-calibrated signal output, a linear relationship between I.sub.Fc/I.sub.MB ratio and concentrations of the exosomes (as shown in FIG. 2B) was fitted to construct a self-calibrating sensor for quantitative detection of the exosomes. A linear detection range of concentrations of the tumor-derived exosomes was 1.3×10.sup.2−2.6×10.sup.5 particles μL.sup.−1, and a detection limit was 60 particles μL.sup.−1.

Embodiment 2

[0022] A self-assembled dual-functional hybrid thin-film based on a cobalt-based metal-organic framework composite doped with BPNSs and Fc according to the embodiment had the same preparation method and detection principle as embodiment 1, and other specific preparation steps are as follows:

[0023] Preparation of a Fc/ZIF-67 composite: A predetermined amount of Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole were weighed in a mixed solvent containing 47 mL of ethanol and 3 mL of deionized water, and magnetically stirred to form an uniform mixed solution. Concentrations of Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole were adjusted to 0.3 and 1.2 mol/L, respectively. The mixed solution was added to an electrolytic cell; with Ag/AgCl as a reference electrode, platinum wire as a counter electrode, and ITO as a working electrode, an Fc/ZIF-67 composite was electrodeposited on the surface of the ITO electrode through cyclic voltammetry at a constant voltage of −8 V for 200 s.

[0024] Preparation of a BPNSs/Fc/ZIF-67 composite: 20 mg of black phosphorus crystals were weighed and added into 50 mL of 1-methyl-2-pyrrolidone, sonicated in an ultrasonic cleaner for 3 h, and then transferred to a probe-type ultrasonic generator and sonicated for 2 h. A product dispersion was centrifuged for 15 min at 12,000 rpm, and an upper dispersion was centrifuged for 15 min at 5,000 rpm. A prepared BPNSs dispersion was added dropwise to the surface of the Fc/ZIF-67 composite, followed by natural drying, and the BPNSs/Fc/ZIF-67 composite was prepared on the surface of the ITO electrode.

[0025] Preparation of an aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film: A BPNSs/Fc/ZIF-67/ITO film electrode was immersed in PBS containing 4 μM of CD63 transmembrane protein corresponding to single-stranded DNA aptamer, and incubated at 37° C. for 50 min; the film electrode was taken out and dried naturally, and an aptamer-BPNSs/Fc/ZIF-67 composite was prepared on the surface of the ITO electrode, i.e., the aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film was prepared.

[0026] Preparation of a hybrid thin-film sensor: A hybrid thin-film as a working electrode was placed in a three-electrode system of an electrochemical workstation, exosomes extracted from breast cancer MCF-7 cells were added in PBS as an electrolyte, and electrochemical square wave voltammetry curves were measured at different concentrations of the exosomes. With MB modified on an aptamer strand as a response signal, Fc doped in the ZIF-67 metal-organic framework as a reference signal, and ratios of peak current intensities I.sub.Fc/I.sub.MB as self-calibrated signal output, a linear relationship between I.sub.Fc/I.sub.MB ratio and concentrations of the exosomes was fitted to construct a self-calibrating sensor for quantitative detection of the exosomes. A linear detection range of concentrations of the tumor-derived exosomes was 1.0×10.sup.2−1.0×10.sup.5 particles μL.sup.−1, and a detection limit was 50 particles μL.sup.−1.

Embodiment 3

[0027] A self-assembled dual-functional hybrid thin-film based on a cobalt-based metal-organic framework composite doped with BPNSs and Fc according to the embodiment had the same preparation method and detection principle as embodiment 1, and other specific preparation steps are as follows:

[0028] Preparation of a Fc/ZIF-67 composite: A predetermined amount of Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole were weighed in a mixed solvent containing 47 mL of ethanol and 3 mL of deionized water, and magnetically stirred to form an uniform mixed solution. Concentrations of Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole were adjusted to 0.5 and 1.5 mol/L, respectively. The mixed solution was added to an electrolytic cell; with Ag/AgCl as a reference electrode, platinum wire as a counter electrode, and ITO as a working electrode, an Fc/ZIF-67 composite was electrodeposited on the surface of the ITO electrode through cyclic voltammetry at a constant voltage of −10 V for 400 s.

[0029] Preparation of a BPNSs/Fc/ZIF-67 composite: 30 mg of black phosphorus crystals were weighed and added into 50 mL of 1-methyl-2-pyrrolidone, sonicated in an ultrasonic cleaner for 5 h, and then transferred to a probe-type ultrasonic generator and sonicated for 4 h. A product dispersion was centrifuged for 15 min at 12,000 rpm, and an upper dispersion was centrifuged for 15 min at 5,000 rpm. A prepared BPNSs dispersion was added dropwise to the surface of the Fc/ZIF-67 composite, followed by natural drying, and the BPNSs/Fc/ZIF-67 composite was prepared on the surface of the ITO electrode.

[0030] Preparation of an aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film: A BPNSs/Fc/ZIF-67/ITO film electrode was immersed in PBS containing 8 μM of CD63 transmembrane protein corresponding to single-stranded DNA aptamer, and incubated at 37° C. for 100 min. The film electrode was taken out and dried naturally, and an aptamer-BPNSs/Fc/ZIF-67 composite was prepared on the surface of the ITO electrode, i.e., the aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film was prepared.

[0031] Preparation of a hybrid thin-film sensor: A hybrid thin-film as a working electrode was placed in a three-electrode system of an electrochemical workstation, exosomes extracted from breast cancer MCF-7 cells were added in PBS as an electrolyte, and electrochemical square wave voltammetry curves were measured at different concentrations of the exosomes. With MB modified on an aptamer strand as a response signal, Fc doped in the ZIF-67 metal-organic framework as a reference signal, and ratios of peak current intensities I.sub.Fc/I.sub.MB as self-calibrated signal output, a linear relationship between I.sub.Fc/I.sub.MB ratio and concentrations of the exosomes was fitted to construct a self-calibrating sensor for quantitative detection of the exosomes. A linear detection range of concentrations of the tumor-derived exosomes was 1.0×10.sup.3−1.0×10.sup.6 particles μL.sup.−1, and a detection limit was 80 particles 0.sup.−1.

[0032] The foregoing descriptions are merely preferred embodiments of the present invention. It should be noted that several variations and modifications can be made by those skilled in the art without departing from the principles of the present invention and should also fall within the protection scope of the invention.