Method for preparing nanohybrid used for ratiometric fluorescence and ratiometric electrochemical sensing simultaneously

Abstract

A method for preparing a nanohybrid used for ratiometric fluorescence and ratiometric electrochemical sensing simultaneously is provided. Surface-aminated (—NH.sub.2) SiO.sub.2 nanospheres encapsulating an electroactive material A or B are prepared and conjugated with surface-carboxylated (—COOH) carbon dots (CDs) or gold nanoclusters (AuNCs) to prepare a conjugate, and the conjugate is conjugated with a DNA aptamer terminated with —NH.sub.2. Ions or biomolecules are added to two types of DNA-conjugate dispersions, and ratiometric florescence sensing is realized by fitting the linear relationship between ratiometric fluorescent peak intensity I.sub.CDs/I.sub.AuNCs and a specific ion concentration or a specific biomolecule concentration. A-SiO.sub.2@CDs-DNA is attached to the surface of a gold electrode based on a DNA terminal —SH and Au—S bonding; B—SiO.sub.2@AuNCs-DNA and ions or biomolecules are added, and ratiometric electrochemical sensing is realized by fitting the linear relationship between the specific ion concentration or the specific biomolecule concentration and the ratiometric current peak intensity I.sub.B/I.sub.A.

Claims

1. A method for preparing a nanohybrid used for ratiometric fluorescence sensing and ratiometric electrochemical sensing simultaneously, comprising the following steps: (1) dissolving an electroactive material in absolute ethanol to obtain a first mixture, stirring the first mixture uniformly with (3-aminopropyl)triethoxysilane (APTS) to obtain a second mixture, and storing the second mixture in a dark environment to avoid light; adding ammonia water and ethanol to the second mixture to obtain a third mixture and stirring the third mixture uniformly, and then adding tetraethyl orthosilicate (TEOS) to the third mixture to stir continuously to obtain a fourth mixture, and then adding the TEOS to the fourth mixture for a first reaction to obtain a first resulting product; subjecting the first resulting product to a first treatment of high-speed centrifugation, ethanol washing, and vacuum drying to obtain SiO.sub.2 nanospheres encapsulating the electroactive material; dispersing the SiO.sub.2 nanospheres encapsulating the electroactive material in a mixed solution of the APTS and acetic acid to obtain a fifth mixture, stirring the fifth mixture at room temperature, and purifying the fifth mixture by the first treatment of high-speed centrifugation, ethanol washing, and vacuum drying to obtain surface-aminated (—NH.sub.2) SiO.sub.2 nanospheres encapsulating the electroactive material; (2) dispersing citric acid and thiourea in dimethylformamide to obtain a sixth mixture, transferring the sixth mixture to a high-pressure microreactor, wherein the high-pressure microreactor contains a polytetrafluoroethylene (PTFE) lining, and stirring the sixth mixture at a predetermined temperature for a second reaction to obtain a second resulting product, cooling the second resulting product to room temperature, followed by a second treatment of high-speed centrifugation, washing with ethanol and water, and vacuum drying on the second resulting product, to obtain surface-carboxylated (—COOH) carbon dots (CDs); (3) dispersing mercaptoundecanoic acid in a NaOH solution to obtain a seventh mixture, adding an aqueous HAuCl.sub.4 solution to the seventh mixture under rapid stirring to obtain an eighth mixture, adjusting the eighth mixture with the NaOH solution until clear to obtain a ninth mixture, adding a NaBH.sub.4 solution to the ninth mixture dropwise to obtain a tenth mixture, stirring the tenth mixture at room temperature for a third reaction to obtain a third resulting product, and subjecting the third resulting product to a third treatment of dialysis, rotary distillation, centrifugation, washing and drying to obtain surface-carboxylated (—COOH) gold nanoclusters (AuNCs); (4) dispersing a first coupling agent N-hydroxythiosuccinimide (NHS) and a second coupling agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride in phosphate buffered saline (PBS) to obtain an eleventh mixture, adding the surface-aminated (—NH.sub.2) SiO.sub.2 nanospheres encapsulating the electroactive material to the eleventh mixture to obtain a twelfth mixture, stirring the twelfth mixture uniformly, performing an ultrasonic treatment on the twelfth mixture in the dark environment to obtain a thirteenth mixture, adding a surface-carboxylated CDs aqueous dispersed solution or an AuNCs aqueous dispersed solution to the thirteenth mixture under a magnetic stirring to obtain a fourteenth mixture, stirring the fourteenth mixture for a fourth reaction to obtain a fourth resulting product, and subjecting the fourth resulting product to a fourth treatment of centrifugation, washing, and drying to obtain two conjugates, wherein the two conjugates are SiO.sub.2@CDs and SiO.sub.2@AuNCs, respectively; (5) add the first coupling agent NHS and the second coupling agent EDC hydrochloride to an aqueous solution of Tris-HCl and NaOH to obtain a fifteenth mixture, adding the SiO.sub.2@CDs or the SiO.sub.2@AuNCs to the fifteenth mixture to obtain a sixteenth mixture, stirring the sixteenth mixture continuously for a fifth reaction to obtain a fifth resulting product, adding a specific single-stranded DNA aptamer to the fifth resulting product, stirring for a sixth reaction at room temperature to obtain a sixth resulting product, and subjecting the sixth resulting product to a fifth treatment of dialysis, rotary distillation, centrifugation, washing, and drying to obtain the nanohybrid, wherein the nanohybrid is SiO.sub.2@CDs-DNA or SiO.sub.2@AuNCs-DNA; (6) adding a specific ion or a specific biomolecule to a nanohybrid aqueous dispersed solution to obtain a seventeenth mixture, determining a fluorescence emission spectrum of the seventeenth mixture, and building a linear relationship between a concentration of the specific ion or a concentration of the specific biomolecule and ratiometric fluorescent peak intensity I.sub.CDs/I.sub.AuNCs to achieve the ratiometric fluorescence sensing of the specific ion or the specific biomolecule; and (7) transferring a nanohybrid-Tris-HCl dispersed solution into an electrolytic cell, wherein the electrolytic cell is equipped with a gold electrode, a surface of the gold electrode is bonded to DNA terminal sulfhydryl groups through Au—S bonds; conjugating the nanohybrid to the surface of the gold electrode, adding the specific ion or the specific biomolecule to obtain an eighteenth mixture, determining a square wave voltammetry curve of the eighteenth mixture through an electrochemical workstation, and building a linear relationship between the concentration of the specific ion or the concentration of the specific biomolecule and ratiometric current peak intensity I.sub.electroactive material B/I.sub.electroactive material A to achieve the ratiometric electrochemical sensing of the specific ion or the specific biomolecule.

2. The method according to claim 1, wherein the electroactive material in step (1) is an electrochemical redox probe molecule, and the electrochemical redox probe molecule is one selected from the group consisting of ferrocene (Fc), methylene blue (MB) and thionine (TH), and the SiO.sub.2 nanospheres encapsulating the electroactive material have an average size of 50-200 nm.

3. The method according to claim 1, wherein a reaction temperature of the second reaction is 100-200° C., and a reaction time of the second reaction is 3-10 h in step (2).

4. The method according to claim 1, wherein a reaction time of the third reaction is 12-48 h in step (3).

5. The method according to claim 1, wherein a mass ratio of the first coupling agent NHS to the second coupling agent EDC hydrochloride is 1:1-1:3 in step (4), and a reaction time of the fourth reaction is 6-12 h in step (4).

6. The method according to claim 1, wherein a reaction time of the fifth reaction and the sixth reaction is 6-18 h in step (5).

7. The method according to claim 1, wherein in steps (6) and (7), the specific ion is one selected from the group consisting of Ag.sup.+, Hg.sup.2+ and Pb.sup.2+, the specific biomolecule is a tumor biomarker, wherein the tumor biomarker is one selected from the group consisting of thrombin, lipopolysaccharide (LPS), carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), and the specific ion or the specific biomolecule has a molar concentration of 1 nM-1 mM.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of a nanohybrid used simultaneously for ratiometric fluorescence and ratiometric electrochemical sensing of the present invention;

(2) FIG. 2 is a schematic diagram of a preparation and a principle of the nanohybrid used for ratiometric fluorescence sensing of ions and biomolecules of the present invention; and

(3) FIG. 3 is a schematic diagram of a preparation and a principle of the nanohybrid used for ratiometric electrochemical sensing of the ions and the biomolecules of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(4) The present invention will be described in detail below with reference to the drawings.

Example 1

(5) The present invention provides a method for preparing a nanohybrid that is used for ratiometric fluorescence and ratiometric electrochemical sensing simultaneously. The preparation process and detection principle are shown in FIGS. 1-3, and the specific preparation steps are as follows:

(6) Ferrocene (Fc) or methylene blue (MB) was dissolved in absolute ethanol, stirred uniformly with (3-aminopropyl) triethoxysilane (APTS), and stored in a dark environment to avoid light. Ammonia water and ethanol were added to stir uniformly, tetraethyl orthosilicate (TEOS) was added to stir and react, and then the TEOS was added to continue the reaction. A resulting product was subjected to centrifugation, washing, and drying to obtain 80 nm SiO.sub.2 nanospheres encapsulating Fc- or MB. A resulting product was dispersed in a mixed solution of the APTS and acetic acid, reacted under stirring at room temperature, and was purified in a similar manner to obtain surface-aminated (—NH.sub.2) SiO.sub.2 nanospheres.

(7) Citric acid and thiourea were dispersed in dimethylformamide, and transferred to a high-pressure microreactor containing a polytetrafluoroethylene (PTFE) lining; reacted for 6 h at 160° C. under stirring. A resulting product was cooled to room temperature, followed by centrifugation, washing with ethanol and water, and drying, to obtain surface-carboxylated (—COOH) carbon dots (CDs).

(8) Mercaptoundecanoic acid was dissolved in a NaOH solution, an aqueous HAuCl.sub.4 solution was added under rapid stirring, a mixed solution was adjusted with NaOH until clear, a NaBH.sub.4 solution was added dropwise, and reacted for 24 h at room temperature under stirring; a resulting product was subjected to dialysis, rotary distillation, centrifugation, washing, and drying to obtain surface-carboxylated (—COOH) AuNCs.

(9) N-hydroxythiosuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride were dispersed in phosphate buffered saline (PBS) in a mass ratio of 1:1, and stirred uniformly with the surface-aminated and SiO.sub.2 nanospheres encapsulating Fc- or MB were added, stirred uniformly, subjected to an ultrasonic treatment in a dark environment to avoid light, and magnetically stirred; the surface-carboxylated CDs or AuNCs were added to a resulting mixture, and the reaction was conducted for 8 h under stirring. A resulting product was subjected to centrifugation, washing, and drying to obtain to obtain two conjugates, Fc-SiO.sub.2@CDs and MB-SiO.sub.2@AuNCs, respectively.

(10) Coupling agents NHS and EDC hydrochloride were added to an aqueous solution of Tris-HCl and NaOH, and stirred with the Fc-SiO.sub.2@CDs or the MB-SiO.sub.2@AuNCs continuously; specific single-stranded DNA aptamer was added, and the reaction was conducted for 12 h at room temperature under stirring. A resulting product was subjected to dialysis, rotary distillation, centrifugation, washing, and drying to obtain the nanohybrid, i.e., Fc-SiO.sub.2@CDs-DNA or MB-SiO.sub.2@AuNCs-DNA.

(11) Ag.sup.+ or thrombin was added to a nanohybrid aqueous dispersed solution, a fluorescence emission spectrum of a mixed solution was determined, and a linear relationship between the Ag.sup.+ or the thrombin concentration and ratiometric fluorescent peak intensity I.sub.CDs/I.sub.AuNcs was built to achieve ratiometric fluorescence sensing of the Ag.sup.+ or the thrombin. The nanohybrid dispersed in the Tris-HCl was transferred into an electrolytic cell equipped with a gold electrode. The surface of the gold electrode was bonded to the DNA terminal sulfhydryl via Au—S bond, and the nanohybrid was attached to the surface of the gold electrode, and the Ag.sup.+ or the thrombin was added. The square wave voltammetry curve was determined by an electrochemical workstation, and a linear relationship between Ag.sup.+ or thrombin concentration and ratiometric current peak intensity I.sub.MB/I.sub.Fc was built to achieve ratiometric electrochemical sensing of the Ag.sup.+ or the thrombin; a concentration of the Ag.sup.+ or the thrombin is 5 nM-0.1 mM.

Example 2

(12) Fc or MB was dissolved in absolute ethanol, stirred uniformly with APTS, and stored in a dark environment to avoid light. Ammonia water and ethanol were added to stir uniformly, the TEOS was added to stir and react, and then the TEOS was added to continue the reaction. A resulting product was subjected to centrifugation, washing, and drying to obtain 100 nm SiO.sub.2 nanospheres encapsulating Fc- or MB. A resulting product was dispersed in a mixed solution of the APTS and acetic acid, reacted under stirring at room temperature, and was purified in a similar manner to obtain surface-aminated (—NH.sub.2) SiO.sub.2 nanospheres.

(13) Citric acid and thiourea were dispersed in dimethylformamide, and transferred to a high-pressure microreactor containing a PTFE lining; reacted for 5 h at 180° C. under stirring. A resulting product was cooled to room temperature, followed by centrifugation, washing with ethanol and water, and drying, to obtain surface-carboxylated (—COOH) carbon dots (CDs).

(14) Mercaptoundecanoic acid was dissolved in a NaOH solution, an aqueous HAuCl.sub.4 solution was added under rapid stirring, a mixed solution was adjusted with NaOH until clear, a NaBH.sub.4 solution was added dropwise, and reacted for 18 h at room temperature under stirring; a resulting product was subjected to dialysis, rotary distillation, centrifugation, washing, and drying to obtain surface-carboxylated (—COOH) AuNCs.

(15) NHS and EDC hydrochloride were dispersed in PBS in a mass ratio of 1:2, and stirred uniformly with the surface-aminated and SiO.sub.2 nanospheres encapsulating Fc- or MB were added, stirred uniformly, subjected to an ultrasonic treatment in a dark environment to avoid light, and magnetically stirred; the surface-carboxylated CDs or AuNCs were added to a resulting mixture, and the reaction was conducted for 10 h under stirring. A resulting product was subjected to centrifugation, washing, and drying to obtain to obtain two conjugates, Fc-SiO.sub.2@CDs and MB-SiO.sub.2@AuNCs, respectively.

(16) Coupling agents NHS and EDC hydrochloride were added to an aqueous solution of Tris-HCl and NaOH, and stirred with the Fc-SiO.sub.2@CDs or the MB-SiO.sub.2@AuNCs continuously; specific single-stranded DNA aptamer was added, and the reaction was conducted for 15 h at room temperature under stirring. A resulting product was subjected to dialysis, rotary distillation, centrifugation, washing, and drying to obtain the nanohybrid, i.e., Fc-SiO.sub.2@CDs-DNA or MB-SiO.sub.2@AuNCs-DNA.

(17) Hg.sup.2+ or lipopolysaccharide (LPS) was added to a nanohybrid aqueous dispersed solution, a fluorescence emission spectrum of a mixed solution was determined, and a linear relationship between the Hg.sup.2+ or the LPS concentration and ratiometric fluorescent peak intensity I.sub.CDs/I.sub.AuNcs was built to achieve ratiometric fluorescence sensing of the Hg.sup.2+ or the LPS. The nanohybrid dispersed in the Tris-HCl was transferred into an electrolytic cell equipped with a gold electrode. The surface of the gold electrode was bonded to the DNA terminal sulfhydryl via Au—S bond, and the nanohybrid was attached to the surface of the gold electrode, and the Hg.sup.2+ or the LPS was added. The square wave voltammetry curve was determined by an electrochemical workstation, and a linear relationship between the Hg′ or the LPS concentration and ratiometric current peak intensity I.sub.MB/I.sub.Fc was built to achieve ratiometric electrochemical sensing of the Hg.sup.2+ or the LPS; a concentration of the Hg.sup.2+ or the LPS is 10 nM-0.5 mM.

Example 3

(18) Fc or MB was dissolved in absolute ethanol, stirred uniformly with APTS, and stored in a dark environment to avoid light. Ammonia water and ethanol were added to stir uniformly, the TEOS was added to stir and react, and then the TEOS was added to continue the reaction. A resulting product was subjected to centrifugation, washing, and drying to obtain 120 nm SiO.sub.2 nanospheres encapsulating Fc- or MB. A resulting product was dispersed in a mixed solution of the APTS and acetic acid, reacted under stirring at room temperature, and was purified in a similar manner to obtain surface-aminated (—NH.sub.2) SiO.sub.2 nanospheres.

(19) Citric acid and thiourea were dispersed in dimethylformamide, and transferred to a high-pressure microreactor containing a PTFE lining; reacted for 3 h at 200° C. under stirring. A resulting product was cooled to room temperature, followed by centrifugation, washing with ethanol and water, and drying, to obtain surface-carboxylated (—COOH) carbon dots (CDs).

(20) Mercaptoundecanoic acid was dissolved in a NaOH solution, an aqueous HAuCl.sub.4 solution was added under rapid stirring, a mixed solution was adjusted with NaOH until clear, a NaBH.sub.4 solution was added dropwise, and reacted for 36 h at room temperature under stirring; a resulting product was subjected to dialysis, rotary distillation, centrifugation, washing, and drying to obtain surface-carboxylated (—COOH) AuNCs.

(21) NHS and EDC hydrochloride were dispersed in PBS in a mass ratio of 1:3, and stirred uniformly with the surface-aminated and SiO.sub.2 nanospheres encapsulating Fc- or MB were added, stirred uniformly, subjected to an ultrasonic treatment in a dark environment to avoid light, and magnetically stirred; the surface-carboxylated CDs or AuNCs were added to a resulting mixture, and the reaction was conducted for 12 h under stirring. A resulting product was subjected to centrifugation, washing, and drying to obtain to obtain two conjugates, Fc-SiO.sub.2@CDs and MB-SiO.sub.2@AuNCs, respectively.

(22) Coupling agents NHS and EDC hydrochloride were added to an aqueous solution of Tris-HCl and NaOH, and stirred with the Fc-SiO.sub.2@CDs or the MB-SiO.sub.2@AuNCs continuously; specific single-stranded DNA aptamer was added, and the reaction was conducted for 18 h at room temperature under stirring. A resulting product was subjected to dialysis, rotary distillation, centrifugation, washing, and drying to obtain the nanohybrid, i.e., Fc-SiO.sub.2@CDs-DNA or MB-SiO.sub.2@AuNCs-DNA.

(23) Pb.sup.2+ or carcinoembryonic antigen (CEA) was added to a nanohybrid aqueous dispersed solution, a fluorescence emission spectrum of a mixed solution was determined, and a linear relationship between the Pb.sup.2+ or the CEA concentration and ratiometric fluorescent peak intensity I.sub.CDs/I.sub.AuNcs was built to achieve ratiometric fluorescence sensing of the Pb.sup.2+ or the CEA. The nanohybrid dispersed in the Tris-HCl was transferred into an electrolytic cell equipped with a gold electrode. The surface of the gold electrode was bonded to the DNA terminal sulfhydryl via Au—S bond, and the nanohybrid was attached to the surface of the gold electrode, and the Pb.sup.2+ or the CEA was added. The square wave voltammetry curve was determined by an electrochemical workstation, and a linear relationship between the Pb.sup.2+ or the CEA concentration and ratiometric current peak intensity I.sub.MB/I.sub.Fc was built to achieve ratiometric electrochemical sensing of the Pb.sup.2+ or the CEA; a concentration of the Pb.sup.2+ or the CEA is 100 nM-1 mM.

(24) The foregoing descriptions are merely preferred examples 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 present invention.