METHOD FOR PREPARING UPCONVERSION-LUMINESCENCE FLEXIBLE HYBRID MEMBRANE FOR VISUAL DETECTION OF TUMOR MARKER

Abstract

A method for preparing an upconversion-luminescence flexible hybrid membrane for visual detection of tumor markers is provided. Metal ion doped black phosphorus quantum dots (M-BPQDs) are prepared by adopting ultrasonic and solvothermal processes; mesoporous SiO.sub.2 grows on the surfaces of the M-BPQDs and amination modification is performed; the M-BPQDs are connected with carboxylated single-stranded DNA1; receptor molecules enter pores; single-stranded DNA2 aptamers and the DNA1 are combined due to base complementation to encapsulate receptors in the pores; and an M-BPQDs probe is prepared. DNA1 terminal-SH and a composite membrane are formed by assembling polymethyl methacrylate-polyimide-gold nanoparticles in a layer-by-layer manner bound by AuS bonds, and the membrane and the probe are connected to construct the flexible hybrid membrane. The new flexible hybrid membrane is simple and inexpensive to prepare and is highly sensitive.

Claims

1. A method for preparing an upconversion-luminescence flexible hybrid membrane for a visual detection of a tumor marker, comprising the following steps: (1) grinding a block black phosphorus crystal into powder, adding the powder to a polar solvent to obtain a first mixture, wherein a metal salt is dissolved in the polar solvent, ultrasonically stripping black phosphorus nanosheets from the first mixture by a probe and a bath, adding a thiol ligand to the black phosphorus nanosheets to obtain a second mixture, and preparing metal ion doped black phosphorus quantum dots (M-BPQDs) from the second mixture by an ultrasonic-assisted solvothermal treatment; (2) growing mesoporous silica (mSiO.sub.2) on surfaces of the M-BPQDs according to a Stober method to obtain a third mixture, conducting an amination (NH.sub.2) modification on the third mixture to prepare M-BPQDs/mSiO.sub.2NH.sub.2, and binding the M-BPQDs/mSiO.sub.2NH.sub.2 to a single-stranded DNA1 (HS-DNA1-COOH) by a carboxy-amine coupling to prepare M-BPQDs/mSiO.sub.2@DNA1; (3) allowing an electron receptor molecule to enter a pore of the mSiO.sub.2 to complete a loading of the electron receptor molecule, adding a specific aptamer single-stranded DNA2 for a complementary base pairing with the single-stranded DNA1 to form a double helix structure, and encapsulating the electron receptor molecule to obtain a nano-hybrid carrier probe M-BPQDs/mSiO.sub.2@DNA1-DNA2@receptor; (4) using polymethyl methacrylate (PMMA) as a layered substrate, bonding a layer of polyimide (PI) on a surface of the PMMA to obtain a PMAA-PI film, using an electrode clip to fix the PMAA-PI film to obtain an electrode clip fixed PMAA-PI film and immersing the electrode clip fixed PMAA-PI film in an electrolyte; using a KCl saturated calomel electrode as a reference electrode, a platinum wire electrode as an auxiliary electrode, and the PMMA-PI film as a working electrode, adding HAuCl.sub.4 to the electrolyte, scanning the reference electrode, the auxiliary electrode and the working electrode by cyclic voltammetry, and electrochemically reducing a surface of the PMAA-PI film to form gold nanoparticles (AuNPs) to prepare a PMMA-PI-AuNPs composite film; and (5) bonding DNA1 (HS-DNA1-COOH) terminal-SH on the nano-hybrid carrier probe and the AuNPs on the PMMA-PI-AuNPs composite film by AuS bonds, and connecting the PMMA-PI-AuNPs composite film and the nano-hybrid carrier probe together to construct a flexible hybrid membrane, wherein when a biological fluid sample contains the tumor marker, the biological fluid sample is dropped onto the flexible hybrid membrane to obtain an infiltrated hybrid membrane, and a color change of an upconversion luminescence of the infiltrated hybrid membrane is observed under a near-infrared light excitation to realize the visual detection of the tumor marker.

2. The method according to claim 1, wherein a metal ion in step (1) is Ag.sup.+, Mn.sup.2+, Co.sup.2+, Ni.sup.2+, and the M-BPQDs are 1-5 nm in size.

3. The method according to claim 1, wherein a thickness of mSiO2 on the surfaces of the M-BPQDs in step (2) is 50-200 nm.

4. The method according to claim 1, wherein the electron receptor molecule in step (3) is 5-fluorouracil, dopamine, rutin, quercetin, and the specific aptamer single-stranded DNA2 is a single-stranded DNA aptamer of the tumor marker, wherein the tumor marker is carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), carbohydrate antigen (CA), and prostate specific antigen (PSA).

5. The method according to claim 1, wherein in step (4), the PMMA is 50-100 nm in thickness, the PI is 1-2 m in thickness, and the AuNPs are 10-100 nm in thickness.

6. The method according to claim 1, wherein in step (5), an emission peak of the upconversion luminescence has a wavelength of 500-600 nm, and the tumor marker has a concentration of 1 nM-1 mM.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is a schematic diagram of the preparation process and principle of metal ion doped black phosphorus quantum dots (M-BPQDs);

[0021] FIG. 2 is a schematic diagram of the preparation process of upconversion-luminescence flexible hybrid membrane based on M-BPQDs and its principle for visual detection of tumor markers.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The present invention will be described in detail below with reference to the accompanying drawings.

Example 1

[0023] The present invention relates to a method for preparing an upconversion-luminescence flexible hybrid membrane for visual detection of tumor markers. The preparation process and detection principle thereof are shown in FIGS. 1 and 2, and the specific preparation steps are as follows:

[0024] Block black phosphorus crystal was ground into a powder, mixed with N-methylpyrrolidone (NMP) in which silver nitrate was dissolved. Black phosphorus nanosheets were ultrasonically stripped by a probe and a bath, and the mercaptopropionic acid ligand was added to prepare Ag-BPQDs with an average size of 2 nm by ultrasonic-assisted solvothermal treatment. According to the Stober method, 50 nm thick mSiO.sub.2 was grown on the surface of Ag-BPQDs, then functionalized by NH.sub.2 to obtain Ag-BPQDs/mSiO.sub.2NH.sub.2, and bound to single-stranded DNA1 (HS-DNA1-COOH) by carboxy-amine coupling to prepare Ag-BPQDs/mSiO.sub.2@DNA1. 5-fluorouracil (5FU) entered the pores of mSiO.sub.2 to complete the molecular loading of the receptor, and specific aptamer single-stranded DNA2 were added to form a double helix structure with the DNA1 to encapsulate the receptor molecule by complementary base pairing, and a nano-hybrid carrier probe Ag-BPQDs/mSiO.sub.2@DNA1-DNA2@5FU was prepared. PMMA was used as a layered substrate measuring 80 nm, and the surface thereof was bonded with a layer of 1.6 m PI, and an electrode clip fixed PMAA-PI film was immersed in an electrolyte. Using a KCl saturated calomel electrode as a reference electrode, a platinum wire electrode as an auxiliary electrode, and the PMAA-PI film as the working electrode, 10 mM HAuCl.sub.4 was added to the electrolyte, and cyclic voltammetry was performed for 50 cycles to electrochemically deposit gold on the surface of the film. The nano-particle AuNPs have a layer thickness of 50 nm, and finally a PMMA-PI-AuNPs composite film is obtained. DNA1 (HS-DNA1-COOH) tail-SH on the probe and AuNPs on the membrane were bonded by AuS bond, and the film and the probe were connected together to form a flexible hybrid membrane. When a human serum sample contains carcinoembryonic antigen (CEA), a trace amount of sample is added dropwise to the hybrid membrane, and under the excitation of 800 nm and 5 mW near-infrared light, changes in intensity of orange-red upconversion luminescence were observed at 550 nm of the visible region of the infiltrated hybrid membrane. As the concentration of CEA in the sample increased from 10 nM to 10 M, the up-conversion luminescence of Ag-BPQDs was gradually enhanced, and the up-conversion luminescence of the tumor marker CEA in human serum samples was visualized.

Example 2

[0025] Block black phosphorus crystal was ground into a powder, mixed with NMP in which nickel nitrate was dissolved, black phosphorus nanosheets were ultrasonically stripped by a probe and a bath, and the mercaptopropionic acid ligand was added to prepare Ni-BPQDs with an average size of 3 nm by ultrasonic-assisted solvothermal treatment. According to the Stober method, 100 nm thick mSiO.sub.2 was grown on the surface of Ni-BPQDs, then functionalized by NH.sub.2 to obtain Ni-BPQDs/mSiO.sub.2NH.sub.2, and bound to single-stranded DNA1 (HS-DNA1-COOH) by carboxy-amine coupling to prepare Ni-BPQDs/mSiO.sub.2@DNA1. Dopamine (DA) entered the pores of mSiO.sub.2 to complete the molecular loading of the receptor, and specific aptamer single-stranded DNA2 were added to form a double helix structure with the DNA1 encapsulated receptor molecule by complementary base pairing, and a nano-hybrid carrier probe Ni-BPQDs/mSiO.sub.2@DNA1-DNA2@DA was prepared. PMMA was used as a layered substrate measuring 50 nm, and the surface thereof was bonded with a layer of 1.5 m PI, and an electrode clip fixed PMAA-PI film was immersed in an electrolyte. Using a KCl saturated calomel electrode as a reference electrode, a platinum wire electrode as an auxiliary electrode, and the PMAA-PI film as the working electrode, 10 mM HAuCl.sub.4 was added to the electrolyte, and cyclic voltammetry was performed for 50 cycles to electrochemically deposit gold on the surface of the film. The nano-particle AuNPs have a layer thickness of 40 nm, and finally a PMMA-PI-AuNPs composite film is obtained. DNA1 (HS-DNA1-COOH) tail-SH on the probe and AuNPs on the membrane were bonded by AuS bond, and the film and the probe were connected together to form a flexible hybrid membrane. When a human urine sample contains alpha-fetoprotein (AFP), a trace amount of sample is added dropwise to the hybrid membrane, and under the excitation of 800 nm and 5 mW near-infrared light, changes in intensity of orange-red upconversion luminescence were observed at 540 nm of the visible region of the infiltrated hybrid membrane. As the concentration of AFP in the sample increased from 10 nM to 100 M, the up-conversion luminescence of Ni-BPQDs was gradually enhanced, and the up-conversion luminescence of the tumor marker AFP in human urine samples was visualized.

Example 3

[0026] Block black phosphorus crystal was ground into a powder, mixed with NMP in which cobalt nitrate was dissolved, black phosphorus nanosheets were ultrasonically stripped by a probe and a bath, and the mercaptopropionic acid ligand was added to prepare Co-BPQDs with an average size of 5 nm by ultrasonic-assisted solvothermal treatment. According to the Stober method, 150 nm thick mSiO.sub.2 was grown on the surface of Co-BPQDs, then functionalized by NH.sub.2 to obtain Co-BPQDs/mSiO.sub.2NH.sub.2, and bound to single-stranded DNA1 (HS-DNA1-COOH) by carboxy-amine coupling to prepare Co-BPQDs/mSiO.sub.2@DNA1. Rutin (LU) entered the pores of mSiO.sub.2 to complete the molecular loading of the receptor, and specific aptamer single-stranded DNA2 were added to form a double helix structure with the DNA1 to execute the encapsulation of the receptor molecule by complementary base pairing, and a nano-hybrid carrier probe Co-BPQDs/mSiO.sub.2@DNA1-DNA2@LU was prepared. PMMA was used as a layered substrate measuring 60 nm, and the surface thereof was bonded with a layer of 1.8 m PI, and an electrode clip fixed PMAA-PI film was immersed in an electrolyte. Using a KCl saturated calomel electrode as a reference electrode, a platinum wire electrode as an auxiliary electrode, and the PMAA-PI film as the working electrode, 10 mM HAuCl.sub.4 was added to the electrolyte, and cyclic voltammetry was performed for 50 cycles to electrochemically deposit gold on the surface of the film. The nano-particle AuNPs have a layer thickness of 20 nm, and finally a PMMA-PI-AuNPs composite film is obtained. DNA1 (HS-DNA1-COOH) tail-SH on the probe and AuNPs on the membrane were bonded by AuS bond, and the film and the probe were connected together to form a flexible hybrid membrane. When a human urine sample contains prostate-specific antigen (PSA), a trace amount of sample is added dropwise to the hybrid membrane, and under the excitation of 800 nm and 5 mW near-infrared light, changes in intensity of orange-red upconversion luminescence were observed at 575 nm of the visible region of the infiltrated hybrid membrane. As the concentration of PSA in the sample increased from 10 nM to 1 mM, the up-conversion luminescence of Co-BPQDs was gradually enhanced, and the up-conversion luminescence of the tumor marker PSA in human urine samples was visualized.

[0027] 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 be construed as within the scope of claims of the present invention.