Preparation method of an optoelectrochemical sensor for detecting transmembrane glycoprotein CD44 on the surface of breast cancer cells

Abstract

The present invention relates to a preparation method of a photoelectrochemical sensor for detecting the transmembrane glycoprotein CD44 on the surface of breast cancer cells. The preparation method is divided into three steps. First, a TiO2 nanoarray is loaded on the surface of FTO conductive glass by a hydrothermal method. Then, a Ag nano-layer is uniformly sputtered on the surface of the TiO2 nanoarray by using a magnetron sputtering technique. Finally, the TiO2-Ag is immersed in a sodium sulfide solution to realize the local sulfidation of Ag to obtain a photoelectric conversion body, a TiO2-Ag-Ag2S nano-composite array. Then, the extraction of the transmembrane glycoprotein CD44 on the surface of breast cancer cells MDA-MB-231 is realized by using the host-guest recognition between hyaluronic acid and the transmembrane glycoprotein CD44 and a DNA strand displacement reaction. Finally, through bioconjugation and covalent bonding, the assembly of the optoelectronic material and the target on the conductive interface is realized. The preparation method of this sensor has stronger controllability compared with other reported methods, especially obtaining a sensing interface with good signal output stability. The scheme and process mentioned in this method have important reference in the fields of material synthesis, photoelectrochemical sensing, cell detection, etc.

Description

Technical Field

[0001] The present invention relates to the fields of inorganic nanomaterial synthesis and photoelectrochemical biosensing, and particularly relates to a preparation method of a photoelectrochemical sensor for detecting transmembrane glycoprotein CD44 on the surface of breast cancer cells. Background Art

[0002] At present, breast cancer, known as the "pink killer", has replaced lung cancer as the number one cancer in the world. It has been confirmed that transmembrane glycoprotein CD44 is closely related to tumor invasion and metastasis, and transmembrane glycoprotein CD44 has been generally found in breast tumors clinically. Therefore, if rapid and sensitive detection of transmembrane glycoprotein CD44 in body fluids can be achieved, it will play an important role in the early diagnosis and treatment of breast cancer. Photoelectrochemical sensors have received extensive attention from researchers due to their good stability, low cost, and high detection efficiency, but the batch-to-batch difference of sensing chips has always been a major defect hindering their wide application.

[0003] Titanium dioxide (TiO2) has received extensive attention in photocatalysis and photoelectrochemical sensing due to its non-toxic, inexpensive, and stable advantages, but its defects are also obvious. Its relatively wide energy band forces it to only have an obvious response to short-wavelength ultraviolet light with higher energy, and its absorption of visible light is poor. In addition, the electron-hole recombination rate of pure TiO2 is relatively fast, which limits its application in different fields. For the modification of TiO2, methods such as quantum dot / dye sensitization, noble metal loading, ion doping, and construction of heterojunctions are widely used at present.

[0004] The heterojunction types of N-type semiconductors are mainly divided into three configurations: Type-I, Type-II, and Z-type. Each configuration has different requirements for the energy band structures of the two semiconductors. Among them, Type-I is composed of two or more components with parallel energy band structures, while both Type-II and Z-type require two or more components with staggered energy band arrangements. Different configurations can be transformed. By recombining the electrons and holes of a semiconductor with a parallel energy band structure through an ohmic contact surface, the energy band of one of the components is elevated, thus realizing the transformation from Type-I to Z-type. There have been research reports on the construction of Ag2S-Ag-TiO2 Z-type heterojunctions. The scheme is to first synthesize Ag nanocubes, in-situ synthesize TiO2 on the Ag surface through the strong bond energy of Ag-O, and finally locally sulfide Ag to obtain an inward-outward Ag2S-Ag-TiO2 structure. The difficulty of this scheme lies in the control of the Ag layer thickness, which only relies on the Ag-O bond to protect the trace Ag in the middle from being oxidized. For this, we propose a new synthesis scheme to prepare the Z-type nanocomposite array heterojunction TiO2-Ag-Ag2S, and use this as a signal source and sensing substrate to achieve the sensitive detection of transmembrane glycoprotein CD44. This scheme effectively improves the signal output stability of the sensor and reduces the batch-to-batch difference of the sensing chip, providing a new method for the early detection and treatment of breast cancer. Summary of the Invention

[0005] A preparation method of a photoelectrochemical sensor for detecting transmembrane glycoprotein CD44 on the surface of breast cancer cells, the method comprising the following process steps:

[0006] (1) Preparation of TiO2-Ag-Ag2S heterojunction:

[0007] Place the FTO glass with an area of 1*1 cm 2 conductive surface facing up flat on the bottom of a 50 mL high-pressure reactor, and pour in a mixed solution containing 8 - 45 mL of deionized water, 8 - 45 mL of concentrated hydrochloric acid, and 0.2 - 1.2 mL of tetrabutyl titanate. Keep it warm at 120 - 160 o °C for 8 - 12 h to obtain TiO2 nanorod arrays with different densities and thicknesses. After washing the product and drying it at room temperature, anneal it in a muffle furnace at 400 - 500 o °C for 3 h to improve the crystallinity;

[0008] Load metal silver particles onto the TiO2 nanorod arrays obtained in the previous step. Use a silver target on a magnetron sputtering coater, adjust the argon pressure to 1 - 5 Pa, the sputtering power to 40 - 80 W, and the sputtering time to 1 - 3 min to obtain TiO2-Ag composites with different silver particle densities;

[0009] Immerse the TiO2-Ag composite material obtained in the above step into an aqueous sodium sulfide solution with a concentration of 1-5 mol / L, take it out after 4-12 h, wash and dry it to obtain a TiO2-Ag-Ag2S nanocomposite array with different Ag layer thicknesses;

[0010] (2)Extract the transmembrane glycoprotein CD44 on the surface of breast cancer cells:

[0011] Take 20-50 mg of hyaluronic acid HA, dissolve it in 20-50 mL of 1×PBS after sterilization by ultraviolet lamp irradiation, mix 1 mL of single-stranded DNA P2 with a concentration of 1-5 mmol / L with 5-10 mL of HA solution to form a bioconjugate HA-P2; mix 10 mL of tyrosine Y with a concentration of 5-10 mmol / L with 1 mL of single-stranded DNA P1 with a concentration of 1-5 mmol / L and shake to form a conjugate Y-P1; mix P2-HA with Y-P1, incubate at 60 o ℃ for 4 h to achieve local base pairing to obtain double-stranded DNA Y-P1-P2-HA; add 200-800 μL of Y-P1-P2-HA to each well of a 24-well plate, and then add breast cancer cells with different concentrations to each well for host-guest recognition for 0.5 h; after centrifugation, redisperse the lower layer substrate with an equal volume of 1×PBS, and then add 50-100 U of restriction endonuclease Nt.BbvCI, 37 o ℃ for 2 h to complete the extraction of transmembrane glycoprotein CD44;

[0012] (3)Construct a photoelectrochemical sensor:

[0013] Use an ion beam sputtering instrument to adjust the current to 10-30 μA and the sputtering time to 30-80 s, and sputter a layer of gold nanoparticles on the surface of the TiO2-Ag-Ag2S nanorod array obtained in step (1) to obtain TiO2-Ag-Ag2S / Au; drop-coat 1 mL of hairpin DNA H1 with a concentration of 5-10 mmol / L on the surface of TiO2-Ag-Ag2S / Au and incubate for 2-5 h, continue to drop 800 μL of 5% 6-mercapto-1-hexanol solution on the electrode surface to block non-specific active sites, and finally drop-coat the solution of transmembrane glycoprotein CD44 carrying a signal label obtained in step (2) on the electrode surface to complete the construction of the sensor.

[0014] The preparation method of a photoelectrochemical sensor for detecting the transmembrane glycoprotein CD44 on the surface of breast cancer cells, and the DNA sequences used are H1: SH-TAC TAT ATT GTG TAA GTA GTC TAG ACG TAG CTG ATTTTA TTA CAC GCC GAA TCC TAG ACT ACTT; P1: AAC CTC AGC TAC GTC TAG ACT ACT TACACAA-NH2; P2: TCT AGA CGT AGC TGA GGTT-NH2.

[0015] The preparation method of a photoelectrochemical sensor for detecting the transmembrane glycoprotein CD44 on the surface of breast cancer cells is characterized in that the type of breast cancer cells is MDA-MB-231 cells.

[0016] Advantages of the present invention

[0017] (1) The present invention prepares a photoelectrochemical sensor for detecting the transmembrane glycoprotein CD44 on the surface of breast cancer cells. For the first time, a photosensitive material, Z-type Ag2S-Ag-TiO2 nanocomposite array, is prepared by combining three new synthesis techniques: hydrothermal synthesis method, magnetron sputtering method, and in-situ sulfidation method as a sensing substrate. By synergistically using DNA strand displacement reaction, host-guest recognition between hyaluronic acid and transmembrane glycoprotein CD44, and specific cleavage sites of restriction endonucleases, sensitive detection of transmembrane glycoprotein CD44 in breast cancer cells MDA-MB-231 is achieved. This method has the advantages of specific selectivity, simple preparation method, low cost, easy surface renewal, and small residual current.

[0018] (2) The photoelectrochemical sensor for detecting the transmembrane glycoprotein CD44 on the surface of breast cancer cells prepared by the present invention shows high selectivity and sensitivity to transmembrane glycoprotein CD44, has specific specificity, and the response current has a good linear relationship with the concentration of breast cancer cells MDA-MB-231. The correlation coefficient R 2 = 0.998, and the detection limit is as low as 230 cells / ml.

[0019] (3) The photoelectrochemical sensor for detecting the transmembrane glycoprotein CD44 on the surface of breast cancer cells prepared by the present invention does not use toxic reagents during the preparation process, is environmentally friendly and green, and the photosensitive material synthesis scheme is applicable to the preparation of other similar structure composite materials.

[0020] Specific embodiments

[0021] To further understand the present invention, the preferred embodiments of the present invention will be described below in conjunction with examples. However, it should be understood that these descriptions are only for further explaining the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0022] Example 1

[0023] (1)Preparation of TiO2-Ag-Ag2S heterojunction:

[0024] Place the FTO glass with an area of 1*1 cm 2 conductive side up flat on the bottom of a 50 mL autoclave, and pour in a mixed solution containing 8 mL of deionized water, 8 mL of concentrated hydrochloric acid and 0.2 mL of tetrabutyl titanate. Keep it warm at 120 o °C for 8 h to obtain TiO2 nanorod arrays with different densities and thicknesses. After washing the product and drying it at room temperature, anneal it in a muffle furnace at 400 o °C for 3 h to improve the crystallinity;

[0025] Load metal silver particles onto the TiO2 nanorod arrays obtained in the above step. Use a silver target on a magnetron sputtering coater, adjust the argon pressure to 1 Pa, the sputtering power to 40 W, and the sputtering time to 1 min to obtain TiO2-Ag composites with different silver particle densities;

[0026] Immerse the TiO2-Ag composites obtained in the above step into an aqueous solution of sodium sulfide with a concentration of 1 mol / L. After 4 h, take it out, wash and dry it to obtain TiO2-Ag-Ag2S nanocomposite arrays with different Ag layer thicknesses;

[0027] (2)Extraction of transmembrane glycoprotein CD44 on the surface of breast cancer cells:

[0028] Take 20 mg of hyaluronic acid HA, sterilize it by ultraviolet lamp irradiation and dissolve it in 20 mL of 1×PBS. Mix 1 mL of 1 mmol / L single-stranded DNA P2 with 5 mL of HA solution to form a bioconjugate HA-P2; Mix 10 mL of 5 mmol / L tyrosine Y with 1 mL of 1 mmol / L DNA single-strand P1 and shake to form a conjugate Y-P1; Mix P2-HA with Y-P1 and keep it warm at 60 o °C for 4 h to achieve local base pairing to obtain double-stranded DNA double-strand Y-P1-P2-HA; Drop 200 μL of Y-P1-P2-HA on each well of a 24-well plate, and then add different concentrations of breast cancer cells to each well for host-guest recognition for 0.5 h; After centrifugation, redisperse the lower substrate with an equal volume of 1×PBS, and then add 50 U of restriction endonuclease Nt.BbvCI, 37 oThe extraction of transmembrane glycoprotein CD44 was completed by storing it at C for 2 h.

[0029] (3)Construct a photoelectrochemical sensor:

[0030] Use an ion beam sputtering instrument to adjust the current to 10 µA and the sputtering time to 30 s, and sputter a layer of gold nanoparticles on the surface of the TiO2-Ag-Ag2S nanorod array obtained in step (1) to obtain TiO2-Ag-Ag2S / Au; Drop 1 mL of 5 mmol / L hairpin DNA H1 on the surface of TiO2-Ag-Ag2S / Au and incubate for 2 h, then continue to drop 800 μL of 5% 6-mercapto-1-hexanol solution on the electrode surface to block non-specific active sites, and finally drop the transmembrane glycoprotein CD44 solution carrying the signal marker obtained in step (2) on the electrode surface to complete the construction of the sensor.

[0031] Example 2

[0032] (1)Prepare a TiO2-Ag-Ag2S heterojunction:

[0033] Place the FTO glass with an area of 1*1 cm 2 conductive side up on the bottom of a 50 mL high-pressure reactor, and pour in a mixed solution containing 25 mL of deionized water, 25 mL of concentrated hydrochloric acid, and 1 mL of tetrabutyl titanate. Keep it at 150 o C for 10 h to obtain TiO2 nanorod arrays with different densities and thicknesses. After washing the product and drying it at room temperature, anneal it in a muffle furnace at 450 o C for 3 h to improve the crystallinity;

[0034] Load metal silver particles onto the TiO2 nanorod array obtained in the previous step. Use a silver target on a magnetron sputtering coater, adjust the argon pressure to 2 Pa, the sputtering power to 60 W, and the sputtering time to 2 min to obtain a TiO2-Ag composite with different silver particle densities;

[0035] Immerse the TiO2-Ag composite obtained in the previous step in an aqueous solution of sodium sulfide with a concentration of 2 mol / L, take it out after 8 h, wash and dry it to obtain a TiO2-Ag-Ag2S nanocomposite array with different Ag layer thicknesses;

[0036] (2)Extract transmembrane glycoprotein CD44 on the surface of breast cancer cells:

[0037] Take 30 mg of hyaluronic acid HA, dissolve it in 30 mL of 1×PBS after sterilization by ultraviolet lamp irradiation, mix 1 mL of 3 mmol / L single-stranded DNA P2 with 8 mL of HA solution to form a bioconjugate HA-P2; mix 10 mL of 8 mmol / L tyrosine Y with 1 mL of 3 mmol / L DNA single-strand P1 and shake to form a conjugate Y-P1; mix P2-HA with Y-P1, and incubate at 60 o Under C for 4 h to achieve local base pairing and form double-stranded DNA Y-P1-P2-HA; add 400 μL of Y-P1-P2-HA to each well of a 24-well plate, and then add different concentrations of breast cancer cells to each well for host-guest recognition for 0.5 h; after centrifugation, redisperse the lower substrate with an equal volume of 1×PBS, and then add 80 U of restriction endonuclease Nt.BbvCI, 37 o Store at C for 2 h to complete the extraction of transmembrane glycoprotein CD44;

[0038] (3)Construct a photoelectrochemical sensor:

[0039] Use an ion beam sputtering instrument to adjust the current to 20 µA and the sputtering time to 40 s, and sputter a layer of gold nanoparticles on the surface of the TiO2-Ag-Ag2S nanorod array obtained in step (1) to obtain TiO2-Ag-Ag2S / Au; drop-coat 1 mL of 7 mmol / L hairpin DNA H1 on the surface of TiO2-Ag-Ag2S / Au and incubate for 3 h, then continue to drop 800 μL of 5% 6-mercapto-1-hexanol solution on the electrode surface to block non-specific active sites, and finally drop-coat the transmembrane glycoprotein CD44 solution carrying the signal label obtained in step (2) on the electrode surface to complete the construction of the sensor.

[0040] Example 3

[0041] (1)Prepare a TiO2-Ag-Ag2S heterojunction:

[0042] Place the FTO glass with an area of 1*1 cm 2 with the conductive side facing up flat on the bottom of a 50 mL high-pressure reaction kettle, and pour in a mixture containing 45 mL of deionized water, 45 mL of concentrated hydrochloric acid and 1.2 mL of tetrabutyl titanate, and incubate at 160 o Under C for 12 h to obtain TiO2 nanorod arrays with different densities and thicknesses. After washing the product and drying it at room temperature, anneal it in a muffle furnace at 500 o Under C for 3 h to improve the crystallinity;

[0043] The TiO2 nanorod arrays obtained in the previous step were loaded with silver metal particles. Using a silver target on a magnetron sputtering coater, the argon pressure was adjusted to 5 Pa, the sputtering power was 80 W, and the sputtering time was 3 min to obtain TiO2-Ag composites with different silver particle densities;

[0044] The TiO2-Ag composites obtained in the previous step were immersed in an aqueous solution of sodium sulfide with a concentration of 5 mol / L. After 12 h, they were taken out, washed, and dried to obtain TiO2-Ag-Ag2S nanocomposite arrays with different Ag layer thicknesses;

[0045] (2)Extract the transmembrane glycoprotein CD44 on the surface of breast cancer cells:

[0046] Take 50 mg of hyaluronic acid HA. After sterilization by ultraviolet lamp irradiation, it was dissolved in 50 mL of 1×PBS. 1 mL of 5 mmol / L single-stranded DNA P2 was mixed with 10 mL of HA solution to form a bioconjugate HA-P2; 10 mL of 10 mmol / L tyrosine Y was mixed with 1 mL of 5 mmol / L DNA single-strand P1 and shaken to form a conjugate Y-P1; P2-HA and Y-P1 were mixed and incubated at 60 o °C for 4 h to achieve local base pairing, DNA double-strand Y-P1-P2-HA; 800 μL of Y-P1-P2-HA was added dropwise to each well of a 24-well plate, and then breast cancer cells with different concentrations were added to each well for host-guest recognition for 0.5 h; after centrifugation, the lower substrate was redispersed with an equal volume of 1×PBS, and then 100 U of restriction endonuclease Nt.BbvCI was added and stored at 37 o °C for 2 h to complete the extraction of transmembrane glycoprotein CD44;

[0047] (3)Construct a photoelectrochemical sensor:

[0048] Using an ion beam sputtering instrument, the current was adjusted to 30 µA and the sputtering time was 80 s. A layer of gold nanoparticles was sputtered on the surface of the TiO2-Ag-Ag2S nanorod arrays obtained in step (1) to obtain TiO2-Ag-Ag2S / Au; 1 mL of 10 mmol / L hairpin DNA H1 was drop-coated on the surface of TiO2-Ag-Ag2S / Au and incubated for 5 h. Then, 800 μL of 5% 6-mercapto-1-hexanol solution was added dropwise to the electrode surface to block non-specific active sites. Finally, the solution of transmembrane glycoprotein CD44 carrying a signal label obtained in step (2) was drop-coated on the electrode surface to complete the construction of the sensor.

[0049] The photoelectrochemical sensor for detecting the transmembrane glycoprotein CD44 on the surface of breast cancer cells prepared by the present invention was successfully used for the detection of breast cancer cells MDA-MB-231, and the recovery rate was as low as 230 cells / mL at the detection limit.

Claims

1. A preparation method of a photoelectrochemical sensor for detecting the transmembrane glycoprotein CD44 on the surface of breast cancer cells, characterized in that, Including the following steps: (1) Preparation of TiO2-Ag-Ag2S heterojunction: Lay the conductive surface of the FTO glass with an area of 1*1 cm 2 flat on the bottom of a 50 mL high-pressure reactor with the conductive surface facing up, and pour in a mixed solution containing 8 - 45 mL of deionized water, 8 - 45 mL of concentrated hydrochloric acid, and 0.2 - 1.2 mL of tetrabutyl titanate. Keep it warm at 120 - 160 o °C for 8 - 12 h to obtain TiO2 nanorod arrays with different densities and thicknesses. Wash the product, dry it at room temperature, and then anneal it in a muffle furnace at 400 - 500 o °C for 3 h to improve the crystallinity; Loading metal silver particles onto the TiO2 nanorod arrays obtained in the previous step. Using a silver target in a magnetron sputtering coater, adjusting the argon pressure to 1 - 5 Pa, the sputtering power to 40 - 80 W, and the sputtering time to 1 - 3 min to obtain TiO2-Ag composites with different silver particle densities; Immersing the TiO2-Ag composites obtained in the previous step into an aqueous sodium sulfide solution with a concentration of 1 - 5 mol / L, taking them out after 4 - 12 h, washing and drying to obtain TiO2-Ag-Ag2S nanocomposite arrays with different Ag layer thicknesses; (2) Extracting the transmembrane glycoprotein CD44 on the surface of breast cancer cells; Take 20 - 50 mg of hyaluronic acid HA, dissolve it in 20 - 50 mL of 1×PBS after sterilization by ultraviolet lamp irradiation, mix 1 mL of single-stranded DNA P2 with a concentration of 1 - 5 mmol / L with 5 - 10 mL of the HA solution to form a bioconjugate HA-P2; mix 10 mL of tyrosine Y with a concentration of 5 - 10 mmol / L with 1 mL of single-stranded DNA P1 with a concentration of 1 - 5 mmol / L and shake to form a conjugate Y-P1; mix P2-HA with Y-P1, incubate at 60 o °C for 4 h to achieve local base pairing to obtain double-stranded DNA Y-P1-P2-HA; add 200 - 800 μL of Y-P1-P2-HA to each well of a 24-well plate, then add different concentrations of breast cancer cells to each well for host-guest recognition for 0.5 h; after centrifugation, redisperse the lower substrate with an equal volume of 1×PBS, and then add 50 - 100 U of restriction endonuclease Nt.BbvCI, and incubate at 37 o °C for 2 h to complete the extraction of transmembrane glycoprotein CD44; (3) Constructing a photoelectrochemical sensor: Adjusting the current to 10 - 30 μA and the sputtering time to 30 - 80 s using an ion beam sputter, sputtering a layer of gold nanoparticles on the surface of the TiO2-Ag-Ag2S nanorod arrays obtained in step (1) to obtain TiO2-Ag-Ag2S / Au; dropping 1 mL of 5 - 10 mmol / L hairpin DNA H1 onto the surface of TiO2-Ag-Ag2S / Au and incubating for 2 - 5 h, then continuously dropping 800 μL of 5% 6-mercapto-1-hexanol solution onto the electrode surface to block non-specific active sites, and finally dropping the transmembrane glycoprotein CD44 solution carrying signal markers obtained in step (2) onto the electrode surface to complete the construction of the sensor.

2. The preparation method of an optoelectrochemical sensor for detecting the transmembrane glycoprotein CD44 on the surface of breast cancer cells according to claim 1, characterized in that, The DNA sequences used in the preparation of the sensor are H1: SH-TAC TAT ATTGTG TAA GTA GTC TAG ACG TAG CTG ATT TTA TTA CAC GCC GAA TCC TAG ACT ACTT; P1: AAC CTC AGC TAC GTC TAG ACT ACT TAC ACAA-NH2; P2: TCT AGA CGT AGC TGA GGTT-NH2.

3. The preparation method of an optoelectrochemical sensor for detecting transmembrane glycoprotein CD44 on the surface of breast cancer cells according to claim 1, characterized in that, The type of breast cancer cells is MDA-MB-231 cells.