CN109387499B

CN109387499B - PD-L1 probe and preparation method and application thereof

Info

Publication number: CN109387499B
Application number: CN201710671215.XA
Authority: CN
Inventors: 冯恩铎; 田阳; 郑婷婷
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2017-08-08
Filing date: 2017-08-08
Publication date: 2021-03-26
Anticipated expiration: 2037-08-08
Also published as: CN109387499A

Abstract

The invention discloses a graphene-metal oxide composite material PD-L1 probe and a preparation method thereof, and the probe is prepared by methods such as chemical synthesis, physical adsorption and the like. The invention also discloses application of the PD-L1 probe in surface-enhanced Raman spectrum sensitive detection of cell membrane surface programmed death receptor ligand 1 (PD-L1). The PD-L1 probe has good SERS activity and sensitivity. The PD-L1 probe can be applied to detection of PD-L1 and is also applicable to other biomarkers in cells.

Description

PD-L1 probe and preparation method and application thereof

Technical Field

The invention relates to a PD-L1 probe, a preparation method and application thereof, in particular to a novel method for detecting programmed death receptor ligand 1(PD-L1) on the surface of a cell membrane by a surface enhanced Raman technology based on a chemical enhancement mechanism, belonging to the technical field of material preparation and biosensing.

Background

Programmed death receptor ligand-1 (PD-L1) is an important immunosuppressive molecule. Programmed death receptor-1 (PD-1) is highly expressed in lymphocytes and some somatic cells, respectively, and is a transmembrane protein closely related to autoimmune response. Normally, the immune system will respond to foreign antigens accumulated in the lymph nodes or spleen, triggering cytotoxic T Cells (CD) with antigen specificity⁸⁺) And (5) hyperplasia. The programmed death receptor-1 (PD-1) can be combined with the ligand thereof to transmit a response inhibition signal and reduce lymph node CD⁸⁺T cell proliferation, and thus avoiding some unnecessary autoimmune reactions, control autoimmune diseases.

In the field of cancer therapy, monoclonal antibodies and polyclonal antibodies targeting PD-L1 and PD-1 have become a popular field of research in recent years. Research shows that the high expression activity of PD-L1 appears on the cell membrane surface of many cancers (such as breast cancer, non-small cell lung cancer, prostate cancer and the like), and CD is enhanced by inhibiting the signal path of PD-1⁸⁺T cell activity, and immune scavenging effect on cancer cells to treat cancer.

The surface enhanced raman spectroscopy is a technique for generating raman signal enhancement on the surface of a specific substrate material by using a specific molecule. The technology can effectively provide high sensitivity, and even can reach the trace detection level. Meanwhile, the half-peak width is narrow, so that the aim of multi-element detection can be fulfilled to a certain extent. Since the discovery of the Surface Enhanced Raman Spectroscopy (SERS), a large number of SERS substrate materials prepared from precious metal materials such as gold, silver, and copper have been widely used in the field of analysis and detection of complex biological samples, and SERS is gradually applied in various fields such as production, life, and scientific research due to its unique multi-element detection capability and ultrahigh sensitivity. In recent years, it has been reported that a two-dimensional material represented by graphene and a metal oxide represented by ferriferrous oxide or titanium dioxide are applied to the SERS field and have good activity. Compared with the traditional noble metal material, the material has obvious advantages in the aspect of molecular selectivity.

The substrates commonly used today for enhancing raman spectrum are mainly noble metal materials such as Au, Ag, Cu, etc., however, these metal substrates usually have great disadvantages in terms of biocompatibility, molecular selectivity, etc.

Graphene, as a representative of two-dimensional materials, has very good SERS activity and very outstanding performance in terms of molecular selectivity. Meanwhile, metal oxides such as titanium dioxide and ferroferric oxide are also commonly used as SERS substrate materials in recent years. The invention creatively combines two substrate materials which generate the SERS effect based on a chemical enhancement mechanism, namely the graphene and the metal oxide, and obtains a better SERS enhancement effect. And can be used for detecting the expression level of PD-L1 on the surface of the cell membrane.

Disclosure of Invention

The invention aims to provide a graphene-metal oxide composite material which is applied to surface enhanced Raman spectroscopy detection of cell membrane surface programmed death receptor ligand 1(PD-L1), and has the advantages of good biocompatibility, high molecular selectivity and high detection sensitivity.

The preparation method of the PD-L1 probe, which is disclosed by the invention, prepares the PD-L1 probe by methods such as chemical synthesis, physical adsorption and the like, and comprises the following steps:

(1) preparation of ferroferric oxide nano particles

In organic solvents, FeCl₃·6H₂And performing hydrothermal reaction on O, a reducing agent and weak base to obtain the ferroferric oxide nano particles.

In the step (1), the organic solvent is one or more selected from ethylene glycol, glycerol and the like; preferably, it is ethylene glycol.

In the step (1), the reducing agent is selected from one or more of sodium citrate, sodium borohydride, ascorbic acid and the like; preferably, sodium citrate.

In the step (1), the weak base is used for adjusting the pH and is selected from one or more of sodium acetate, ammonia water, sodium bicarbonate and the like; preferably, it is sodium acetate.

In the step (1), the temperature of the hydrothermal reaction is 150-250 ℃; preferably, it is 200 ℃.

In the step (1), the hydrothermal reaction time is 6-12 h; preferably, it is 10 h.

In the step (1), the FeCl₃·6H₂The mass ratio of O, the reducing agent and the weak base is (2-4): (0.8-1.5): (4-8); preferably, it is 3.25: 1.3: 6. when FeCl is added₃·6H₂When the dosage of O is 2-4g, the dosage of the organic solvent is 50-100 ml; preferably, 100 ml; such as FeCl₃·6H₂When the amount of O is 3.25g, the volume of ethylene glycol is 100 ml.

In a specific embodiment, the preparation of the ferroferric oxide nanoparticles comprises the following steps: FeCl is added₃·6H₂O is dissolved in ethylene glycol, then sodium citrate and sodium acetate are added under magnetic stirring, and stirring is continued for 30 minutes. The resulting yellow solution was transferred to a 100ml teflon reaction kettle and reacted for 10 hours. Finally, slowly cooling to room temperature. The obtained black solid is washed three times with ultrapure water to obtain the product.

Wherein FeCl₃·6H₂The using amount of O is 3.25g, the using amount of sodium citrate is 1.3g, the using amount of sodium acetate is 6g, and the temperature of the reaction kettle is 200 ℃.

(2) Preparation of graphene oxide

And carrying out oxidation reaction on graphite and potassium chlorate in concentrated sulfuric acid and fuming nitric acid to obtain the graphene oxide.

In the step (2), the concentrated sulfuric acid is used as an oxidant; the fuming nitric acid acts as an oxidizing agent; the potassium chlorate acts as an oxidant.

In the step (2), the temperature of the oxidation reaction is 0-30 ℃; preferably, it is room temperature 25 ℃.

In the step (2), the time of the oxidation reaction is 48-120 h; preferably, it is 96 h.

In the step (2), the dosage ratio of the concentrated sulfuric acid, the fuming nitric acid, the graphite and the potassium chlorate is (50 ml-100 ml): (30 ml-50 ml): (3 g-8 g): (40 g-60 g); preferably, 87.5 ml: 45 ml: 5 g: 55 g.

In the step (2), after the reaction is completed, a step of adding cold water is further included for the purpose of quenching the reaction and diluting the reaction system.

In a specific embodiment, the preparation of the graphene oxide comprises: concentrated sulfuric acid and fuming nitric acid are placed in an ice bath to be uniformly mixed, and then graphite is added into the mixture to be continuously stirred and uniformly mixed. In a fume hood, potassium chlorate was added very slowly to the above system. Stirring was continued at room temperature for 96 hours. And finally, pouring the mixture into 4L of cold water, and filtering to obtain the graphene oxide.

Wherein the volume of the concentrated sulfuric acid is 87.5ml, the volume of the fuming nitric acid is 45ml, the consumption of the graphite is 5g, and the consumption of the potassium chlorate is 55 g.

(3) Preparation of ferroferric oxide/graphene oxide composite material

And (3) carrying out ultrasonic treatment on the ferroferric oxide nanoparticles, PDDA and graphene oxide in a solution to obtain the ferroferric oxide/graphene oxide composite material.

In the step (3), the solution is selected from one or more of water, ethanol, methanol and the like; preferably, it is water.

In the step (3), the PDDA is used for adjusting the surface charge of the ferroferric oxide, and the concentration is 1%.

In the step (3), the ultrasonic condition is 120 kW.

In the step (3), the temperature of the ultrasonic wave is 0-50 ℃; preferably, it is room temperature 25 ℃.

In the step (3), the ultrasonic treatment time is 15-60 min; preferably, it is 60 min. In the step (3), preferably, the ferroferric oxide nanoparticles and the PDDA are mixed and subjected to ultrasonic treatment for 30 minutes, then the graphene oxide is added, and the ultrasonic treatment is continued for 30 minutes. The nano particles and the graphene are uniformly compounded by electrostatic adsorption by adopting a layer-by-layer self-assembly method.

In the step (3), the dosage ratio of the ferroferric oxide nanoparticles to the PDDA to the graphene oxide is (5 ml-20 ml): (1 ml-10 ml): (3 ml-10 ml); preferably, 15 ml: 5 ml: 5 ml.

In the step (3), after the reaction is completed, the method further comprises the steps of centrifugally separating the mixture, and respectively washing and purifying the sample by using ethanol and ultrapure water; among them, the number of washing is preferably 3.

In a specific embodiment, the preparation of the ferroferric oxide/graphene oxide composite material comprises the following steps: adding PDDA solution into 15ml of ferroferric oxide-containing nano particle solution sample, and carrying out ultrasonic treatment on the mixed solution for 30 minutes. And (3) adding the graphene oxide solution prepared in the step (2) into the solution, and continuing to perform ultrasonic treatment for 30 min. And finally, centrifugally separating the mixture, and washing the mixture with ethanol and ultrapure water for three times respectively to obtain a product. Wherein the concentration of PDDA is 1% (mass percent), the volume is 5ml, and the dosage of graphene oxide is 5 ml.

Wherein the dosage of PDDA is 1%, the volume is 5ml, and the dosage of graphene oxide is 5 ml.

(4) Preparation of ferroferric oxide/graphene oxide/titanium dioxide composite material

And (4) dispersing the ferroferric oxide/graphene oxide composite material prepared in the step (3) in an organic solvent, and adding ammonia water into the organic solvent. The mixture was subjected to sonication. Tetrabutyl titanate (TBOT) is added into the system for reaction, stirred, dried and calcined to obtain the titanium dioxide coated ferroferric oxide/graphene oxide composite material, namely the ferroferric oxide/graphene oxide/titanium dioxide composite material.

In the step (4), the organic solvent is selected from one or more of absolute ethyl alcohol, methanol and the like; preferably, it is absolute ethanol.

In the step (4), the ammonia water is used for adjusting the pH value and providing an amino environment; the concentration of ammonia was 28%.

In the step (4), tetrabutyl titanate (TBOT) is added, and the reaction temperature is 30-60 ℃; preferably, it is 45 ℃.

In the step (4), tetrabutyl titanate (TBOT) is added, and the reaction time is 12-36 h; preferably 24 h.

In the step (4), the ultrasonic condition is 120 kW.

In the step (4), the temperature of the ultrasonic wave is 15-30 ℃; preferably, it is 25 ℃.

In the step (4), the time of the ultrasonic treatment is 5min-30min, preferably 15 min.

In the step (4), the reaction temperature is 30-60 ℃; preferably, it is 45 ℃.

In the step (4), the reaction time is 12-36 h; preferably 24 h.

In the step (4), the drying condition is 60-120 ℃; preferably, it is dried at 100 ℃ overnight.

In the step (4), the calcining temperature is 400-600 ℃; preferably, it is 500 ℃. The calcining time is 1h-3 h; preferably, it is 2 hours.

In the step (4), the dosage ratio of the ferroferric oxide/graphene oxide composite material to the absolute ethyl alcohol to the ammonia water to the tetrabutyl titanate (TBOT) is (1 ml-3 ml): (10 ml-30 ml): (40. mu.L-80. mu.L): (0.05 ml-0.2 ml); preferably, 2 ml: 20 ml: 60 μ L of: 0.15 ml.

In the step (4), the tetrabutyl titanate is preferably added dropwise.

In the step (4), the purification mode of the product can be selected from centrifugal separation.

In a specific embodiment, the preparation of the ferroferric oxide/graphene oxide/titanium dioxide composite material comprises the following steps: and (4) re-dispersing about 2ml of the ferroferric oxide/graphene oxide composite material prepared in the step (3) in absolute ethyl alcohol, and adding ammonia water into the absolute ethyl alcohol. The mixture was exposed to ultrasound for 15 minutes. The resulting solution was placed in a water bath at a constant temperature, and TBOT (tetrabutyl titanate) was added dropwise to the reaction system over 5 minutes. Subsequently, the reaction system was stirred. The product was isolated by centrifugation and washed 3 times with ethanol and water, respectively. The product was dried overnight at 100 ℃. And transferring the mixture to a muffle furnace to calcine the mixture to obtain a product.

Wherein the volume of the absolute ethyl alcohol is 20ml, the concentration of the ammonia water is 28 percent, and the volume is 60 mu L. The reaction temperature was 45 ℃ and the amount of TBOT used was 0.15 ml. The reaction time was 24 hours. The calcination temperature was 500 ℃ for 2 hours.

(5) Characterization of SERS performance of ferroferric oxide/graphene oxide/titanium dioxide composite material

Mixing and shaking the Raman spectrum signal molecule solution and the titanium dioxide coated ferroferric oxide/graphene oxide composite material. The resulting mixture was centrifuged and then redispersed in ultrapure water. And dropwise adding the solution onto a silicon wafer, drying, and testing a Raman spectrum signal under laser excitation.

In the step (5), the Raman spectrum signal molecule is selected from copper phthalocyanine and/or crystal violet and/or R6G and the like; preferably, copper phthalocyanine.

In the step (5), the concentration of the Raman spectrum signal molecule solution is 5mM-20 mM; preferably, it is 10 mM.

In the step (5), the volume ratio of the Raman spectrum signal molecular solution to the titanium dioxide-coated ferroferric oxide/graphene oxide composite material to ultrapure water is 0.5ml-2 ml: 2ml-8 ml: 1ml-3 ml: preferably, 1 ml: 5 ml: 2 ml.

In the step (5), the shaking time of the mixture is 30-90 min; preferably, it is 1 hour.

In the step (5), the wavelength of the laser is 532nm, 633nm and 780 nm; preferably 633 nm.

In a specific embodiment, the step of characterization of the SERS performance of the titanium dioxide-coated ferroferric oxide/graphene oxide composite material comprises: and mixing and shaking the copper phthalocyanine solution and the titanium dioxide coated ferroferric oxide/graphene oxide composite material for 1 hour. The resulting mixture was re-dispersed by centrifugation in 2ml of ultrapure water. And dropwise adding the solution onto a silicon wafer, drying at room temperature, and testing a Raman spectrum signal under laser excitation.

Wherein the concentration of the copper phthalocyanine solution is 10mM, and the volume is 1 ml. The volume of the nanomaterial is 5 ml. The laser wavelength was 633 nm.

(6) Preparation of PD-L1 Probe

(6-1) adding a ferroferric oxide/graphene oxide/titanium dioxide composite material into the Raman spectrum signal molecule solution, incubating, centrifugally separating, and drying the solid sample in vacuum to obtain blue solid powder.

(6-2) then incubating the obtained blue solid powder with PD-L1 antibody at constant temperature to obtain the PD-L1 probe.

In the step (6-1), the Raman spectrum signal molecule is selected from copper phthalocyanine and/or crystal violet and/or R6G and the like; preferably, copper phthalocyanine.

In the step (6-1), the concentration of the Raman spectrum signal molecule solution is 5mM-20 mM; preferably, it is 10 mM.

In the step (6-1), the incubation temperature is 25-45 ℃; preferably, it is 37 ℃.

In the step (6-1), the incubation time is 1h-5 h; preferably, it is 3 h.

In the step (6-1), the temperature of vacuum drying is 40-80 ℃; preferably, it is 60 ℃.

In the step (6-1), the vacuum drying time is 1-5 h; preferably, it is 2 h.

In the step (6-2), the incubation temperature at constant temperature is 20-45 ℃; preferably, it is 37 ℃.

In the step (6-2), the incubation time at constant temperature is 2-5 h; preferably, it is 3 h.

In the step (6-2), the concentration of the PD-L1 antibody is 20 to 200. mu.M; preferably, it is 100. mu.M.

In the step (6-2), the dosage ratio of the blue solid powder to the PD-L1 antibody is 1ml-3 ml: 10-30 μ L; preferably, 1.5 ml: 20 μ L.

In the step (6), the dosage ratio of the Raman spectrum signal molecule, the ferroferric oxide/graphene oxide composite material coated by titanium dioxide and PD-L1 is (100-300 muL): (1 ml-3 ml): (10. mu.L-30. mu.L); preferably, 200 μ L: 2 ml: 20 μ L.

In a specific embodiment, the PD-L1 probe is prepared by the steps of: 5ml of nanoparticles were added to the copper phthalocyanine solution for incubation. The product was centrifuged and the resulting solid sample was dried in vacuo. The blue solid powder obtained was incubated with 1ml of PD-L1 antibody at constant temperature.

Wherein the concentration of copper phthalocyanine is 10mM, the volume is 1ml, the incubation temperature is 37 ℃, and the incubation time is 1 hour. The vacuum drying temperature was 60 ℃. The antibody concentration was 100. mu.M, the incubation temperature was 37 ℃ and the time was 3 hours.

The invention also provides a PD-L1 probe prepared by the preparation method.

The invention also provides a preparation method of the silicon slice modified by the SYL3C aptamer, which comprises the following steps:

(i) mixing APTES (3-aminopropyltriethoxysilane) and a solvent, and immersing a silicon wafer into the solution to obtain an amino modified silicon wafer;

(ii) and re-immersing the silicon wafer into the aptamer solution, then dropwise adding the SYL3C aptamer on the silicon wafer, incubating at constant temperature, and modifying the silicon substrate under the action of a coupling agent to obtain the SYL3C aptamer modified silicon wafer.

In the step (i), the APTES (3-aminopropyltriethoxysilane) is used for modifying NH on the silicon substrate₂。

In step (i), the solvent is selected from one or more of ethanol, methanol, water and the like; preferably, it is ethanol.

In step (i), the volume ratio of the APTES to the solvent is 1-10: 1 to 20; preferably, it is 1: 15.

In step (i), the immersion temperature is 25-45 ℃; preferably, it is 37 ℃.

In the step (i), the immersion time is 1-6 h; preferably, it is 4 h.

In step (ii), the coupling agent is a coupling molecule reacted with amino and is selected from glutaraldehyde, malondialdehyde and succinaldehyde; preferably, it is glutaraldehyde.

In step (ii), the sequence of SYL3C is 5^’-NH₂-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3’ 。

In step (ii), the SYL3C aptamer acts to specifically capture cells.

In the step (ii), the concentration of the coupling agent is 1% -5%; preferably, it is 2.5%.

In step (ii), the concentration of SYL3C was 10. mu.M.

In step (ii), when the wafer is 50mm by 50mm in size, the volume of the SYL3C aptamer is 5-20 μ L.

In step (ii), the immersion temperature is 10-30 ℃; preferably, it is 25 ℃.

In the step (ii), the immersion time is 1h-3 h; preferably, it is 2 h.

In step (ii), the incubation temperature is 25-45 ℃; preferably, it is 37 ℃.

In the step (ii), the incubation time at constant temperature is 1h-3 h; preferably, it is 2 h.

In a specific embodiment, the preparation of the SYL3C aptamer-modified silicon wafer comprises: after APTES (3-aminopropyltriethoxysilane) was mixed with 15ml of ethanol, the silicon wafer was immersed in the above solution and shaken at a constant temperature, and then the silicon wafer was washed twice with ultrapure water and ethanol to remove the residual solution, to obtain an amino-modified silicon wafer, which was again immersed in a glutaraldehyde solution. Residual glutaraldehyde solution was removed by washing with ultrapure water. And finally, dropwise adding a solution containing the SYL3C aptamer on the silicon wafer, and incubating at constant temperature to obtain the SYL3C aptamer modified silicon wafer.

Wherein the volume of the APTES is 1ml, the APTES and the silicon chip are incubated for 4 hours at the constant temperature of 37 ℃. Glutaraldehyde concentration was 2.5% and incubation time was 2 hours. Sequence of SYL3C is 5^’-NH₂The concentration of CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3' was 10. mu.M, the volume was 10. mu.L, and the incubation time with the silicon wafer was 2 hours at 37 ℃.

The invention also provides a silicon slice modified by the SYL3C aptamer prepared by the preparation method.

The invention also provides application of the PD-L1 probe in detecting the programmed death receptor ligand 1(PD-L1) on the surface of the cell membrane by surface enhanced Raman spectroscopy.

The invention also provides a method for detecting the content of PD-L1 in cells, which comprises the following steps

(a) Cell capture

(a-1) taking cells in a logarithmic growth phase, centrifuging, and dispersing in a coupling buffer solution to obtain a cell suspension;

(a-2) dropping the cell suspension onto the SYL3C aptamer-modified silicon wafer, and incubating together.

(b) PD-L1 Raman immunoassay

Mixing the silicon chip containing the cells in the step (a) with a PD-L1 probe, incubating and then carrying out Raman detection.

In step (a-1), the cells are selected from MB-MDA-231, MCF-7; preferably, it is MB-MDA-231.

In step (a-1), the rotational speed of the centrifugal separation of the cells may be, but is not limited to, 800rpm for 3 minutes.

In step (a-1), the composition of the coupling buffer comprises 4.5 g L⁻¹ glucose，5 mM MgCl₂，0.1 mg mL⁻¹ tRNA，1 mg mL⁻¹BSA, calcium chloride, magnesium chloride, PBS and the balance of water.

In the step (a-2), when the wafer is 50mm by 50mm in size, the amount of the cell suspension dropped on the wafer is 50 to 200. mu.L.

In the step (a-2), the temperature of the co-incubation is 25-45 ℃; preferably, it is 37 ℃.

In the step (a-2), the co-incubation time is 30min-90 min; preferably, it is 1 h.

In step (b), the volume of the PD-L1 probe is 5 to 20. mu.L when the wafer is 50mm by 50mm in size.

In a specific embodiment, the method for detecting the content of PD-L1 in the cells comprises the following steps:

(a) cell capture: centrifuging the cells in the logarithmic growth phase at the rotating speed of 800rpm for 3 minutes; dispersing cells in a coupling buffer solution to obtain a cell suspension; the cell suspension was added dropwise to the SYL3C aptamer-modified silicon wafer and incubated together.

Wherein the formula of the coupling buffer solution is 4.5 g L⁻¹ glucose, 5 mM MgCl₂, 0.1 mg mL⁻¹tRNA, and 1 mg mL⁻¹BSA was dissolved in PBS buffer containing calcium chloride and magnesium chloride.

The cell suspension was used in an amount of 100. mu.L, and the co-incubation time was 1 hour at 37 ℃.

(b) PD-L1 Raman immunoassay

The silicon wafer containing cells prepared in (a) was mixed with the PD-L1 probe prepared above and incubated. The surface of the wafer was then washed with PBS to remove the non-specifically bound probes. And finally carrying out Raman detection.

The volume of the PD-L1 probe was 10. mu.L. The incubation temperature was 37 ℃ for 1 hour.

In the invention, cells with different concentrations are captured and subjected to Raman linear detection, so that a cell detection linear relation can be obtained.

In the invention, the cells of the same species, the same batch and the same concentration are respectively taken, different amounts of gamma IFN are respectively added into a culture medium, the incubation is carried out for 24 hours, the Raman detection is carried out according to the method, and the cell stimulation response experiment can be carried out.

In one embodiment of the invention, the method for detecting telomerase activity in stem cells by using the surface enhanced raman spectroscopy substrate material based on a chemical enhancement mechanism comprises the following steps:

(1) preparing ferroferric oxide nano particles: FeCl is added₃·6H₂O was dissolved in 100ml of ethylene glycol, followed by addition of sodium citrate and sodium acetate under magnetic stirring, and stirring was continued for 30 minutes. The resulting yellow solution was transferred to a 100ml teflon reaction kettle and reacted for 10 hours. Finally, slowly cooling to room temperature. The obtained black solid is washed three times with ultrapure water to obtain the product.

Wherein FeCl₃·6H₂3.25g of O and lemonThe amount of sodium acid is 1.3g, the amount of sodium acetate is 6g, and the temperature of the reaction kettle is 200 ℃.

(2) Preparing graphene oxide: concentrated sulfuric acid and fuming nitric acid are placed in an ice bath to be uniformly mixed, and then graphite is added into the mixture to be continuously stirred and uniformly mixed. In a fume hood, potassium chlorate was added very slowly to the above system. Stirring was continued at room temperature for 96 hours. And finally, pouring the mixture into 4L of cold water, and filtering to obtain the graphene oxide.

(3) Preparing a ferroferric oxide/graphene oxide composite material: adding PDDA solution into 15ml of ferroferric oxide-containing nano particle solution sample, and carrying out ultrasonic treatment on the mixed solution for 30 minutes. And (3) adding the graphene oxide solution in the step (2) into the solution, and continuing to perform ultrasonic treatment for 30 min. And finally, centrifugally separating the mixture, and washing the mixture with ethanol and ultrapure water for three times respectively to obtain a product.

(4) And (3) titanium dioxide wrapping: redispersing about 2ml of the product obtained in (3) in absolute ethanol. And ammonia water was added thereto. The mixture was exposed to ultrasound for 15 minutes. The resulting solution was placed in a water bath at a constant temperature, and TBOT (tetrabutyl titanate) was added dropwise to the reaction system over 5 minutes. Subsequently, the reaction system was stirred. The product was isolated by centrifugation and washed 3 times with ethanol and water, respectively. The product was dried overnight at 100 ℃. And transferring the mixture to a muffle furnace to calcine the mixture to obtain a product.

(5) Characterization of SERS performance of materials: the copper phthalocyanine solution is mixed with the nanomaterial and shaken for 1 hour. The resulting mixture was re-dispersed by centrifugation in 2ml of ultrapure water. And dropwise adding the solution onto a silicon wafer, drying at room temperature, and testing a Raman spectrum signal under laser excitation.

(6) Preparation of PD-L1 Probe: 5ml of nanoparticles were added to the copper phthalocyanine solution for incubation. The product was centrifuged and the resulting solid sample was dried in vacuo. The blue solid powder obtained was incubated with 1ml of PD-L1 antibody at constant temperature.

Wherein the concentration of copper phthalocyanine is 10mM, the volume is 1ml, the incubation temperature is 37 ℃, and the incubation time is one hour. The vacuum drying temperature was 60 ℃. The antibody concentration was 100. mu.M, the incubation temperature was 37 ℃ and the time was 3 hours.

(7) Silicon substrate modification

After APTES (3-aminopropyltriethoxysilane) was mixed with 15ml of ethanol, the silicon wafer was immersed in the above solution and shaken at a constant temperature, and then the silicon wafer was washed twice with ultrapure water and ethanol to remove the residual solution, to obtain an amino-modified silicon wafer, which was again immersed in a glutaraldehyde solution. Residual glutaraldehyde solution was removed by washing with ultrapure water. And finally, dropwise adding a solution containing the SYL3C aptamer on the silicon wafer, and incubating at constant temperature to obtain the SYL3C aptamer modified silicon wafer.

Wherein the volume of the APTES is 1ml, and the APTES is incubated with the silicon chip for 4 hours at the constant temperature of 37 ℃. Glutaraldehyde concentration was 2.5% and incubation time was 2 hours. Sequence of SYL3C is 5^’-NH₂The concentration of CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3' was 10. mu.M, the volume was 10. mu.L, and the incubation time with the silicon wafer was 2 hours at 37 ℃.

(8) Cell capture

Cells in logarithmic growth phase were centrifuged, redispersed in coupling buffer, and the cell suspension was dropped onto the silicon wafer as described in (7), and after co-incubation, the cells that had not been trapped were washed out with PBS.

Wherein the rotation speed of cell centrifugation is 800rpm, and the time is 3 minutes. The formulation of the coupling buffer was 4.5 g L⁻¹glucose, 5 mM MgCl₂, 0.1 mg mL⁻¹ tRNA, and 1 mg mL⁻¹BSA was dissolved in PBS buffer containing calcium chloride and magnesium chloride. The cell suspension was used in an amount of 100. mu.L, and the co-incubation time was 1 hour at 37 ℃.

(9) PD-L1 Raman immunoassay

Mixing the silicon wafer containing the cells in (8) with the PD-L1 probe in (6), and incubating. The surface of the wafer was then washed with PBS to remove the non-specifically bound probes. And finally carrying out Raman detection.

Wherein the volume of the PD-L1 probe was 10. mu.L. The incubation temperature was 37 ℃ for 1 hour.

(10) Cell detection linearity test

Capturing cells at different concentrations by the method mentioned in (8), and performing Raman linear detection on the cells at different concentrations according to (9).

(11) Cell stimulus response experiment

Respectively taking the same cell with the same concentration in the same batch, respectively adding different amounts of gamma IFN into the culture medium, and incubating for 24 hours. And (4) carrying out Raman detection on the obtained cells according to the method in the step (9).

The graphene-metal oxide substrate material based on the chemical enhancement mechanism is prepared. Compared with pure graphene, the composite material maintains the selectivity of graphene and metal oxide to the molecular structure, shows better SERS activity, and greatly improves the sensitivity of a PD-L1 probe. The PD-L1 probe is successfully applied to the application of detecting the expression quantity of the PD-L1 receptor on the surface of the cell by combining with a Raman reporter molecule and applying to a PD-L1 antibody. In addition, the PD-L1 probe can be applied to detection of PD-L1 and detection of other biomarkers (HER 2, IFN and the like) in cells.

Drawings

FIG. 1 is a TEM image of ferroferric oxide nanoparticles prepared in example 1.

Fig. 2 is a TEM image of the ferriferrous oxide/graphene oxide composite material prepared in example 1.

Fig. 3 is a TEM image of the ferroferric oxide/graphene oxide/titanium dioxide composite material prepared in example 1.

Fig. 4 is an XRD pattern of the ferroferric oxide/graphene oxide/titanium dioxide composite material prepared in example 1.

Fig. 5 is a comparison of raman spectra obtained by copper phthalocyanine mentioned in example 1 and copper phthalocyanine loaded on the surface of ferroferric oxide/graphene oxide/titanium dioxide composite material.

FIG. 6 is a graph showing the relationship between the number of cells and the intensity of Raman peaks in the case of detecting the expression level of PD-L1 on the cell surface at different concentrations in example 2 using the biosensor.

FIG. 7 is a Raman linear graph of the expression quantity of cell surface PD-L1 under the stimulation of gamma IFN detected by the biosensor in example 2.

FIG. 8 is a Raman image of the expression quantity of PD-L1 on the cell surface under the stimulation of detecting gamma IFN by using the biosensor in example 2.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art, and the present invention is not particularly limited, except for the contents specifically mentioned below.

Example 1 preparation and Performance characterization of nanoprobes

(1) Preparing ferroferric oxide nano particles: 3.25g of FeCl₃·6H₂O was dissolved in 100ml of ethylene glycol, followed by addition of 1.3g of sodium citrate and 6.0g of sodium acetate under magnetic stirring, and stirring was continued for 30 minutes. The resulting yellow solution was transferred to a 100ml teflon reaction kettle and maintained at 200 ℃ for 10 hours. Finally, slowly cooling to room temperature. Washing the obtained black solid with ultrapure water for three times to obtain the ferroferric oxide nano particles.

As can be seen from FIG. 1, the particle size of the ferroferric oxide nanoparticles is about 100nm, the particles are dispersed, the particle size is uniform, and the size uniformity and the dispersibility are good.

(2) Preparing graphene oxide: 87.5ml of concentrated sulfuric acid and 45ml of fuming nitric acid are placed in an ice bath and mixed uniformly, and then 5g of graphite is added into the mixture and mixed uniformly with continuous stirring. In a fume hood, 55g of potassium chlorate was slowly added to the system. Stirring was continued at room temperature for 96 hours. And finally, pouring the mixture into 4L of cold water, and filtering to obtain the graphene oxide.

(3) Preparing a ferroferric oxide/graphene oxide composite material: 5ml of 1 percent PDDA solution is added into 15ml of ferroferric oxide-containing nano particle solution, and the mixed solution is subjected to ultrasonic treatment for 30 minutes. And (3) adding 5ml of the graphene oxide solution prepared in the step (2) into the solution, and continuing to perform ultrasonic treatment for 30 min. And finally, centrifugally separating the mixture, and respectively washing the mixture with ethanol and ultrapure water for three times to obtain the ferroferric oxide/graphene oxide composite material.

The folds of the graphene can be seen from fig. 2, which proves the successful preparation of the graphene, and ferroferric oxide nano particles are uniformly dispersed in the middle of the graphene, which proves the successful preparation of ferroferric oxide/graphene. The graphene is successfully wrapped on the outer surface of the ferroferric oxide.

And (3) titanium dioxide wrapping: dispersing about 2ml of the ferroferric oxide/graphene oxide composite material obtained in the step (3) in 20ml of absolute ethyl alcohol, and adding 60 mu L of ammonia water (28%). Placing the mixture under ultrasonic conditions for 15 minutes, placing the obtained solution in a water bath at 45 ℃ for constant temperature, and dropwise adding 0.15ml of TBOT (tetrabutyl titanate) into the reaction system within 5 minutes; subsequently, the reaction system was left to stir at 45 ℃ for 24 hours. Carrying out centrifugal separation; and repeatedly washing with ethanol and water for 3 times, respectively; the product is dried at 100 ℃ overnight and finally transferred to a muffle furnace to be calcined at 500 ℃ for 2 hours to obtain the product.

Fig. 3 shows that the titanium dioxide nanoparticles coated on the surface of the substance are uniformly coated on the outer surface of the graphene, which proves the successful preparation of the ferroferric oxide/graphene oxide/titanium dioxide nanostructure.

FIG. 4 is an XRD comparison diagram of characteristic XRD spectrum peaks and a standard spectrum of three nano materials of ferroferric oxide, titanium dioxide and graphene in the preparation process. The figure shows that the characteristic XRD spectrum peaks of the ferroferric oxide, the titanium dioxide and the graphene further prove that the ferroferric oxide/graphene oxide/titanium dioxide nano structure is successfully prepared.

(5) SERS performance characterization of ferroferric oxide/graphene oxide/titanium dioxide nano material

1ml of copper phthalocyanine solution (10 mM) and 5ml of ferroferric oxide/graphene oxide/titanium dioxide nano material are mixed and shaken for 1 hour. The resulting mixture was centrifuged and then redispersed in 2ml of ultrapure water. And dropwise adding the solution onto a silicon wafer, drying at room temperature, and testing a Raman spectrum signal under the excitation of 633nm laser.

As shown in FIG. 5, when the ferroferric oxide/graphene oxide/titanium dioxide composite material is used as an SERS substrate material, the standard Raman peak type of copper phthalocyanine is remarkably improved, which indicates that the copper phthalocyanine has good SERS activity, and compared with other separate materials, the ferroferric oxide/graphene oxide/titanium dioxide composite material has stronger SERS signal and more complete spectrum peak.

(6) Preparation of PD-L1 Probe: to 1ml of copper phthalocyanine solution (10 mM), 5ml of nanoparticles were added and incubated at 37 ℃ for 1 hour. The mixture was centrifuged and the resulting solid was dried under vacuum at 60 ℃. The resulting blue solid powder was then incubated with 1ml of about 100. mu.M PD-L1 antibody at a constant temperature of 37 ℃ for 3 hours to give the PD-L1 probe.

Example 2 detection of expression level of PD-L1 on cell surface by PD-L1 Probe

(1) Silicon substrate modification

Mixing 1ml of APTES (3-aminopropyltriethoxysilane) with 15ml of ethanol, immersing the silicon wafer into the solution, and shaking at the constant temperature of 37 ℃ for 4 hours; and then washing the silicon wafer twice by using ultrapure water and ethanol to remove residual solution to obtain the silicon wafer modified by the amino group, and immersing the silicon wafer into 2.5% glutaraldehyde solution again for 2 hours. Residual glutaraldehyde solution was removed by washing with ultrapure water. Finally, 10. mu.L of SYL3C aptamer (10. mu.M) was added dropwise to the above silicon wafer, and incubation was carried out again at 37 ℃ for 2 hours to obtain a silicon wafer modified with SYL3C aptamer.

(2) Cell capture

Taking MCF-7 cells in the logarithmic growth phase, centrifuging at 800rpm for 3 minutes, and then re-dispersing in a coupling buffer solution; mu.L of the above cell suspension was added dropwise to the SYL3C aptamer-modified silicon wafer described in step (1), incubated at 37 ℃ for 1 hour, and the uncaptured cells were washed with PBS.

Wherein the composition of the coupling buffer is 4.5 g L⁻¹Glucose, 5mM MgCl₂，0.1 mg mL⁻¹tRNA and 1 mg mL⁻¹BSA, calcium chloride, magnesium chloride, PBS. From glucose, MgCl₂tRNA and BSA were dissolved in PBS buffer containing calcium chloride and magnesium chloride.

(3) PD-L1 Raman immunoassay

The silicon wafer containing cells described in step (2) was mixed with 10. mu.L of the PD-L1 probe prepared in example 1 and incubated at 37 ℃ for 1 h. Then, washing the surface of the silicon wafer by PBS (phosphate buffer solution), and removing probes which are not specifically bound; and finally carrying out Raman detection.

(4) Cell detection linearity test

And (3) repeating the steps (2) and (3) to capture cells with different concentrations and performing Raman linear detection.

As can be seen from FIG. 6, the values are 100 to 10⁶In the range of individual cells/ml, the PD-L1 probe has a good linear response relationship. The probe is proved to be used for detecting the expression level of PD-L1 on the surface of the cell.

(5) Cell stimulus response experiment

Respectively taking the same kind of cells, the same batch of cells and the same concentration, respectively adding different amounts of 20ng/ml gamma IFN into the culture medium, and incubating for 24 hours. And (4) carrying out Raman detection on the obtained cells according to the method in the step (3).

From FIG. 7 and FIG. 8, it can be seen that the expression level of PD-L1 is gradually increased under the stimulation of gamma IFN. And the expression level of cell PD-L1 is in linear relation with the concentration of stimulus gamma IFN along with the change of the addition of gamma IFN.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.

<110> university of east China

<120> PD-L1 probe, and preparation method and application thereof

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 48

<212> DNA

<213> Artificial sequence

<400> 1

CACTACAGAG GTTGCGTCTG TCCCACGTTG TCATGGGGGG TTGGCCTG 48

Claims

1. A preparation method of a PD-L1 probe, which is characterized by comprising the following steps:

(1) preparation of ferroferric oxide nano particles

In organic solvents, FeCl₃·6H₂Carrying out hydrothermal reaction on O, a reducing agent and weak base to obtain the ferroferric oxide nano particles;

(2) preparation of graphene oxide

In concentrated sulfuric acid and fuming nitric acid, carrying out oxidation reaction on graphite and potassium chlorate to obtain the graphene oxide;

(3) preparation of ferroferric oxide/graphene oxide composite material

In a solution, carrying out ultrasonic treatment on ferroferric oxide nanoparticles, PDDA and graphene oxide to obtain the ferroferric oxide/graphene oxide composite material;

Dispersing the ferroferric oxide/graphene oxide composite material prepared in the step (3) in an organic solvent, and adding ammonia water into the organic solvent; subjecting the mixture to ultrasonication; adding tetrabutyl titanate (TBOT) into a system for reaction, stirring, drying and calcining to obtain the ferroferric oxide/graphene oxide/titanium dioxide composite material;

Mixing and shaking the Raman spectrum signal molecule solution and the titanium dioxide coated ferroferric oxide/graphene oxide composite material; centrifuging the resulting mixture and then redispersing it in ultrapure water; dropwise adding the solution onto a silicon wafer, drying, and testing a Raman spectrum signal under laser excitation;

(6) preparation of PD-L1 Probe

(6-1) adding a ferroferric oxide/graphene oxide/titanium dioxide composite material into the Raman spectrum signal molecule solution, incubating, centrifugally separating, and drying a solid sample in vacuum to obtain blue solid powder;

2. The method according to claim 1, wherein in the step (1), the organic solvent is selected from the group consisting of ethylene glycol and/or glycerol; and/or the reducing agent is selected from one or more of sodium citrate, sodium borohydride and ascorbic acid; and/or the weak base is selected from one or more of sodium acetate, ammonia water and sodium bicarbonate; and/or the temperature of the hydrothermal reaction is 150-250 ℃; and/or, the FeCl₃·6H₂The mass ratio of O, the reducing agent and the weak base is (2-4): (0.8-1.5): (4-8).

3. The method according to claim 1, wherein in the step (2), the temperature of the oxidation reaction is 0 to 30 ℃; and/or the time of the oxidation reaction is 48-120 h; and/or the dosage ratio of the concentrated sulfuric acid, the fuming nitric acid, the graphite and the potassium chlorate is (50 ml-100 ml): (30 ml-50 ml): (3 g-8 g): (40 g-60 g).

4. The method according to claim 1, wherein in the step (3), the solution is selected from one or more of water, ethanol, and methanol; and/or the concentration of the PDDA is 1%; and/or the condition of the ultrasound is 120 kW; and/or the temperature of the ultrasound is 0-50 ℃; and/or the ultrasonic time is 15min-60 min; and/or the dosage ratio of the ferroferric oxide nanoparticles to the PDDA to the graphene oxide is (5 ml-20 ml): (1 ml-10 ml): (3 ml-10 ml).

5. The method according to claim 1, wherein in the step (4), the organic solvent is selected from the group consisting of absolute ethanol and/or methanol; and/or the concentration of the ammonia water is 28 percent; and/or after tetrabutyl titanate (TBOT) is added, the reaction temperature is 30-60 ℃; and/or after tetrabutyl titanate (TBOT) is added, the reaction time is 12-36 h; and/or the condition of the ultrasound is 120 kW; and/or the temperature of the ultrasound is 15-30 ℃; and/or the ultrasonic time is 5min-30 min; and/or the temperature of the reaction is 30-60 ℃; and/or the drying condition is 60-120 ℃; and/or, the temperature of the calcination is 400 ℃ to 600 ℃; the calcining time is 1h-3 h; and/or the dosage ratio of the ferroferric oxide/graphene oxide composite material to the absolute ethyl alcohol, the ammonia water and the tetrabutyl titanate (TBOT) is (1 ml-3 ml): (10 ml-30 ml): (40. mu.L-80. mu.L): (0.05 ml-0.2 ml).

6. The method according to claim 1, wherein in the step (5), the raman spectrum signal molecule is selected from copper phthalocyanine and/or crystal violet and/or R6G; and/or the concentration of the Raman spectrum signal molecule solution is 5mM-20 mM; and/or the volume ratio of the Raman spectrum signal molecule solution to the titanium dioxide coated ferroferric oxide/graphene oxide composite material to ultrapure water is 0.5-2 ml: 2ml-8 ml: 1ml-3 ml; and/or the shaking time of the mixture is 30min-90 min; and/or the wavelength of the laser is 532nm, 633nm and 780 nm.

7. The method according to claim 1, wherein in the step (6-1), the raman spectrum signal molecule is selected from copper phthalocyanine and/or crystal violet and/or R6G; and/or the concentration of the Raman spectrum signal molecule solution is 5mM-20 mM; and/or the incubation temperature is 25-45 ℃; and/or the incubation time is 1h-5 h; and/or the temperature of the vacuum drying is 40-80 ℃; and/or the vacuum drying time is 1-5 h.

8. The method according to claim 1, wherein in the step (6-2), the incubation temperature is 20-45 ℃; and/or the constant-temperature incubation time is 2-5 h; and/or the concentration of the PD-L1 antibody is 20-200 μ M; and/or the dosage ratio of the blue solid powder to the PD-L1 antibody is 1ml-3 ml: 10-30 μ L; and/or in the step (6), the dosage ratio of the Raman spectrum signal molecule, the titanium dioxide coated ferroferric oxide/graphene oxide composite material and the PD-L1 is (100-300 muL): (1 ml-3 ml): (10. mu.L-30. mu.L).

9. A PD-L1 probe produced by the production method according to any one of claims 1 to 8.