CN113049823A - Preparation method of ultrasensitive SERS (surface enhanced Raman scattering) immunosensor and application of ultrasensitive SERS immunosensor in cervical cancer serum biomarker detection - Google Patents

Preparation method of ultrasensitive SERS (surface enhanced Raman scattering) immunosensor and application of ultrasensitive SERS immunosensor in cervical cancer serum biomarker detection Download PDF

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CN113049823A
CN113049823A CN202110285129.1A CN202110285129A CN113049823A CN 113049823 A CN113049823 A CN 113049823A CN 202110285129 A CN202110285129 A CN 202110285129A CN 113049823 A CN113049823 A CN 113049823A
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卢丹
曹小卫
冉梦林
刘依帆
史宏灿
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Yangzhou University
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Abstract

The immunosensor not only has good specificity recognition performance, but also has low detection limit and good repeatability. The fluorescent probe is successfully applied to detection of survivin and OPN in blood serum of healthy subjects, CIN 1, CIN 2 and CIN 3 and cervical cancer patients, and the reliability of detection results is verified by ELISA. In a word, the sensitive SERS immunosensor has wide application prospect in early screening of cervical cancer.

Description

Preparation method of ultrasensitive SERS (surface enhanced Raman scattering) immunosensor and application of ultrasensitive SERS immunosensor in cervical cancer serum biomarker detection
Technical Field
The invention relates to a preparation method of an ultrasensitive SERS immunosensor and application of the ultrasensitive SERS immunosensor in cervical cancer serum biomarker detection, and belongs to the technical field of cervical cancer detection.
Background
Cervical cancer is one of the most common gynecological cancers and poses a serious threat to women's health. Early screening and accurate diagnosis of cervical cancer are closely related to effective treatment, thereby improving the survival rate of patients. However, cervical cancer patients without typical clinical symptoms are difficult to diagnose at an early stage. Therefore, there is an urgent need for new diagnostic tools for early screening with high sensitivity and specificity. Today, detection technology has advanced significantly, and monitoring changes in tumor markers allows for the screening and diagnosis of cancer. Survivin (survivin) is a member of the apoptotic protein family, which not only inhibits apoptosis of tumor cells, but also promotes their ability to proliferate and invade. Osteopontin (OPN) is a secreted glycoprotein that plays an important role in a variety of physiological and pathological processes, including the development and progression of tumors. According to the biological properties of survivin and OPN, the serum levels of survivin and OPN change in the process of tumorigenesis and can be considered as biomarkers for tumor screening and diagnosis.
Currently, enzyme-linked immunosorbent assays (ELISAs), colorimetries, chemiluminescent immunoassays (CLIA), and Localized Surface Plasmon Resonances (LSPR) have been developed to detect tumor markers. Despite some advantages of these methods, their clinical use is limited by the high cost, time consuming and complex reaction conditions. Surface Enhanced Raman Scattering (SERS) is a convenient, fast and ultra-sensitive sensing technology that has been widely used for quantitative analysis of biomarkers due to its unique spectral fingerprint, high selectivity and sensitivity at the single molecule level. Particularly, the narrow-linewidth SERS spectral characteristic peak is beneficial to simultaneously collecting signals of different analytes, so that the detection efficiency is improved, and the detection cost is saved. The Yang group reported the practical application of SERS-based immunoassays that enable the quantitative detection of ferritin antigens. Ma et al developed a complex of Fe3O4@TiO2The SERS immune structure composed of the @ Ag core-shell nanoparticle probe and the gold nanowire substrate is used for detecting PSA and AFP in a serum sample of a cancer patient, and the result is accurate to pg/mL. These indicate that SERS techniques are very promising in detecting biomarkers.
The great enhancement of the Raman signal is the key for realizing high-sensitivity detection by the SERS technology. At present, it is generally believed that the amplification of SERS signals is due to the plasma nanomaterial surfaceThe local electromagnetic field excited by Surface Plasmon Resonance (SPR) is enhanced. The areas where the electromagnetic field is strongest are called "hot spots". The nanoscale gaps between the metal nanoparticles create strong local electromagnetic fields, which are particularly beneficial for obtaining SERS "hot spots". To achieve a high density of "hot spots," researchers have assembled nanomaterials in various morphologies into substrates. These SERS substrates are mainly classified into: disordered or highly ordered. Reproducibility is another important factor in quantitative detection methods. Compared to a disordered SERS substrate, a highly ordered SERS substrate guarantees reliability of data due to its excellent signal uniformity and repeatability. In addition, the detection of biomarkers has other requirements on the biosensing activity of SERS substrates compared to the detection of inorganic substances (antibiotics, pesticides, etc.). SiO22The microspheres and gold nanoparticles (AuNPs) have the advantages of low cost, simple preparation and high biocompatibility. By assembling them into an ordered SERS substrate, not only is the density of "hot spots" increased, but further functionalization of the immunologically active proteins is also facilitated. The specificity of SERS probes benefits from the unique fingerprint of raman reporter molecules, which allows SERS technologies to achieve simultaneous analysis of different analytes. The Au-Ag nanoshell (Au-AgNSs) is a hollow non-porous spherical structure, and has SPR effect on the inner surface and the outer surface, which is beneficial to enhancing the surface electromagnetic field and shows excellent SERS performance. In addition, the chemical stability of the Au-Ag alloy structure can ensure the SERS activity. The Au-AgNSs has a very high application prospect in the preparation of the SERS probe.
Disclosure of Invention
The invention aims to provide a SERS immunosensor based on a high-density hot spot Au @ SiO2 array substrate and an Au-Ag nanoshell probe and an application thereof in ultra-sensitive detection of cervical cancer serum biomarkers, aiming at the defects of the prior art.
The invention uses AuNPs @ SiO2Microsphere (Au @ SiO)2) An array immune substrate and two Au-AgNSs immune probes develop a novel SERS immunosensor which is used for ultra-sensitive detection of survivin and OPN in clinical serum (figure 1). In the preparation of Au @ SiO2For array substrate, Langmuir-Blodgett (LB) was used) Method for obtaining single-layer SiO2Microsphere array, then using oil-water interface self-assembly method to make SiO2The surface of the microsphere is covered with a single-layer AuNPs film. The enhanced electromagnetic field of the substrate is simulated by Finite Difference Time Domain (FDTD). SiO22The electromagnetic field on the microsphere surface and the gap between adjacent microspheres were both enhanced, demonstrating that Au @ SiO2The array substrate has a high density of "hot spots". The Raman peaks of 4-aminothiophenol (4-ATP) and 5,5' -dithiobis- (2-nitrobenzoic acid) (DTNB) are readily distinguishable and were therefore selected to prepare two probes. They can not only hold Au-AgNS by-SH bond, but also bind to the antibody by forming amide bond. Anti-survivin antibody and anti-OPN antibody were reacted with Au @ SiO2After the array substrate is connected with Au-AgNSs, the SERS immunosensor consisting of the immunoprobe and the immunopotentiator is prepared.
After survivin and OPN were captured by the immune substrate, two immunoprobes were added for the formation of a sandwich immune complex. Next, quantitative analysis of these biomarkers was performed by monitoring the SERS signal intensity of the immunosensor. It exhibits good reproducibility, excellent sensitivity and low detection limit. Finally, healthy subjects and cervical intraepithelial neoplasia 1(CIN 1), CIN 2, CIN 3 and survivin and OPN in the serum of cervical cancer patients were tested and results consistent with ELISA were obtained. It is conceivable that this extremely attractive SERS immunosensor has excellent clinical applications in early diagnosis of cervical cancer.
Specifically, the preparation method of the SERS immunosensor provided by the invention comprises the following steps:
step 1) collection and processing of blood samples:
the serum samples are divided into five groups of healthy people, CIN 1, CIN 2, CIN 3 and cervical cancer patients, 5mL of venous blood is extracted before breakfast, the obtained serum samples are stored at-80 ℃ after centrifugation treatment at the rotating speed of 3000r/min at 4 ℃ for 10 min.
Step 2) preparation of immune substrate:
2.1) Synthesis of AuNPs
First, 25mL of 1mM HAuCl was added4The solution was added to a 100mL two-necked flask and heated to boiling(ii) a 4mL of 38mM trisodium citrate dihydrate aqueous solution is added dropwise, and after the mixture is stirred vigorously at the rotating speed of 700r/min at 100 ℃ for 15min, the color of the solution is changed from light yellow to wine red; finally, after the solution is naturally cooled to room temperature, the preparation of the AuNPs solution is finished;
2.2)SiO2synthesis of microspheres
Synthesis of SiO following the Sol-gel method2Microspheres; mixing 10mL of ammonium hydroxide, 50mL of ethanol and 6mL of ultrapure water and heating to 30 ℃; then 10mL of ethyl orthosilicate is added into the mixed solution dropwise, and the color of the solution gradually changes into milk white; after 12h of reaction, SiO is prepared2Microspheres; finally, unreacted raw materials were removed by centrifugation at 4000r/min for 10min, and they were dispersed in a methanol solution to prepare SiO at a concentration of 5 wt%2A suspension of microspheres;
2.3) preparation of hydrophilic silicon wafers
Treating the silicon wafer with a piranha solution to obtain a hydrophilic silicon wafer; adding hydrogen peroxide into concentrated sulfuric acid in a volume ratio of 3:7 to prepare a piranha solution; then, cutting the silicon slice into small pieces with the size of 10 multiplied by 15mm, immersing the silicon slice into the piranha solution for 30min, and then ultrasonically cleaning the silicon slice for 3 times by using ultrapure water and absolute ethyl alcohol;
2.4) Single SiO layer2Preparation of microsphere arrays
The single-layer SiO2 microsphere array is manufactured by a Langmuir-Bloadgate gas-liquid interface assembly method; chloroform was added to the microsphere suspension at a volume ratio of 2:3, and then 5. mu.L of SiO was taken each time2The microsphere suspension is dropped on the water surface; SiO22The microspheres spontaneously diffuse on the water surface until the entire water surface is covered; next, by dropping 100. mu.L of a 2 wt% aqueous SDS solution onto the water surface, SiO was formed in a close arrangement2A monolayer array of microspheres, which is then transferred to a clean hydrophilic silicon wafer; finally, a single layer of SiO2Drying the microsphere array silicon chip for further use;
2.5)Au@SiO2preparation of array substrate
Firstly, 4mL of AuNPs colloidal solution and 2mL of hexane were sequentially added to a beaker, and then 2mL of ethanol was added dropwise; after addition of ethanol, AuNPs are in the oil-water boundaryForming a film with metallic luster on the surface; secondly, using the assembled single layer SiO2Picking up the Au film by the microsphere array silicon chip, and drying to obtain Au @ SiO2An array substrate having a single SiO layer on the bottom2The microsphere array structure is provided with a tightly packed single-layer AuNPs on the top;
2.6) anti-survivin antibody and anti-OPN antibody conjugated Au @ SiO2Preparation of array substrate
Modification of anti-survivin antibody (coating) and anti-OPN antibody (coating) to Au @ SiO2Preparing an immune substrate on the array substrate; firstly, Au @ SiO2The array substrate was immersed in 5mL of 2 mM DMSA solution for 2h, then washed twice with water;
next, the carboxyl group (-COOH) of DMSA modified on the substrate was activated with 5mL of a mixed solution of 150mM EDC and 30mM NHS, reacted for 0.5h and then washed with PBS; next, 20. mu.L of a mixture of 200. mu.g/mL anti-survivin antibody (coating) and 200. mu.g/mL anti-OPN antibody (coating) was added to the DMSA-modified Au @ SiO2A surface of an array substrate; finally, the substrate was incubated at 37 ℃ for 4h, then washed twice with PBS, and then dried naturally.
Step 3) synthesis of Au-AgNS:
in the first step, 1.5mL of 0.01M AgNO was added30.225 mL0.3M C6H5Na3O7·2H2O and 0.3mL of 0.005M PVP were mixed together and water was added to make a volume of 120 mL;
in the second step, after the solution mixture was magnetically stirred at room temperature for 15min, 0.5mL of 0.05M NaBH was added4The reaction mixture was injected dropwise and then 2 μ L of 0.3M NaOH was added to the solution to a pH above 7; after the color of the reaction mixture changed from colorless to yellow, the resulting solution was stored at room temperature in the dark for 2 h;
third, using a light output of 0.5mW cm-2And a green LED with the central wavelength of 520nm is used for irradiation, and 20mL of the obtained solution is used for irradiation for 55min each time; when the color of the solution is changed into blue, the preparation of the Ag seeds is finished;
the fourth step, 150. mu.L of 0.3M NaOH and 5.5mL of 100mM L-AA is sequentially added into 50mL of Ag seed solution; next, 100. mu.L of 1mM HAuCl4Slowly adding the mixture into the obtained solution, and stirring vigorously for 1 h; finally, the product was collected by centrifugation at 5000r/min for 15min and dispersed in water.
Step 4) preparation of Au-AgNSs immune probe:
firstly, 200 mu L of 1mM 4-ATP ethanol solution is added into 5mL Au-AgNSs solution, and the solution is vibrated for 1h at room temperature, so that a covalent bond is formed between 4-ATP and Au-AgNSs; centrifuging at the rotating speed of 5000r/min for 5min, discarding the supernatant, and then re-dispersing the supernatant in 5mL PBS; then, 50. mu.L of 200. mu.g/mL of an anti-survivin antibody (label) was added to the solution, and then 50. mu.L of 150mM EDC was added to activate the carboxyl group (-COOH) of the anti-survivin antibody; incubating the obtained solution at 37 ℃ for 1h, then purifying the solution by centrifuging at the rotating speed of 5000r/min for 5min, and dispersing the prepared survivin-AgNSs immunoprobe in PBS solution with the same volume; for the preparation of OPNAu-AgNSs immunoprobe, 200. mu.L of 1mM DTNB ethanol solution and 5mL Au-AgNSs solution were reacted at room temperature for 1h, and after purification by centrifugation, re-dispersed in 5mL PBS, followed by addition of 50. mu.L of 150mM EDC and 50. mu.L of 30mM NHS in that order; next, the carboxyl group (-COOH) was activated by shaking for 1.5h, and 50. mu.L of 200. mu.g/mL of an anti-OPN antibody (labeled) was added to the solution; finally, after incubation at 37 ℃ for 2h, OPNAu-AgNSs immunoprobes were synthesized.
Cervical cancer is a common cause of cancer-related deaths of women worldwide, and quantitative detection of serum tumor markers has practical significance for early diagnosis of cervical cancer. The invention provides an immunosensor for simultaneously detecting survivin (survivin) and Osteopontin (OPN) in serum of a cervical cancer patient based on Surface Enhanced Raman Scattering (SERS), which is composed of AuNPs @ SiO2Microsphere (Au @ SiO)2) The array immune substrate and the Au-Ag nanoshell (Au-AgNSs) immune probe. Single layer AuNP thin film and single layer SiO2The substrate formed by the microsphere array not only ensures uniformity, but also improves SERS performance. A high density of "hot spots" on the substrate surface was confirmed by finite difference time domain simulations. Experimental results show that the constructed SERS immunosensor has satisfactory performanceThe selectivity and the reproducibility of the compound are improved, and the detection limit of survivin and OPN in human serum is reduced to 0.908pg/mL and 0.813 pg/mL. Finally, the effectiveness of the kit in detecting real human serum samples (healthy subjects; cervical intraepithelial neoplasia 1(CIN 1), CIN 2, CIN 3 and cervical cancer patients) is verified, and the detection result is well matched with the result of enzyme-linked immunosorbent assay. The immunosensor has excellent performance and is expected to be used for early screening of cancers.
In conclusion, the invention develops the Au @ SiO2The SERS immunosensor comprises an array immune substrate and two Au-AgNSs immune probes marked by Raman signal molecules, and the content of survivin and OPN in human serum is detected simultaneously by measuring the SERS signal intensity of 4-ATP and DTNB in the immune probes. The experimental result shows that the gold film and the ordered SiO2Au @ SiO assembled by microsphere arrays2The array substrate has good uniformity and high SERS activity. The immunosensor not only has good specificity recognition performance, but also has low detection limit and good repeatability. Finally, the detection method is successfully applied to the detection of survivin and OPN in the serum of healthy subjects, CIN 1, CIN 2 and CIN 3 and cervical cancer patients, and the reliability of the detection result is verified by ELISA. In a word, the sensitive SERS immunosensor has wide application prospect in early screening of cervical cancer.
Drawings
FIG. 1 is a schematic diagram of a SERS-based immunosensor for simultaneous detection of survivin and OPN; preparing an immunological substrate (a); two immunoprobes (B); the prepared SERS immunosensor (C) is used for quantitatively analyzing survivin and OPN;
FIG. 2: (A) single-layer AuNPs are obtained through self-assembly of an oil-water interface; (B) single-layer SiO prepared by L-B method2A microsphere substrate; (C) au @ SiO2SEM images of the array substrate, the top right corner of each image being a corresponding digital photograph; (D) adsorption on Au @ SiO2Raman spectra of 4-ATP molecules on an array substrate, 4-ATP-labeled monolayers of AuNPs and 4-ATP;
FIG. 3: (A) by applying on Au @ SiO240 x 40 μm on array substrate2Measured on an area of 1083cm-14-AT of (2)P signal intensity to obtain SERS mapping; (B) 4-ATP-labeled Au @ SiO2Average SERS spectra after 1 day, 5 days, 10 days, and 15 days of storage of the array substrate and (C) at 1083cm-1The peak intensity of (d);
FIG. 4: (A) FDTD simulates Au @ SiO from perspective model2The electromagnetic field distribution of the array substrate; (B) a side view of an FDTD simulated electric field profile of the substrate; top view of FDTD simulated electric field distribution of the substrate at (C) z 225nm and (D) z 450 nm;
FIG. 5: (A) SEM image of Au-AgNSs; (B) a TEM image; (C) HRTEM image and (D) partial magnified view; (E) SAED pattern and (F) STEM HAADF image; wherein (G) Au and (H) Ag are mapped; (I) the ultraviolet visible absorption spectrum and the corresponding digital photo of Au-AgNSs;
FIG. 6 FT-IR spectra of SERS immunosensors at each preparation step: (A) DMSA-labeled Au @ SiO2(ii) a (B) An immunological substrate; (C) 4-ATP-labeled Au-AgNSs; (D) an immunoprobe for measuring survivin; (E) DTNB-labeled Au-AgNSs; and (F) is an immunoprobe for the determination of OPN;
FIG. 7 SERS spectra of different species of biomarker samples and blank samples, illustrating selectivity of the prepared SERS immunosensor;
FIG. 8(A) the immunosensor detects SERS spectra of survivin and OPN dispersed in PBS at different concentrations; according to different concentrations of survivin and OPN at 1083cm-1And 1329cm-1Obtaining the dose response curves of (B) survivin and (C) OPN in PBS; (D) the method comprises the steps that an immunosensor detects SERS spectra of survivin and OPN with different concentrations in serum of a blank person; dose response curves for (E) survivin and (F) OPN in serum from human blanks were derived at different concentrations of survivin and OPN at 1083cm-1And 1329cm-1SERS signal intensity of (d);
FIG. 9 SERS spectra of the same human serum sample (100ng/mL) containing survivin and OPN detected with 10 different batches of SERS immunosensors, and (B)1083cm per spectrum-1And (C)1329cm-1SERS signal intensity histogram of (a);
FIG. 10(A) mean SERS spectra of each group of clinical samples, and corresponding (B)108cm-1And (C)1329cm-1SERS intensity histogram of (a).
Detailed Description
First, preparation method
1. Material
All materials were used directly without further purification. Chloroauric acid tetrahydrate (HAuCl)4) Silver nitrate (AgNO)3) Sodium borohydride (NaBH)4) Sodium hydroxide (NaOH), trisodium citrate dihydrate (C)6H5Na3O7·2H2O), ammonium hydroxide (NH)4OH), ethyl orthosilicate (C)8H20O4Si, chloroform (HCl), hydrogen peroxide (H)2O2) Sulfuric acid (H)2SO4) Hexane (C)6H14) Potassium carbonate (K)2CO3) Sodium Dodecyl Sulfate (SDS), polyvinylpyrrolidone (PVP), L-ascorbic acid (L-AA), absolute ethyl alcohol, methanol and silicon wafers are all purchased from Yangzhou Fei chemical Co., Ltd (China). 4-aminothiophenol (4-ATP), 5' -dithiobis- (2-nitrobenzoic acid) (DTNB), dimercaptosuccinic acid (DMSA), N-hydroxysuccinimide (NHS), and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Nokia chemical Co., Ltd, Yangzhou (China). survivin, OPN, anti-survivin antibodies, anti-OPN antibodies and ELISA kits were purchased from Sangon Biotech, shanghai (china). In all experiments, water was supplied directly from Milli-Q (resistivity)>18M Ω. cm, millipore, usa).
2. Blood sample collection and processing
The serum samples were divided into five groups of healthy population, CIN 1, CIN 2, CIN 3 and cervical cancer patients, all from Yangzhou university clinical medical college. 5mL of venous blood were drawn before breakfast by the volunteers, and after centrifugation at 3000r/min for 10min at 4 ℃ the serum samples obtained were stored at-80 ℃. The experiment was approved by the ethical committee of the clinical medical college at Yangzhou university, signed with informed consent with volunteers, and followed the moral principles of medical research involving human subjects according to the declaration of Helsinki.
3. Preparation of immune substrates
3.1 Synthesis of AuNPs
First, 25mL of 1mM HAuCl was added4The solution was added to a 100mL two-necked flask and heated to boiling. 4mL of 38mM trisodium citrate dihydrate in water are subsequently added dropwise, and after vigorous stirring at 100 ℃ at a speed of 700r/min for 15min, the solution changes its color from pale yellow to reddish brown. And finally, after the solution is naturally cooled to room temperature, the preparation of the AuNPs solution is finished.
3.2、SiO2Synthesis of microspheres
Synthesis of SiO following the Sol-gel method2And (3) microspheres. Briefly, 10mL of ammonium hydroxide, 50mL of ethanol, and 6mL of ultrapure water were mixed and heated to 30 ℃. Then, 10mL of ethyl orthosilicate was added dropwise to the mixed solution, and the color of the solution gradually changed to milky white. After 12h of reaction, SiO is prepared2And (3) microspheres. Finally, unreacted raw materials were removed by centrifugation at 4000r/min for 10min, and they were dispersed in a methanol solution to prepare SiO at a concentration of 5 wt%2A suspension of microspheres.
3.3 preparation of hydrophilic silicon wafer
The silicon wafer was treated with piranha solution to obtain a hydrophilic silicon wafer. Briefly, hydrogen peroxide (30%) was added to concentrated sulfuric acid at a 3:7 volume ratio to prepare a piranha solution. Then, the silicon wafer was cut into 10X 15mm pieces, immersed in the piranha solution for 30min, and then ultrasonically cleaned 3 times with ultrapure water and absolute ethanol.
3.4, single SiO layer2Preparation of microsphere arrays
Single layer SiO2The microsphere array was fabricated by Langmuir-Bloadgate (LB) gas-liquid interface assembly method. Chloroform was added to the microsphere suspension at a volume ratio of 2:3, and then 5. mu.L of SiO was taken each time2The microsphere suspension is dropped on the water surface. SiO22The microspheres spontaneously diffuse across the surface of the water until they cover the entire surface of the water. Next, by dropping 100. mu.L of a 2 wt% aqueous SDS solution onto the water surface, SiO was formed in a close arrangement2A monolayer array of microspheres, which is then transferred to a clean hydrophilic silicon wafer. Finally, a single layer of SiO2The microsphere array silicon wafer was dried for further use.
3.5、Au@SiO2Preparation of array substrate
First, 4mL of AuNPs colloidal solution and 2mL of hexane were sequentially added to a beaker, and then 2mL of ethanol was added dropwise. After the ethanol is added, AuNPs form a metallic film on an oil-water interface. Secondly, using the assembled single layer SiO2Picking up the Au film by the microsphere array silicon chip, and drying to obtain Au @ SiO2An array substrate having a single SiO layer on the bottom2The microsphere array structure has a close-packed monolayer AuNPs on the top.
3.6 Au @ SiO conjugated anti-survivin and anti-OPN antibodies2Preparation of array substrate
Modification of anti-survivin antibody (coating) and anti-OPN antibody (coating) to Au @ SiO2Array substrate to prepare immune substrate. Firstly, Au @ SiO2The array substrate was immersed in 5mL of 2 mM DMSA solution for 2h, then washed twice with water. Next, the carboxyl group (-COOH) of DMSA modified on the substrate was activated with 5mL of a mixed solution of 150mM EDC and 30mM NHS, and the substrate was washed with PBS after reacting for 0.5 h. Next, 20. mu.L of a mixture of 200. mu.g/mL anti-survivin antibody (coating) and 200. mu.g/mL anti-OPN antibody (coating) was added to the DMSA-modified Au @ SiO2A surface of an array substrate. Finally, the substrate was incubated at 37 ℃ for 4h, then washed twice with PBS, and then dried naturally.
4. Synthesis of Au-AgNS
The synthesis of Au-AgNSs is divided into four steps.
In the first step, 1.5mL of 0.01M AgNO was added30.225 mL0.3M C6H5Na3O7·2H2O and 0.3mL of 0.005M PVP were mixed together and water was added to make a volume of 120 mL.
In the second step, after the solution mixture was magnetically stirred at room temperature for 15min, 0.5mL of 0.05M NaBH was added4The reaction mixture was injected dropwise and then 2 μ L of 0.3M NaOH was added to the solution to a pH above 7. After the color of the reaction mixture changed from colorless to yellow, the resulting solution was stored at room temperature in the dark for 2 h.
Third, using a light output of 0.5mW cm-2And green with center wavelength of 520nmIrradiation was performed with a color LED (light emitting diode), and 20mL of the resulting solution was irradiated for 55min each time. When the color of the solution turned blue, the Ag seed preparation was completed.
In the fourth step, 150. mu.L of 0.3M NaOH and 5.5mL of 100mM L-AA were added sequentially to 50mL of Ag seed solution at room temperature under magnetic stirring. Next, 100. mu.L of 1mM HAuCl4The resulting solution was added slowly and stirred vigorously for 1 h. Finally, the product was collected by centrifugation at 5000r/min for 15min and dispersed in water.
5. Preparation of Au-AgNSs immune probe
First, 200. mu.L of 1mM 4-ATP in ethanol was added to 5mL of Au-AgNSs solution and shaken at room temperature for 1h, and a covalent bond was formed between 4-ATP and Au-AgNSs. After centrifugation at 5000r/min for 5min, the supernatant was discarded and then redispersed in 5mL PBS. Then, 50. mu.L of 200. mu.g/mL of anti-survivin antibody (label) was added to the solution, and then 50. mu.L of 150mM EDC was added to activate the carboxyl group (-COOH) of the anti-survivin antibody. The obtained solution was incubated at 37 ℃ for 1h, and then the solution was purified by centrifugation at 5000r/min for 5min, and the prepared survivin au-AgNSs immunoprobe was dispersed in an equal volume of PBS solution. For the preparation of OPNAu-AgNSs immunoprobes, 200. mu.L of 1mM DTNB ethanol solution and 5mL Au-AgNSs solution were reacted at room temperature for 1h, purified by centrifugation and then redispersed in 5mL PBS, followed by the addition of 50. mu.L of 150mM EDC and 50. mu.L of 30mM NHS in that order. Next, the carboxyl group (-COOH) was activated by shaking for 1.5h, and 50. mu.L of 200. mu.g/mL of an anti-OPN antibody (labeled) was added to the solution. Finally, after incubation at 37 ℃ for 2h, OPNAu-AgNSs immunoprobes were synthesized.
6. Simultaneous detection
The detection process for survivin and OPN is as follows: first, 20 μ L of an antigen solution comprising survivin and OPN was added to the prepared immune substrate and incubated at 37 ℃ for 1 h. Next, a mixed solution containing 10. mu.L of survivin Au-AgNSs immunoprobe and 10. mu.L of LOPNAu-AgNSs immunoprobe was added dropwise, and the reaction was continued at 37 ℃ for 1 hour. After rinsing off excess reactants with PBS solution, SERS measurements were performed.
7. Characterization of
The UV-vis absorption spectrum of the nanomaterials was monitored using an UV-vis spectrophotometer (Cary UV-5000, Agilent, usa). Fourier transform Infrared Spectroscopy (FT-IR) analysis was performed on a microscope infrared spectrometer (Cary 610/670, Varian, USA). The morphology and structure of the nanomaterials were observed using transmission electron microscopy (Tecnai 12, Philips, the Netherlands) and Field Emission Scanning Electron Microscopy (FESEM) (S-4800 II, Hitachi, Japan). Selected Area Electron Diffraction (SAED) images and High Resolution Transmission Electron Microscopy (HRTEM) images of the nanomaterials were measured using S-twin transmission electron microscopy (Tecnai G2F 30, FEI, USA). The Renysha Raman spectrometer measures the SERS spectrum of a sample at room temperature by using a 50 multiplied objective lens, 5mw laser power and 10s acquisition time under an excitation wavelength of 785 nm.
8. Finite Difference Time Domain (FDTD) simulation
FDTD simulation software (version 8.9) from Lumerical Solutions corporation for simulating electromagnetic field distribution and enhancing Au @ SiO @2Array substrate (SEM image). The excitation was carried out with incident light having a wavelength of 785 nm. A Perfect Matching Layer (PML) was used to intercept the analog domain in the x, y, z directions and set the background index of refraction to 1.
Secondly, the invention has the advantages
1、Au@SiO2Characterization of array substrates
FIG. 1A shows the fabrication process of a gold array substrate film vividly. When normal hexane is added into the AuNPs solution, an oil-water interface is formed due to density difference, and the addition of ethanol reduces the surface charge density of AuNPs, so that the AuNPs are self-assembled on the oil-water interface to form a single-layer AuNPs film. Fig. 2A is a digital photograph and SEM image of AuNPs thin film. In this digital photograph, a uniform Au thin film located on the oil-water interface can be clearly seen. From SEM images, the Au film is arranged by AuNPs with uniform appearance and diameter of about 20nm, and the small gap between the AuNPs is beneficial to improving the SERS performance. Fig. 2B is an SEM image and optical photograph of the substrate assembled by the L-B method. Orderly arranging SiO with the diameter of about 450nm on a hydrophilic silicon wafer2And (3) microspheres. Since there is only one layer of SiO2Microspheres, photo-micrographs, appear pale gray. Combining these two ordered structures together will create a new oneA substrate as shown in fig. 2C. The optical color of the substrate (top right of fig. 2C) changed from light gray to a uniform dark yellow color due to the AuNPs film covering the substrate. As can be seen from the SEM image, a large number of AuNPs are densely and orderly covered on SiO2Microsphere surface, although there were some minor defects, indicating Au @ SiO2The preparation of the array substrate was successful. SERS performance of different substrates was evaluated using 4-ATP as Raman signal molecule, and FIG. 2D shows 4-ATP molecule (10)-6M) adsorption on Au @ SiO2An array substrate; 4-ATP-labeled (10)-6M) monolayer AuNPs and 4-ATP (10)-2M) raman spectrum. The Raman signal of unlabeled 4-ATP is almost negligible, and both structures amplify the Raman signal of 4-ATP. Au @ SiO can be easily found2The enhancement effect of the array substrate is more remarkable, which indicates that the 'hot spot' is more. To further evaluate the SERS activity of the substrate, we used EF ═ (I)SERS/CSERS)/(IRaman/CRaman) Enhancement Factors (EF) were calculated for both substrates. FIG. 2D presents 1083cm in Raman spectra-1Wherein C represents the concentration of 4-ATP. Au @ SiO2The EF values of the array substrate and the single-layer AuNPs are respectively 4.23 multiplied by 106And 3.65X 105. The calculation result shows that the combination of the two ordered structures further improves the substrate SERS performance.
In addition to excellent reinforcement properties, uniformity and stability of the substrate are also required. Therefore, at 40X 40 μm2Measured on a randomly selected area of (2) 4-ATP-labeled Au @ SiO2SERS intensity profile of the array substrate. As shown in FIG. 3A, each pixel in the mapped image represents 1083cm-1Signal intensity of 4-ATP. Although there are a small number of yellow and blue regions in the image, most of the regions in the image are relatively uniformly green, reflecting the high signal uniformity of the substrate. FIG. 3B is 4-ATP labeled Au @ SiO measured when stored at room temperature for 1 day, 5 days, 10 days, and 15 days2Average SERS spectrum of the array substrate. The spectrum does not change significantly except for the reduced signal strength. To more intuitively understand the signal change, FIG. 3C shows 1593cm in each cycle-1The SERS peak intensity of (c). After 15 days of storage, SEThe RS intensity is reduced by 11.86%, which indicates that the substrate has stable SERS enhancement effect.
2. FDTD simulation
To confirm the Au @ SiO prepared under irradiation of a linearly polarized light beam2The spatial electric field distribution of the array substrate, FDTD simulation is an effective method. The simulated geometric parameters were consistent with the average actual size of the samples shown in the SEM images (fig. 2D). Fig. 4A is a simulation model of a perspective view, which clearly reveals the propagation and vibration directions of the simulation light. Total Field Scattered Field (TFSF) linearly polarized light waves are injected along the-z direction, and frequency domain field profile monitors located at z 225nm and 450nm in the x-z plane (side view) and the x-y plane (top view) are respectively used for collecting electric field distribution data of the AuNPs-Si surface and the inside under the condition that the excitation wavelength is kept 785 nm. FIG. 4B shows a typical distribution of the local electromagnetic field of the substrate in the x-z plane, found along SiO2And the enhanced electromagnetic field is distributed on the surface of the microsphere. In addition, adjacent SiO2The gaps between the microspheres have higher local electromagnetic fields than other regions. This phenomenon is due to SiO2The distance between AuNPs on the surface of the microsphere is gradually reduced, so that the LSPR effect is gradually increased. As shown in fig. 4C and 4D, the electromagnetic field distribution of the substrate in the x-y plane is when z is 225nm and z is 450 nm. Adjacent SiO in the x-y plane at z 225nm2A strong local electromagnetic field exists in the gap between the microspheres. When z is 450nm, it is located on top of the microsphere. Although the distance between AuNPs becomes large and the LSRP effect is reduced, the electromagnetic field is still significantly enhanced. By means of simulation results from different viewing angles, it can be predicted that the substrate surface has been covered by a strong local electromagnetic field, which is crucial for the formation of high density "hot spots". To this end, Au @ SiO was further confirmed2Excellent SERS performance of the array substrate.
3. Characterization of Au-AgNS
FIG. 5 shows the composition and structural features of the synthesized Au-AgNS. As shown in the SEM image (fig. 5A), Au-AgNS having an intact structure and a relatively uniform morphology was prepared. From the TEM image, it can be more clearly observed that Au-AgNSs has a particle size of about 40nm, a hollow structure with a smooth surface and dense walls, as shown in FIG. 5B. The HRTEM image in fig. 5C shows more details of Au-AgNS, further confirming the hollow feature. The thickness of the shell layer is about 5nm, which is advantageous for enhancing the SPR effect. Fig. 5D is a partial magnified view of the HRTEM image, and the lattice structure with 0.232nm pitch is clearly visible. As shown in FIG. 5E, the bright diffraction rings in the SAED pattern correspond to the {111}, {200}, {220} and {311} planes, respectively, indicating that the prepared Au-AgNSs are polycrystalline. STEM-HAADF images confirm from another perspective that Au-AgNSs is a special hollow shell structure, as shown in fig. 5F. In addition, the distribution of Au and Ag can be more intuitively understood by measuring the elemental mapping of Au-AgNS. Fig. 5G and 5H show elemental mapping of Au and Ag. The random distribution of the Au and Ag elements forms a structurally and chemically stable alloy structure, further confirming the synthesis of Au-AgNS. The alloy structure not only has good thermodynamic stability, but also has strong SERS activity. As shown in FIG. 5I, the Au-AgNSs solution is dark blue in the visible range, and the corresponding UV-vis absorption spectrum has the strongest absorption peak at 612 nm. The above results indicate that homogeneous Au-AgNSs were successfully prepared.
4. FT-IR characterization of SERS immunosensors
Chemical bonds and functional groups on the surface of the material have corresponding absorption peaks in an FT-IR spectrum, and the preparation process of the SERS immunosensor is characterized by FT-IR. During the preparation of the immunosensor, interference from excess reagents (EDC, NSH, etc.) is avoided by washing. DMSA has thiol and carboxyl groups and no raman signal, which is a good choice for the preparation of immune substrates. FIG. 6A is a coupling to Au @ SiO via a thiol group2FT-IR Spectroscopy of DMSA on array substrate, 1664cm-1The peak at (a) is due to stretching of C ═ O in the carboxyl group, and modification of the anti-survivin antibody and anti-OPN antibody on the substrate was achieved by an amide bond (-CONH-) formed by reacting the amino group on the antibody with the carboxyl group of DMSA. As shown in FIG. 6B, at 1287cm-1、1539cm-1And 1642cm-1The peaks of (a) are from C-N, N-H and C ═ O in the amide bond (-CONH-) respectively. The appearance of these peaks confirmed that the antibody had been successfully coupled to Au @ SiO2An array substrate. FIG. 6C shows Au-FT-IR spectrum of AgNS and appears at 1286cm-1The peak at (A) is attributed to C-N stretching. FIG. 6D is a FT-IR spectrum of an anti-survivin antibody coupled with 4-ATP labeled Au-AgNS, wherein 1290cm-1The peak at (a) is related to C-N stretching; 1562cm-1The peak at (a) is associated with N-H amide II stretching; 1662cm-1The peak at (a) is due to C ═ O amide I stretching; the appearance of these characteristic peaks confirms the formation of amide bonds. Similarly, after capture of Au-AgNSs by DTNB, at 1660cm-1The peak at (b) belongs to C ═ O in the carboxyl group (fig. 6E). Furthermore, when anti-OPN antibodies were coupled to DTNB-labeled Au-AgNSs, characteristic peaks for CN, NH and CO appeared due to the corresponding FT-IR spectra (FIG. 6F), and the amide bonds formed were located at 1223cm each-1、1564cm-1And 1664cm-1To (3). These peaks clearly demonstrate the existence of a strong chemical bond between the antibody and the nanomaterial, indicating that the SERS immunosensor has been successfully prepared.
5. Selectivity of SERS immunosensor
In the process of detecting a real sample, due to the existence of other biomarkers, accurate identification of the target by the SERS immunosensor is very important, and false positive can be avoided due to good selectivity of the SERS immunosensor. After the immune substrate captures the antigen, an immune probe is added to form a sandwich immune complex, and the content of the antigen is detected by monitoring SERS signals of 4-ATP and DTNB in the probe. CEA, APF, survivin, OPN and survivin/OPN samples were tested at the same concentration (1. mu.g/mL) with a blank sample as a control. The corresponding spectrum is shown in FIG. 7, and it is clearly seen that only survivin (1083 cm)-1) Or OPN (1329 cm)-1) The sample (2) shows a strong characteristic peak, while the other samples show no obvious characteristic peak. spectra of survivin/OPN samples were co-represented at 1083cm-1And 1329cm-1The peaks of (a) not only confirm the good selectivity of the SERS immunosensor, but also confirm the capability of detecting two antigens simultaneously. The excellent performance of the SERS immunosensor lays a foundation for subsequent experiments.
6. Simultaneous detection of survivin and OPN in PBS and human serum
The PBS solution in which survivin and OPN were dissolved was continuously dilutedAnd obtaining a sample with a concentration gradient of 1pg/mL to 1 mu g/mL. FIG. 8A is the average SERS spectra of samples of different concentrations after 5 SERS immunosensor detections. The results show that the SERS signal decreases with decreasing survivin and OPN concentrations, and the weak signal of the blank sample is negligible. The dose response curve more intuitively illustrates SERS signal intensity and survivin (1083 cm)-1) And OPN (1329 cm)-1) See fig. 8B and 8C. In addition, the survivin has the corresponding linear equation of y being 25928.374+2101.102x, and the correlation coefficient R is20.988, and the corresponding linear equation for OPN is y 29294.255+2370.933x, R20.992. Finally, limit of detection (LOD) for survivin and OPN were calculated to be 0.783pg/mL and 0.764pg/mL, respectively, with reference to the blank sample.
To verify its practical applicability, we used SERS immunosensors to analyze human serum samples containing different concentrations of survivin and OPN. Similarly, survivin and OPN were dissolved in human serum blanks and serially diluted to give samples with a concentration gradient of 1pg/mL to 1. mu.g/mL. Fig. 8D shows the average spectra of 5 measurements for each type of sample, and similarly, the measured SERS signal decreases with decreasing survivin and OPN concentrations in the serum of the blank. Fig. 8E and fig. 8F are dose response curves corresponding to survivin and OPN in serum of a blank human, respectively, and the linear regression equations are: 23250.310+1876.532x, 26556.096+2142.8933x, R2＝0.987,R20.990. By taking a blank serum sample as a reference, the detection limits of survivin and OPN in human serum are respectively calculated to be 0.908pg/mL and 0.813pg/mL according to a linear equation.
In addition, the reproducibility was evaluated by testing the same sample with 10 different batches of SERS immunosensors. FIG. 9A shows spectra of human serum samples with survivin and OPN concentrations of 100 ng/mL. The peak shapes of different spectra are not obviously different, and the peak intensities are similar. To more intuitively understand the fluctuation of SERS signals, FIGS. 9B and 9C show the spectra at 1083cm-1And 1329cm-1A histogram of the signal strength. The Relative Standard Deviation (RSD) was 6.23% (1083 cm), respectively-1) And 5.85% (1329 cm)-1) This demonstrates the design of SERS immunotransductionThe sensors have satisfactory repeatability.
7. Clinical application
The practical operation performance of the constructed SERS immunosensor is further verified through the analysis of clinical serum samples. Fig. 10A shows the average SERS spectra of serum samples from 40 healthy subjects, 38 CIN 1, 35 CIN 2, 33 CIN 3, and 35 cervical cancer patients. The characteristic peak of each spectrum is 1083cm-1And 1340cm-1Indicating the presence of survivin and OPN in the serum sample. From the characteristic peaks of the histograms corresponding to the signal intensities, it can be found that the two antigens are closely related to the occurrence and development of the disease, as shown in fig. 10B and C. The signal intensity of the characteristic peak is taken as a linear regression equation obtained by being included in human serum, and the content of survivin and OPN in the corresponding sample is calculated. The SERS detection result is compared with the ELISA result, and the specific data are shown in Table 1, so that the reliability of the SERS immunosensor is verified. Therefore, the SERS immunosensor can be used for early screening of cervical cancer by monitoring changes of serum survivin and OPN.
Table 1 mean serum levels of survivin and OPN in clinical samples.
Figure BDA0002980141960000171
Third, conclusion
In conclusion, the invention develops the Au @ SiO2The SERS immunosensor comprises an array immune substrate and two Au-AgNSs immune probes marked by Raman signal molecules, and the content of survivin and OPN in human serum is detected simultaneously by measuring the SERS signal intensity of 4-ATP and DTNB in the immune probes. The experimental result shows that the gold film and the ordered SiO2Au @ SiO assembled by microsphere arrays2The array substrate has good uniformity and high SERS activity. The immunosensor not only has good specificity recognition performance, but also has low detection limit and good repeatability. Finally, the method is successfully applied to the detection of survivin and OPN in the serum of healthy subjects, CIN 1, CIN 2 and CIN 3 and cervical cancer patients, and the detection is verified by ELISAAnd (4) reliability of detection results. In a word, the sensitive SERS immunosensor has wide application prospect in early screening of cervical cancer.

Claims (7)

1. The preparation method of the ultrasensitive SERS immunosensor is characterized in that Au @ SiO is prepared2In the process of array substrate, a single-layer SiO is obtained by using a Langmuir-Blodgett method2Microsphere array, then using oil-water interface self-assembly method to make SiO2Covering a monolayer of AuNPs on the surface of the microsphere; simulating an enhanced electromagnetic field of the substrate through a finite difference time domain; SiO22The electromagnetic field on the surface of the microsphere and the gap between the adjacent microspheres are enhanced, which proves that Au @ SiO2The array substrate has a high density of "hot spots";
the raman peaks of 4-aminothiophenol (4-ATP) and 5,5' -dithiobis- (2-nitrobenzoic acid) (DTNB) are readily distinguishable and selected to prepare two probes that can not only grasp Au-AgNS by the-SH bond but also bind to the antibody by forming an amide bond; anti-survivin antibody and anti-OPN antibody were reacted with Au @ SiO2After the array substrate is connected with Au-AgNSs, the SERS immunosensor consisting of the immunological substrate and the immunological probe is manufactured.
2. The method for preparing the ultrasensitive SERS immunosensor according to claim 1, wherein the method comprises the following steps:
1) collecting and processing a blood sample;
2) preparing an immune substrate;
2.1) synthesizing AuNPs;
2.2)SiO2synthesizing microspheres;
2.3) preparing a hydrophilic silicon wafer;
2.4) Single SiO layer2Preparing a microsphere array;
2.5)Au@SiO2preparing an array substrate;
2.6) anti-survivin antibody and anti-OPN antibody conjugated Au @ SiO2Preparing an array substrate;
3) synthesizing Au-AgNS;
4) and preparing an Au-AgNSs immune probe.
3. The method for preparing an ultrasensitive SERS immunosensor according to claim 2, wherein the step 1) of collecting and processing the blood sample comprises the following steps: the serum samples are divided into five groups of healthy people, CIN 1, CIN 2, CIN 3 and cervical cancer patients, 5mL of venous blood is extracted before breakfast, the obtained serum samples are stored at-80 ℃ after centrifugation treatment at the rotating speed of 3000r/min at 4 ℃ for 10 min.
4. The method for preparing an ultrasensitive SERS immunosensor according to claim 3, wherein the step 2) of preparing the immunological substrate comprises the following steps:
2.1) Synthesis of AuNPs
First, 25mL of 1mM HAuCl was added4Adding the solution into a 100mL double-neck flask, and heating to boil; 4mL of 38mM trisodium citrate dihydrate aqueous solution is added dropwise, and after the mixture is stirred vigorously at the rotating speed of 700r/min at 100 ℃ for 15min, the color of the solution is changed from light yellow to wine red; finally, after the solution is naturally cooled to room temperature, the preparation of the AuNPs solution is finished;
2.2)SiO2synthesis of microspheres
Synthesis of SiO following the Sol-gel method2Microspheres; mixing 10mL of ammonium hydroxide, 50mL of ethanol and 6mL of ultrapure water and heating to 30 ℃; then 10mL of ethyl orthosilicate is added into the mixed solution dropwise, and the color of the solution gradually changes into milk white; after 12h of reaction, SiO is prepared2Microspheres; finally, unreacted raw materials were removed by centrifugation at 4000r/min for 10min, and they were dispersed in a methanol solution to prepare SiO at a concentration of 5 wt%2A suspension of microspheres;
2.3) preparation of hydrophilic silicon wafers
Treating the silicon wafer with a piranha solution to obtain a hydrophilic silicon wafer; adding hydrogen peroxide into concentrated sulfuric acid in a volume ratio of 3:7 to prepare a piranha solution; then, cutting the silicon slice into small pieces with the size of 10 multiplied by 15mm, immersing the silicon slice into the piranha solution for 30min, and then ultrasonically cleaning the silicon slice for 3 times by using ultrapure water and absolute ethyl alcohol;
2.4) Single SiO layer2Preparation of microsphere arrays
Single layer SiO2The microsphere array is manufactured by a Langmuir-Bloadgate gas-liquid interface assembly method; chloroform was added to the microsphere suspension at a volume ratio of 2:3, and then 5. mu.L of SiO was taken each time2The microsphere suspension is dropped on the water surface; SiO22The microspheres spontaneously diffuse on the water surface until the entire water surface is covered; next, by dropping 100. mu.L of a 2 wt% aqueous SDS solution onto the water surface, SiO was formed in a close arrangement2A monolayer array of microspheres, which is then transferred to a clean hydrophilic silicon wafer; finally, a single layer of SiO2Drying the microsphere array silicon chip for further use;
2.5)Au@SiO2preparation of array substrate
Firstly, 4mL of AuNPs colloidal solution and 2mL of hexane were sequentially added to a beaker, and then 2mL of ethanol was added dropwise; after ethanol is added, AuNPs form a metallic film on an oil-water interface; secondly, using the assembled single layer SiO2Picking up the Au film by the microsphere array silicon chip, and drying to obtain Au @ SiO2An array substrate having a single SiO layer on the bottom2The microsphere array structure is provided with a tightly packed single-layer AuNPs on the top;
2.6) anti-survivin antibody and anti-OPN antibody conjugated Au @ SiO2Preparation of array substrate
Modification of anti-survivin antibody (coating) and anti-OPN antibody (coating) to Au @ SiO2Preparing an immune substrate on the array substrate; firstly, Au @ SiO2The array substrate was immersed in 5mL of 2 mM DMSA solution for 2h, then washed twice with water;
next, the carboxyl group (-COOH) of DMSA modified on the substrate was activated with 5mL of a mixed solution of 150mM EDC and 30mM NHS, reacted for 0.5h and then washed with PBS; next, 20. mu.L of a mixture of 200. mu.g/mL anti-survivin antibody (coating) and 200. mu.g/mL anti-OPN antibody (coating) was added to the DMSA-modified Au @ SiO2A surface of an array substrate; finally, the substrate was incubated at 37 ℃ for 4h, then washed twice with PBS, and then dried naturally.
5. The method for preparing the ultrasensitive SERS immunosensor according to claim 4, wherein the Au-AgNS is synthesized in the step 3):
in the first step, 1.5mL of 0.01M AgNO was added30.225 mL0.3M C6H5Na3O7·2H2O and 0.3mL of 0.005M PVP were mixed together and water was added to make a volume of 120 mL;
in the second step, after the solution mixture was magnetically stirred at room temperature for 15min, 0.5mL of 0.05M NaBH was added4The reaction mixture was injected dropwise and then 2 μ L of 0.3M NaOH was added to the solution to a pH above 7; after the color of the reaction mixture changed from colorless to yellow, the resulting solution was stored at room temperature in the dark for 2 h;
third, using a light output of 0.5mW cm-2And a green LED with the central wavelength of 520nm is used for irradiation, and 20mL of the obtained solution is used for irradiation for 55min each time; when the color of the solution is changed into blue, the preparation of the Ag seeds is finished;
fourthly, 150 mu L of 0.3M NaOH and 5.5mL of 100mM L-AA are sequentially added into 50mL of Ag seed solution at room temperature under magnetic stirring; next, 100. mu.L of 1mM HAuCl4Slowly adding the mixture into the obtained solution, and stirring vigorously for 1 h; finally, the product was collected by centrifugation at 5000r/min for 15min and dispersed in water.
6. The method for preparing the ultrasensitive SERS immunosensor according to claim 5, wherein the Au-AgNSs immunosensor is prepared in the step 4):
firstly, 200 mu L of 1mM 4-ATP ethanol solution is added into 5mL Au-AgNSs solution, and the solution is vibrated for 1h at room temperature, so that a covalent bond is formed between 4-ATP and Au-AgNSs; centrifuging at the rotating speed of 5000r/min for 5min, discarding the supernatant, and then re-dispersing the supernatant in 5mL PBS; then, 50. mu.L of 200. mu.g/mL of an anti-survivin antibody (label) was added to the solution, and then 50. mu.L of 150mM EDC was added to activate the carboxyl group (-COOH) of the anti-survivin antibody; incubating the obtained solution at 37 ℃ for 1h, then purifying the solution by centrifuging at the rotating speed of 5000r/min for 5min, and dispersing the prepared survivin-AgNSs immunoprobe in PBS solution with the same volume; for the preparation of OPNAu-AgNSs immunoprobe, 200. mu.L of 1mM DTNB ethanol solution and 5mL Au-AgNSs solution were reacted at room temperature for 1h, and after purification by centrifugation, re-dispersed in 5mL PBS, followed by addition of 50. mu.L of 150mM EDC and 50. mu.L of 30mM NHS in that order; next, the carboxyl group (-COOH) was activated by shaking for 1.5h, and 50. mu.L of 200. mu.g/mL of an anti-OPN antibody (labeled) was added to the solution; finally, after incubation at 37 ℃ for 2h, OPNAu-AgNSs immunoprobes were synthesized.
7. The application of the ultrasensitive SERS immunosensor in cervical cancer serum biomarker detection according to claim 1, wherein two immunosobes are added after survivin and OPN are captured by immune substrates to form a sandwich immune complex; next, quantitative analysis is carried out on the biomarkers by monitoring the SERS signal intensity of the immunosensor; it exhibits good reproducibility, excellent sensitivity and low detection limit; finally, healthy subjects and cervical intraepithelial neoplasia 1(CIN 1), CIN 2, CIN 3 and survivin and OPN in the serum of cervical cancer patients were tested and results consistent with ELISA were obtained.
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田晖艳;刘羽;黄姣祺;谢凤欣;黄国荣;廖璞;府伟灵;张阳;: "表面增强拉曼散射技术在核酸检测中的研究进展及应用", 光谱学与光谱分析, no. 10, 13 October 2020 (2020-10-13) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117250345A (en) * 2023-11-20 2023-12-19 重庆医科大学绍兴柯桥医学检验技术研究中心 In-situ detection method of biomolecules in organ chip
CN117250345B (en) * 2023-11-20 2024-02-13 重庆医科大学绍兴柯桥医学检验技术研究中心 In-situ detection method of biomolecules in organ chip

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