CN115420727A - Method for detecting colon cancer biomarkers by using surface enhanced Raman spectroscopy - Google Patents
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Abstract
The invention relates to a method for detecting a colon cancer biomarker carcinoembryonic antigen (CEA) by using surface enhanced Raman spectroscopy. The invention prepares a two-dimensional silver plate modified by 4-mercaptobenzoic acid (4-MPBA) as an SERS substrate for capturing glycoprotein in blood plasma. Use 4MBN as a no biological interference's raman reporter, prepare Au @4MBN @ Ag NPs as the SERS probe to modify Au @4MBN @ Ag NPs nucleocapsid nanoprobe with the CEA antibody, realize accurate discernment to CEA glycoprotein with this. The method provided by the invention can carry out high-selectivity and high-sensitivity detection on the colon cancer biomarker carcinoembryonic antigen (CEA) in the blood plasma, and the detection range is 10 ‑14 M‑10 ‑8 M, the recovery rate of clinical serum samples is 105.33% -112.00%, and early screening can be realized under simpler operation, so that a new idea is provided for future application in clinical diagnosis.
Description
Technical Field
The invention relates to a method for detecting colon cancer biomarkers by using surface enhanced Raman spectroscopy, belonging to the technical field of biomedical detection.
Background
CEA, known as carcinoembryonic antigen, is the carcinoembryonic antigen, abbreviated as CEA. Carcinoembryonic antigen is glycoprotein, belongs to common tumor markers, and mainly exists in tissues such as colon, pancreas, liver and the like. Due to their reliability for quantitative analysis, glycoproteins have been used as biomarkers for disease diagnosis and prognosis. For example, alpha-fetoprotein (AFP) is used as an early diagnosis biomarker of hepatocellular carcinoma (HCC), prostate Specific Antigen (PSA) and alpha-fetoprotein (AFP) are used as early diagnosis biomarkers of prostate cancer and primary liver cancer, respectively, and haptoglobin (Hp) and carcinoembryonic antigen (CEA) are used as prognosis biomarkers of ovarian cancer and colon cancer, respectively, and have important clinical values.
The main analysis methods for detecting CEA in the traditional complex sample include an electrochemical method, a fluorescence method, an enzyme-linked immunosorbent assay and the like, wherein the enzyme-linked immunosorbent assay is considered as a 'gold standard'. However, these methods require complicated sample preparation and expensive equipment. Therefore, a method with low cost and good sensitivity and accuracy is urgently needed for the detection of CEA.
In recent years, surface Enhanced Raman Spectroscopy (SERS) is an emerging vibrational spectroscopy technology, and has become an ultra-sensitive biological detection platform due to its advantages. SERS is an important analysis method, and is suitable for nondestructive data acquisition and testing due to the fact that the SERS has a fingerprint spectrum, so that researchers can record data under the condition that materials are not damaged. Meanwhile, the SERS immunosensor has the remarkable advantages of stability, sensitivity and the like, so that the SERS immunosensor has important significance in monitoring tumor markers. However, because the environment of CEA in vivo is very complex and the content of CEA is very low, there is a great challenge in quantitative detection, and therefore, it is necessary to improve the signal intensity of CEA and exclude the interference of other signals through specific recognition.
At present, two main detection principles aiming at CEA are provided, one is to detect the value of carcinoembryonic antigen through hematology, the other is to detect through an immunohistochemical method, and whether tumor cells or detected lesion parts have positive expression or not is observed according to the principle of combining antigen and antibody. The patent CN102636649B discloses a kit for detecting carcinoembryonic antigen based on antibody functionalized magnetic nano material and upconversion fluorescent nano material, and the method mainly utilizes the light conversion performance of rare earth upconversion fluorescent nano material based on the principle of antigen-antibody combination to realize qualitative and quantitative detection of CEA. The method has the advantages of good detection stability, small interference of background light and autofluorescence, and high specificity. However, the method needs near infrared ray excitation in the detection process, the detection equipment cost is high, and the problems of low yield of the up-conversion luminescence fluorescence quantum, difficulty in preparing the small-particle-size up-conversion fluorescent material and the like exist at present. In patent CN101609096B, a preparation method of immunochromatographic test paper for detecting lung cancer markers is disclosed, and the method detects CEA by preparing an immunochromatographic test paper, which has the advantages of simple operation and low cost, but it is difficult to realize relatively accurate quantitative detection, and the detection sensitivity is different from that of the SERS method.
Disclosure of Invention
In order to solve the problems in the prior art, the invention prepares a two-dimensional immunoassay substrate, and constructs a surface-enhanced Raman scattering sandwich immunoassay system for detecting CEA with high sensitivity and effectiveness by combining Au @4MBN @ Ag NPs as a surface-enhanced Raman scattering probe. The two-dimensional silver plate modified by 4-MPBA is used as a substrate to capture glycoprotein in plasma, the Au @4MBN @ Ag NPs core-shell nano probe modified by the CEA antibody is used for specifically recognizing the CEA glycoprotein, and the detection of the CEA is realized through the amplified signal of an internal standard molecule. And the interference of other glycoprotein macromolecules in the environment can be avoided through a calibration model of the 4-MPBA and 4MBN dual-signal molecules, so that the quantitative detection of the CEA is realized.
The technical scheme of the invention is as follows:
a method for detecting biomarkers for colon cancer using surface enhanced raman spectroscopy, comprising the steps of: fully contacting a colon cancer biomarker CEA solution with a two-dimensional silver plate modified by 4-MPBA, adding Au @4MBN @ Ag NPs modified by CEA antibody functionalization as a SERS probe of CEA for incubation, finally washing the two-dimensional silver plate by PBS, and performing SERS detection after the two-dimensional silver plate is naturally dried.
Further, the synthesis method of the Ag NRs comprises the following steps:
35mg of AgNO 3 Adding 200mL of water, heating to boiling, adding 10mL of 1% trisodium citrate, stirring for reacting for 1 hour to obtain a gray-green Ag NRs solution, centrifuging the obtained solution, washing twice, and dispersing with ultrapure water for later use.
Further, the method for modifying the two-dimensional silver plate by the 4-MPBA comprises the step of soaking the prepared two-dimensional silver plate in the 4-MPBA solution for a specified time to obtain the 4-MPBA-loaded two-dimensional silver plate.
Further, the synthesis method of Au @4MBN @ Ag NPs is to rapidly add sodium citrate (Na 3 Ct) into boiling HAuCl 4 And stirring vigorously for 15min to generate Au nuclei. Adding 4-cyano thiophenol (4 MBN) to form Au-S covalent bond, magnetically stirring for 30min, centrifuging, re-dispersing, and dropwise adding AgNO 3 And AA, stirring for 60min to obtain Au @4MBN @ Ag NPs, centrifuging, and re-dispersing for later use.
Further, the CEA antibody modified Au @4MBN @ Ag NPs core-shell nanoprobe is prepared by adding 1mL Au @4MBN @ Ag NPs into SH-PEG-COOH solution, stirring for reaction for 30min, performing first centrifugation, then adding 20 μ L10mg/mL 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 10 μ L10mg/mL N-hydroxysuccinimide (NHS) solution to activate carboxyl, adding CEA antibody after centrifugal redispersion PBS, continuously shaking for 2 hours at room temperature, incubating for 24 hours at 4 ℃, and performing second centrifugal redispersion to obtain Au @4MBN @ Ag NPs-COOH-CEA antibody solution;
further, the specific method of Raman detection is that a colon cancer biomarker CEA sample is fully contacted with a 4-MPBA modified two-dimensional silver plate, then Au @4MBN @ Ag NPs functionally modified by a CEA antibody is added to serve as a SERS probe of CEA for co-incubation, finally PBS is used for washing the two-dimensional silver plate, and SERS detection is carried out after the two-dimensional silver plate is naturally dried.
Further, the first centrifugation speed is 10000rpm, and the centrifugation time is 10 minutes; the second centrifugation speed is 12000rpm, and the centrifugation time is 10 minutes; the third centrifugation speed is 4000rpm, and the centrifugation time is 8 minutes; the fourth centrifugation speed is 9500rpm, and the centrifugation time is 12 minutes; the fifth centrifugation speed was 8600rpm and the centrifugation time was 10 minutes.
Furthermore, the optimal dosage of HS-PEG-COOH is 10 mu L, and the optimal parameter of CEA antibody is 50 mu L.
Further, the concentration of the 4-MPBA solution is 10 -6 M, modification time 24 hours.
Further, in the step (3), the CEA standard sample or plasma is contacted with the two-dimensional silver plate for 3 hours, the amount of the BSA solution added is 50 μ L, the concentration is 0.2%, and the incubation time is 2 hours. The laser used for Raman detection is 785nm, the power is 2.38mW, and the integration time is 10s.
The invention has the beneficial effects that:
1. according to the method provided by the invention, the two-dimensional silver plate modified by 4-MPBA is selected as the SERS substrate, so that the glycoprotein in the blood plasma can be effectively captured.
2. The method provided by the invention selects 4MBN as an internal standard molecule which is 2226cm -1 The characteristic peak of the biological silencing band is positioned, so that the interference caused by other possible Raman signals is effectively avoided, and the 4MBN is embedded between the gold layer and the silver layer, so that the exposure to the detection environment is avoided, and competitive adsorption with impurities cannot be formed, so that the stability of the signals is influenced.
3. According to the method provided by the invention, the Au @4MBN @ Ag NPs core-shell nano probe is modified by the CEA antibody, the Au @4MBN @ Ag NPs-COOH-CEA antibody probe can be used for accurately identifying CEA glycoprotein, a CEA-containing sample is deposited on a two-dimensional silver plate, and after the blocking by Bovine Serum Albumin (BSA), the CEA antigen of the SERS probe can be combined with the corresponding antigen specificity immune recognition, so that the selectivity is greatly improved, and the interference of other proteins in the sample is effectively avoided. Meanwhile, the construction of a nuclear-satellite structure is completed after antigen-antibody combination pairing in the co-incubation process, so that a large number of hot spot areas are created, the internal standard signals are greatly enhanced, the detection sensitivity is superior to that of the single substrate for detecting CEA, and the repeatability is good.
4. The method provided by the invention does not need to carry out complex pretreatment on the sample, is simple, convenient, quick and high in sensitivity, does not cause damage to the organism, and the calibration model of the 4-MPBA and 4MBN double signal molecules can avoid the interference of other glycoprotein macromolecules in the environment, thereby realizing the quantitative detection of CEA, and can directly detect the blood plasma to provide a basis for early screening of colon cancer and judge the development degree of the cancer.
Drawings
FIG. 1A is a comparison of Raman enhancement effect of Au @4MBN @ Ag NPs carboxyl functionalization with different amounts of HS-PEG-COOH added;
FIG. 1B is a comparison of Raman enhancement effect of Au @4MBN @ Ag NPs modified with carboxyl functional by adding different amounts of CEA antibody;
FIG. 2 is a graph showing UV-Vis spectra, zeta potential and DLS spectra (FIG. 2A, 2B, 2C) of the probe at different stages in the preparation of Au @4MBN @ Ag NPs-COOH-CEA antibody probe in example 2;
FIG. 3 is a comparison of contact angle test results for substrates prepared under different operating conditions;
FIG. 4 is a comparison of Raman enhancement effect and uniformity of a two-dimensional silver plate prepared by setting different modification times in the process of surface modification of the two-dimensional silver plate by 4-MPBA;
FIG. 5A is a scanning electron micrograph of a two-dimensional silver plate before CEA is added, FIG. 5B is a scanning electron micrograph of a two-dimensional silver plate after CEA is added, FIG. 5C is a SERS spectrogram obtained by detecting CEA standards with different concentrations by using the sandwich-type immunoassay method of example 2, and FIG. 5D is a working curve with concentration logarithm as abscissa plotted according to the SERS spectrogram of FIG. 5C;
FIG. 6A is a SERS spectrum obtained by detecting CEA, TYR, TNF-a, PSA, IDO, and BSA as materials respectively to evaluate the CEA selectivity of the detection method of example 2, and FIG. 6B is a SERS spectrum of 2226cm for different materials in FIG. 6A -1 Comparing the intensities of characteristic peaks;
FIG. 7 is a flow chart of the detection according to the present invention.
Detailed Description
The invention will be further described with reference to preferred embodiments.
The two-dimensional silver plate is a glass sheet with silver nano particles deposited on the surface by utilizing a silane coupling agent; APTES is 3-aminopropyl triethoxysilane; ag NPs are nano silver spheres; 4-MPBA is 4-mercaptobenzoic acid; au @4MBN @ Ag NPs are silver-coated gold core-shell structure nanoparticles taking 4MBN as an internal standard; SH-PEG-COOH is sulfhydryl-polyethylene glycol-carboxyl; EDC is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide; NHS for N-hydroxy succinimide; CEA is carcinoembryonic antigen; BSA is bovine serum albumin; 4MBN is 4-mercaptobenzonitrile.
Example 1
A method for detecting biomarkers for colon cancer using surface enhanced raman spectroscopy, comprising the steps of:
1. preparation of 4-MPBA modified two-dimensional silver plate
Firstly 35mg AgNO 3 Adding 200mL of water, heating to boiling, adding 10mL of 1% trisodium citrate, stirring for reacting for 1 hour to obtain a gray-green Ag NRs solution, centrifuging the obtained solution at 12000rpm for 10min, washing twice, and dispersing with ultrapure water for later use. And then placing the cut glass pieces (5 mm multiplied by 1 mm) in absolute ethyl alcohol for ultrasonic treatment for 15min, drying the glass pieces, reacting the dried glass pieces with the piranha solution for 50 min at the temperature of 90 ℃, and then repeatedly washing the glass pieces by using ethyl alcohol and water to remove the redundant piranha solution. Immersing the reacted glass sheet in 15% APTES solution, reacting at room temperature for 24 hr, taking out the glass sheet, washing with ethanol and water repeatedly, placing in an oven, condensing at 120 deg.C for 40min, immersing the glass sheet in 1mL Ag NPs solution, standing for 24 hr, taking out, washing with ultrapure water to remove excessive Ag NPs to obtain two-dimensional silver plate, and soaking the two-dimensional silver plate in 10% APTES solution -6 And soaking the mixture in a 4-MPBA solution of M for 24 hours to modify the 4-MPBA on a two-dimensional silver plate.
2. Preparation of Au @4MBN @ Ag NPs-COOH-CEA antibody Probe
First, 2mL 1wt% sodium citrate (Na 3 Ct) was added rapidly to boiling 100mL 0.01wt% HAuCl 4 And stirring vigorously for 15min to generate Au nuclei. Then 100. Mu.L of 0.1M 4-nitrilothiophenol (4 MBN) was added to form an Au-S covalent bond, the mixture was magnetically stirred for 30min, and the mixture was centrifuged at 9500rpm for 12min to remove the impuritiesThe 4MBN was separated and then redispersed in 10mL of ultrapure water to give Au-4MBN colloid, followed by dropwise addition of 1mL of 1mM AgNO 3 And 400. Mu.L of 10mM AA to 10mL of Au-4MBN colloid, stirred for 60min and provided Au @4MBN @ Ag NPs, centrifuged at 8600rpm for 10min and redispersed in 10mL of ultrapure water for use. Then, 1mL of Au @4MBN @ Ag NPs was added to 5. Mu.L of 10 -4 And (3) stirring and reacting SH-PEG-COOH solution of M for 30min. Followed by centrifugation at 10000rpm for 10 minutes, and 20. Mu.L of 10mg/mL1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 10. Mu.L of 10mg/mL N-hydroxysuccinimide (NHS) solution were added to activate the carboxyl group, respectively. Excess NHS and EDC were then separated from the activated Au @4MBN @ Au NPs, centrifuged at 10000rpm for 10min, and then re-dispersed in PBS (10mM, pH 7.4). The above solution was added to 50. Mu.L of CEA antibody solution, and after shaking continuously at room temperature for 2 hours, the mixture was transferred to 4 ℃ and incubated for 24 hours, centrifuged at 4000rpm for 8min to remove excess antibody, and redispersed in PBS for further use.
3. Raman detection
After fully contacting the 4-MPBA modified two-dimensional silver plate with the prepared CEA standard sample or CEA-containing plasma for 3h, washing the plate with PBS for 2 times, adding 50 μ L of 0.2 BSA solution for reaction for 30min, then adding Au @4MBN @ Ag NPs-COOH-CEA antibody probe for co-incubation for 2h, then washing the two-dimensional silver plate with PBS for 2 times again to remove SERS probes not bound with CEA, and after waiting for natural drying, performing SERS test under the conditions of 785nm laser, 2.38mW power and 10s integration time.
Example 2
A method for detecting biomarkers of colon cancer using surface enhanced raman spectroscopy, comprising the steps of:
1. preparation of 4-MPBA modified two-dimensional silver plate
Firstly 35mg AgNO 3 Adding into 200mL of water, heating to boil, adding 10mL of 1% trisodium citrate, stirring and reacting for 1 hour to obtain a grayish green Ag NRs solution, centrifuging the obtained solution at 12000rpm for 10min, washing twice, and dispersing with ultrapure water for later use. Placing the cut glass pieces (5 mm × 5mm × 1 mm) in anhydrous ethanol, performing ultrasonic treatment for 15min, oven drying, and reacting with piranha solution at 90 deg.C for 50 minAfter that, the glass slides were repeatedly rinsed with ethanol and water to remove excess piranha solution. Immersing the reacted glass pieces in 15% APTES solution, reacting at room temperature for 24 hours, taking out the glass pieces, washing with ethanol and water repeatedly, placing in an oven, condensing at 120 deg.C for 40min, immersing the glass pieces in 1mL of Ag NPs solution, standing for 24 hours, taking out, washing with ultrapure water to remove excess Ag NPs, obtaining a two-dimensional silver plate, and immersing the two-dimensional silver plate in 10% APTES solution -6 And soaking the mixture in a 4-MPBA solution of M for 24 hours to modify the 4-MPBA on a two-dimensional silver plate.
2. Preparation of Au @4MBN @ Ag NPs-COOH-CEA antibody probe
First, 2mL 1wt% sodium citrate (Na 3 Ct) was quickly added to boiling 100mL 0.01wt% HAuCl 4 And stirring vigorously for 15min to generate Au nuclei. Then, 100. Mu.L of 0.1M 4-nitrilothiophenol (4 MBN) was added to form an Au-S covalent bond, magnetic stirring was carried out for 30min, the mixture was centrifuged at 9500rpm for 12min to remove free 4MBN, and then redispersed in 10mL of ultra-pure water to obtain Au-4MBN colloid, followed by dropwise addition of 1mL of 1mM AgNO 3 And 400. Mu.L of 10mM AA to 10mL of Au-4MBN colloid, stirred for 60min and provided Au @4MBN @ Ag NPs, centrifuged at 8600rpm for 10min and redispersed in 10mL of ultrapure water for use. Then, 1mL Au @4MBN @ Ag NPs was added to 10. Mu.L 10 -4 M SH-PEG-COOH solution, stirring and reacting for 30min. Followed by centrifugation at 10000rpm for 10 minutes, and 20. Mu.L of 10mg/mL1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 10. Mu.L of 10mg/mL N-hydroxysuccinimide (NHS) solution were added to activate the carboxyl group, respectively. Excess NHS and EDC were then separated from the activated Au @4MBN @ Au NPs, centrifuged at 10000rpm for 10min, and then re-dispersed in PBS (10mM, pH 7.4). Adding 50 μ L CEA antibody solution into the above solution, shaking at room temperature for 2 hr, transferring to 4 deg.C, incubating for 24 hr, centrifuging at 4000rpm for 8min, removing excess antibody, and dispersing with PBS for further use.
3. Raman detection
After fully contacting the 4-MPBA modified two-dimensional silver plate with the prepared CEA standard sample or CEA-containing plasma for 3h, washing the plate with PBS for 2 times, adding 50 μ L of 0.2 BSA solution for reaction for 30min, then adding Au @4MBN @ Ag NPs-COOH-CEA antibody probe for co-incubation for 2h, then washing the two-dimensional silver plate with PBS for 2 times again to remove SERS probes not bound with CEA, and after waiting for natural drying, performing SERS test under the conditions of 785nm laser, 2.38mW power and 10s integration time.
Example 3
A method for detecting biomarkers for colon cancer using surface enhanced raman spectroscopy, comprising the steps of:
1. preparation of 4-MPBA modified two-dimensional silver plate
Firstly 35mg AgNO 3 Adding 200mL of water, heating to boiling, adding 10mL of 1% trisodium citrate, stirring for reacting for 1 hour to obtain a gray-green Ag NRs solution, centrifuging the obtained solution at 12000rpm for 10min, washing twice, and dispersing with ultrapure water for later use. And then placing the cut glass pieces (5 mm multiplied by 1 mm) in absolute ethyl alcohol for ultrasonic treatment for 15min, drying the glass pieces, reacting the dried glass pieces with the piranha solution for 50 min at 90 ℃, and then repeatedly washing the glass pieces by using ethyl alcohol and water to remove the redundant piranha solution. Immersing the reacted glass sheet in 15% APTES solution, reacting at room temperature for 24 hr, taking out the glass sheet, washing with ethanol and water repeatedly, placing in an oven, condensing at 120 deg.C for 40min, immersing the glass sheet in 1mL Ag NPs solution, standing for 24 hr, taking out, washing with ultrapure water to remove excessive Ag NPs to obtain two-dimensional silver plate, and soaking the two-dimensional silver plate in 10% APTES solution -6 And soaking the mixture in a 4-MPBA solution of M for 24 hours to modify the 4-MPBA on a two-dimensional silver plate.
2. Preparation of Au @4MBN @ Ag NPs-COOH-CEA antibody Probe
First, 2mL 1wt% sodium citrate (Na 3 Ct) was added rapidly to boiling 100mL 0.01wt% HAuCl 4 And stirring vigorously for 15min to generate Au nuclei. Then, 100. Mu.L of 0.1M 4-nitrilothiophenol (4 MBN) was added to form an Au-S covalent bond, magnetic stirring was carried out for 30min, the mixture was centrifuged at 9500rpm for 12min to remove free 4MBN, and then re-dispersed in 10mL of ultrapure water to obtain Au-4MBN colloid, followed by dropwise addition of 1mL of 1mM AgNO 3 And 400. Mu.L of 10mM AA to 10mL of Au-4MBN colloid, stirred for 60min and provided Au @4MBN @ Ag NPs, centrifuged at 8600rpm for 10min and redispersed in 10mL of ultrapure water for use. Then will be1mL Au @4MBN @ Ag NPs was added 20. Mu.L of 10 -4 And (3) stirring and reacting SH-PEG-COOH solution of M for 30min. Followed by centrifugation at 10000rpm for 10 minutes, and 20. Mu.L of 10mg/mL1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 10. Mu.L of 10mg/mL N-hydroxysuccinimide (NHS) solution were added to activate the carboxyl group, respectively. Excess NHS and EDC were then separated from the activated Au @4MBN @ Au NPs, centrifuged at 10000rpm for 10min, and then re-dispersed in PBS (10mM, pH 7.4). Adding 50 μ L CEA antibody solution into the above solution, shaking at room temperature for 2 hr, transferring to 4 deg.C, incubating for 24 hr, centrifuging at 4000rpm for 8min, removing excess antibody, and dispersing with PBS for further use.
3. Raman detection
After fully contacting the 4-MPBA modified two-dimensional silver plate with the prepared CEA standard sample or CEA-containing plasma for 3h, washing the plate with PBS for 2 times, adding 50 μ L of 0.2 BSA solution for reaction for 30min, then adding Au @4MBN @ Ag NPs-COOH-CEA antibody probe for co-incubation for 2h, then washing the two-dimensional silver plate with PBS for 2 times again to remove SERS probes not bound with CEA, and after waiting for natural drying, performing SERS test under the conditions of 785nm laser, 2.38mW power and 10s integration time.
Example 4
A method for detecting biomarkers for colon cancer using surface enhanced raman spectroscopy, comprising the steps of:
1. preparation of 4-MPBA modified two-dimensional silver plate
Firstly 35mg AgNO 3 Adding into 200mL of water, heating to boil, adding 10mL of 1% trisodium citrate, stirring and reacting for 1 hour to obtain a grayish green Ag NRs solution, centrifuging the obtained solution at 12000rpm for 10min, washing twice, and dispersing with ultrapure water for later use. And then placing the cut glass pieces (5 mm multiplied by 1 mm) in absolute ethyl alcohol for ultrasonic treatment for 15min, drying the glass pieces, reacting the dried glass pieces with the piranha solution for 50 min at 90 ℃, and then repeatedly washing the glass pieces by using ethyl alcohol and water to remove the redundant piranha solution. Immersing the reacted glass sheet in 15% APTES solution, reacting at room temperature for 24 hr, taking out the glass sheet, washing with ethanol and water repeatedly, placing in an oven, and shrinking at 120 deg.CAfter the synthetic reaction is carried out for 40min, the glass sheet is immersed in 1mL of Ag NPs solution and placed for 24h, then the glass sheet is taken out and washed by ultrapure water to remove redundant Ag NPs, a two-dimensional silver plate is obtained, and the two-dimensional silver plate is immersed in 10 -6 And soaking the mixture in a 4-MPBA solution of M for 24 hours to modify the 4-MPBA on a two-dimensional silver plate.
2. Preparation of Au @4MBN @ Ag NPs-COOH-CEA antibody Probe
First, 2mL 1wt% sodium citrate (Na 3 Ct) was added rapidly to boiling 100mL 0.01wt% HAuCl 4 And stirring vigorously for 15min to generate Au nuclei. Then, 100. Mu.L of 0.1M 4-nitrilothiophenol (4 MBN) was added to form an Au-S covalent bond, magnetic stirring was carried out for 30min, the mixture was centrifuged at 9500rpm for 12min to remove free 4MBN, and then redispersed in 10mL of ultra-pure water to obtain Au-4MBN colloid, followed by dropwise addition of 1mL of 1mM AgNO 3 And 400. Mu.L of 10mM AA to 10mL of Au-4MBN colloid, stirred for 60min and provided Au @4MBN @ Ag NPs, centrifuged at 8600rpm for 10min and redispersed in 10mL of ultrapure water for use. Then, 1mL Au @4MBN @ Ag NPs was added to 30. Mu.L 10 -4 M SH-PEG-COOH solution, stirring and reacting for 30min. Followed by centrifugation at 10000rpm for 10 minutes, and then activation of the carboxyl group by the addition of 20. Mu.L of 10mg/mL1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 10. Mu.L of 10mg/mL N-hydroxysuccinimide (NHS) solution, respectively. Excess NHS and EDC were then separated from activated Au @4MBN @ Au NPs, centrifuged at 10000rpm for 10 minutes, and then re-dispersed in PBS (10mM, pH 7.4). The above solution was added to 50. Mu.L of CEA antibody solution, and after shaking continuously at room temperature for 2 hours, the mixture was transferred to 4 ℃ and incubated for 24 hours, centrifuged at 4000rpm for 8min to remove excess antibody, and redispersed in PBS for further use.
3. Raman detection
After fully contacting the 4-MPBA modified two-dimensional silver plate with the prepared CEA standard sample or CEA-containing plasma for 3h, washing the plate with PBS for 2 times, adding 50 μ L of 0.2 BSA solution for reaction for 30min, then adding Au @4MBN @ Ag NPs-COOH-CEA antibody probe for co-incubation for 2h, then washing the two-dimensional silver plate with PBS for 2 times again to remove SERS probes not bound with CEA, and after waiting for natural drying, performing SERS test under the conditions of 785nm laser, 2.38mW power and 10s integration time.
Example 5
A method for detecting biomarkers for colon cancer using surface enhanced raman spectroscopy, comprising the steps of:
1. preparation of 4-MPBA modified two-dimensional silver plate
Firstly 35mg AgNO 3 Adding 200mL of water, heating to boiling, adding 10mL of 1% trisodium citrate, stirring for reacting for 1 hour to obtain a gray-green Ag NRs solution, centrifuging the obtained solution at 12000rpm for 10min, washing twice, and dispersing with ultrapure water for later use. And then placing the cut glass pieces (5 mm multiplied by 1 mm) in absolute ethyl alcohol for ultrasonic treatment for 15min, drying the glass pieces, reacting the dried glass pieces with the piranha solution for 50 min at 90 ℃, and then repeatedly washing the glass pieces by using ethyl alcohol and water to remove the redundant piranha solution. Immersing the reacted glass sheet in 15% APTES solution, reacting at room temperature for 24 hr, taking out the glass sheet, washing with ethanol and water repeatedly, placing in an oven, condensing at 120 deg.C for 40min, immersing the glass sheet in 1mL Ag NPs solution, standing for 24 hr, taking out, washing with ultrapure water to remove excessive Ag NPs to obtain two-dimensional silver plate, and soaking the two-dimensional silver plate in 10% APTES solution -6 And soaking the mixture in a 4-MPBA solution of M for 24 hours to modify the 4-MPBA on a two-dimensional silver plate.
2. Preparation of Au @4MBN @ Ag NPs-COOH-CEA antibody probe
First, 2mL 1wt% sodium citrate (Na 3 Ct) was added rapidly to boiling 100mL 0.01wt% HAuCl 4 The Au core is generated after 15min of moderate and violent stirring. Then, 100. Mu.L of 0.1M 4-nitrilothiophenol (4 MBN) was added to form an Au-S covalent bond, magnetic stirring was carried out for 30min, the mixture was centrifuged at 9500rpm for 12min to remove free 4MBN, and then re-dispersed in 10mL of ultrapure water to obtain Au-4MBN colloid, followed by dropwise addition of 1mL of 1mM AgNO 3 And 400. Mu.L of 10mM AA to 10mL of Au-4MBN colloid, stirred for 60min and provided Au @4MBN @ Ag NPs, centrifuged at 8600rpm for 10min and redispersed in 10mL of ultrapure water for use. Then adding 1mL Au @4MBN @ Ag NPs into 40 μ L10 μ L -4 And (3) stirring and reacting SH-PEG-COOH solution of M for 30min. Followed by centrifugation at 10000rpm for 10 minutes, and then 20. Mu.L of 10mg/mL1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 10. Mu.L of 10mg/mL N-hydroxysuccinimide (NHS) solution were added to activate the carboxyl group. Excess NHS and EDC were then separated from activated Au @4MBN @ Au NPs, centrifuged at 10000rpm for 10 minutes, and then re-dispersed in PBS (10mM, pH 7.4). The above solution was added to 50. Mu.L of CEA antibody solution, and after shaking continuously at room temperature for 2 hours, the mixture was transferred to 4 ℃ and incubated for 24 hours, centrifuged at 4000rpm for 8min to remove excess antibody, and redispersed in PBS for further use.
3. Raman detection
After fully contacting the 4-MPBA modified two-dimensional silver plate with the prepared CEA standard sample or CEA-containing plasma for 3h, washing the plate with PBS for 2 times, adding 50 μ L of 0.2 BSA solution for reaction for 30min, then adding Au @4MBN @ Ag NPs-COOH-CEA antibody probe for co-incubation for 2h, then washing the two-dimensional silver plate with PBS for 2 times again to remove SERS probes not bound with CEA, and after waiting for natural drying, performing SERS test under the conditions of 785nm laser, 2.38mW power and 10s integration time.
The Au @4MBN @ Ag NPs-COOH-CEA antibody probes prepared by reacting the same concentration of different volumes of HS-PEG-COOH solution with 1mL Au @4MBN @ Ag NPs according to the above examples 1-5 were compared as shown in FIG. 1.
As can be seen from FIG. 1A, the Raman signal intensity of 4MBN reaches a maximum value when the addition amount of HS-PEG-COOH is 10. Mu.L, and then the Raman signal intensity drops sharply as the volume of the HS-PEG-COOH solution increases. This indicates that when the HS-PEG-COOH chain density is too low, the stability of Au @4MBN @ Ag NPs in solution cannot be maintained. In contrast, as HS-PEG-COOH was increased, the HS-PEG-COOH chain density on the surface of Au @4MBN @ Ag NPs was increased, but due to the excess volume of HS-PEG-COOH, the excess PEG coating on the surface of Au @4MBN @ Ag NPs might lead to a decrease in Raman intensity of the internal reporter. Therefore, for best analytical performance, the optimum amount of HS-PEG-COOH was 10. Mu.L.
Since the amount of CEA antibody affects the antigen-antibody coupling efficiency, we examined the modification effect of the appropriate amount of CEA antibody on carboxyl-functionalized Au @4MBN @ Ag NPs.
FIG. 1B shows that the best SERS response was obtained when the CEA antibody was 20 μ L in volume. Less antibody addition may result in non-specific binding, while too much CEA antibody accumulates on the surface of carboxylated Au @4MBN @ Ag NPs, possibly destroying the stability of Au @4MBN @ Ag NPs, e.g., 50-70. Mu.L of CEA antibody. Therefore, for optimal assay performance, the optimal parameter for CEA antibody is 50 μ L.
Whether the surface of Au @4MBN @ Ag NPs is successfully modified with carboxyl and then coupled with CEA antibody is a key step of the proposed CEA detection scheme. The synthesis of the nanoparticles is illustrated in fig. 2. The analysis of UV-Vis spectra (FIG. 2A), zeta potential (FIG. 2B) and DLS (FIG. 2C) confirmed that Au @4MBN @ Ag NPs have successfully attached HS-PEG-COOH and successfully modified the CEA antibody.
As shown in FIG. 2B, the net charge on the surface of the particles changed after modification of negatively charged-COOH with Au @4MBN @ Ag NPs, the zeta potential value was changed from-28.14 mV to-34.76 mV, which was-21.52 mV after coupling to CEA antibody.
Contact angle is a simple and commonly used method to characterize the chemical or physical properties of a modified interface. During the preparation of the two-dimensional silver plate, the contact angle measurement results are related to the modification of different functional groups on the surface of the glass. As shown in fig. 3, generally, the introduction of hydrophilic or hydrophobic groups significantly changes the contact angle of the glass surface. According to the experimental results, the contact angle of the original glass sheet is 33.25 degrees, the contact angle of the hydroxyl modified glass sheet is 15.90 degrees, the contact angle of the amino modified glass sheet is 83.95 degrees and the contact angle of the silver plate is 69.20 degrees in different steps, which indicates that the relative hydrophobicity of the glass surface is in the order of amino > silver plate > original glass sheet > hydroxyl. These results are consistent with the published reports. The successful preparation of the two-dimensional silver plate is proved.
FIG. 4 is a graph showing that in order to investigate the saturation of 4-MPBA on the surface of a two-dimensional silver plate, when SERS is used to scan a selected area of the two-dimensional silver plate prepared at different modification times (1, 3,8, 12, 24, 48 h) of 4-MPBA on a silver film, the Raman characteristic peak intensity of 4-MPBA at 999cm-1 increases first with the increase of the modification time, and the intensity reaches the maximum and then decreases after 24 hours. In addition to the enhancement effect, the degree of uniformity of the distribution of the two-dimensional silver substrate at different modification times was also observed. We can see that RSD gradually decreases with increasing modification time. The minimum was reached at 24 hours with RSD of 9.62%, evenly distributed, and then RSD increased. These results demonstrate that the two-dimensional silver substrate reaches saturation state when 4-MPBA is modified for 24 hours.
FIG. 5 is a scanning electron micrograph before and after CEA addition in the detection step, and FIG. 5A is a scanning electron micrograph of a two-dimensional silver plate, and it can be seen that the silver nanoparticles on the prepared two-dimensional silver plate are substantially in a monomolecular distribution. FIG. 5B shows that when CEA is finally added, many nanoparticles larger than the size of the silver colloid are added on the surface compared with the initial two-dimensional silver plate electron microscope image, because the CEA antibody on the Au @4MBN @ Ag-COOH-Ab probe is combined with the CEA antigen and adsorbed on the two-dimensional silver plate. The method can specifically identify the CEA from scanning electron micrographs presented before and after the CEA is added. FIG. 5C is a SERS spectrum of CEA standard sample detected at different concentrations (10-8, 10-9, 10-10, 10-11, 10-12, 10-13, 10-14M) by the sandwich immunoassay method. The spectrum was calibrated using the characteristic peak of 4-MPBA 999cm-1 on a two-dimensional silver plate as a standard peak. We can observe that the SERS signal of 2226cm-1 is stronger with increasing CEA concentration. This indicates that the more CEA is added, the more CEA probes are adsorbed on the two-dimensional silver plate. FIG. 5D is a working curve with the logarithm of concentration as the abscissa, after calibration of the 999cm-1 peak. The linear equation is y =0.45185x-0.122044, R2 is 0.98234, the linearity is good, and the working curve can be used as the basis for detecting CEA in real samples.
To evaluate the selectivity of this method, TYR, TNF-a, PSA, IDO, BSA were used as materials and the selectivity of this sensor for CEA detection was evaluated. As shown in FIG. 6, there was no significant change in SERS intensity at 2226cm-1 for the same concentration of 10-10M of TYR, TNF-a, PSA, IDO, BSA. Whereas the SERS signal is stronger in the presence of CEA. Indicating that the capture antibody is selective for CEA.
To verify the utility of the invention, 1ml of plasma samples were taken and different amounts (10-8M, 10-9M, 10-11M) of CEA were added and SERS measurements were performed using the detection procedure of example 2. The obtained working curve is substituted into the measured result and the measured result is calculated to obtain the table 1, the recovery rate is 105.33% -127.00%, and the recovery rate is in a normal interval, which shows that the method provided by the invention has sensitivity, reliability and practical application value.
Watch (CN)
1 recovery of CEA in plasma samples
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A method for detecting biomarkers of colon cancer by surface enhanced Raman spectroscopy, comprising the steps of: fully contacting a colon cancer biomarker CEA standard solution with a two-dimensional silver plate modified by 4-MPBA, adding Au @4MBN @ Ag NPs modified by CEA antibody functionalization as a SERS probe of CEA for incubation, finally washing the two-dimensional silver plate by PBS, and performing SERS detection after the two-dimensional silver plate is naturally dried.
2. The method for detecting biomarkers of colon cancer by surface enhanced Raman spectroscopy according to claim 1, comprising the steps of:
(1) Preparing a 4-MPBA modified two-dimensional silver plate;
(2) Preparing an Au @4MBN @ Ag NPs-COOH-CEA antibody probe;
(3) Fully contacting a 4-MPBA modified two-dimensional silver plate with a prepared CEA standard sample, washing with PBS, adding BSA solution, reacting for 30min, adding Au @4MBN @ Ag NPs-COOH-CEA antibody probe, incubating together, washing the two-dimensional silver plate with PBS again to remove SERS probes which are not combined with CEA, waiting for natural drying, performing SERS test, and obtaining a working curve;
(4) And (3) detecting a plasma sample, namely fully contacting the 4-MPBA modified two-dimensional silver plate with the plasma sample containing CEA, washing the sample by PBS, adding BSA solution to react for 30min, then adding Au @4MBN @ Ag NPs-COOH-CEA antibody probe to incubate together, washing the two-dimensional silver plate by PBS again to remove the SERS probe which is not combined with the CEA, waiting for natural drying, carrying out an SERS test, and substituting the corresponding characteristic peak intensity into the working curve obtained in the step (3) to calculate the CEA content in the plasma sample.
3. The method for detecting the biomarkers of the colon cancer by using the surface enhanced Raman spectroscopy as claimed in claim 2, wherein the step (1) comprises the following specific steps: preparing a "piranha" solution according to the ratio of V (H2 SO 4) to V (H2O 2) =7, ultrasonically cleaning and drying glass sheets, reacting the glass sheets with the piranha "solution at 90 ℃ for 50 minutes, repeatedly washing the glass sheets by using ethanol and water, and removing the redundant piranha" solution; immersing the reacted glass sheet in 15% of APTES solution, reacting at room temperature for 24 hours, taking out the glass sheet, repeatedly washing the glass sheet with ethanol and water, placing the glass sheet in an oven, performing condensation reaction at 120 ℃ for 40 minutes, immersing the glass sheet in 1mL of Ag NPs solution, standing the glass sheet for 24 hours, taking out the glass sheet, washing the glass sheet with ultrapure water to remove redundant Ag NPs, obtaining a two-dimensional silver plate, immersing the two-dimensional silver plate in 4-MPBA solution for a specified time, and modifying the 4-MPBA on the two-dimensional silver plate.
4. The method for detecting the colon cancer biomarkers by using surface enhanced Raman spectroscopy according to claim 2 or 3, wherein the step (2) comprises the following specific steps: adding 1mL Au @4MBN @ Ag NPs into the SH-PEG-COOH solution, stirring for reaction for 30min, performing primary centrifugation, adding 20 mu L10mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 10 mu L10mg/mL N-hydroxysuccinimide solution to activate carboxyl, performing secondary centrifugation, redispersing in PBS, adding the CEA antibody, continuously shaking for 2 hours at room temperature, transferring to 4 ℃, incubating for 24 hours, and performing centrifugal redispersion to obtain the Au @4MBN @ Ag NPs-COOH-CEA antibody solution.
5. The method of claim 4, wherein the biomarker of colon cancer is detected by surface enhanced Raman spectroscopyCharacterized in that the Ag NRs synthesis steps are as follows: 35mg of AgNO 3 Adding 200mL of water, heating to boiling, adding 1% trisodium citrate 10mL, stirring and reacting for 1 hour to obtain a grayish green Ag NRs solution, centrifuging the obtained solution, washing twice, and dispersing with ultrapure water for later use.
6. The method of claim 5, wherein the biomarker for colon cancer is detected by Surface Enhanced Raman Spectroscopy (SERS), wherein the method comprises: the synthesis method of the Au @4MBN @ Ag NPs comprises the following steps: adding sodium citrate to the boiling HAuCl 4 Stirring vigorously for 15min to generate gold core; adding 4-cyano thiophenol to form Au-S covalent bond, magnetically stirring for 30min, centrifuging, re-dispersing, and dropwise adding AgNO 3 The solution and ascorbic acid are stirred for 60min and centrifuged to obtain Au @4MBN @ Ag NPs.
7. The method of claim 6, wherein the biomarker for colon cancer is detected by Surface Enhanced Raman Spectroscopy (SERS), wherein the method comprises: the first centrifugation is carried out at the speed of 10000rpm for 10 minutes, and the second centrifugation is carried out at the speed of 4000rpm for 8 minutes; the centrifugation of claim 5 at 12000rpm for 10 minutes; the first centrifugation of claim 6 at 9500rpm for 12 minutes and the second centrifugation at 8600rpm for 10 minutes.
8. The method for detecting biomarkers of colon cancer using surface enhanced raman spectroscopy according to claim 7, wherein: the optimal dosage of the HS-PEG-COOH is 10 mu L, and the optimal parameter of the CEA antibody is 50 mu L.
9. The method of claim 8, wherein the biomarker for colon cancer is detected by Surface Enhanced Raman Spectroscopy (SERS), wherein the method comprises: the concentration of the 4-MPBA solution is 10 -6 M, modification time 24 hours.
10. The method for detecting biomarkers of colon cancer using surface enhanced raman spectroscopy according to claim 9, wherein: in the steps (3) and (4), the CEA standard sample or the plasma is contacted with the two-dimensional silver plate for 3 hours, the adding amount of the BSA solution is 50 mu L, the concentration is 0.2%, the co-incubation temperature is 37 ℃, and the time is 2 hours.
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| CN104697977A (en) * | 2015-03-23 | 2015-06-10 | 苏州大学 | Silicon-based SERS multifunctional chip and preparation method thereof |
| WO2017066537A1 (en) * | 2015-10-16 | 2017-04-20 | The Johns Hopkins University | Multiplexed detection of circulating tumor antigens and epigenetic markers using plasmon-enhanced raman spectroscopic assays |
| CN112505017A (en) * | 2020-11-19 | 2021-03-16 | 福建师范大学 | Method for detecting IL-6 in blood based on SERS technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104697977A (en) * | 2015-03-23 | 2015-06-10 | 苏州大学 | Silicon-based SERS multifunctional chip and preparation method thereof |
| WO2017066537A1 (en) * | 2015-10-16 | 2017-04-20 | The Johns Hopkins University | Multiplexed detection of circulating tumor antigens and epigenetic markers using plasmon-enhanced raman spectroscopic assays |
| CN112505017A (en) * | 2020-11-19 | 2021-03-16 | 福建师范大学 | Method for detecting IL-6 in blood based on SERS technology |
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| QIONG WU 等: ""A target-triggered and self-calibration aptasensor based on SERS for precise detection of a prostate cancer biomarker in human blood"" * |
| TING ZHOU 等: ""Hypersensitive detection of IL-6 on SERS substrate calibrated by dual model"" * |
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