CN110618093B - Application of nano probe complex system in tumor marker detection kit - Google Patents
Application of nano probe complex system in tumor marker detection kit Download PDFInfo
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- CN110618093B CN110618093B CN201910758392.0A CN201910758392A CN110618093B CN 110618093 B CN110618093 B CN 110618093B CN 201910758392 A CN201910758392 A CN 201910758392A CN 110618093 B CN110618093 B CN 110618093B
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Abstract
The invention discloses an application of a nano probe comprehensive system in a tumor marker detection kit, wherein the nano probe comprehensive system comprises a glucose oxidase marked ELISA system, a silicon dioxide coated silver coated gold nanorod combined with the glucose oxidase marked ELISA system, and a silver ion fluorescent probe. The invention applies the nano probe comprehensive system to the tumor marker detection kit, and can simultaneously realize the colorimetric/fluorescent/photoacoustic three-output detection of the tumor marker. Furthermore, the detection system has a two-step amplification design: firstly, a large amount of glucose oxidase molecules are loaded on the surface of magnetic beads; second H produced by enzyme catalysis2O2Etching the silver layer to form a large amount of Ag+Therefore, the detection sensitivity of the probe comprehensive system to the target analyte is ensured, and the method has good application prospect in the field of tumor marker detection.
Description
Technical Field
The invention relates to the field of nano material medicine application, in particular to application of a nano probe complex in a tumor marker detection kit.
Background
Tumor markers are chemical substances which reflect the existence of tumors, are mainly present in blood, urine or tumor tissues, and the existence or content change of the chemical substances can indicate the properties of the tumors so as to help the diagnosis, classification, prognosis judgment and curative effect observation of the tumors. Therefore, the detection of tumor markers is crucial for the early diagnosis of cancer. The common clinical detection means at present are: radioimmunoassay, chemiluminescence immunoassay, Enzyme-linked immunosorbent assay (ELISA), liquid biopsy, protein chip technology, and the like. Among them, ELISA has the advantages of less sample demand, low analysis cost, simple operation, high sensitivity, good selectivity and the like, so that it becomes the most widely used means in clinical tumor marker detection.
With the rapid development of nanotechnology, many nanomaterials, such as gold nanomaterials, magnetic nanoparticles, quantum dots, carbon nanotubes, etc., are widely developed as detection sensors for tumor markers due to their large specific surface area, unique optical, magnetic and electrical properties. The nano materials are combined with a traditional ELISA system to establish a composite analysis system, and a new opportunity is provided for the practical application of tumor marker detection. Existing studies combine GNPs with traditional ELISA systems, where the growth of GNPs is controlled by catalase adsorbed in the ELISA system, which produces colored solutions with different colors when the analyte is present. In this reaction scheme, when the concentration of the target analyte is high, the non-aggregated colloidal suspension of GNPs grows and the solution appears red; when the concentration of the target analyte is low, the GNPs are aggregated and grow, and the solution presents blue, so that the colorimetric detection of the tumor marker with low concentration can be realized. This novel detection system can be combined with catalase as an enzyme label into any existing ELISA system to improve naked eye detection of low concentrations of analyte. The colorimetry is a method for determining the content of a component to be detected by comparing or measuring the color depth of a colored substance solution, and the method for detecting a tumor marker by the colorimetry is single and has limited application fields.
Accordingly, the prior art is yet to be improved and developed.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide the application of a nano probe complex system in a tumor marker detection kit, and aims to solve the problems of single detection method of the tumor marker and limited application field.
The technical scheme of the invention is as follows:
an application of a nano probe complex system in a tumor marker detection kit, wherein the nano probe complex system comprises a glucose oxidase-labeled ELISA system, a silica-coated silver-coated gold nanorod combined with the glucose oxidase-labeled ELISA system, and a silver ion fluorescent probe.
The application of the nano probe complex system in a tumor marker detection kit is characterized in that the glucose oxidase-labeled ELISA system comprises bovine serum albumin, a specific antibody Ab1 and magnetic beads combined with glucose oxidase and the specific antibody Ab 2.
The application of the nano probe complex system in a tumor marker detection kit is provided, wherein the diameter of the silicon dioxide coated silver-coated gold nanorod is 80-100 nm.
The application of the nano probe complex in a tumor marker detection kit is provided, wherein the silver ion fluorescent probe comprises rhodamine B.
The application of the nano probe complex system in a tumor marker detection kit is provided, wherein the tumor marker is selected from one or more of serum carcinoembryonic antigen, alpha fetoprotein and prostate specific antigen.
Has the advantages that: the invention applies the nano probe comprehensive system to the tumor markerIn the marker detection kit, the colorimetric/fluorescent/photoacoustic three-output detection of the tumor marker can be realized simultaneously. Furthermore, the detection system has a two-step amplification design: firstly, a large amount of glucose oxidase molecules are loaded on the surface of magnetic beads; second H produced by enzyme catalysis2O2Etching the silver layer to form a large amount of Ag+Therefore, the detection sensitivity of the probe comprehensive system to the target analyte is ensured, and the method has good application prospect in the field of tumor marker detection.
Drawings
FIG. 1 shows GNR @ Ag @ SiO in example 1 of the present invention2Synthetic route to nanoparticles.
FIG. 2 is the GNR @ Ag @ SiO in example 1 of the present invention2A TEM image of (a).
FIG. 3 is the GNR @ Ag @ SiO in example 1 of the present invention2UV-vis-NIR absorption spectrum of (A).
FIG. 4 is a synthesis scheme of a silver ion fluorescent probe in example 2 of the present invention.
FIG. 5 is a mass spectrometry analysis of the silver ion fluorescent probe in example 2 of the present invention.
FIG. 6 shows the hydrogen spectrum analysis of the silver ion fluorescent probe in example 2 of the present invention.
FIG. 7 is a graph showing the fluorescence change of silver ion fluorescent probes incubated with different concentrations of silver ions in example 2 of the present invention.
Fig. 8 is a schematic diagram of colorimetric/fluorescent/photoacoustic three-output detection of the nanoprobe integrated system in example 3 of the present invention.
FIG. 9 shows GNR @ Ag @ SiO in example 5 of the present invention2Color change profile of nanoparticles after incubation with different concentrations of Prostate Specific Antigen (PSA) and UV-vis-NIR absorption spectra.
FIG. 10 is a graph showing the fluorescence change of the silver ion fluorescent probe in example 6 after incubation with Prostate Specific Antigen (PSA) at different concentrations.
FIG. 11 shows GNR @ Ag @ SiO in example 7 of the present invention2Graph of photoacoustic signal changes of nanoparticles after incubation with different concentrations of Prostate Specific Antigen (PSA).
Detailed Description
The invention provides an application of a nano probe complex in a tumor marker detection kit, and the invention is further detailed below in order to make the purpose, technical scheme and effect of the invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
The embodiment of the invention provides application of a nano probe integrated system in a tumor marker detection kit, wherein the nano probe integrated system comprises a glucose oxidase-labeled ELISA system and a silica-coated silver-coated gold nanorod (GNR @ Ag @ SiO) combined with the glucose oxidase-labeled ELISA system2) And silver ion fluorescent probes.
This example first combines GNR @ Ag @ SiO2The nanoparticle, the silver ion fluorescent probe and the traditional ELISA system marked by glucose oxidase are organically combined to construct a composite detection system, the detection system can be used for fluorescence/photoacoustic/colorimetric three-output detection of tumor markers, the detection mode is diversified, the accuracy and penetration depth are higher, and the sensitivity and selectivity are good, wherein GNR @ Ag @ SiO2The preparation of the nano-particles and the silver ion fluorescent probe is simple and repeatable.
The nano probe comprehensive system provided by the embodiment is prepared by inducing a target substance to obtain GNR @ Ag @ SiO2The color of the detection system, the fluorescence intensity of the silver ion fluorescent probe and the photoacoustic signal of the detection system are changed due to the etching of the silver layer of the nano particles, so that the colorimetric/fluorescent/photoacoustic detection of the tumor marker is realized. The detection system has a two-step amplification design: firstly, a large amount of glucose oxidase (GOx) molecules are loaded on the surface of a magnetic bead; second H produced by enzyme catalysis2O2Etching the silver layer to form a large amount of Ag+. Therefore, the sensitivity of the system to the target analyte is ensured, and the method has good application prospect in the field of tumor marker detection.
In some embodiments, the glucose oxidase-labeled ELISA system comprises bovine serum albumin, specific antibody Ab1, magnetic beads that bind glucose oxidase to specific antibody Ab 2. Specifically, the preparation of the glucose oxidase labeled ELISA system comprises the following steps:
firstly, 1.0mg/mL of specific antibody Ab2 is dissolved in 100 mu L of ELISA antibody dilution; then mixed with NHS-DBCO (6.5. mu.L, 1mM) and incubated in a shaker at room temperature for 30 minutes, the reaction was stopped by adding a quenching buffer (50mM Tris-HCl, pH8), and purified by centrifugation to give Dibenzocyclooctyl (DBCO) conjugated Ab2(DBCO-Ab 2);
adding 100 μ L of GOx (30mg/mL) PBS into the EP tube containing the magnetic beads, incubating the mixture in a shaker for 2 hours, and centrifuging to collect glucose oxidase-bound magnetic beads (GOx-MB); finally, azide-modified GOx-MB was mixed with 20 μ L DBCO-Ab2 and incubated for 2 hours at room temperature; after the reaction was completed, the supernatant was magnetically aspirated and then dispersed into PBS (10mM, pH 7.4) to obtain the final glucose oxidase-labeled ELISA system.
In some embodiments, the silica-coated silver-clad gold nanorods have a diameter of 80 to 100 nm. Specifically, the preparation of the silicon dioxide coated silver-coated gold nanorod comprises the following steps:
mixing 1.5% tetraethyl orthosilicate (TEOS) solution and gold seed solution (50 μ L TEOS solution/mL GNR solution), stirring, and centrifuging to obtain silicon dioxide coated gold nanorods (GNR @ SiO)2) Redispersed to 30mL AgNO3To the ethylene glycol solution (1.2mM), 600. mu.L of an ethylene glycol solution of ethanolamine (10mM) was added, stirred for 1 hour (200rpm), washed with an equal volume of acetone, and centrifuged to obtain silica-coated silver-coated gold nanorods (GNR @ Ag @ SiO)2)。
In some embodiments, the silver-ion fluorescent probe comprises rhodamine B. Specifically, the preparation of the silver ion fluorescent probe comprises the following steps:
5g of rhodamine B (dissolved in ethanolamine) was mixed with 7.5mL of methanol, stirred at 70 ℃ for 48 hours, and then cooled to room temperature. Then adding 200mL of ethyl acetate and water for extraction, and purifying by a column to obtain a product 1;
dissolving the product 1 in 30mL of dichloromethane, gradually adding triethylamine and methylsulfonyl chloride, stirring at room temperature overnight, and drying to obtain a product 2; dissolving the product 2 in 20mL of anhydrous acetone, adding 0.456g of NaI, performing reflux reaction for 12 hours under the protection of nitrogen, and finally obtaining the final silver ion probe through rotary evaporation and recrystallization at 75 ℃.
In some embodiments, the nanoprobe integration system when applied to tumor marker detection comprises the following steps: first, Ab1 (4. mu.g/mL, 50. mu.L) dispersed in ELISA coating was added to the wells of a 96-well polystyrene plate and incubated overnight at 4 ℃. After washing the well plate three times with ELISA wash solution, 200. mu.L of 1% BSA blocking solution was added to block the well plate for 1 hour at 37 ℃. Subsequently, the plates were washed three times with ELISA wash buffer and 50 μ Ι _ of different concentrations of PSA were added to the well plates. After 1 hour incubation at 37 deg.C, the plates were washed three times with wash buffer and incubated for 1 hour at 37 deg.C with 50. mu.L of prepared Ab 2-GOx-MB. Then, 50. mu.L of 20mM glucose solution was added and incubated for 30 minutes. After the reaction is finished, the supernatant is magnetically absorbed with 50 mu L of GNR @ Ag @ SiO2Mix for 1 hour and take a photograph to record the color change of the well plate. Then, 100. mu.L of silver ion fluorescent probe (10. mu.M) was added and incubated for 2 hours, and the fluorescence spectrum thereof (excitation wavelength 530nm, emission wavelength 585nm) was detected with a microplate reader. And finally, performing photoacoustic detection on the solution in the pore plate by using a photoacoustic system.
The embodiment adopts the comprehensive system of nanoprobes to detect tumor markers, firstly carries out colorimetric detection, then adds silver ion probes to carry out fluorescence detection, and finally can use a photoacoustic system to carry out photoacoustic detection on the solution.
In some embodiments, the Ab1, tumor marker, Ab2-GOx-MB, glucose solution and GNR @ Ag @ SiO added2The volume of the silver ion fluorescent probe is 50 mu L, and the volume of the silver ion fluorescent probe is 100 mu L.
In some embodiments, the tumor marker is selected from one or more of serum carcinoembryonic antigen (CEA), Alpha Fetoprotein (AFP), and Prostate Specific Antigen (PSA), but is not limited thereto.
In some preferred embodiments, the tumor marker is PSA as prostate cancerSpecific marker of (2), for pre-proAdenocarcinomaIs up to 90% -97%, considered to be the most valuable for prostate cancerTumor markerIt is widely applied to screening, diagnosis and post-treatment monitoring of prostate cancer.
The nano probe comprehensive system prepared by the preparation method can realize colorimetric/fluorescent/photoacoustic three-output detection of Prostate Specific Antigen (PSA), so the nano probe comprehensive system has good application prospect in the field of tumor marker diagnosis. Furthermore, the detection system has a two-step amplification design: firstly, loading a large amount of GOx molecules on the surface of magnetic beads; second H produced by enzyme catalysis2O2Etching the silver layer to form a large amount of Ag+. Therefore, the sensitivity of the system to the target analyte is ensured, and meanwhile, the preparation process is simple, does not need complex and expensive equipment, and is easy to realize industrial production.
The technical solution of the present invention is further explained by the following specific examples:
example 1
Synthesis of GNR @ Ag @ SiO2Nano-particles:
FIG. 1 is a scheme for synthesizing GNR @ Ag @ SiO2The route of the nanoparticles is schematically shown as follows:
preparation of GNR: preparing gold seed solution, and mixing HAuCl4(17.16. mu.L, 0.04856M) and 616.1. mu.L of purified water were added to 2.5mL of a CTAB (0.1M) solution, stirred for 1 minute, and then 200. mu.L of ice NaBH4The (0.01M) solution was stirred vigorously for 2 minutes and incubated at 37 ℃ for two hours to obtain a gold seed solution. Subsequently, a growth solution was prepared by adding 103. mu.L of HAuCl4(0.04856M)、200μL H2SO4(0.5M)、80μL AgNO3(10mM) 10mL of CTAB (0.1M) solution was added sequentially and stirred for 1 minute. Then 80. mu.L ascorbic acid (0.1M) is added and stirred vigorously for two minutes until the solution turns yellow to be clear, then 24. mu.L gold seed solution is added and stirred for two minutes, and reaction is carried out at 37 ℃ overnight to obtain GNR solution.
Preparation of GNR @ SiO2: the resulting GNR was dispersed in the original volume of water after centrifugation (11000rpm,10 minutes) once. Adjusting pH to about 10 with 10% ammonia water, addingAdding 1.5% TEOS ethanol solution (50 μ L TEOS solution/mL GNR solution), shaking uniformly, reacting at 37 deg.C and 100rpm for 24 hr, and centrifuging once to obtain GNR @ SiO2。
Preparation of GNR @ Ag @ SiO2: resulting GNR @ SiO2Centrifuged once (10000rpm,10 min) and redispersed into an equal volume of ethanol solution. Taking 10mL of GNR @ SiO2Adding into 45mL MPS ethanol solution with 1.5% w/w, stirring for 20 hr, centrifuging with ethanol, washing with water twice, and dispersing to 30mL AgNO3(1.2mM) in ethylene glycol. Then 600. mu.L of ethanolamine (10mM) in ethylene glycol was added, stirred for 1 hour (200rpm), centrifuged once with an equal volume of acetone solution, then dispersed in ethanol and centrifuged once, and finally stored in aqueous solution.
FIG. 2 is a synthetic GNR @ Ag @ SiO2TEM image of the nanoparticles, from left to right, GNR @ SiO, respectively2And GNR @ Ag @ SiO2. As shown in TEM image, the prepared GNRs, GNR @ SiO2And GNR @ Ag @ SiO2The nano particles have uniform size and consistent appearance. FIG. 3 is a synthetic GNR @ Ag @ SiO2The UV-vis-NIR absorption spectrogram of the nano-particles is GNR @ Ag @ SiO from left to right2GNR and GNR @ SiO2. As can be seen from the UV-vis-NIR absorption spectrum, after a layer of silicon dioxide is coated on the surface of GNRs, the ultraviolet absorption spectrum is red-shifted by about 20nm, and after a silver layer is grown, the ultraviolet absorption spectrum is obviously blue-shifted, and the position of a longitudinal SPR peak is blue-shifted from 800nm to 680 nm.
Example 2
Synthesizing a silver ion fluorescent probe:
the synthetic route of the silver ion fluorescent probe is shown in figure 4. 5g of rhodamine B was dissolved in 12.5mL of ethanolamine solution, and the solution was added to a 50mL three-necked round-bottomed flask containing 7.5mL of methanol solution, stirred at 70 ℃ for 48 hours, and then cooled to room temperature. Then, 200mL of ethyl acetate and water were added for extraction, and the remaining water after extraction was removed by adding anhydrous sodium sulfate, and the solvent was removed by spin-drying. Then, the mixture is stirred with ethyl acetate: hexane: the resulting product was purified by column chromatography using an eluent of triethylamine 10:10:1 to give product 1 (C1).
0.2g C1(0.41mmol) was dissolved in 30mL of dichloromethane, and then 0.863mL of triethylamine (0.62mmol) was added to the solution and reacted in an ice bath under nitrogen protection for 5 minutes. To the solution was added dropwise 144. mu.L of methanesulfonyl chloride, and after the color turned red, the reaction was allowed to warm to room temperature and stirred overnight. After the reaction was completed, the reaction product was washed three times with water, dried over anhydrous sodium sulfate, and then the solvent was dried by rotary evaporator to obtain product 2 (C2).
Acetone was first dried over anhydrous sodium sulfate. An appropriate amount of C2 was dissolved in 20mL of anhydrous acetone, 0.456g of anhydrous NaI was added to the solution, and the mixture was refluxed for 12 hours at 65 ℃ under the protection of nitrogen. Cooled to room temperature, the solvent was spun dry on a rotary evaporator, redissolved in dichloromethane and washed with 10mL 10% sodium thiosulfate and 10mL brine, respectively. And recovering the organic layer, drying the organic layer by using anhydrous sodium sulfate, adding ethanol after the solvent is dried in a spinning mode, heating the mixture to 75 ℃, recrystallizing the mixture, drying the mixture in vacuum to obtain a target product silver ion fluorescent probe, and performing mass spectrum and nuclear magnetism characterization on the structure of the target product silver ion fluorescent probe.
FIG. 5 and FIG. 6 are the mass spectrum and nuclear magnetic resonance spectrum of silver ion fluorescent probe, respectively, and prove that a relatively pure target with a molecular weight of 596.1 is obtained successfully.
FIG. 7 shows that Ag concentrations were investigated+The experiment shows that the probe and Ag have influence on the fluorescence spectrum of the probe+After 2 hours of reaction, with Ag+The increase in concentration, the fluorescence signal of the probe solution at 585nm increased gradually. And it is to Ag+The detection limit of (2) is 0.001. mu.M, which is based on GNR @ Ag @ SiO2The ELISA system provides a certain guarantee for the sensitivity of PSA detection.
Example 3
The method for detecting the Prostate Specific Antigen (PSA) by the colorimetric/fluorescent/photoacoustic three-output of the nano probe comprehensive system comprises the following steps:
designs and prepares a silicon dioxide coated silver-coated gold nanorod (GNR @ Ag @ SiO)2) Three-layer composite nano-particles, which are combined with an ELISA system marked by Glucose oxidase (GOx) and successfully used for Prostate Specific Antigen (PSA), which is a commonly used prostate cancer organismMarker) is detected. GNR @ Ag @ SiO2The nanoparticles have almost no absorption at 680-970nm, so the photoacoustic signal in the wavelength window is very weak, and when the target is present in the system, GOx adsorbed on the Magnetic Beads (MBs) can catalyze glucose to generate hydrogen peroxide (H)2O2) The silver layer on the nano-particles can be catalyzed by enzyme to generate H2O2Etching, and remaining silicon dioxide gold-coated nano rod (GNR @ SiO)2) The absorption peak of the optical signal is red-shifted to the range of 680-970nm, and the optical signal is relatively strong, so that OFF-ON of the optical signal can be realized. At the same time, due to the etching of the silver layer, GNR @ Ag @ SiO2The color of the fluorescent probe changes from green to red, and the colorimetric detection of the tumor marker can be realized. In addition, Ag is generated after the silver layer is etched+Can make Ag+The fluorescence signal of the fluorescent probe is OFF to ON, and the fluorescence detection of the tumor marker can be realized. The sensitivity of the detection system is based on two-step amplification: firstly, a large amount of GOx molecules can be loaded on the surface of MBs; second H produced by enzyme catalysis2O2Etching the silver layer to form a large amount of Ag+See fig. 8 for design.
Example 4
Evaluation of Hydrogen peroxide on GNR @ Ag @ SiO2Effect of nanoparticles:
adding 20mM hydrogen peroxide into the solution filled with GNR @ Ag @ SiO2The solution was placed in a cuvette and the change in absorbance of the mixed solution was recorded using an ultraviolet spectrophotometer. GNR @ Ag @ SiO2In the presence of H2O2Thereafter, the UV-vis-NIR absorption spectrum is red-shifted with time and remains unchanged after 45 minutes, with the position of the longitudinal SPR peak red-shifted from 680nm to 780 nm. At the same time, the color of the solution also changes from green to red. This is subsequently based on GNR @ Ag @ SiO2The ELISA system is used for colorimetric detection of PSA and provides guarantee.
Example 5
Prostate Specific Antigen (PSA) pairs GNR @ Ag @ SiO at different concentrations2Evaluation of the effect of the color of the nanoparticles and the UV-vis-NIR absorption spectrum:
after Ab2-GOx-MB is synthesized, the application of the Ab2-GOx-MB is based on GNR @ Ag @ SiO2ELISA of (1)The system is used to detect PSA. Here, different concentrations of PSA (0-1000ng/mL) were prepared in PBS (10mM, pH 7.4) and 10mM PBS solution was set as blank. After the GOx enzyme labeling-ELISA system is established, 20mM glucose solution is added into the pore plates with PSA with different concentrations to promote the enzyme catalytic reaction to generate different amounts of H2O2. As shown in FIG. 9, orifice plates with different concentrations of PSA present, due to GNR @ Ag @ SiO2The reaction solutions differ in the degree of etching, producing a visible distinction between the solutions. In the concentration range of 0-1000ng/mL, the color of the detection system shows a trend from green to red with the increase of the PSA concentration. It can also be seen from their UV-vis-NIR absorption spectra that the higher the PSA concentration, the more red-shifted the absorption spectrum, i.e. the indication GNR @ Ag @ SiO2The more strongly the nanoparticle silver layer is etched.
Example 6
Evaluation of the effect of Prostate Specific Antigen (PSA) at different concentrations on silver ion fluorescent probes:
and adding a silver ion fluorescent probe with the final concentration of 10 mu M into each hole, incubating for two hours, and detecting the fluorescence spectrum of each sample by using a microplate reader. As shown in FIG. 10, in the range of 0-100ng/mL concentration, the fluorescence intensity increased with the increase of PSA concentration, and when the PSA concentration was increased to 100-1000ng/mL, the fluorescence intensity at 585nm was substantially unchanged. In addition, the concentration of PSA in the range of 0-10ng/mL is linear with fluorescence intensity.
Example 7
Prostate Specific Antigen (PSA) pairs GNR @ Ag @ SiO at different concentrations2Evaluation of the effect of the photoacoustic signal of the nanoparticles:
the solution in the pore plate is filled into an imaging tube to detect the photoacoustic signal, and as shown in FIG. 11, the intensity of the photoacoustic signal increases with the increase of the PSA concentration in the concentration range of 0-1000 ng/mL. And data analysis shows that the logarithm of PSA concentration and the change value of the photoacoustic signal intensity at 780nm show a good linear relationship in the range of 0.1-1000 ng/mL.
In conclusion, the invention combines GNR @ Ag @ SiO2Nanoparticles, silver ion fluorescent probe and traditional glucose oxidase-enzyme-linked immunosorbent assay (ELISA) systemOrganically combined to construct a comprehensive detection system for three-output detection of tumor markers. The detection mechanism can be briefly described as follows: GNR @ Ag @ SiO prepared by target induction2The nano-particle silver layer is etched to cause the color change of a detection system, the fluorescence intensity change of the silver ion fluorescent probe and the photoacoustic signal change of the detection system, thereby realizing the colorimetric/fluorescent/photoacoustic detection of the PSA. The detection system has good sensitivity, the detection limit of the detection system to PSA is 0.1ng/mL, and meanwhile, the PSA concentration and the fluorescence intensity are in a linear relation in the range of 0-10 ng/mL. Within the range of 0.1-1000ng/mL, the logarithm of the PSA concentration and the photoacoustic signal intensity are in a linear relationship; the comprehensive detection system can be expanded to the detection of other target objects.
The nano probe comprehensive system prepared by the preparation method can realize colorimetric/fluorescent/photoacoustic three-output detection of the tumor marker, so the nano probe comprehensive system has a good application prospect in the field of tumor marker diagnosis. Furthermore, the detection system has a two-step amplification design: firstly, loading a large amount of GOx molecules on the surface of magnetic beads; second H produced by enzyme catalysis2O2Etching the silver layer to form a large amount of Ag+. Thus ensuring the sensitivity of the system to the target analyte. Meanwhile, the preparation process is simple, complex and expensive equipment is not needed, and industrial production is easy to realize.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (7)
1. An application of a nano probe comprehensive system in preparing a tumor marker detection kit is characterized in that the nano probe comprehensive system comprises: glucose oxidase (GOx) -labeled ELISA system, and silica-coated silver-coated gold nanorod (GNR @ Ag @ SiO) combined with glucose oxidase-labeled ELISA system2) And a silver ion fluorescent probe;
the working method of the nano probe comprehensive system comprises the following steps:
separately preparing silica-coated silver-coated gold nanorods (GNR @ Ag @ SiO)2) An ELISA system marked by glucose oxidase and a silver ion fluorescent probe;
capturing a tumor marker by using the ELISA system marked by the glucose oxidase;
the tumor marker is combined with glucose oxidase and glucose to be incubated together, and after the incubation is finished, supernatant liquid and a silicon dioxide coated silver coated gold nanorod (GNR @ Ag @ SiO) are magnetically absorbed2) Mixing, and finally adding a silver ion fluorescent probe to obtain a nano probe comprehensive system for detecting the tumor marker.
2. The use according to claim 1, characterized in that the glucose oxidase-labeled ELISA system consists of Bovine Serum Albumin (BSA), specific antibody (Ab 1), Magnetic Beads (MB) binding glucose oxidase (GOx) and anti-specific antibody (Ab 2).
3. Use according to claim 1, wherein said silica-coated silver-coated gold nanorods (GNR @ Ag @ SiO @)2) The diameter is 80-100 nm.
4. The use of claim 1, wherein the silver ion probe body is rhodamine B.
5. The use of claim 1, wherein the tumor marker is detected by colorimetric/fluorescent/photoacoustic three-output detection.
6. The use according to claim 1, wherein the detection sequence is colorimetric detection first, followed by addition of a silver ion probe for fluorescence detection, and finally photoacoustic detection of the solution using a photoacoustic system.
7. The use of claim 1, wherein said tumor marker is selected from the group consisting of serum carcinoembryonic antigen (CEA), Alpha Fetoprotein (AFP), Prostate Specific Antigen (PSA).
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