CN111650379A - Preparation and immunochromatography application of gold shell magnetic Raman-enhanced nano tag - Google Patents
Preparation and immunochromatography application of gold shell magnetic Raman-enhanced nano tag Download PDFInfo
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Abstract
The invention discloses preparation of a gold shell magnetic Raman enhanced nano tag and application of immunochromatography. The gold shell magnetic Raman enhanced nano-label is based on self-assembly of cationic polymer polyethyleneimine, and adsorbs 3nm colloidal gold to form seeded Fe3O4And nanoparticles, namely generating gold-shell modified Raman signal molecules DTNB through a seed growth method and coupling the DTNB with the antibody. The invention also discloses application of the gold shell magnetic Raman-enhanced nano-label in immunochromatography detection. In a short time, the low-concentration SAA and CRP antigens can be directly and simultaneously detected in an untreated blood sample, so that the detection sensitivity is improved. The magnetic SERS tag has good dispersibility, strong magnetic responsiveness and stabilityThe kit has the advantages of high strength, good biocompatibility, difficult oxidation and easy mass preparation, and can be used for capturing, enriching and quantitatively detecting target substances in a complex sample system.
Description
Technical Field
The invention relates to the technical field of novel magnetic nano materials, in particular to preparation and immunochromatography application of a gold shell magnetic Raman enhanced nano label.
Background
Common infectious diseases are mainly caused by bacteria and viruses, have high mortality rate, and are always serious public health problems threatening all mankind. In recent years, a series of inflammation biomarkers such as C-reactive protein (CRP), Serum Amyloid A (SAA), Procalcitonin (PCT), interleukin-6 (IL-6) and the like are found to be closely related to infectious diseases, and can be used as a key index for infection diagnosis. Since the clinical manifestations of CRP and SAA are significantly different after bacterial and viral infections, blood testing in combination with SAA and CPR is expected to give satisfactory results. However, the conventional methods for diagnosing infectious diseases include blood sedimentation, pathogen culture, white blood cell counting, polymerase chain reaction, etc.; the methods have the defects of long detection time, complex sample pretreatment, expensive equipment or technicians and the like, so that the requirements of field detection cannot be met. The rapid and accurate detection of SAA and CRP is important for identifying bacterial and viral infection and guiding the selection of appropriate treatment and prognosis.
The SERS immunochromatographic assay is an immunoassay technology which combines SERS with labeling immunology, utilizes the high sensitivity and spectral selectivity of SERS and combines the specific reaction of an antibody and an antigen. The combination of the SERS marker detection technology and the immunochromatography detection technology has become a hot point of research in related fields in recent years. However, the traditional immunochromatography technology relying on gold/silver colloid as a substrate has the defects of low detection sensitivity and capability of only qualitative or semi-quantitative detection. The magnetic nano material has the advantages of good stability, low toxicity, good biocompatibility, strong magnetism, capability of avoiding centrifugation or filtration through magnetic separation, and the like, and can replace the traditional gold/silver colloid SERS label as a magnetic SERS label for immunochromatography detection.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention provides a novel magnetic composite nano label with strong magnetic responsiveness and monodispersity. The preparation of the novel gold shell magnetic Raman enhanced nano label is based on self-assembly of the Polyethyleneimine (PEI), and the preparation process is simple, efficient and repeatable. The invention also provides the application of the gold shell magnetic Raman enhancement nano-label as a magnetic SERS label in the detection of an immunochromatographic system.
In order to achieve the purpose, the invention adopts the technical scheme that: a gold shell magnetic Raman enhanced nano-label comprises magnetic Fe3O4The core, the PEI self-assembly layer, the shell formed by reduction of gold ions and the DTNB modified and then coupled with the antibody part.
As a general inventive concept, the present application also provides a method for preparing a gold shell magnetic raman enhanced nano-tag, comprising the steps of:
(1) mixing Fe3O4Adding magnetic particles into a PEI solution, and carrying out intense ultrasonic treatment to enable PEI to be in Fe3O4The magnetic particle surface self-assembles to form Fe with strong positive electricity on the surface3O4@ PEI particles;
(2) fe synthesized in the step (1)3O4Adding @ PEI particles into 3nm colloidal gold with negative electricity, and carrying out violent ultrasonic reaction to enable the Fe to be reacted3O4The surface of the @ PEI particle is adsorbed with a dense layer of colloidal gold particles to obtain seeded magnetic Fe3O4Magnetic composite (Fe)3O4-Au)；
(3) Fe obtained in the step (2)3O4The Au nano-particles take PVP as a protective agent, chloroauric acid as a raw material and hydroxylamine hydrochloride as a reducing agent, and gold ions are quickly reduced and deposited on the surfaces of the adsorbed gold seeds to form continuous and rough nano-gold shells (Fe)3O4@Au)。
(4) Fe obtained in the step (3)3O4The @ Au nanoparticle is connected with 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) through an Au-S bond under an ultrasonic condition, and a rear terminal carboxyl group is directly coupled with SAA and a CRP antibody (capture antibody) under the activation of a carbodiimide solution and an N-hydroxysuccinimide solution to serve as a multifunctional magnetic SERS immune tag.
Preparation method of gold shell magnetic Raman enhanced nano-labelIn the step (1), Fe3O4The particles are prepared by a solvent thermal synthesis method, and the surfaces of the particles are negatively charged; the concentration of the PEI solution is 0.2 mg/mL-5 mg/mL, preferably 1 mg/mL; the ultrasonic time is 10-60 min, preferably 15 min.
In the preparation method of the gold-shell magnetic Raman-enhanced nano-tag, the 3nm colloidal gold in the step (2) is excessive; the ultrasonic reaction time is 10-60 min, preferably 30 min.
In the preparation method of the gold-shell magnetic Raman-enhanced nano-tag, in the step (3), the concentration of hydroxylamine hydrochloride is preferably 0.5 mg/mL; the ultrasonic time is 10-60 min, preferably 30 min; and the cleaning times after the magnetic enrichment are 2-3 times.
In the preparation method of the gold-shell magnetic Raman-enhanced nano tag, in the step (4), the concentration of DTNB is preferably 40 μ M, the addition amounts of the carbodiimide solution and the N-hydroxysuccinimide solution are preferably 50 μ L and 10 μ L respectively, the ultrasonic reaction time is 30-90 min, preferably 60min, and the activation reaction time is 10-30 min, preferably 15 min.
As a general inventive concept, the present application further provides an application of the gold-shell magnetic raman-enhanced nano-tag as a magnetic SERS tag of an immunochromatography detection system in an unprocessed blood sample. The method comprises the following steps: will be based on Fe3O4Throwing the SERS immune label of @ Au into an untreated blood sample for oscillation reaction for 20min, and rapidly enriching the magnetic label/SAA and CRP antigen complex through an external magnetic field; 70 microliters of sample loading solution is used for resuspending the magnetic label/SAA and CRP antigen complex, then an immunochromatographic test strip is inserted (the two detection lines are respectively marked with the SAA and CRP detection antibodies), and a Raman signal at the detection line of the immunochromatographic test strip is read after 10 minutes, so that the SAA and CRP can be simultaneously and rapidly detected in an untreated blood sample in a high-sensitivity and quantitative manner.
Compared with the prior art, the invention has the advantages that:
(1) the gold shell magnetic Raman enhanced nano-tag provided by the invention has excellent performance and can provide an ultra-strong Raman signal; with adjustable Fe3O4The magnetic core has strong magnetic responsiveness and is convenient for rapid magnetic recovery; in addition, the material has good dispersibility and strong stability, and is the magnetic SERS label material with the most advanced comprehensive performance at present.
(2) The gold shell magnetic Raman enhanced nano label is prepared by adopting a PEI self-assembly method, the preparation method is simple and mature, the repeatability is good, and the mass production can be realized.
(3) The gold shell magnetic Raman-enhanced nano-tag provided by the invention has wide application prospects and can be used in the fields of biochemical analysis, biosensing, rapid detection and the like. The gold shell magnetic Raman enhancement nano-label can be used as a high-performance magnetic SERS immune label for rapid capture and magnetic separation of a target object in untreated blood. The magnetic separation process avoids centrifugation or filtration, and has convenient operation, good stability and difficult oxidation.
(4) The gold shell magnetic Raman enhanced nano-tag provided by the invention is used as a multifunctional magnetic SERS tag for immunochromatography detection, can enrich target substances and provide a super-strong Raman signal, so that the sensitivity of SERS immunochromatography detection can be effectively improved, and the gold shell magnetic Raman enhanced nano-tag is particularly suitable for rapid quantitative detection of low-concentration samples.
In conclusion, the gold-shell magnetic Raman enhanced nano-label disclosed by the invention has the advantages of simple preparation route, high efficiency and repeatability. The prepared gold shell magnetic Raman enhanced nano-label has the performances of monodispersity, strong magnetic responsiveness, difficult oxidation and the like, has uniform and controllable particle size, and has wide application prospect in the field of untreated blood sample detection, particularly high-sensitivity SERS immunochromatography detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
Fig. 1 is a schematic diagram of a preparation method of a gold-shell magnetic raman-enhanced nano-tag of the present invention.
FIG. 2 shows the Transmission Electron Microscope (TEM) and the element surface scanning results of the components of the magnetic substrate during the preparation of the gold-shell magnetic Raman-enhanced nano-tag, wherein (a) is Fe3O4Particles, (b) picture is Fe3O4Au particles, (c) diagram is Fe3O4@ Au, and (d) diagram is Fe3O4@ Au element plane scan.
FIG. 3 shows Fe in the preparation process of the gold-shell magnetic Raman-enhanced nano-tag of embodiment 1 of the present invention3O4Particles of Fe3O4Hysteresis curves of @ Au particles.
Fig. 4 is an experimental flowchart of the combination of the magnetic SERS tag of embodiment 2 of the present invention as a high-performance magnetic SERS tag and an immunochromatographic system for simultaneously detecting SAA and CRP.
Fig. 5 shows the results of the immunochromatographic system for magnetic SERS tags of example 2 of the present invention for detecting various blood biomarkers.
FIG. 6A is a test strip showing the test results of samples of different concentrations of SAA and CRP in the detection buffer according to example 2 of the present invention.
FIG. 6B is the average SERS spectra of two test lines of different concentrations of SAA and CRP in the buffer provided in example 2 of the present invention.
Fig. 6C is a calibration curve of raman signal based on magnetic SERS tags in buffer provided in example 2 of the present invention.
Fig. 7A is a test strip of example 2 of the present invention for testing samples with different concentrations of SAA and CRP in real samples.
Fig. 7B is an average SERS spectrum result of two test lines with different concentrations of SAA and CRP in an actual sample provided in example 2 of the present invention.
Fig. 7C is a calibration curve of a raman signal based on a magnetic SERS tag in an actual sample according to example 2 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The gold shell magnetic Raman enhanced nano tag comprises magnetic Fe3O4The core, the PEI self-assembly layer, the shell formed by reduction of gold ions and the DTNB modified and then coupled with the antibody part. Raman signal molecules DTNB are firmly combined through Au-S bonds, and terminal carboxyl of DTNB can be directly coupled with an antibody to serve as a multifunctional magnetic SERS immune label. The final particle size of the magnetic composite nano material is about 200nm, the magnetic composite nano material has good dispersibility and magnetic responsiveness, and the magnetic saturation value is 49.82 emu/g.
A method for preparing the gold-shell magnetic raman-enhanced nano tag of the embodiment, as shown in fig. 1, includes the following steps:
(1) preparation of Fe3O4@ PEI particle:
0.1g of prepared Fe was weighed3O4MNPs were added to 10mL of an aqueous PEI solution (1mg/mL) and sonicated for 15 min. The product was magnetically enriched and washed 2 times with deionized water and resuspended in 5mL of deionized water for use.
(2) Preparation of Fe3O4-Au seeds particles:
fe to be prepared3O4Adding the @ PEI solution into 50mL of colloidal gold solution, and carrying out intense ultrasonic treatment for 30 minutes to obtain Fe with strong positive charges3O4@ PEI adsorbs a large amount of negatively charged colloidal gold to form Fe3O4-Auseeds particles. And magnetically enriching the product, washing twice with deionized water, and suspending in deionized water for later use.
(3) Preparation of Fe3O4@ Au particles:
fe to be prepared3O4Au seeds solution (2mL) was added to 100mL deionized water containing 300mg PVP and 0.5mg/mL hydroxylamine hydrochloride, and after 15 minutes of vigorous sonication, 40. mu.M chloroauric acid was added and vigorous sonication continued for 15 minutes. The product was magnetically enriched and washed 3 times with deionized waterThen, the suspension is resuspended in 5mL of deionized water for later use.
(4) 5mL of Fe3O4Mixing @ Au with 30 μ M DTNB, performing vigorous ultrasonic reaction for 1 hr, washing with anhydrous ethanol for 1 time, suspending in 5mL ethanol solution, collecting 2mLFe3O4@ Au-DTNB solution was subjected to magnetic enrichment, and 1mL of MES buffer (100mM) containing 50. mu.L of carbodiimide solution (0.01M) and 10. mu. L N-hydroxysuccinimide solution (0.1M) was added to conduct ultrasonic reaction for 15 minutes to activate Fe3O4@ Au-DTNB surface carboxyl groups; then magnetic enrichment is carried out to recover Fe3O4@ Au-DTNB, resuspended in 200. mu.L of PBST solution (0.01M, pH 7.4); adding 15 μ g antibody, shaking for 2h at room temperature, adding 80 μ L BSA (10%), continuing to block for 1h, magnetic-enriching the product and washing with PBST for 2 times, and resuspending in 100 μ L PBS for use.
FIG. 2 shows Fe obtained in step (1) of this example3O4Magnetic core particle, Fe produced in step (3)3O4-Auseeds particles, Fe obtained in step (4)3O4Transmission Electron Micrograph (TEM) of @ Au particles and Fe obtained in step (4)3O4Elemental surface scans of the @ Au particles. Wherein (a) is 160nm Fe3O4TEM images of magnetic core particles; (b) it is shown as Fe3O4TEM image of Au seeds particles; (c) it is shown as Fe3O4TEM image of @ Au particles; (d) it is shown as Fe3O4Elemental surface scans of the @ Au particles. According to the TEM and the element surface scanning results, the gold shell magnetic Raman enhanced nano label has uniform and well-arranged structure, and the shell formed by reducing gold ions is wrapped in Fe3O4The product has excellent magnetic property and SERS property at the same time due to the surface of the particles.
FIG. 3 shows 160nm Fe prepared in step (1) of this example3O4Magnetic core and Fe3O4Hysteresis curve of @ Au. From FIG. 3, it can be seen that the magnetic nanomaterial prepared in the two stages is superparamagnetic, and the final product Fe3O4The magnetic saturation value of @ Au can still reach 49.82 emu/g. Shows that the gold shell magnetic Raman enhanced nano-label prepared by the invention has the advantages of having the effect of an external magnetic fieldSufficient magnetic response capability, and convenient magnetic enrichment.
Example 2
The gold shell magnetic Raman enhancement nano-tag provided by the invention can be used as a multifunctional magnetic SERS (surface enhanced Raman scattering) tag, can effectively capture a target object and rapidly separate through the magnetic enrichment effect, and can also be used as a high-performance magnetic SERS nano-tag for detection of an immunochromatographic system. In the embodiment, the magnetic SERS label modified by the SAA and CRP antibody is combined with an immunochromatography system to detect the SAA and CRP samples with different concentrations in a complex sample. Fig. 4 is a flowchart of an experiment for simultaneously and rapidly detecting SAA and CRP by using the magnetic SERS tag in combination with an immunochromatographic system shown in this example. Fig. 5 shows the results of the immunochromatographic system for magnetic SERS tags of the present embodiment detecting various blood biomarkers. From a in FIG. 5, it can be seen that in the detection of the biomarkers CEA, PSA, AFP, IL-6, PCT and blank items, the test lines of the immunochromatographic test strip are not developed, and only the test lines of the CRP and SAA are developed; b in fig. 5 is the above reading of the raman spectrometer at the test line of the corresponding test strip. The result shows that the magnetic SERS label modified with the SAA and CRP antibodies only recognizes and combines the SAA and CRP, and the phenomenon that the Raman signal is obviously enhanced is shown, so that the gold-shell magnetic Raman enhanced nano label is proved to have higher accuracy and specificity. FIGS. 6A, 6B, and 6C are the results of a test assay based on a magnetic SERS immunochromatographic system for simultaneous detection of SAA and CRP in buffer. FIG. 6A shows the test strip results (785nm excitation wavelength) of samples (500ng/mL-0ng/mL) tested for different concentrations of SAA and CRP. As can be seen from the figure, the degree of visualization on the test strip detection line (T line) gradually decreases with the decrease of the target concentration, and the visual sensitivity is SAA: 5ng/mL, CRP: 0.5ng/mL, the detection limit of the reading value of the Raman spectrometer can respectively reach SAA: 0.1ng/mL, CRP: 0.01 ng/mL. FIG. 6B is an average SERS spectrum of two test lines at different concentrations of SAA and CRP. Fig. 6C is a calibration curve based on the raman signal of the magnetic SERS tag. The error line is obtained by measuring five times, and the correlation coefficients of the detection result are both R20.99. FIGS. 7A, 7B and 7C are illustrations of the operation of an established magnetic SERS tag-based immunochromatographic system in untreated blood samplesThe situation is. The result shows that the magnetic SERS label has excellent stability, can realize high-sensitivity detection of the target object in the untreated blood sample, and the detection result is basically consistent with that in PBST. The results show that the immunochromatographic system based on the magnetic SERS tag has the remarkable advantages of high sensitivity, good stability, suitability for low-concentration detection of SAA and CRP and the like, and provides a powerful method for clinically distinguishing bacterial and viral infections.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. The preparation method of the gold-shell magnetic Raman-enhanced nano tag is characterized in that the gold-shell magnetic Raman-enhanced nano tag is prepared by assembling polymer polyethyleneimine and adsorbing 3nm colloidal gold to form seeded Fe3O4The gold shell is generated by the nano particles through a seed growth method, the Raman signal molecule DTNB is firmly combined on the surface of the gold shell through an Au-S bond, and carboxyl at the tail end of the DTNB is coupled with the SAA and the CRP antibody to form the gold shell magnetic Raman enhanced nano label.
2. The method of claim 1, comprising the steps of:
(1) mixing Fe3O4Adding magnetic particles into the polyethyleneimine solution to carry out violent ultrasonic treatment to ensure that the polyethyleneimine is Fe3O4The magnetic particle surface self-assembles to form Fe with strong positive electricity on the surface3O4@ PEI particles;
(2) mixing the polyethyleneimine modified magnetic nanoparticles obtained in the step (1) with 3nm colloidal gold, and then carrying out ultrasonic reaction, wherein the colloidal gold is obtained in the processUniformly fixing the mixture on the surface of a polyethyleneimine modified magnetic nanoparticle through electrostatic adsorption, and finally enriching the mixture by using a magnet to obtain seeded Fe3O4Nanoparticle Fe3O4-Au；
(3) The seeded Fe obtained in the step (2)3O4The nano-particles take PVP as a protective agent, chloroauric acid as a raw material and hydroxylamine hydrochloride as a reducing agent, and gold ions are quickly reduced and deposited on the surface of the adsorbed seeds to form continuous and rough nano-gold shell structure Fe3O4@Au；
(4) Fe obtained in the step (3)3O4The @ Au nanoparticle is connected with DTNB through Au-S bond under the ultrasonic condition, and then is directly coupled with SAA and CRP antibody as a gold shell magnetic Raman enhanced nano-label through activation of terminal carboxyl in carbodiimide solution and N-hydroxysuccinimide solution, so that the effect of the multifunctional magnetic SERS immune label is realized.
3. The preparation method of the gold-shell magnetic Raman-enhanced nano-tag according to claim 2, wherein in the step (4), the concentration of DTNB is preferably 40 μ M, the addition amounts of the carbodiimide solution and the N-hydroxysuccinimide are preferably 50 μ L and 10 μ L respectively, the reaction time of the ultrasonic treatment is 30-90 min, preferably 60min, and the reaction time of the activation is 10-30 min, preferably 15 min.
4. The gold-shell magnetic Raman enhancement nano-tag prepared by the preparation method of any one of claims 1 to 3.
5. The immunochromatography application of the gold shell magnetic Raman enhancement nano-label is characterized by comprising the following steps: will be based on Fe3O4Throwing the SERS immune label of @ Au into an untreated blood sample for oscillation reaction for 20min, and rapidly enriching the magnetic label/SAA and CRP antigen complex through an external magnetic field; resuspending the magnetic label/SAA and CRP antigen complex with 70 microliters of sample loading solution, inserting an immunochromatographic test strip, and reading a Raman signal at the detection line of the immunochromatographic test strip after 10 minutes, thereby realizingSimultaneous quantitative detection of SAA and CRP in an untreated blood sample;
two detection lines of the immunochromatographic test strip are respectively marked with SAA and CRP detection antibodies.
6. The immunochromatography application of gold-shelled magnetic Raman-enhanced nano-labels according to claim 5, wherein in the step, the blood sample added with the SAA and CRP antigen is not subjected to any treatment; the concentration of the SAA antibody coated on the immunochromatographic test strip is 0.5-1.5 mg/mL, and preferably 1.0 mg/mL; the concentration of the CRP antibody is 0.5-1.5 mg/mL, preferably 0.8 mg/mL.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251232A (en) * | 2020-10-22 | 2021-01-22 | 中国人民解放军军事科学院军事医学研究院 | Difunctional quantum dot microsphere composite nanomaterial, preparation method and application thereof |
| CN112326623A (en) * | 2020-10-23 | 2021-02-05 | 中国人民解放军军事科学院军事医学研究院 | Silicon-core-silver-shell composite nano label marked by double-layer Raman molecules, preparation method and immunochromatography application |
| 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 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2814110A1 (en) * | 2011-06-20 | 2012-05-03 | Pbd Biodiagnostics, Llc | Methods, kits and arrays for screening for, predicting and identifying donors for hematopoietic cell transplantation, and predicting risk of hematopoietic cell transplant (hct) toinduce graft vs. host disease (gvhd) |
| CN106997799A (en) * | 2016-01-22 | 2017-08-01 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of preparation method and its SERS application of high-performance gold shell magnetic microballoon |
| CN107941779A (en) * | 2017-09-30 | 2018-04-20 | 中国人民解放军总医院 | The method of gold shell magnetic nanoparticles and nanometer label surface enhancing Raman scattering detection bacterium based on vancomycin modification |
| CN110506207A (en) * | 2017-03-20 | 2019-11-26 | 由联邦材料研究和检测机构主席所代表的经济与能源部长所代表的德意志联邦共和国 | Indicator for detecting the analyte in food discharges system, its test-strips and analysis method |
| CN110887962A (en) * | 2019-12-05 | 2020-03-17 | 中国人民解放军军事科学院军事医学研究院 | Preparation and immunochromatography application of magnetic composite nanomaterial with double-layer quantum dot shell structure |
-
2020
- 2020-06-01 CN CN202010487039.6A patent/CN111650379A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2814110A1 (en) * | 2011-06-20 | 2012-05-03 | Pbd Biodiagnostics, Llc | Methods, kits and arrays for screening for, predicting and identifying donors for hematopoietic cell transplantation, and predicting risk of hematopoietic cell transplant (hct) toinduce graft vs. host disease (gvhd) |
| CN106997799A (en) * | 2016-01-22 | 2017-08-01 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of preparation method and its SERS application of high-performance gold shell magnetic microballoon |
| CN110506207A (en) * | 2017-03-20 | 2019-11-26 | 由联邦材料研究和检测机构主席所代表的经济与能源部长所代表的德意志联邦共和国 | Indicator for detecting the analyte in food discharges system, its test-strips and analysis method |
| CN107941779A (en) * | 2017-09-30 | 2018-04-20 | 中国人民解放军总医院 | The method of gold shell magnetic nanoparticles and nanometer label surface enhancing Raman scattering detection bacterium based on vancomycin modification |
| CN110887962A (en) * | 2019-12-05 | 2020-03-17 | 中国人民解放军军事科学院军事医学研究院 | Preparation and immunochromatography application of magnetic composite nanomaterial with double-layer quantum dot shell structure |
Non-Patent Citations (1)
| Title |
|---|
| XIAOXIAN LIU ET AL: "Fe3O4@Au SERS tags-based lateral flow assay for simultaneous detection ofserum amyloid A and C-reactive protein in unprocessed blood sample", 《SENSORS AND ACTUATORS B: CHEMICAL》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251232A (en) * | 2020-10-22 | 2021-01-22 | 中国人民解放军军事科学院军事医学研究院 | Difunctional quantum dot microsphere composite nanomaterial, preparation method and application thereof |
| CN112326623A (en) * | 2020-10-23 | 2021-02-05 | 中国人民解放军军事科学院军事医学研究院 | Silicon-core-silver-shell composite nano label marked by double-layer Raman molecules, preparation method and immunochromatography application |
| CN112326623B (en) * | 2020-10-23 | 2024-05-14 | 中国人民解放军军事科学院军事医学研究院 | Silicon core silver shell composite nano-tag with double-layer Raman molecular marker, preparation method and immunochromatography application |
| 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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