CN108896756B - Colloidal gold nano-polyaniline-gold nano composite microsphere and preparation method and application thereof - Google Patents
Colloidal gold nano-polyaniline-gold nano composite microsphere and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of immunodetection, and discloses colloidal gold nano-polyaniline-gold nano composite microspheres and a preparation method and application thereof. In the composite microsphere, the Au nano particles are wrapped by polyaniline, and the colloidal gold nano particles are coupled on the surface of the polyaniline. According to the invention, the colloidal gold nano-polyaniline-gold nano composite microspheres with a three-layer structure replace conventionally applied colloidal gold combined with the glycocholic acid monoclonal antibody, so that the monoclonal antibody is multiplied, the detection sensitivity can be increased when the immunochromatography detection is carried out, the detection limit is reduced to a better degree of 0.2mg/L, and the monoclonal antibody can be directly observed by naked eyes.
Description
Technical Field
The invention relates to the technical field of immunodetection, in particular to colloidal gold nano-polyaniline-gold nano composite microspheres and a preparation method and application thereof.
Background
The damage and accumulation of the liver cells cause liver diseases, the damage degree of the liver cells is detected, and the liver function is evaluated in real time, so that the method has important significance for early diagnosis, prevention and treatment of the liver diseases. Glycocholic acid is one of the main components of bile acid, is formed by combining cholesterol and glycine in the liver, has sensitivity to liver disease diagnosis in content determination, and is an important index for evaluating the liver function. Relevant researches indicate that bile acid is obviously excreted through the kidney and is related to the degree of liver and gall diseases, so that the detection of glycocholic acid in urine has certain clinical guiding value, and the clinical detection of glycocholic acid mostly takes venous blood, while the detection of glycocholic acid in urine of some children patients and patients who are inconvenient to take venous blood has convenience and guiding significance for disease diagnosis.
The prior patent CN105481877A discloses an immunity detection reagent of glycocholic acid based on an anti-glycocholic acid specific antibody and a preparation method thereof, wherein the time-resolved immunochromatographic test paper prepared by a glycocholic acid specific monoclonal antibody obtained by immunizing animals with the glycocholic acid specific hapten-coupled protein can rapidly determine the content of glycocholic acid in a sample, and improve the specificity and accuracy of the prior glycocholic acid clinical diagnosis.
However, the existing scheme can achieve the detection limit of 0.5mg/L only by means of a time-resolved fluorescence immunochromatography instrument, rapid qualitative judgment cannot be directly carried out by naked eyes, and the detection limit is high due to poor sensitivity.
Disclosure of Invention
In view of the above, the present invention provides a colloidal gold nano-polyaniline-gold nano composite microsphere, which can be directly observed by naked eyes when combined with a glycocholic acid monoclonal antibody for immunoassay, and has a detection limit of 0.1-0.2mg/L, and a very high sensitivity;
another object of the present invention is to provide a method for preparing the above composite microsphere;
the invention also aims to provide the related application of the composite microspheres in the preparation of qualitative detection glycocholic acid products.
In order to achieve the above purpose, the invention provides the following technical scheme:
a colloidal gold nano-polyaniline-gold nano composite microsphere is provided, wherein Au nano particles are wrapped by polyaniline, and the surface of the polyaniline is coupled with the colloidal gold nano particles.
Wherein the coupling is via-Au-NH-bond and enwraps polyaniline to form emeraldine salt as follows:
in an acidic medium, aniline and HAuCl4In between, a redox reaction can occur. Initially, HAuCl reduced from aniline4Resulting in the formation of metallic Au0 atoms. More new gold atoms are generated in this system over time, and nucleation occurs when the gold atom concentration reaches a critical supersaturation. The nuclei are grown to the nanopores by further addition of gold atoms. Finally, the primary particles can be clustered together to form larger gold nanospheres with a relatively small size distribution. Meanwhile, aniline monomer is transferred into polyaniline, and the surface of the gold nanospheres is coated with a coating.
Aiming at the defects that the existing immunochromatography technology for detecting glycocholic acid needs a time-resolved fluorescence immunochromatography instrument and is low in sensitivity, the immunochromatography detection method uses the colloidal gold nano-polyaniline-gold nano composite microspheres to replace the conventionally used colloidal gold combined with the glycocholic acid monoclonal antibody for immunochromatography detection, can directly observe the detection result by naked eyes, and is high in sensitivity.
The preparation method of the composite microsphere is divided into two stages, firstly HAuCl is added4The aqueous solution and HCl solution containing aniline monomer are stirred to react until the color is dark green, at which time HAuCl4Reducing the polyaniline into Au nano particles as an oxidant, wrapping the Au nano particles in polyaniline, and performing centrifugal separation to obtain polyaniline microspheres wrapping the Au nano particles by precipitation; the surface of the prepared polyaniline microsphere can not be coupled with the glycocholic acid monoclonal antibody because no group capable of coupling protein exists, so the second step of the preparation method of the inventionAnd the section is to resuspend the polyaniline microspheres and stir the polyaniline microspheres and the colloidal gold nanoparticles for reaction overnight, and then to resuspend and precipitate after centrifugation to obtain the polyaniline wrapped with the Au nanoparticles and the composite microspheres coupled with the colloidal gold nanoparticles on the surface of the polyaniline (the flow schematic diagram is shown in figure 1).
The colloidal gold nanoparticles are mainly used for being connected to the surface of polyaniline and glycocholic acid monoclonal antibodies, and the surface of the polyaniline microspheres prepared in the first stage can be connected with a plurality of colloidal gold nanoparticles, so that the coupled glycocholic acid monoclonal antibodies are multiplied, the sensitivity is high, and the degree of identification by naked eyes can be achieved.
Preferably, the preparation method comprises the following steps:
1.0mmol/L HAuCl4Dripping 0.1mol/L HCl solution containing 0.1mmol/L aniline monomer into the aqueous solution, and uniformly stirring; when the color of the mixture turns into dark green, the precipitate obtained by centrifugal separation is polyaniline microspheres wrapped with Au nano particles; and (3) resuspending the polyaniline microspheres by using deionized water, slowly adding colloidal gold nanoparticles with the Au concentration of 24 +/-1 mol/L, slowly stirring at room temperature overnight, centrifuging, precipitating and resuspending to obtain dark green polyaniline wrapping the Au nanoparticles, and coupling the colloidal gold nanoparticles on the surface of the polyaniline to obtain the composite microspheres.
Wherein, the HAuCl4The volume ratio of the aqueous solution to the HCl solution containing aniline monomer is 3: 10; the volume ratio of the deionized water to the colloidal gold nanoparticles is 1: 3.
In the specific implementation method of the invention, the preparation method specifically comprises the following steps:
3mL of HAuCl410mL of an HCl solution (0.1mol/L) containing 0.1mmol/L of an aniline monomer was added dropwise to the aqueous solution (1.0mmol/L), and the mixture was stirred uniformly. When the color of the mixture turns into dark green, centrifugally separating the obtained precipitate (namely the polyaniline microspheres coated with the Au nano particles); after being resuspended in 1mL of deionized water, 3mL of colloidal gold nanoparticles (C (Au) ═ 24 +/-1 mol/L) are slowly added, the mixture is slowly stirred at room temperature overnight, the mixture is centrifuged at 12000rpm at 4 ℃ for 20 minutes, 1.0mL of deionized water is added for resuspension, and dark green colloidal gold nano-polyaniline-gold nano composite microspheres can be obtained and stored at 4 DEG C。
According to the beneficial effects brought by the composite microspheres, the invention provides the application of the composite microspheres or the composite microspheres prepared by the preparation method in glycocholic acid detection and/or glycocholic acid product preparation. Preferably, the product is an immunochromatographic test strip.
In a specific embodiment of the invention, the invention provides an immunochromatographic test strip for detecting glycocholic acid, which comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the gold-labeled pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped; the gold label pad is adsorbed with the compound microsphere or the glycocholic acid monoclonal antibody marked by the compound microsphere prepared by the preparation method; the nitrocellulose membrane is scribed with a detection line and a quality control line, the detection line is glycocholic acid polyclonal antibody, the quality control line is goat anti-mouse IgG, and the schematic diagram is shown in figure 2.
In a specific embodiment of the present invention, the glycocholic acid mab labeled with the composite microsphere or the composite microsphere prepared by the preparation method can be labeled and coupled by the following method:
with Na2CO3The pH value of 1mL of composite microsphere solution is adjusted to 9.0-9.5 by using aqueous solution (2 mmol/L). Slowly dripping 100uL of glycocholic acid monoclonal antibody into the composite microsphere solution. After shaking slowly for 5min, the cells were transferred to a refrigerator overnight at 4 ℃. Centrifuging the solution at 12000 speed for 20min, and adding 1mL of Na2CO3Resuspend the aqueous solution (2mmol/L), slowly add 20ul 10% BSA dropwise to block the excess residues on the surface of the composite microspheres, mix well and react for 30 minutes. Centrifuging at 9000rpm at 4 ℃ for 15 minutes, removing the supernatant, and obtaining the precipitate as the marked glycocholic acid monoclonal antibody.
In a specific embodiment of the invention, the glycocholic acid monoclonal antibody and the polyclonal antibody are prepared by taking glycocholic acid coupled with bovine serum albumin as an immunogen; the glycocholic acid conjugated with bovine serum albumin has a structure of formula 1 (n ═ 1):
the preparation method comprises the following steps: coupling glycocholic acid and bovine serum albumin by adopting a mixed anhydride method, and dialyzing and purifying to obtain an immune antigen; immunizing New Zealand white rabbit with the immunizing antigen, collecting blood from ear vein of rabbit, centrifuging at 4 deg.C, collecting supernatant as polyclonal antibody, and storing at-20 deg.C. Immunizing Balb/c mice with the immune antigen, taking spleen cells for fusion, preparing a glycocholic acid hybridoma cell strain, preparing ascites from the abdominal cavity of the sensitized mice, and purifying by using an octanoic acid-ammonium sulfate method to obtain a glycocholic acid monoclonal antibody;
in order to obtain a monoclonal antibody with better specificity, the glycocholic acid hybridoma cell strain capable of secreting the monoclonal antibody with higher specificity is screened out through multiple preparation of hybridoma cell strains and monoclonal antibodies, is named as CG-706, and has a preservation number of CGMCC No. 15292.
When the immunochromatographic test strip is used for detecting glycocholic acid standard substances with different concentrations, the result shows that the chromogenic result of the detection line can be directly distinguished by naked eyes when the concentration is 0.1-0.2mg/L, the content of glycocholic acid in a normal human body is 0.4-2.98mg/L, the existing detection requirements can be met, and the specific detection result is shown in the figure 3.
According to the technical scheme, the colloidal gold nano-polyaniline-gold nano composite microspheres with a three-layer structure replace conventionally applied colloidal gold combined with the glycocholic acid monoclonal antibody, so that the monoclonal antibody is multiplied, the detection sensitivity can be increased when the immunochromatography detection is carried out, the detection limit is reduced to a better degree of 0.2mg/L, and the monoclonal antibody can be directly observed by naked eyes.
Biological deposited material information description
CG-706, Classification nomenclature: glycocholic acid hybridoma cell lines; the strain is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 1 month and 25 days, and the address is No. 3 of West Lu No.1 of the morning area of the south of the republic of Beijing, and the preservation number is CGMCC No. 15292.
Drawings
FIG. 1 is a flow chart showing the preparation of the colloidal gold nano-polyaniline-gold nano composite microsphere according to the present invention;
FIG. 2 is a schematic structural diagram of the immunochromatographic test strip of the present invention;
FIG. 3 is a schematic diagram showing the positive, negative and invalid results of the immunochromatographic test strip of the present invention;
FIG. 4 shows the results of detection of glycocholic acid standards at different concentrations; wherein A-D respectively represent the test results of 1, 0.5, 0.2 and 0.1mg/L glycocholic acid standard substance, and E represents the test result of PBS buffer solution.
Detailed Description
The invention discloses a colloidal gold nano-polyaniline-gold nano composite microsphere and a preparation method and application thereof, and a person skilled in the art can realize the preparation by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the composite microspheres and methods of making and using the same of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that the techniques of the present invention may be practiced and used with modification, or with appropriate modification, and combinations of the composite microspheres and methods of making and using the same without departing from the spirit, scope, and spirit of the invention.
The colloidal gold nano-polyaniline-gold nano composite microsphere provided by the invention, and the preparation method and application thereof are further described below.
Example 1: preparation of colloidal gold nano-polyaniline-gold nano composite microspheres
3mL of HAuCl410mL of an HCl solution (0.1mol/L) containing 0.1mmol/L of an aniline monomer was added dropwise to the aqueous solution (1.0mmol/L), and the mixture was stirred uniformly. When the color of the mixture turned dark green, the resulting precipitate (i.e., polyaniline-protected gold nanoparticles) was centrifuged; after being resuspended by 1mL of deionized water, 3mL of colloidal gold nanoparticles (C (Au) ≈ 24mol/L) are slowly added, the mixture is slowly stirred at room temperature overnight, the mixture is centrifuged at 12000rpm for 20 minutes at 4 ℃, 1.0mL of deionized water is added for resuspension, and dark green polyaniline-nano composite microspheres can be obtained, the particle size is 30-40nm, and the mixture is stored at 4 ℃.
Example 2: preparing the immunochromatographic test strip
1. Synthesis of immune antigens
Weighing 10-40mg of glycocholic acid and 10-30mg of N, N-dimethylformamide, dissolving in 1-3mL of DMF, adding 20-40mg of DCC, stirring overnight at 4 ℃, centrifuging the reactant for 10min at 10000rpm, taking the supernatant, adding the supernatant into a phosphate buffer solution containing 100-150mg of bovine serum albumin, and stirring for reaction for 12 hours at 4 ℃. And after the reaction, centrifuging again, taking supernatant, putting the supernatant into a dialysis bag, dialyzing for three days at 4 ℃ by using phosphate buffer saline solution, and replacing the solution once every 12 hours to obtain the immune antigen.
2. Production of glycocholic acid polyclonal antibody
Immunization was performed by multiple subcutaneous injections. 2 New Zealand white rabbits, female, 3 months old and 1.5-2.5 kg of body weight are adopted. The immunizing antigen is prepared into 1mg/ml by using the phosphate buffer solution prepared in situ, 1ml of immunizing antigen solution and equal volume of Freund's complete adjuvant are respectively taken, and an emulsifier is used for emulsifying so as to completely mix the immunizing antigen solution and the Freund's complete adjuvant into one phase. Adopting an immunization mode of subcutaneous multipoint injection, and injecting 2ml into each rabbit; two weeks after the first immunization, performing first boosting immunization, mixing and emulsifying 1mg/ml immune antigen with equivalent volume of Freund incomplete adjuvant, performing subcutaneous multipoint injection immunization on each 2ml, and performing boosting immunization once every two weeks; after 4 times of immunization, blood is taken from ear veins of the rabbits after one week of the last immunization, centrifugal separation is carried out for 10min at 4 ℃ with the rotating speed of 10000rpm, and supernatant is taken and stored at-20 ℃ for later use.
3. Preparation of glycocholic acid monoclonal antibody
Immunization was performed by multiple subcutaneous injections. 3 Bal/c, female, 3 months old and 25-30g of body weight are adopted. Preparing 1mg/ml of immune antigen by using the prepared phosphate buffer solution, taking 1ml of immune antigen solution and Freund's complete adjuvant with the same volume, and emulsifying by using an emulsifier to completely mix the immune antigen solution and the Freund's complete adjuvant into one phase. Adopting an immunization mode of subcutaneous multipoint injection, and injecting 0.2ml into each mouse; two weeks after the first immunization, performing first boosting immunization, mixing and emulsifying 1mg/ml immune antigen with equivalent volume of Freund incomplete adjuvant, performing subcutaneous multipoint injection immunization on each 2ml, and performing boosting immunization once every two weeks; 4 times after the boost immunization, tail harvest was performed on antigen immunized mice one week after the last immunizationBlood, using ELISA method to determine antiserum titer, taking splenocyte of mouse with better serum titer, using fusion agent PEG4000 to fuse splenocyte with myeloma cell cultured in vitro, using indirect ELISA to detect cell supernatant positive and competitive inhibition condition, using limiting dilution method to make subclone, after 3 times of subcloning are all positive, taking hybridoma cell concentration as 106-1080.5mL of abdominal cavity of a mouse injected with the ascites/mL, centrifuging and separating the ascites for 10min at 4 ℃ after 14 days at 10000rpm, and taking supernatant to obtain a large amount of monoclonal antibodies, and storing the monoclonal antibodies at-20 ℃ for later use.
According to the method, the monoclonal antibody secreted by the hybridoma cell strain with the preservation number of CGMCC No.15292 is selected.
3. Preparation of gold label pad
With Na2CO3The pH value of 1mL of polyaniline-nano microsphere solution is adjusted to 9.0-9.5 by using aqueous solution (2 mmol/L). Slowly dripping 100uL of glycocholic acid monoclonal antibody into the polyaniline-nano microsphere solution. After shaking slowly for 5min, the cells were transferred to a refrigerator overnight at 4 ℃. Centrifuging the solution at 12000 speed for 20min, and adding 1mL of Na2CO3Resuspending the aqueous solution (2mmol/L), slowly adding 20ul 10% BSA dropwise to block the redundant residues on the surface of the polyaniline-nanospheres, and uniformly mixing and reacting for 30 minutes. Centrifuging at 9000rpm at 4 deg.C for 15 min, discarding supernatant, adding 200ul of diluent into the obtained precipitate, uniformly spreading on glass fiber membrane at 15ul/cm on gold spraying and membrane scribing instrument, and freeze drying;
4. preparation of nitrocellulose membranes
Diluting the prepared glycocholic acid polyclonal antibody to a concentration of 2mg/mL as a detection line, diluting goat anti-mouse IgG to a concentration of 2mg/mL as a quality control line, uniformly spraying the diluted glycocholic acid polyclonal antibody on a nitrocellulose membrane by using a gold spraying membrane scribing instrument at a concentration of 1ul/cm, wherein the distance between the detection line and the front part of the quality control line is 0.4cm, and drying the diluted glycocholic acid polyclonal antibody in a constant-temperature oven at 37 ℃ for later use;
5. preparation of test paper strip
Paste sample pad, the gold mark pad, cellulose nitrate membrane and water absorption pad top-down in proper order on plastics PVP floor, wherein the water absorption pad is pasted to the top of cellulose nitrate membrane, mutual overlap 0.2mm is pasted to the top of cellulose nitrate membrane, paste glass fiber membrane (gold mark pad) and sample pad in proper order in the below of cellulose nitrate membrane, mutual overlap 0.3mm is pasted to cellulose nitrate membrane and gold mark pad, mutual overlap 2mm is pasted to gold mark pad and sample pad, then cut into 4 mm's rectangular with the test paper strip, install the test paper strip in the detection card shell of rectangular flat shell form at last, obtain the immunochromatography test paper strip.
Example 3: sensitivity (detection limit) detection
The glycocholic acid standard substance is diluted with PBS to the concentration of 1, 0.5, 0.2, 0.1mg/L, after mixing uniformly, 50ul of each concentration standard substance is added on the sample pad for detection, and the result is shown in figure 4.
As can be seen from FIG. 4, the detection limit of the present invention is between 0.2mg/L (C in FIG. 4) without the aid of an instrument and visible to the naked eye. Therefore, the detection line of the immunochromatographic test strip for detecting the hepatocholic acid can be defined to be 0.2mg/L, and the content of the glycocholic acid in a normal human body is 0.4-2.98mg/L, so that the existing detection requirements can be met.
Example 4: clinical sample testing
Adding 50ul of 28 morning urine samples from hospital on the sample pad, repeating the test for 3 times, laying flat for 3-5min to obtain whether glycocholic acid in urine sample is out of standard, and performing synchronous detection on the 28 samples by using the existing enzyme-linked immunoassay method, wherein the result is shown in Table 1.
TABLE 1
Note: + positive (out of standard), + negative (not out of standard),/ineffective
As can be seen from the table 1, according to the common sense that the content of glycocholic acid in a normal human body is 0.4-2.98mg/L, whether the glycocholic acid in a detected sample exceeds the standard can be accurately and qualitatively detected by the immunochromatographic test strip, and the result of quantitative detection is consistent.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. The application of the colloidal gold nano-polyaniline-gold nano composite microspheres in the preparation of an immunochromatography test strip for detecting glycocholic acid is realized, wherein the polyaniline in the composite microspheres wraps Au nano particles, and the surfaces of the polyaniline are coupled with the colloidal gold nano particles;
the preparation method of the composite microsphere comprises the following steps: adding HAuCl4Stirring and reacting the aqueous solution and HCl solution containing aniline monomer until the aqueous solution is dark green, performing centrifugal separation to obtain polyaniline microspheres which are deposited to wrap Au nano particles, then re-suspending the polyaniline microspheres and stirring and reacting with the colloidal gold nano particles overnight, and re-suspending and depositing after centrifugation to obtain the polyaniline wrapped Au nano particles and the composite microspheres which are coupled with the colloidal gold nano particles on the surface of the polyaniline.
2. The application of claim 1, comprising:
1.0mmol/L HAuCl4Dripping 0.1mol/L HCl solution containing 0.1mmol/L aniline monomer into the aqueous solution, and uniformly stirring; when the color of the mixture turns into dark green, the precipitate obtained by centrifugal separation is polyaniline microspheres wrapped with Au nano particles; and (3) resuspending the polyaniline microspheres by using deionized water, slowly adding colloidal gold nanoparticles with Au concentration =24 +/-1 mol/L, slowly stirring at room temperature overnight, centrifuging, precipitating and resuspending to obtain dark green polyaniline wrapping the Au nanoparticles, and coupling the colloidal gold nanoparticles on the surface of the polyaniline to obtain the composite microspheres.
3. Use according to claim 2, wherein the HAuCl is4The volume ratio of the aqueous solution to the HCl solution containing aniline monomer was 3: 10.
4. The use according to claim 2, wherein the volume ratio of the deionized water to the colloidal gold nanoparticles is 1: 3.
5. An immunochromatography test strip is characterized by comprising a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped; adsorbing the glycocholic acid monoclonal antibody marked by the composite microspheres in the application of claim 1 on the gold label pad; the nitrocellulose membrane is scribed with a detection line and a quality control line, the detection line is glycocholic acid polyclonal antibody, and the quality control line is goat anti-mouse IgG.
6. The immunochromatographic test strip of claim 5, wherein the glycocholic acid monoclonal antibody and the polyclonal antibody are prepared by using glycocholic acid coupled with bovine serum albumin as an immunogen.
7. The immunochromatographic test strip of claim 5 or 6, wherein the glycocholic acid monoclonal antibody is secreted from hybridoma with the preservation number of CGMCC No. 15292.
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| CN102175735A (en) * | 2010-12-31 | 2011-09-07 | 甘肃省科学院传感技术研究所 | Au NPs-CeO2@PANI (polyaniline) nanocomposite, preparation method thereof and glucose biological sensor manufactured by utilizing nanocomposite |
| CN203101405U (en) * | 2013-03-22 | 2013-07-31 | 通辽天和世纪科技有限公司 | Human chorionic gonadotropin test paper |
| CN104360071A (en) * | 2014-12-05 | 2015-02-18 | 重庆乾德生物技术有限公司 | Kit for quickly determining content of glycocholic acid (CG) in serum or blood plasma |
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| US7214427B2 (en) * | 2002-03-21 | 2007-05-08 | Aviva Biosciences Corporation | Composite beads comprising magnetizable substance and electro-conductive substance |
| US20130177836A1 (en) * | 2011-12-13 | 2013-07-11 | Georgia Tech Research Corporation | Polyaniline-supported atomic gold electrodes and methods of making and using same |
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| CN102175735A (en) * | 2010-12-31 | 2011-09-07 | 甘肃省科学院传感技术研究所 | Au NPs-CeO2@PANI (polyaniline) nanocomposite, preparation method thereof and glucose biological sensor manufactured by utilizing nanocomposite |
| CN203101405U (en) * | 2013-03-22 | 2013-07-31 | 通辽天和世纪科技有限公司 | Human chorionic gonadotropin test paper |
| CN104360071A (en) * | 2014-12-05 | 2015-02-18 | 重庆乾德生物技术有限公司 | Kit for quickly determining content of glycocholic acid (CG) in serum or blood plasma |
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