CN107576702B - Preparation method of electrochemical sensor for detecting concentration of galectin-3 in serum - Google Patents
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Abstract
The invention relates to a preparation method and application of an electrochemical sensor for detecting blood concentration level of an insulin sensitivity-reducing early diagnosis biomarker-galectin-3 (Gal-3) causing type 2 diabetes, belonging to the technical field of electrochemical detection. The method is characterized in that: firstly, respectively synthesizing N-GNRs-Fe-MOFs @ AuPNs sensor substrate modified nano composite material and AuPt-MB nano composite signal material, and then mixing a detection antibody capable of being specifically combined with Gal-3 with the AuPt-MB composite material to prepare the biological signal probe. Then, a Gal-3 specific capture antibody is jointed on a substrate platform modified by an N-GNRs-Fe-MOFs @ AuPNs composite material so as to specifically capture the target object Gal-3. And finally, preparing the electrochemical sensor for detecting the blood concentration level of the Gal-3 through the specific binding of the Gal-3 and the detection antibody and the capture antibody thereof, wherein the sensor is successfully used for detecting the Gal-3 in serum. The invention has the advantages of high sensitivity, strong specificity, rapid and convenient detection. The invention provides early diagnosis basis for the insulin resistance of the type 2 diabetes.
Description
The technical field is as follows:
the invention relates to a preparation method and application of an electrochemical sensor for detecting blood concentration level of insulin sensitivity-reduced early diagnosis biomarker galectin-3(Gal-3) causing type 2 diabetes, in particular to a sandwich type immune biosensor prepared by taking AuPt-MB nano composite material as a novel signal probe, which is used for detecting galectin-3(Gal-3) concentration in serum, and belongs to the field of electrochemical detection.
Background art:
diabetes mellitus is rapidly increasing worldwide and has posed a serious threat to human health. Particularly type 2 diabetes (T2D) caused by reduced insulin sensitivity can lead to fatal cardiovascular complications and account for 90% of the prevalence of diabetes. In recent studies, it was found that galectin-3(Gal-3) can directly bind to insulin receptor to inhibit downstream Insulin Receptor (IR) signaling and link inflammation to reduce insulin sensitivity, which ultimately leads to insulin resistance and glucose intolerance, and thus, type 2 diabetes mellitus is initiated. Therefore, monitoring changes in the blood level of Gal-3 concentrations is particularly important for the prevention of T2D and its cardiovascular complications.
At present, the main traditional methods for detecting Gal-3 are as follows: enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and "conventional Gal-3" assay (Yapei diagnosis). However, these conventional methods have relatively high cost, time consuming, semi-quantitative, skilled operators, limited sensitivity and narrow linear range. In particular, the clinical need for early detection cannot be met. Therefore, it is extremely important to explore a simple, rapid and sensitive detection method for Gal-3. Compared with the traditional detection method, the electrochemical immunosensor detection method has the advantages of high sensitivity, strong specificity, rapid detection and the like, and has more and more attention paid to the detection of clinical protein biomarkers in recent years. Meanwhile, the electrochemical biosensor based on the nano material is widely applied to the detection of biological samples due to the advantages of simplicity, rapidness, low cost, high sensitivity and the like. Therefore, a sandwich-type electrochemical immune biosensor constructed based on the nanocomposite provides a new idea for realizing low-concentration high-sensitivity detection of Gal-3 in serum.
Methylene Blue (MB) is a derivative of a phenothiazine dye, and is widely used in the fields of catalysis, sensing, redox indication, and the like as a new electrochemical redox active substance due to the presence of electron-rich sulfur and nitrogen heteroatoms in its structure. However, its use in immunosensor signaling probes is limited due to its heterogeneous morphology. In order to solve the problem, the AuPt bimetal nano material with large specific surface area, strong conductivity and good biomolecule compatibility is innovatively introduced to carry out oxidative polymerization reaction with MB, so that the AuPt-MB nano composite material with a uniform rod-shaped form is obtained, and the AuPt-MB nano composite material shows obvious electrochemical activity. In the invention, the AuPt-MB nano composite material is combined with a Gal-3 specific detection antibody Ab2, and BSA is used as a blocking agent to prepare a novel nano signal probe.
In order to further increase the sensitivity and stability of the present sandwich-type immunosensor. A novel nano composite material nitrogen-doped graphene nanoribbon (N-GNRs) -iron metal organic framework (Fe-MOFs) -gold nanoparticle (AuNPs) - (N-GNRs-Fe-MOFs @ AuNPs) is introduced to serve as a sensor substrate modification material. Metal-organic frameworks (MOFs) are zeolite-like crystalline porous materials that have the advantages of large surface area, flexible porosity, easy customization of compositional components and many active sites, and as novel multifunctional materials they have been widely used in the fields of gas storage/separation, catalysis and sensing. The Fe-MOFs introduced in the invention has the characteristics of good stability, large specific surface area, low toxicity and the like. Because the antibody has large specific surface area, a large amount of AuNPs nanoparticles can be combined on the surface of the antibody through Au-N chemical bonds to increase the conductivity of the antibody, and the antibody Ab is beneficial to the next step of solid-borne combination of Gal-3 specific capture antibody1. In order to further increase the conductivity, N-GNRs and Fe-MOFs @ AuNPs are mixed and reacted to obtain the N-GNRs-Fe-MOFs @ AuNPs nanocomposite. N-GNRs are synthesized from N-doped multi-walled carbon nanotubes (N-MWCNTs) that are widely used to improve the electrical conductivity of modified electrode surfaces. Compared with carbon nanotubes, N-GNR has a reactive boundary and an inherent terrace and step structure, and can enhance chemical activity and adsorption capacity. Finally, the N-GNRs-Fe-MOFs @ AuNPs nano composite material not only has the advantages of the N-GNRs, the Fe-MOFs and the AuNPs, but also shows a remarkable comprehensive effect in the aspect of conductivity. Thus, it is possible to provideThe novel nanocomposite is applied to substrate modification of the electrochemical immunosensor to further enhance the sensitivity and stability of the sensor.
The invention constructs a novel nano signal probe based on an AuPt-MB nano composite material and takes an N-GNRs-Fe-MOFs @ AuNPs nano composite material as a substrate modification material of an immunosensor, establishes a preparation method and application of an electrochemical sensor for detecting the concentration of galectin-3(Gal-3) in serum, and provides an early diagnosis basis for insulin resistance causing type 2 diabetes.
The invention content is as follows:
the invention aims to provide a preparation method and application of an electrochemical sensor for detecting concentration of galectin-3(Gal-3) in serum, which is characterized by comprising the following steps of:
(1) preparing a nitrogen-doped graphene nanoribbon (N-GNRs) -iron metal organic framework (Fe-MOFs) -gold nanoparticle (AuNPs) sensor substrate modified nanocomposite.
(2) Gold platinum bimetal (AuPt) -Methylene Blue (MB) -Gal-3 specific detection antibody (Ab)2) And (3) preparing a nano signal probe.
(3) Establishing an electrochemical immune biosensor, detecting Gal-3 in serum, and drawing a standard curve.
The preparation process of the N-GNRs-Fe-MOFs @ AuNPs compound specifically comprises the following steps, and is characterized by comprising the following steps:
(1) preparation of N-GNRs material:
0.1g of nitrogen-doped multiwall carbon nanotubes (N-MWCNTs) was weighed out and added to 10mL of H2SO4And H3PO4In mixed solution (H)2SO4∶H3PO4Mixing at ratio of 9: 1). Heating the mixed solution in a high pressure reaction kettle to 140 deg.C under magnetic stirring for 10min, transferring the reaction mixed solution to 65 deg.C oil bath, and adding 0.25g KMnO under stirring4The reaction was continued for 8 min. And after the reaction is finished, controlling the temperature of the solution to be reduced to room temperature, centrifuging the obtained solution for 5min at 10000r/min, and cleaning the solution for 3 times by using ultrapure water. The obtained precipitate is dried in vacuum for 24h and stored at 4 ℃ for later use.
(2) Preparation of Fe-MOFs material
0.187g FeCl was weighed out separately3·6H2O and 0.126g of 2-aminoterephthalic acid were added to 15mL of a Dimethylformamide (DMF) solution and mixed well. The mixed solution was heated in 120 ℃ silicone oil for 4 hours, and 200. mu.L of glacial acetic acid was added to the mixed solution 15min after the start of heating. After heating, the solution is controlled to be cooled to room temperature, the obtained solution is centrifuged for 5min at 10000r/min, and is respectively washed 3 times by DMF, absolute ethyl alcohol and ultrapure water. The obtained precipitate is dried in vacuum for 24h and stored at 4 ℃ for later use.
(3) Preparation of Fe-MOFs @ AuNPs nano composite material
2mL of HAuCl was taken4·4H2O (1%) solution was added to the prepared 2mLFe-MOFs (1mg mL)-1) After vigorous sonication for 30 minutes in solution, 4mL of NaBH was added slowly dropwise to the above solution4(0.1M) and placing on a magnetic stirrer to perform stirring reaction for 30min, centrifuging the obtained solution for 5min at 10000r/min, and cleaning with ultrapure water for 3 times. The precipitate obtained is redispersed in 4mL of deionized water and stored at 4 ℃ until use.
(4) Preparation of N-GNRs-Fe-MOFs @ AuNPs nano composite material
0.2mg of N-GNRs is weighed and ultrasonically dissolved in 2mL of deionized water, and the deionized water and 4mL of Fe-MOFs @ AuNPs compound solution are ultrasonically mixed for 30min and stirred overnight under the condition of magnetic stirring at 500 r/min. The resulting solution was centrifuged at 8000r/min for 5min and washed 3 times with ultrapure water. The precipitate obtained is redispersed in 4mL of deionized water and stored at 4 ℃ until use.
The AuPt-MB-Ab of the invention2The preparation process of the nano signal probe specifically comprises the following steps, and is characterized by comprising the following steps:
(1) preparation of AuPt-MB nanocomposite:
600 μ L of HCl (0.1M) and 6mL of dodecyltrimethylammonium bromide (DTAB 1.14mM) were added to 1mL of MB (9.8mM) solution, and stirred vigorously for 10 min. Then 100. mu.L of HAuCl was added simultaneously4(4 wt.%) and 100. mu. L H2PtCl6(4 wt%,), stirring for 6h at room temperature. The obtained solution is subjected to8000r/min, centrifuging for 10min, and cleaning with ultrapure water for 3 times. The precipitate obtained was redispersed in 2ml PBS buffer (0.1M pH7.0) and stored at 4 ℃ until use.
(2)AuPt-MB-Ab2Preparing a nano signal probe:
take 100. mu.L of Gal-3 specific detection antibody Ab2Added to 2mL of AuPt-MB nanocomposite solution and stirred gently at 4 ℃ for 12 h. Add 40. mu.L of bovine serum albumin BSA (1 wt%) and keep stirring gently at 4 ℃ for 6 h. The resulting solution was centrifuged at 8000r/min for 10min, and the precipitate was redispersed in 2ml PBS buffer (0.1M pH7.0) and stored at 4 ℃ for further use.
The invention relates to an electrochemical immune biosensor which is established for detecting Gal-3 in serum and drawing a standard curve, and is characterized by comprising the following steps:
(1) with 0.3 and 0.05 μm Al, respectively2O3Polishing the electrode into a mirror surface by using powder, then respectively carrying out ultrasonic treatment on the electrode for 5min according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use;
(2) 6 mu L of the prepared N-GNRs-Fe-MOFs @ AuNPs nano composite material solution is dripped on the surface of the electrode and dried at room temperature.
(3) 6 μ L of Gal-3 specific capture antibody Ab was added dropwise1And (4) adding the solution to the surface of the modified electrode, and incubating for 12h at 4 ℃.
(4) The incubated electrode was rinsed with double distilled water, 6. mu.L BSA (1 wt%) blocking agent was added dropwise to the electrode surface, and blocking was carried out at room temperature for 30 min.
(5) The BSA blocked electrode was washed with a washing buffer (10mM Na)2HPO4，2mM KH2PO437mM NaCl,2.7mM KCl, pH 7.4) was rinsed clean and dried under nitrogen.
(6) mu.L of target Gal-3 at various concentrations was added dropwise to the electrode surface, incubated at 37 ℃ for 1h, rinsed with washing buffer and dried under nitrogen.
(7) After drying, 10. mu.L of the prepared AuPt-MB-Ab was added dropwise to the surface of the electrode2The nanophase signal probe solution was incubated at 37 ℃ for 1h, rinsed with washing buffer and dried under nitrogen.
(8) The electrodes were characterized in 5mL,0.1M PBS (pH 7.0), and the peak of the current change current response was measured.
(9) And drawing a working curve according to the linear relation between the current change current response peak value and the concentration of Gal-3.
Compared with the prior art, the preparation method and the application of the electrochemical sensor for detecting the concentration of galectin-3(Gal-3) in serum have the outstanding characteristics that:
(1) the N-GNRs-Fe-MOFs @ AuNPs nano composite material is used for modifying the substrate of the electrochemical immune biosensor, so that a large amount of active site immobilized capture antibodies are provided, the electron conduction rate is promoted, and the sensitivity and the biocompatibility of the electrochemical immune biosensor are improved;
(2) a signal probe constructed by a nano composite material based on AuPt-MB is introduced into the preparation of an electrochemical immune biosensor, wherein the AuPt bimetal nano material not only has the function of amplifying an electrochemical signal, but also increases the biocompatibility of the AuPt bimetal nano material to a detection antibody.
(3) The electrochemical immunosensor prepared by the method can provide a new idea for detecting the Gal-3 blood concentration level causing the reduction of the insulin sensitivity, and provides an early diagnosis basis for the prevention of type 2 diabetes.
(4) The specificity and high-sensitivity detection of various protein biomarkers can be realized by using the completely same nano materials and modification methods and utilizing the specificity recognition of the capture antibody, the signal probe and the target protein only by changing the detection antibody of the probe and the capture antibody immobilized on the substrate.
Description of the drawings:
FIG. 1 is a schematic diagram of the construction of the electrochemical immunosensor of the present invention.
FIG. 2 is the scanning electron microscope image of N-GNRs, Fe-MOFs @ AuNPs and N-GNRs-Fe-MOFs @ AuNPs of the present invention, and the transmission electron microscope and EDS image of AuPt-MB.
FIG. 3 is an EDS diagram of Fe-MOFs @ AuNPs
FIG. 4 is a linear relationship between the peak value of the current response and the logarithm of the concentration of the differential pulse voltammetry obtained when the electrochemical immunosensor of the present invention detects Gal-3.
FIG. 5 shows the reproducibility, specificity and stability of the sensor.
The specific implementation mode is as follows:
the invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
Example 1
And 3, weighing 0.2mg of N-GNRs, ultrasonically dissolving the N-GNRs in 2mL of deionized water, ultrasonically mixing the N-GNRs and 4mL of Fe-MOFs @ AuNPs compound solution for 30min, and stirring overnight under the condition of magnetic stirring of 500 r/min. The resulting solution was centrifuged at 8000r/min for 5min and washed 3 times with ultrapure water. Obtaining N-GNRs-Fe-MOFs @ AuNPs, re-dispersing the obtained N-GNRs-Fe-MOFs @ AuNPs precipitate into 4mL of deionized water, and storing at 4 ℃ for later use.
and 7, dripping 6 mu L of the prepared N-GNRs-Fe-MOFs @ AuNPs nano composite material solution on the surface of the electrode, and drying at room temperature.
And 9, washing the incubated electrode with double distilled water, dropwise adding 6 mu LBSA (1 wt%) sealing agent on the surface of the electrode, and sealing for 30min at room temperature.
And 11, dripping 6 mu L of target Gal-3 with different concentrations onto the surface of the electrode, incubating at 37 ℃ for 1h, washing by using a washing buffer solution, and drying in nitrogen.
Step 12, dripping 10 mu L of prepared AuPt-MB-Ab on the surface of the dried electrode2Nano signal probe solutionIncubate at 37 ℃ for 1h, rinse clean with wash buffer and dry under nitrogen.
And step 13, placing the electrode in 5mL of 0.1M PBS (PH 7.0) for characterization, and measuring the current change current response peak value of the electrode.
And step 14, drawing a working curve according to the linear relation between the obtained current change current response peak value and the concentration of Gal-3. The measurement result shows that the concentration of Gal-3 is 100fg mL-1-50ng mL-1The linear relation is formed in the range, the linear correlation coefficient is 0.99502, and the detection limit is 33.33fg mL-1(S/N＝3)。
step 16, taking 5 immune biosensors prepared in the same batch, and treating 1ng mL of the biosensors under the same condition-1Gal-3 of (2) was measured separately, and 3 times of measurement were made for each electrode, and the relative standard deviation of the current response values was 2.75%, indicating that the reproducibility of the sensor was good.
Step 17, using the sensor of the invention to detect 100ng mL-1Different interferents and 1ng mL-1The relative standard deviation of the detection result of the mixture of Gal-3 is 1.55%, which indicates that the interferent has little interference effect on the detection result of the sensor and the specificity of the sensor is good.
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 (2)
1. A preparation method of an electrochemical sensor for detecting the concentration of galectin-3 in serum is characterized by comprising the following steps:
(1) preparing a nitrogen-doped graphene nanoribbon:
0.1g of nitrogen-doped multi-walled carbon nanotubes are weighed inTo 10mL of H2SO4And H3PO4Uniformly mixing the mixed solution, placing the mixed solution in a high-pressure reaction kettle, heating to 140 ℃ for 10min under the condition of magnetic stirring, transferring the reaction mixed solution into a 65 ℃ oil bath, adding 0.25g of potassium permanganate under the condition of stirring, continuously reacting for 8min, controlling the solution to cool to room temperature after the reaction is finished, carrying out 10000r/min on the obtained solution, centrifuging for 5min, washing for 3 times by using ultrapure water, carrying out vacuum drying on the obtained precipitate for 24h, and storing at 4 ℃ for later use;
(2) preparation of iron metal organic framework:
weighing 0.187g of ferric chloride hexahydrate and 0.126g of 2-amino terephthalic acid, adding into 15mL of dimethylformamide solution, uniformly mixing, placing the mixed solution into 120 ℃ silicone oil, heating for 4h, adding 200 mu L of glacial acetic acid into the mixed solution at the 15 th min after the heating is started, controlling the solution to cool to room temperature after the heating is finished, centrifuging the obtained solution for 5min at 10000r/min, respectively washing precipitates obtained by centrifuging for 3 times with DMF (dimethyl formamide), absolute ethyl alcohol and ultrapure water, finally drying the obtained precipitates in vacuum for 24h, and storing at 4 ℃ for later use;
(3) preparing an iron metal organic framework @ gold nanoparticle composite material:
2mL of 1% chloroauric acid tetrahydrate solution was added to 2mL,1mg mL of the prepared-1After intense ultrasonic treatment is carried out for 30 minutes, 4mL of 0.1M sodium borohydride solution is slowly added into the iron metal organic framework solution dropwise, the solution is placed on a magnetic stirrer to be stirred and reacted for 30 minutes, the obtained solution is subjected to 10000r/min and centrifugation for 5 minutes, is washed for 3 times by ultrapure water, and the obtained precipitate is re-dispersed into 4mL of deionized water and is stored for later use at 4 ℃;
(4) preparing a nitrogen-doped graphene nanoribbon-iron metal organic framework @ gold nanoparticle composite material:
weighing 0.2mg of nitrogen-doped graphene nanoribbon, ultrasonically dissolving the nitrogen-doped graphene nanoribbon in 2mL of deionized water, ultrasonically mixing the nitrogen-doped graphene nanoribbon with 4mL of prepared iron metal organic framework @ gold nanoparticle compound solution for 30min, stirring overnight under the condition of magnetic stirring of 500r/min, centrifuging the obtained solution for 5min at 8000r/min, cleaning the solution for 3 times by using ultrapure water, re-dispersing the obtained precipitate in 4mL of deionized water, and storing the precipitate at 4 ℃ for later use;
(5) preparation of gold platinum-methylene blue:
adding 600 mu L of 0.1M hydrochloric acid and 6mL of 1.14mM dodecyl trimethyl ammonium bromide into 1mL of 9.8mM methylene blue solution, stirring vigorously for 10min, adding 100 mu L of 4 wt% chloroauric acid solution and 100 mu L of 4 wt% chloroplatinic acid solution, stirring continuously at room temperature for 6h, subjecting the obtained solution to 8000r/min, centrifuging for 10min, washing with ultrapure water for 3 times, re-dispersing the obtained precipitate into 2mL of 0.1M PBS buffer solution with pH of 7.0, and storing at 4 ℃ for later use;
(6) preparation of platinum-methylene blue-secondary antibody:
adding 100 mu L of galectin-3 specificity detection secondary antibody into 2mL of gold platinum-methylene blue nano composite material solution, stirring gently at 4 ℃ for 12 hours, adding 40 mu L of bovine serum albumin with the concentration of 1 wt%, stirring gently at 4 ℃ for 6 hours, centrifuging the obtained solution at 8000r/min for 10 minutes, re-dispersing the obtained precipitate into 2mL of PBS (phosphate buffer solution) with the concentration of 0.1M and the pH value of 7.0, and storing at 4 ℃ for later use;
(7) establishing an electrochemical sensor:
a. polishing the electrodes into mirror surfaces by using 0.3 and 0.05 mu m aluminum oxide powder respectively, then carrying out ultrasonic treatment on the electrodes for 5min respectively according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use;
b. dripping 6 mu L of the prepared nitrogen-doped graphene nanoribbon-iron metal organic framework @ gold nanoparticle composite material solution on the surface of an electrode, and drying at room temperature;
c. dripping 6 mu L of galectin-3 specific capture first antibody solution to the surface of the modified electrode, and incubating for 12h at 4 ℃;
d. washing the incubated electrode with double distilled water, dripping 6 mu L of 1 wt% BSA on the surface of the electrode, and sealing at room temperature for 30 min;
e. the BSA solution was blocked and the electrode was used with 10mM Na2HPO4,2mM KH2PO437mM NaCl,2.7mM KCl, pH7.4 wash buffer and dry under nitrogen.
2. The method for the quantitative detection of galectin-3 by the sensor obtained by the preparation method according to claim 1, comprising the steps of:
(1) dripping 6 mu L of target galectin-3 with different concentrations on the surface of an electrode, incubating at 37 ℃ for 1h, washing with a cleaning buffer solution, and drying in nitrogen;
(2) dripping 10 μ L of the prepared gold platinum-methylene blue-second antibody solution on the surface of the dried electrode, incubating at 37 ℃ for 1h, washing with a washing buffer solution, and drying in nitrogen;
(3) placing the electrode in 5mL of PBS (phosphate buffer solution) with the concentration of 0.1M and the pH value of 7.0 for characterization, and measuring the current change current response peak value of the electrode;
(4) and drawing a working curve according to the linear relation between the current change current response peak value and the concentration of the galectin-3.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102914648A (en) * | 2012-10-09 | 2013-02-06 | 重庆医科大学 | Electrochemical immunosensor for detecting toxoplasma gondii IgM antibody and preparation method thereof |
| CN106290521A (en) * | 2016-09-30 | 2017-01-04 | 重庆医科大学 | A kind of electrochemical sensor preparation method for ADRB1 1165G ＞ C genetic polymorphism detection |
| CN106483176A (en) * | 2016-09-30 | 2017-03-08 | 重庆医科大学 | A kind of electrochemical sensor preparation method for FGFR3 1138G ＞ A genetic polymorphism detection |
-
2017
- 2017-09-06 CN CN201710803421.1A patent/CN107576702B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102914648A (en) * | 2012-10-09 | 2013-02-06 | 重庆医科大学 | Electrochemical immunosensor for detecting toxoplasma gondii IgM antibody and preparation method thereof |
| CN106290521A (en) * | 2016-09-30 | 2017-01-04 | 重庆医科大学 | A kind of electrochemical sensor preparation method for ADRB1 1165G ＞ C genetic polymorphism detection |
| CN106483176A (en) * | 2016-09-30 | 2017-03-08 | 重庆医科大学 | A kind of electrochemical sensor preparation method for FGFR3 1138G ＞ A genetic polymorphism detection |
Non-Patent Citations (5)
| Title |
|---|
| A novel non-invasive detection method for the FGFR3 gene mutation in maternal plasma for a fetal achondroplasia diagnosis based on signal amplification by hemin-MOFs/PtNPs;Jun Chen,et al.;《Biosensors and Bioelectronics》;20161101;第91卷;892-899 * |
| A sensitive sandwich-type immunosensor for the detection of galectin-3 based on N-GNRs-Fe-MOFs@AuNPs nanocomposites and a novel AuPt-methylene blue nanorod;Zhiyong Tang,et al.;《Biosensors and Bioelectronics》;20171016;第101卷;253-259 * |
| D-(+)-Galactose-Conjugated Single-Walled Carbon Nanotubes as New Chemical Probes for Electrochemical Biosensors for the Cancer Marker Galectin-3;Young Kum Park,et al.;《Int. J. Mol. Sci.》;20110505;第12卷;2946-2957 * |
| Highly sensitive electrochemical immunosensor for the detection of alpha fetoprotein based on PdNi nanoparticles and N-doped graphene nanoribbons;Na Li,et al.;《Biosensors and Bioelectronics》;20150722;第74卷;786-791 * |
| Novel metal-organic nanocomposites：Poly(methylene blue)-Au and its application for an ultrasensitive electrochemical immunosensing platform;Jiao Shan,et al.;《Sensors and Actuators B》;20160627;第237卷;666-671 * |
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