高胰岛素处理相关核酸适体的筛选及基于核酸适体技术的胰岛素检测
发布时间：2018-08-11 11:34
【摘要】：第一部分建立针对肝细胞的指数级富集的配体系统进化技术 目的：建立针对肝细胞的指数级富集的配体系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment, SELEX),并获得能识别肝细胞表面膜蛋白的核酸适体(Aptamer)。 方法：设计DNA文库,两端各一段长20bp的引物序列,引物1用异硫氰酸荧光素(fluorescein isothiocyanate, FITC)标记,引物2用生物素biotin标记,中间20bp为随机序列。待培养皿(100mm)中人肝细胞癌细胞株HepG2生长丰度达到95%左右时,弃去细胞培养基,用4℃洗涤缓冲液洗涤。取20nmole DNA文库,溶于1ml结合缓冲液中,与细胞在4℃孵育1h后弃去上清液,洗涤缓冲液洗涤两次后,用细胞刮子收集细胞,用1ml蒸馏水洗涤,并将细胞悬液收集在1.5ml离心管中,在95℃加热15min后离心,取上清液,此即首轮筛选的母液,其内包含与HepG2结合的序列。经过聚合酶链式反应(PCR)扩增所得序列,扩增后的产物为双链DNA,包含目标链及其互补链。将PCR产物与链亲素(Streptavidin)覆盖的琼脂糖珠孵育后滤过上清液,用2%NaOH打开PCR双链结构,目标链则溶解于NaOH溶液中,将该溶液通过NAP-5脱盐柱脱去NaOH,测定单链DNA (ssDNA)浓度以确定该轮筛选终产物总量,并将其甩干。从上一轮筛选终产物中取100pmole,并将其溶于500ul含有10%胎牛血清的结合缓冲液中,将其与培养皿(60mm)中生长丰度达到95%左右的HepG2细胞孵育,按照上述方法完成每一轮筛选。每3-5轮筛选后需要检查每轮产物与细胞的结合情况。待细胞生长丰度达95%左右时,用非酶消化液消化细胞,将贴壁生长的细胞配成单个细胞悬液,用洗涤缓冲液洗涤后将细胞溶于结合缓冲液中,每5×105个细胞与25pmole的筛选产物于4℃孵育30min,筛选产物的浓度为250nM。同时将细胞与同等浓度的DNA文库孵育,作为阴性对照。孵育结束后洗涤细胞,用流式细胞仪分析筛选产物与细胞的结合程度。随着筛选轮数的增加,产物与细胞间的结合程度越来越高,直至达到平台期。选取结合程度最高的三轮产物进行测序,所得序列即针对靶细胞的核酸适体。用DNA合成仪合成所得序列并用流式细胞仪检测各条序列与靶细胞的结合程度,从而确定本筛选最终所得的核酸适体。分别用胰酶以及蛋白酶K处理细胞,处理时间从5min-30min不等,观察酶处理前后各核酸适体与细胞结合的情况。同时,将各核酸适体与不同种类的细胞孵育(包括LH86、Huh7、WT、IRS/KO、M7617、Ramos、CEM、H23、H69、A549、HBE、H661、TOV-21G、CAOV3等细胞),观察各序列的结合情况。 结果：本文通过应用SELEX对HepG2细胞展开筛选,经过测序分析发现了4条针对HepG2细胞的核酸适体：IR01、IR03、IR04以及IR06。其中IR01、IR04与靶细胞的亲和性最佳,其解离常数分别为11.287±3.786nM和88.849±22.339nM; IR03虽然与HepG2细胞结合程度较低,解离常数较高(129.513±47.924nM),但其特异性最好,能特异性与HepG2细胞结合,而与其它种类细胞几乎无交叉反应；IR01、IR04、IR06对大部分肝脏来源的细胞株均有较好结合；IR01是本实验所得的核酸适体当中与靶细胞结合程度最强的一条序列,其仅与肝脏来源的细胞有结合,而与其他组织来源的细胞无交叉反应,并且该条核酸适体与细胞的结合不容易被蛋白酶的作用所破坏。相反,IR03, IR04, IR06三条核酸适体与靶细胞的结合均容易被酶的消化而破环,胰酶或者蛋白酶K处理HepG2细胞5min后上述三条核酸适体与靶细胞的结合即被完全破坏。 结论：通过SELEX,可以获得与靶细胞高亲和性以及高特异性结合的核酸适体,这些核酸适体通过识别靶细胞膜上所表达的某种生物分子从而达到识别表达相同生物分子的不同细胞株。 第二部分筛选针对肝细胞表面与高胰岛素处理相关膜蛋白的核酸适体 目的：在第一部分实验的基础上筛选出针对肝细胞表面与高胰岛素处理相关膜蛋白的核酸适体(Aptamer)。 方法：待HepG2细胞生长丰度达70%左右后血清饥饿12h,然后分成四组,第一组继续予以不含胰岛素的培养基培养,第二组予以加入生理剂量的胰岛素(0.1nM)的培养基培养,第三组予以加入高胰岛素(100nM)的培养基培养,第四组予以加入超高胰岛素(500nM)的培养基培养。同时,予以高胰岛素(100nM)处理胎鼠肝细胞野生型(Wild type, WT)与胰岛素受体底物2基因(Insulin receptor substrate 2, IRS2)敲除后的胎鼠肝细胞(IRS2/KO),并设立正常培养组(不加入胰岛素处理)作为对照。上述各组细胞的培养基中均不含血清。24h后用非酶消化液消化细胞,将贴壁生长的细胞配成单个细胞悬液,用洗涤缓冲液洗涤后将细胞溶于结合缓冲液中,每5×105个细胞与浓度为250nM的核酸适体25pmole于4℃孵育30min。孵育结束后洗涤细胞,用流式细胞仪分析各核酸适体与上述各细胞的结合程度在高胰岛素处理前后有无变化。 结果：IR04与HepG2细胞的结合明显受到100nM胰岛素的抑制；进一步加大胰岛素的剂量(500nM)则抑制作用更加明显,经过500nM胰岛素处理后IR04与HepG2的结合几乎被完全消减；但IR04与HepG2细胞的结合不受生理剂量胰岛素的影响。同样,高胰岛素处理也能减弱IR04与WT细胞的结合；但IR04与IRS2/KO细胞的结合程度不受高胰岛素处理的影响。而IR01、IR03、IR06与细胞的结合均不受高胰岛素处理的影响。 结论：IR04与靶细胞的结合被高胰岛素处理所减弱,且其减弱的程度与高胰岛素的浓度呈正相关；但生理浓度范围内的胰岛素不影响IR04与靶细胞的结合；IR04可能的靶标为肝细胞表面某种与胰岛素作用相关的膜蛋白,此种膜蛋白在高胰岛素处理过程中被下调,但具体机制有待进一步研究；IR04所针对的靶物质在人、鼠肝细胞的表达具有高度同源性,且其受高胰岛素影响而发生下调的机理可能与人类相似。 第三部分基于核酸适体修饰石墨烯的胰岛素检测 目的：应用核酸适体修饰后的氧化石墨烯(GO)来进行胰岛素的检测,并通过加入催化剂(DNA酶)来实现其检测信号的放大。 方法：将天然石墨粉和氯化钠结晶研磨从而减小石墨颗粒的体积。去掉氯化钠后,将研磨过的石墨粉加入到浓硫酸中,强力搅拌下加入高锰酸钾,并用体积分数3%的双氧水还原剩余的高锰酸钾和二氧化锰,使其变为无色可溶的硫酸锰。在双氧水的处理下,悬浮液变成亮黄色。最后,过滤、洗涤3次,然后超声处理4h,离心后取上层溶液用于下一步实验。 异硫氰酸荧光素(FITC)标记的胰岛素核酸适体(insulin binding aptamer, IBA)稀释成100nM的胰岛素缓冲液。将IBA与GO按照摩尔浓度1：1于室温条件下混匀,避光孵育30min。此即本实验的工作溶液。由于GO能淬灭吸附在其表面的IBA所携带的荧光,此时溶液中仅存在一个较弱的荧光信号。当溶液中存在胰岛素时,与GO结合的IBA则从GO表面解离,与胰岛素结合,FITC标记的IBA游离到溶液中,GO对FITC的淬灭作用大大减弱,从而使得溶液中的荧光信号增加；进一步在工作溶液中加入DNA酶,加入不同浓度的胰岛素(胰岛素的加入量从5nM到50μM)后孵育2h。此时与胰岛素结合的IBA将被DNA酶消化成片段,从而失去与胰岛素结合的能力,胰岛素重新游离于工作液中；而与GO结合的IBA由于被GO保护,不被DNA酶消化,但将会从GO表面解离,并与游离的胰岛素结合,从而被DNA酶消化成片段。理论上该反应可以无限循环至IBA耗尽。终止反应后检测溶液中荧光浓度。同时设立阴性对照组(生物素、链霉亲和素、牛血清蛋白)以明确上述胰岛素检测体系的特异性。 结果：本实验通过将胰岛素核酸适体修饰在氧化石墨烯单层表面,成功构建了胰岛素的生物感应器,实现了胰岛素的便捷检测,检测下限为500nM；通过进一步加入DNA酶来实现信号放大后,胰岛素的检测下限降低到5nM。 结论：胰岛素核酸适体修饰后的氧化石墨烯可以作为胰岛素检测的良好工具,通过加入DNA酶实现检测信号的放大,将检测下限降低了100倍。
[Abstract]:The first part is to establish a system of phylogeny for exponential enrichment of hepatocytes.
OBJECTIVE: To establish a ligand system evolution technique (SELEX) for exponential enrichment of hepatocytes, and to obtain an aptamer (Aptamer) for recognizing hepatocyte surface membrane proteins.
METHODS: DNA libraries were designed with 20 BP primer sequences at each end. Primer 1 was labeled with fluorescein isothiocyanate (FITC), primer 2 was labeled with biotin, and the intermediate 20 BP was a random sequence. The cells were washed twice with the washing buffer. The cells were collected with a cell scraper and washed with 1 ml distilled water. The cell suspension was collected in a 1.5 ml centrifugal tube. The supernatant was centrifuged at 95 C for 15 minutes. The mother liquor of the round-screening contains the sequence bound to HepG2. The amplified product is double-stranded DNA containing target and complementary chains. The PCR product is incubated with streptavidin-coated agarose beads and the supernatant is filtered. The double-stranded structure of the PCR is opened with 2% NaOH, and the target chain is dissolved. NaOH was removed from NaOH solution by NAP-5 desalination column. Single-stranded DNA (ssDNA) concentration was determined to determine the total amount of the final screening product and then dried. 100 pmole was extracted from the final screening product and dissolved in a combination buffer containing 10% fetal bovine serum at 500 ul. The growth abundance of the final product in the culture dish (60mm) was 95%. After every 3-5 rounds of screening, we need to check the binding of the products to the cells. When the cell growth abundance reaches about 95%, the cells are digested with non-enzymatic digestive juice. The adherent cells are prepared into a single cell suspension and washed with a washing buffer to dissolve the cells into a combination. In the buffer solution, the screening products of 25 pmole and 5 *105 cells were incubated at 4 C for 30 min and the concentration of screening products was 250 nM. At the same time, the cells were incubated with DNA libraries of the same concentration as negative control. After incubation, the cells were washed and the binding degree between screening products and cells was analyzed by flow cytometry. Three rounds of products with the highest binding degree were selected for sequencing, and the sequence was aptamer for target cells. The DNA synthesized by DNA synthesizer and the binding degree between each sequence and target cells was detected by flow cytometry to determine the final nucleic acid. Aptamers were incubated with trypsin and protease K for 5 min to 30 min, respectively. The aptamers were observed before and after treatment. At the same time, aptamers were incubated with different kinds of cells (including LH86, Huh7, WT, IRS/KO, M7617, Ramos, CEM, H23, H69, A549, HBE, H661, TOV-21G, CAOV3, etc.) and observed. The combination of sequences.
RESULTS: Four aptamers, IR01, IR03, IR04 and IR06, were identified by SELEX. Among them, IR01, IR04 had the best affinity with target cells, and their dissociation constants were 11.287 (+ 3.786nM) and 88.849 (+ 22.339nM), respectively. The dissociation constant was high (129.513 47.924nM), but its specificity was the best, and it could bind to HepG2 cells with specificity and hardly cross-react with other kinds of cells; IR01, IR04, IR06 had good binding to most of the hepatic cell lines; IR01 was the most binding sequence between the aptamer and the target cells. In contrast, the binding of three aptamers IR03, IR04 and IR06 to target cells is easily broken by enzymatic digestion, and the binding of aptamers to target cells is treated by trypsin or protease K. The binding of these three aptamers to target cells was completely destroyed after HepG2 cells 5min.
Conclusion: High affinity and specific binding aptamers with target cells can be obtained by SELEX. These aptamers can identify different cell lines expressing the same biological molecules by recognizing some biological molecules expressed on the target cell membrane.
The second part screened aptamers targeting hepatocyte surface associated with high insulin processing membrane proteins.
AIM: To screen aptamers (Aptamers) targeting the membrane proteins associated with hyperinsulinemia on the surface of hepatocytes.
Methods: After the growth of HepG2 cells was about 70%, the serum was starved for 12 hours, and then divided into four groups. The first group continued to be cultured in insulin-free medium, the second group was cultured in a physiological dose of insulin (0.1nM), the third group was cultured in a high insulin (100nM) medium, and the fourth group was cultured in a super-high insulin (100nM) medium. At the same time, the wild type (WT) and insulin receptor substrate 2 (IRS2) knockout fetal rat hepatocytes (IRS2 / KO) were treated with high insulin (100nM) and the normal culture group (without insulin) was set up as control. No serum was found in the medium. After 24 hours, the adherent cells were digested with non-enzymatic digestive solution. The adherent cells were prepared into a single cell suspension. After washing with washing buffer, the cells were dissolved in the binding buffer. Every 5 *105 cells and 250 nM aptamer 25 pmole were incubated at 4 C for 30 minutes. The binding degree of aptamers to these cells was analyzed before and after high insulin treatment.
RESULTS: The binding of IR04 to HepG2 cells was inhibited by 100nM insulin, but the binding of IR04 to HepG2 cells was almost completely reduced after 500nM insulin treatment. Similarly, the binding of IR04 to HepG2 cells was not affected by physiological dose of insulin. The binding of IR04 to WT cells was also weakened by hormone treatment, but the binding of IR04 to IRS2/KO cells was not affected by high insulin treatment, while the binding of IR01, IR03 and IR06 to WT cells was not affected by high insulin treatment.
CONCLUSION: The binding of IR04 to target cells is weakened by hyperinsulinemia, and the degree of reduction is positively correlated with the concentration of hyperinsulinemia; however, insulin in the physiological concentration range does not affect the binding of IR04 to target cells; the possible target of IR04 is a membrane protein associated with insulin action on the surface of hepatocytes, which is high in content. Insulin is down-regulated during insulin treatment, but the specific mechanism remains to be further studied; the target substance IR04 is highly homologous to human and mouse hepatocytes, and its mechanism may be similar to human.
The third part is insulin detection based on aptamer modified graphene.
AIM: To detect insulin with aptamer modified graphene oxide (GO) and amplify the detection signal by adding a catalyst (DNA enzyme).
After removing sodium chloride, the grinded graphite powder was added into concentrated sulfuric acid, potassium permanganate was added under strong stirring, and the remaining potassium permanganate and manganese dioxide were reduced by 3% hydrogen peroxide to form colorless and soluble manganese sulfate. After treatment with hydrogen peroxide, the suspension turns bright yellow. Finally, the suspension is filtered and washed three times, and then treated by ultrasonic wave for 4 hours.
An insulin binding aptamer (IBA) labeled with fluorescein isothiocyanate (FITC) was diluted into a 100nM insulin buffer. IBA and GO were mixed at room temperature at a molar concentration of 1:1 to avoid light incubation for 30 minutes. This is the working solution of this experiment. Because GO can quench the fluorescence carried by IBA adsorbed on its surface, at this time the fluorescence can be quenched. There is only a weak fluorescence signal in the solution. When there is insulin in the solution, the IBA bound with GO dissociates from the GO surface, binds with insulin, and the IBA labeled with FITC dissociates into the solution. The quenching effect of GO on FITC is greatly weakened, so that the fluorescence signal in the solution is increased. Different concentrations of insulin (insulin dosage from 5nM to 50mu M) were incubated for 2h. At this time, the insulin-bound IBA was digested into fragments by DNA enzymes, thus losing the ability to bind to insulin, and insulin was re-dissociated in the working fluid; while the GO-bound IBA was protected from digestion by DNA enzymes, but would be dissociated from the GO surface, and In theory, the reaction can be circulated indefinitely until IBA is exhausted. Fluorescence concentration in the solution is detected after termination of the reaction. A negative control group (biotin, streptavidin, bovine serum protein) is set up to determine the specificity of the above insulin detection system.
RESULTS: By modifying the aptamer of insulin nucleic acid on the surface of graphene oxide monolayer, the biosensor of insulin was successfully constructed, and the detection limit of insulin was 500 nM. The detection limit of insulin was reduced to 5 nM by adding DNA enzyme to amplify the signal.
Conclusion: The graphene oxide modified with aptamer insulin nucleic acid can be used as a good tool for insulin detection. The detection limit can be reduced 100 times by adding DNA enzyme to amplify the detection signal.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346
本文编号：2176884
[Abstract]:The first part is to establish a system of phylogeny for exponential enrichment of hepatocytes.
OBJECTIVE: To establish a ligand system evolution technique (SELEX) for exponential enrichment of hepatocytes, and to obtain an aptamer (Aptamer) for recognizing hepatocyte surface membrane proteins.
METHODS: DNA libraries were designed with 20 BP primer sequences at each end. Primer 1 was labeled with fluorescein isothiocyanate (FITC), primer 2 was labeled with biotin, and the intermediate 20 BP was a random sequence. The cells were washed twice with the washing buffer. The cells were collected with a cell scraper and washed with 1 ml distilled water. The cell suspension was collected in a 1.5 ml centrifugal tube. The supernatant was centrifuged at 95 C for 15 minutes. The mother liquor of the round-screening contains the sequence bound to HepG2. The amplified product is double-stranded DNA containing target and complementary chains. The PCR product is incubated with streptavidin-coated agarose beads and the supernatant is filtered. The double-stranded structure of the PCR is opened with 2% NaOH, and the target chain is dissolved. NaOH was removed from NaOH solution by NAP-5 desalination column. Single-stranded DNA (ssDNA) concentration was determined to determine the total amount of the final screening product and then dried. 100 pmole was extracted from the final screening product and dissolved in a combination buffer containing 10% fetal bovine serum at 500 ul. The growth abundance of the final product in the culture dish (60mm) was 95%. After every 3-5 rounds of screening, we need to check the binding of the products to the cells. When the cell growth abundance reaches about 95%, the cells are digested with non-enzymatic digestive juice. The adherent cells are prepared into a single cell suspension and washed with a washing buffer to dissolve the cells into a combination. In the buffer solution, the screening products of 25 pmole and 5 *105 cells were incubated at 4 C for 30 min and the concentration of screening products was 250 nM. At the same time, the cells were incubated with DNA libraries of the same concentration as negative control. After incubation, the cells were washed and the binding degree between screening products and cells was analyzed by flow cytometry. Three rounds of products with the highest binding degree were selected for sequencing, and the sequence was aptamer for target cells. The DNA synthesized by DNA synthesizer and the binding degree between each sequence and target cells was detected by flow cytometry to determine the final nucleic acid. Aptamers were incubated with trypsin and protease K for 5 min to 30 min, respectively. The aptamers were observed before and after treatment. At the same time, aptamers were incubated with different kinds of cells (including LH86, Huh7, WT, IRS/KO, M7617, Ramos, CEM, H23, H69, A549, HBE, H661, TOV-21G, CAOV3, etc.) and observed. The combination of sequences.
RESULTS: Four aptamers, IR01, IR03, IR04 and IR06, were identified by SELEX. Among them, IR01, IR04 had the best affinity with target cells, and their dissociation constants were 11.287 (+ 3.786nM) and 88.849 (+ 22.339nM), respectively. The dissociation constant was high (129.513 47.924nM), but its specificity was the best, and it could bind to HepG2 cells with specificity and hardly cross-react with other kinds of cells; IR01, IR04, IR06 had good binding to most of the hepatic cell lines; IR01 was the most binding sequence between the aptamer and the target cells. In contrast, the binding of three aptamers IR03, IR04 and IR06 to target cells is easily broken by enzymatic digestion, and the binding of aptamers to target cells is treated by trypsin or protease K. The binding of these three aptamers to target cells was completely destroyed after HepG2 cells 5min.
Conclusion: High affinity and specific binding aptamers with target cells can be obtained by SELEX. These aptamers can identify different cell lines expressing the same biological molecules by recognizing some biological molecules expressed on the target cell membrane.
The second part screened aptamers targeting hepatocyte surface associated with high insulin processing membrane proteins.
AIM: To screen aptamers (Aptamers) targeting the membrane proteins associated with hyperinsulinemia on the surface of hepatocytes.
Methods: After the growth of HepG2 cells was about 70%, the serum was starved for 12 hours, and then divided into four groups. The first group continued to be cultured in insulin-free medium, the second group was cultured in a physiological dose of insulin (0.1nM), the third group was cultured in a high insulin (100nM) medium, and the fourth group was cultured in a super-high insulin (100nM) medium. At the same time, the wild type (WT) and insulin receptor substrate 2 (IRS2) knockout fetal rat hepatocytes (IRS2 / KO) were treated with high insulin (100nM) and the normal culture group (without insulin) was set up as control. No serum was found in the medium. After 24 hours, the adherent cells were digested with non-enzymatic digestive solution. The adherent cells were prepared into a single cell suspension. After washing with washing buffer, the cells were dissolved in the binding buffer. Every 5 *105 cells and 250 nM aptamer 25 pmole were incubated at 4 C for 30 minutes. The binding degree of aptamers to these cells was analyzed before and after high insulin treatment.
RESULTS: The binding of IR04 to HepG2 cells was inhibited by 100nM insulin, but the binding of IR04 to HepG2 cells was almost completely reduced after 500nM insulin treatment. Similarly, the binding of IR04 to HepG2 cells was not affected by physiological dose of insulin. The binding of IR04 to WT cells was also weakened by hormone treatment, but the binding of IR04 to IRS2/KO cells was not affected by high insulin treatment, while the binding of IR01, IR03 and IR06 to WT cells was not affected by high insulin treatment.
CONCLUSION: The binding of IR04 to target cells is weakened by hyperinsulinemia, and the degree of reduction is positively correlated with the concentration of hyperinsulinemia; however, insulin in the physiological concentration range does not affect the binding of IR04 to target cells; the possible target of IR04 is a membrane protein associated with insulin action on the surface of hepatocytes, which is high in content. Insulin is down-regulated during insulin treatment, but the specific mechanism remains to be further studied; the target substance IR04 is highly homologous to human and mouse hepatocytes, and its mechanism may be similar to human.
The third part is insulin detection based on aptamer modified graphene.
AIM: To detect insulin with aptamer modified graphene oxide (GO) and amplify the detection signal by adding a catalyst (DNA enzyme).
After removing sodium chloride, the grinded graphite powder was added into concentrated sulfuric acid, potassium permanganate was added under strong stirring, and the remaining potassium permanganate and manganese dioxide were reduced by 3% hydrogen peroxide to form colorless and soluble manganese sulfate. After treatment with hydrogen peroxide, the suspension turns bright yellow. Finally, the suspension is filtered and washed three times, and then treated by ultrasonic wave for 4 hours.
An insulin binding aptamer (IBA) labeled with fluorescein isothiocyanate (FITC) was diluted into a 100nM insulin buffer. IBA and GO were mixed at room temperature at a molar concentration of 1:1 to avoid light incubation for 30 minutes. This is the working solution of this experiment. Because GO can quench the fluorescence carried by IBA adsorbed on its surface, at this time the fluorescence can be quenched. There is only a weak fluorescence signal in the solution. When there is insulin in the solution, the IBA bound with GO dissociates from the GO surface, binds with insulin, and the IBA labeled with FITC dissociates into the solution. The quenching effect of GO on FITC is greatly weakened, so that the fluorescence signal in the solution is increased. Different concentrations of insulin (insulin dosage from 5nM to 50mu M) were incubated for 2h. At this time, the insulin-bound IBA was digested into fragments by DNA enzymes, thus losing the ability to bind to insulin, and insulin was re-dissociated in the working fluid; while the GO-bound IBA was protected from digestion by DNA enzymes, but would be dissociated from the GO surface, and In theory, the reaction can be circulated indefinitely until IBA is exhausted. Fluorescence concentration in the solution is detected after termination of the reaction. A negative control group (biotin, streptavidin, bovine serum protein) is set up to determine the specificity of the above insulin detection system.
RESULTS: By modifying the aptamer of insulin nucleic acid on the surface of graphene oxide monolayer, the biosensor of insulin was successfully constructed, and the detection limit of insulin was 500 nM. The detection limit of insulin was reduced to 5 nM by adding DNA enzyme to amplify the signal.
Conclusion: The graphene oxide modified with aptamer insulin nucleic acid can be used as a good tool for insulin detection. The detection limit can be reduced 100 times by adding DNA enzyme to amplify the detection signal.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346
【参考文献】
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1 陈俊意;黄爱龙;徐莉;陈典全;余虹;朱照静;黄祖春;杨宗发;陈立书;谭涛;;HBV亚基因型B和C体外重组中间体的检测(英文)[J];中南大学学报(医学版);2011年02期
，本文编号：2176884
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