研究报告：鳗弧菌（Vibrio anguillarum）核酸适配体的筛选及其结合蛋白的分离鉴定

目的鳗弧菌（Vibrio anguillarum）是水产养殖中的重要条件致病菌,每年给水产养殖业造成巨大的经济损失,研究其致病机制、对其进行快速的检测鉴定是其病害防治的前提和基础。核酸适配体因其高亲和力、高特异性等多种优点,在微生物的靶标分析、检测鉴定以及致病机制等多个领域都呈现出较好的应用潜力。因此,筛选鳗弧菌的核酸适配体,利用核酸适配体对鳗弧菌相关位点进行分析鉴定,不仅能为鳗弧菌的检测鉴定提供一个新的手段,对于探索鳗弧菌相关位点在其病害防治中的作用也具有重要意义。方法以鳗弧菌为靶目标,采用每轮测序的SELEX筛选方法,从高频序列中筛选鳗弧菌的核酸适配体;采用单链DNA浓度法测定核酸适配体的亲和力,研究核酸适配体对鳗弧菌的亲和特异性;采用Origin软件、选择反比例函数（Hyperbola函数）进行非线性拟合,获得核酸适配体的亲和常数（K_d）和最大亲和力（A_m）;采用磁分离技术和聚丙烯酰胺凝胶电泳分离纯化出核酸适配体H5的结合蛋白,通过质谱对该蛋白质进行分析鉴定,并利用Prabi、Phyre2、Psortb 3.0等在线网站分析该结合蛋白的...     

适配体结合蛋白质靶标的亲和力表征方法及其在疾病诊断中的应用

核酸适配体是通过体外指数富集配体系统进化（SELEX）技术筛选获得,并能够和蛋白质靶标高特异性、高亲和力结合的单链寡核苷酸。核酸适配体不但具有抗体的识别特性,而且具有自己独特的优良性能,目前已应用于分析检验、食品安全和生物医药等各个领域。蛋白质具有多种多样的生物功能以及临床诊断价值。因此,核酸适配体针对蛋白质靶标并在蛋白质相关的基础研究领域受到广泛的关注。核酸适配体应用性能的优劣取决于与其靶标蛋白质的亲和力与特异性。本文主要综述核酸适配体对蛋白质靶标的亲和力表征方法,以及在药物研发、肿瘤检测、生物成像以及生物传感器方面的应用。     

基于荧光染料PicoGreen和核酸适配体的伏马毒素B1检测方法

伏马毒素B1是主要存在于玉米及玉米制品中的一种可以引起癌症的霉菌毒素。针对霉菌毒素精准检测技术的开发对于保障食品安全至关重要。本研究利用核酸适配体与伏马毒素B1结合后不再结合其互补核酸序列的选择性以及Pico Green与双链DNA结合的特异性,开发了一种快速检测伏马毒素B1的适配体方法。Pico Green与双链DNA反应15 min后激发产生的荧光达到峰值(激发波长为480 nm,发射波长为520 nm)。该方法的最低检测限为0.1μg/L(0.1 ppb),线性范围为0.1-1μg/L(0.1-1 ppb),整个检测流程可在40 min内完成。特异性试验显示伏马毒素B1适配体与黄曲霉毒素B1、赭曲霉毒素、桔毒素和玉米赤霉烯酮等常见霉菌毒素无交叉反应。结果表明适配体方法与基于抗体的检测伏马毒素B1商品化ELISA试剂盒相当,Kappa值为0.857。由于核酸适配体比抗体成本低,检测时间短,因此基于核酸适配体的方法比基于抗体的ELISA方法更具有推广应用价值。     

全细菌SELEX法筛选粪肠球菌特异性适配体

肠球菌（Enterococcus）是内源性和外源性医院感染的第二大病原菌,检出率仅次于大肠杆菌,从分子水平上发展靶标的高亲和力分子探针对肠球菌的识别和检测具有非常重要的意义。本研究以粪肠球菌为靶标,运用全细菌指数富集的配体系统进化技术（whole-bacteria systematic evolution of ligands by exponential enrichment, whole-bacteria SELEX）,从全长为79个核苷酸包含35个随机碱基序列的单链DNA文库中筛选与靶标高亲和力、高特异性结合的适配体,利用荧光分析法监控筛选过程中不同轮次所得次级文库与粪肠球菌的结合力,经12轮筛选和克隆测序,获得了39条适配体序列。进一步对筛选得到的适配体进行序列比对、二级结构分析、流式细胞分析、解离常数（Kd）测定及特异性验证,最终获得一条与粪肠球菌能特异性结合的适配体Apt 21,其Kd值为549.2 ± 147.4 nmol/L。该适配体可作为粪肠球菌检测的识别元件,为建立基于适配体的新型粪肠球菌检测方法奠定了基础。     

综述与专论: 核酸适配体筛选与亲和力评价技术主要研究方向、进展与挑战

核酸适配体是一类具有特异性分子识别能力的单链DNA或者RNA分子,通过指数富集的配体系统进化技术（SELEX）筛选得到。核酸适配体相比抗体具有热稳定性高、便于化学合成与修饰、免疫原性低等优点,在生物分析、生物医学、生物技术等众多领域引起广泛关注。高质量的核酸适配体是应用的基础,然而目前能够满足实际应用的核酸适配体数量还非常有限。如何获得高亲和力、高特异性、高体内稳定性的核酸适配体是核酸适配体领域的技术瓶颈。本文首先简单介绍了SELEX技术的基本原理和核酸库的设计、筛选过程监控、次级文库制备、测序和候选适配体筛选等关键步骤。接着归纳总结了30多年来核酸适配体筛选技术的6个主要研究方向、研究进展和局限性。这6个主要研究方向分别是提高适配体特异性的筛选方法、提高适配体稳定性（抗核酸酶降解能力）的筛选方法、快速筛选方法、复杂靶标适配体筛选方法、小分子靶标适配体筛选方法、提高适配体亲和力的筛选方法。其中快速筛选技术是长期以来持续关注的研究方向,几乎所有物理分离手段都已用于提高SELEX的筛选效率。最近,高效化学反应与SELEX技术的结合为核酸适配体的快速筛选提供了新的策略。本文随后对适合小分子靶标核酸适配体筛选的3类方法进展和存在的问题进行了重点评述。这3类方法分别是基于靶标固定的筛选技术、基于文库固定的筛选技术（捕获-SELEX,Capture-SELEX）和均相筛选技术（氧化石墨烯-SELEX,GO-SELEX）。基于靶标固定的筛选技术尽管存在空间位阻等众多问题,由于其操作的简单性,目前依然应用广泛。近年来Capture-SELEX应用广泛。结合36种靶标适配体的筛选实验条件（文库设计、正筛靶标浓度、负筛靶标的选择和浓度）和所获得的适配体的亲和力（K_D,解离常数,dissociation constant）和特异性,对Capture-SELEX的实验条件与适配体性能的关系进行了讨论。统计数据表明,降低正筛靶标浓度有利于提高适配体的亲和力,但不是必要条件。负筛选是目前提高适配体特异性的主要技术手段,但适配体的特异性还不能满足实际需求。负筛选靶标及其浓度的选择差异很大,而且36种靶标中有20种靶标的适配体筛选没有进行负筛选。如何提高核酸适配体的特异性是目前小分子靶标核酸适配体所面临的难题,急需寻找新的策略。本文还列表归纳了近三年利用GO-SELEX进行的13种小分子靶标的实验条件和所获得的适配体的K_D和特异性。统计数据表明,GO-SELEX比Capture-SELEX所需要的筛选轮数少,两种方法所获得的适配体的亲和力多在纳摩尔每升水平。Capture-SELEX相对较低的筛选效率应该主要由于文库的自解离问题。核酸适配体的亲和力评价是候选核酸适配体结构与性能评价的重要组成部分。常用的核酸适配体亲和力评价技术包括基于分离、基于固定、均相体系三大类十多种方法。假阳性和假阴性是各种评价技术都有可能存在的问题。本文以纳米金比色法和等温热滴定技术为例评述技术进展,讨论导致不同亲和力评价技术结果不一致性问题的根本原因。本文最后对核酸适配体筛选技术、亲和力评价技术和技术的标准化的未来发展趋势进行了展望。     

基于高分辨质谱及分子对接技术研究适配体与倍氯米松的相互作用

倍氯米松(beclomethasone)是一种有效的糖皮质激素,而倍氯米松适配体是对倍氯米松具有亲和力与特异性的单链DNA分子。目前对两者的相互作用仍不清楚,研究适配体与药物的相互作用对适配体的应用具有一定的意义。本研究采用高分辨傅里叶变换离子回旋共振质谱仪（FT-MS）及分子对接软件计算模拟研究适配体与倍氯米松的相互作用。首先,在优化的高分辨质谱条件下,通过电喷雾离子源负离子扫描模式对适配体与倍氯米松复合物进行检测。测得复合物多为-8价离子。在饱和前,适配体与倍氯米松化学结合计量数之比为1∶1,其解离常数Kd值为1.01±0.23 μmol/L。经分子对接软件模拟,获得适配体与倍氯米松的化学结合计量数之比为1∶1,两者结合自由能为-24 kcal/mol,主要以氢键作用力相结合。最后,使用超微量紫外检测两者结合前后的吸收波长变化以验证两者的结合,并获得适配体与倍氯米松复合物的解离常数Kd值为1.67 ± 0.35 μmol/L,其结果与高分辨FT-MS结果相近。高分辨FT-MS检测与分子对接模拟不仅提供了适配体与倍氯米松的亲和力,也提供了两者的化学结合计量数之比等其它相互作用信息,对深化适配体的应用具有一定的意义。     

研究报告: 基于分子对接和步进序列群的蓖麻毒素核酸适配体序列优化

目的 有效结合分子对接预测和表面等离子体共振实验评价技术，获得亲和力更强、序列最短的最优适配体。方法 针对前期筛选出的靶向蓖麻毒素的3条80 nt单链DNA适配体（L14、P3、L7），在明确各自二维随机区茎环序列与靶蛋白结合能力的基础上，以H-DOCK分子对接为指导，分别确定蓖麻毒素适配体随机区的最短结合单元，从而构建两端延长步进序列群，以表面等离子体共振技术测定序列群序列的亲和力和动力学参数，明确适配体的结合关键结构，从而筛选得到最优适配体。结果 3条全长适配体的随机区适配体L14r、P3r、L7r均可形成一定的茎环结构，其中L14r较L14的亲和力增强9倍、L7r增强2倍、P3r基本不变。对随机区适配体和蓖麻毒素进行分子对接，结果显示，L14r、P3r、L7r的对接分数值皆优于阴性序列40T，结合关键氨基酸个数分别为11、8、9个，存在距离小于5 ?的预测结合位点分别为20、12、15个，具有良好的与蓖麻毒素的结合能力。进一步明确了蓖麻毒素活性口袋所容纳的适配体最短结合单元L14rm、P3rm、L7rm的序列构成，在此基础上构建出两端延长步进序列群。针对该步进群，基于结合关键氨基酸个数、结合位点个数、对接得分等参数的变化和表面等离子体共振测定结果筛选出最优适配体。所获得的最优适配体L14rm、L7rm-2亲和力继续增强了1~2倍。结论 随机区适配体能有效地与蓖麻毒素结合，较之全长适配体亲和力更强，分子对接结合步进序列群设计，仅使用17条序列，便有效获得了3条最优适配体并明确其结合作用。3条结合蓖麻毒素的最优适配体——L14rm、P3r、L7rm-2的K_D值分别为（64±30）、（167±19）、（120±1）nmol/L，亲和力提高到全长适配体的14、1、4倍。     

适配体与病毒性感染诊断及治疗

适配体（Aptamers）是通过指数富集的配体系统进化（systematic evolution of ligands by exponential enrichment,SELEX）技术,从随机核酸文库中筛选出来的单链寡核苷酸,已在临床医疗及其他领域得到日益广泛的应用.与抗体相比,适配体具有很多优点,如高亲和力、高特异性、分子量小、几乎无免疫排斥反应、结构稳定、易于合成等.可用于适配体筛选的靶标范围非常广,包括有机小分子、蛋白、完整细胞及病毒颗粒等.迅速可靠的病原检测对于病毒性传染病的成功预防和治疗具有重要意义.随着严格筛选和快速分离技术的进步,适配体在病毒感染的检测治疗中显示出巨大的潜力.本文概括介绍了适配体在病毒研究方面的最新应用进展及未来前景.     

金黄色葡萄球菌毒性休克综合征毒素1核酸适配体的筛选和鉴定

目的:筛选能特异性识别毒性休克综合征毒素1(TSST-1)的DNA适配体,为金黄色葡萄球菌感染的治疗奠定实验基础。方法:体外合成含有25个随机序列全长为63个碱基的单链DNA(ss DNA)文库,以TSST-1为靶标,利用指数富集的配体系统进化技术(SELEX),从ss DNA文库中筛选TSST-1适配体,利用生物信息学方法对适配体进行序列分析和结构预测,荧光定量法分析适配体的亲和力及特异性。结果:经过8轮筛选,文库的亲和力不断升高,获得了能识别TSST-1的DNA适配体T-7,它可选择性地与TSST-1结合,并测定其Kd值为103.8 nmol/L。结论:新型适配体T-7能选择性识别TSST-1,在金黄色葡萄球菌感染的治疗和诊断方面具有应用前景。     

小分子靶标与其核酸适配体亲和力的表征方法

核酸适配体是指通过SELEX筛选得到的能与靶标高特异性、高亲和力结合的单链DNA或RNA。目前国内外具有高亲和性和高特异性结合的小分子靶标的核酸适配体依然很少,究其原因,一方面是因为小分子靶标的适配体难以筛选,另一方面是小分子靶标与其候选适配体亲和力表征方法难以确定。亲和力表征是确定适配体筛选成功与否的关键步骤,就现有的小分子靶标与其相应适配体亲和力表征方法进行了总结,包括纳米金比色法、等温滴定量热法、表面等离子共振、圆二色谱法、石英晶体微天平法、微量热泳动法和SYBR Green I染料检测法等,并分析了这些方法的优缺点及改进建议,以期有助于提高适配体表征效率。     

Probing high affinity sequences of DNA aptamer against VEGF165

Vascular endothelial growth factor (VEGF(165)) is a potent angiogenic mitogen commonly overexpressed in cancerous cells. It contains two main binding domains, the receptor-binding domain (RBD) and the heparin-binding domain (HBD). This study attempted to identify the specific sequences of the VEa5 DNA aptamer that exhibit high binding affinity towards the VEGF(165) protein by truncating the original VEa5 aptamer into different segments. Using surface plasmon resonance (SPR) spectroscopy for binding affinity analysis, one of the truncated aptamers showed a >200-fold increase in the binding affinity for HBD. This truncated aptamer also exhibited high specificity to HBD with negligible binding affinity for VEGF(121), an isoform of VEGF lacking HBD. Exposing colorectal cancer cells to the truncated aptamer sequence further confirmed the binding affinity and specificity of the aptamer to the target VEGF(165) protein. Hence, our approach of aptamer truncation can potentially be useful in identifying high affinity aptamer sequences for the biological molecules and targeting them as antagonist for cancer cell detection.     

鳗弧菌(Vibrio anguillarum)核酸适配体的筛选及其结合蛋白的分离鉴定

目的 鳗弧菌(Vibrio anguillarum)是水产养殖中的重要条件致病菌,每年给水产养殖业造成巨大的经济损失,研究其致病机制、对其进行快速的检测鉴定是其病害防治的前提和基础.核酸适配体因其高亲和力、高特异性等多种优点,在微生物的靶标分析、检测鉴定以及致病机制等多个领域都呈现出较好的应用潜力.因此,筛选鳗弧菌的核...     

Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices

An aptamer is an artificial functional oligonucleic acid, which can interact with its target molecule with high affinity and specificity. Enzyme linked aptamer assay (ELAA) is developed to detect cocaine using aptamer fragment/cocaine configuration based on the affinity interaction between aptamer fragments with cocaine. The aptasensor was constructed by cleaving anticocaine aptamer into two fragments: one was assembled on a gold electrode surface, while the other was modified with biotin at 3'-end, which could be further labelled with streptavidin-horseradish peroxidase (SA-HRP). Upon binding with cocaine, the HRP-labelled aptamer fragment/cocaine complex formed on the electrode would increase the reduction current of hydroquinone (HQ) in the presence of H(2)O(2). The sensitivity and the specificity of the proposed electrochemical aptasensor were investigated by differential pulse voltammetry (DPV). The results indicated that the DPV signal change could be used to sensitively detect cocaine with the dynamic range from 0.1 μM to 50 μM and the detection limit down to 20 nM (S/N=3). The proposed aptasensor has the advantages of high sensitivity and low background current. Furthermore, a new configuration for ELAA requiring only a single aptamer sequence is constructed, which can be generalized for detecting different kinds of targets by cleaving the aptamers into two suitable segments.     

Fluorescence detection of adenosine triphosphate through an aptamer-molecular beacon multiple probe

An aptamer-molecular beacon (MB) multiple fluorescent probe for adenosine triphosphate (ATP) assay is proposed in this article. The ATP aptamer was used as a molecular recognition part, and an oligonucleotide (short strand, SS) partially complementary with the aptamer and an MB was used as the other part. In the presence of ATP, the aptamer bound with it, accompanied by the hybridization of MB and SS and the fluorescence recovering. Wherever there is only very weak fluorescence can be measured in the absence of ATP. Based on the relationship of recovering fluorescence and the concentration of ATP, a method for quantifying ATP has been developed. The fluorescence intensity was proportional to the concentration of ATP in the range of 10 to 500 nM with a detection limit of 0.1 nM. Moreover, this method was able to detect ATP with high selectivity in the presence of guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). This method is proved to be simple with high sensitivity, selectivity, and specificity.     

Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX

The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K _d value) of 292.8 ± 53.1 nM with 47.27 ± 5.58% cells fluorescent (bound) in a 1.48-μM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25–36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1–5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets.     

Development of HBsAg-binding aptamers that bind HepG2.2.15 cells via HBV surface antigen

Hepatitis B virus surface antigen(HBsAg),a specific antigen on the membrane of Hepatitis B virus (HBV)-infected cells,provides a perfect target for therapeutic drugs.The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection.Herein,we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes.One high affinity aptamer,HBs-A22,was isolated from an initial 115 mer library of ~1.1×10 15 random-sequence RNA molecules using the SELEX procedure.The selected aptamer HBs-A22 bound specifically to hepatoma cell line HepG2.2.15 that expresses HBsAg but did not bind to HBsAg-devoid HepG2 cells.This is the first reported RNA aptamer which could bind to a HBV specific antigen.This newly isolated aptamer could be modified to deliver imaging,diagnostic,and therapeutic agents targeted at HBV-infected cells.     

A Y2 receptor mimetic aptamer directed against neuropeptide Y.

Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that exerts its activity by at least five different receptor subtypes that belong to the family of G-protein-coupled receptors. We isolated an aptamer directed against NPY from a nuclease-resistant RNA library. Mapping experiments with N-terminally, C-terminally, and centrally truncated analogues of NPY revealed that the aptamer recognizes the C terminus of NPY. Individual replacement of the four arginine residues at positions 19, 25, 33, and 35 by l-alanine showed that arginine 33 is essential for binding. The aptamer does not recognize pancreatic polypeptide, a highly homologous Y4 receptor-specific peptide of the gut. Furthermore, the affinity of the aptamer to the Y5 receptor-selective agonist [Ala(31),Aib(32)]NPY and the Y1/Y5 receptor-binding peptide [Leu(31),Pro(34)]NPY was considerably reduced, whereas Y2 receptor-specific NPY mutants were bound well by the aptamer. Accordingly, the NPY epitope was recognized by the Y2 receptor, and the aptamer was highly similar. This Y2 receptor mimicking effect was further confirmed by competition binding studies. Whereas the aptamer competed with the Y2 receptor for binding of [(3)H]NPY with high affinity, a low affinity displacement of [(3)H]NPY was observed at the Y1 and the Y5 receptors. Consequently, competition at the Y2 receptor occurred with a considerably lower K(i) value compared with the Y1 and Y5 receptors. These results indicate that the aptamer mimics the binding of NPY to the Y2 receptor more closely than to the Y1 and Y5 receptors.     

Aptamer from whole-bacterium SELEX as new therapeutic reagent against virulent Mycobacterium tuberculosis

Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. One-third of the world's population is infected with Mycobacterium tuberculosis (MTB), the etiologic agent of TB. Because of the global health problems of TB, the development of potent new anti-TB drugs without cross-resistance with known antimycobacterial agents is urgently needed. In this study, we have applied a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process to identify a single aptamer (NK2) that binds to virulent strain M. tuberculosis (H37Rv) with high affinity and specificity. We have found that this aptamer improves CD4(+)T cells to produce IFN-gamma after binding to H37Rv. The different component between H37Rv and BCG was identified as some membrane protein. Moreover, the survival rates of mice challenged with i.v. H37Rv have been prolonged after treatment with single injection of aptamer NK2. The bacterial numbers were significantly lower in the spleen of mice treated with aptamer NK2. The histopathological examination of lung biopsy specimens showed lesser pulmonary alveolar fusion and swelling in the presence of the aptamer. These results suggest that aptamer NK2 has inhibitory effects on M. tuberculosis and can be used as antimycobacterial agent.     

A DNA aptamer with high affinity and specificity for therapeutic anthracyclines

We describe the characterization of a DNA aptamer that displays high affinity and specificity for the anthracyclines daunomycin and doxorubicin, both of which are frequently used in chemotherapy. Aptamers were isolated from a pool of random sequences using a semiautomated procedure for magnetic beads. All selected aptamers displayed high affinity for the target molecule daunomycin. One aptamer was further characterized and exhibited a dissociation constant (KD) of 20 nM. To examine the aptamer's binding properties and clarify its applicability for diagnostic assays, its performance under various buffer conditions was evaluated. The aptamer proved to be very robust and not dependent on the presence of specific ions. It also tolerated a wide pH range and immobilization via 5'-biotinylation. Furthermore, a competition assay for sensitive daunomycin detection was established. This not only allows the determination of the aptamer's specificity but also allows the quantification of as little as 8.4 microg/L daunomycin and doxorubicin.     

Rapid and sensitive detection of Nampt (PBEF/visfatin) in human serum using an ssDNA aptamer-based capacitive biosensor

A single-stranded DNA (ssDNA) aptamer was successfully developed to specifically bind to nicotinamide phosphoribosyl transferase (Nampt) through systematic evolution of ligands by exponential enrichment (SELEX) and successfully implemented in a gold-interdigitated (GID) capacitor-based biosensor. Surface plasmon resonance (SPR) analysis of the aptamer revealed high specificity and affinity (K(d)=72.52nM). Changes in surface capacitance/charge distribution or dielectric properties in the response of the GID capacitor surface covalently coupled to the aptamers in response to changes in applied AC frequency were measured as a sensing signal based on a specific interaction between the aptamers and Nampt. The limit of detection for Nampt was 1ng/ml with a dynamic serum detection range of up to 50ng/ml; this range includes the clinical requirement for both normal Nampt level, which is 15.8ng/ml, and Nampt level in type 2 diabetes mellitus (T2DM) patients, which is 31.9ng/ml. Additionally, the binding kinetics of aptamer-Nampt interactions on the capacitor surface showed that strong binding occurred with increasing frequency (range, 700MHz-1GHz) and that the dissociation constant of the aptamer under the applied frequency was improved 120-240 times (K(d)=0.3-0.6nM) independent on frequency. This assay system is an alternative approach for clinical detection of Nampt with improved specificity and affinity.