Name: phthalic acid ester-binding dna aptamer selection, characterization, and application to an electrochemical aptasensor
Uploaded: 2018-03-21T12:30:00.000+00:00
Duration: 9 min 33 s
Description: Watch this Scientific Journal Video about Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor at JoVE.com

我们感谢国家自然科学基金 (21675112)、北京教育委员会科技计划重点项目 (KZ201710028027) 和首都师范大学燕京青年学者项目的资助。

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作者声明没有竞争的财政利益。

适配子的一个显著好处是, 它们通过体外方法 SELEX 识别, 而抗体是通过体内免疫生成的. 因此, 在设计良好的实验条件下, 适配子可以选择理想的靶向特异性, 而抗体仅限于生理条件。
为了便于从自由序列分离绑定序列, 最近报告了几种修改后的 SELEX, 其中毛细管电泳30, 微流体 31, 磁性/丙烯酸珠/琼脂糖珠 14, ...

随着经济的快速发展, 工业化的加速, 城市建设的不断加快, 环境污染问题也日益严重。典型的环境污染物包括重金属离子、毒素、抗生素、杀虫剂、内分泌干扰物和持久性有机污染物 (pop)。除了金属离子和毒素, 其他污染物是小分子, 通常由各种同源物组成。例如, 最有毒的持久性有机污染物包括多氯联苯、多环芳烃 (多环芳烃)、多溴联联苯醚 (二苯醚)、聚氯氯苯并 (PCDDs)、多氯氧芴 (呋喃) 和苯二甲酸酸酯 (PAEs)1,2, 它们都由许多同源项组成。小分子检测主要是以色谱/质谱技术为基础进行的, 因为其应用的多样性3,4,5,6。对于现场检测, 最近开发了基于抗体的方法7,8,9。但是, 由于这些方法对某些同类具有高度的特异特性, 因此必须执行多个测试。更严重的是, 新的同源生长得如此之快, 以至于他们的抗体不能及时生成。因此, 在一项试验中, 开发组特定的生物传感器来监测所有同源物的总水平, 可为评估环境污染状况提供一个宝贵的指标。
近年来, 核酸适配子在各种生物传感平台中被广泛应用为识别元素, 因为它们能够识别各种各样的目标, 从离子和小分子到蛋白质和细胞10, 11 ,12。适配子是通过一个体外方法来识别的, 所谓的配位系统进化的指数富集 (SELEX) 13, 14.SELEX 从随机合成单链寡核苷酸库开始, 其中包含大约 1014-1015序列。随机库的大小确保了 RNA 或 DNA 候选结构的多样性。典型的 SELEX 过程包括多轮的浓缩, 直到图书馆丰富的序列, 高度亲和性和特异性的目标。最后的浓缩池然后排序, 离解常数 (Kd) 和选择性反对潜在的干扰物质是由不同的技术, 如过滤器结合, 亲和层析, 表面等离子共振 (SPR),等。15
由于水的溶解度极差, 且缺乏用于表面固定的功能基团, 因此在理论上很难核酸适体选择持久性有机污染物。SELEX 的重大进展加速了适配子的发现。但是, 尚未报告持久性有机污染物的组特定适配子的选择。到目前为止, 只有对某些同类的高特异性的 PCB 结合 DNA 适配子被识别为 16.PAEs 主要应用于聚氯乙烯材料中, 将聚氯乙烯从硬塑料改成弹性塑料, 从而充当增塑剂。一些 PAEs 已被认定为内分泌干扰物质, 可对肝肾功能造成严重损害, 减少男性精子的运动, 并可能导致畸形精子形态和睾丸癌的发生17。未报告复合类或组特定的 PAE 绑定适配子。
这项工作的目标是提供一个代表性的协议, 以选择组特定的 DNA 适配子高疏水性小分子, 如 PAEs, 一个代表性的持久性有机污染物组。我们还演示了选定的核酸适体在环境污染检测中的应用。该协议为核酸适体发现其他疏水小分子提供了指导和见解。

<table><tbody><tr><td>UV-2550</td><td>Shimadzu,Japan</td><td></td><td>protocol, section 3.8.2</td></tr><tr><td>DNA Engnine Thermal cycler,PTC0200</td><td>BIO-RAD</td><td></td><td>section 3.5.1.2 and 3.5.2</td></tr><tr><td>C1000 Touch</td><td>BIO-RAD</td><td></td><td>section 5.3.6 and 6.3</td></tr><tr><td>VMP3 multichannel potentiostat</td><td>Bio-Logic Science, Claix, France</td><td></td><td>section 7.4,7.8 and 7.11</td></tr><tr><td>Epoxy-activated Sepharose 6B</td><td>GE Healthcare (Piscataway, NJ, USA)</td><td>10220020</td><td>argarose beads, section 2.3 and 3.3</td></tr><tr><td>Dynabeads M-270 carboxylic acid magnetic beads</td><td>Invitrogen, USA</td><td>420420</td><td>magnetic beads,section 5.2. and 5.3</td></tr><tr><td>Premix Taq Hot Start Version</td><td>Takara,Dalian,China</td><td>R028A</td><td>polymerase, section 3.5.1.1</td></tr><tr><td>PARAFILM Sealing Membrane</td><td>Bemis, USA</td><td>PM-996</td><td>section 3.6.5</td></tr><tr><td>Lambda Exonuclease</td><td>Invitrogen, USA</td><td>EN0561</td><td>section3.7.1.2.The 10 &amp;times; reaction buffer is provided along with &amp;lambda; exonuclease by the provider.</td></tr><tr><td>Dr. GenTLE&lt;br/&gt; Precipitation Carrier</td><td>Takara,Dalian,China</td><td>9094</td><td>section 3.6.2 and 3.8.1</td></tr><tr><td>UNIQ-10 PAGE DNA recovery kit</td><td>Sangon Biotech (Shanghai)</td><td>B511135</td><td>section 4.2</td></tr><tr><td>SYBR Gold nucleic acid gel stain</td><td>Invitrogen, USA</td><td>1811838</td><td>nucelic acid stain dye, section 3.5.1.5</td></tr><tr><td>SYBR Premix Ex Taq II</td><td>Takara,Dalian,China</td><td>RR820A</td><td>polymerase mix contaning polymerase and dNTPs, section 5.3.5</td></tr><tr><td>2-(N-Morpholino)ethanesulfonic acid (MES)</td><td>Sigma-Aldrich</td><td>CAS: 1132-61-2</td><td>section 5.2.1</td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)</td><td>Invitrogen, USA</td><td>CAS: 25952-53-8</td><td>section 5.2.2</td></tr><tr><td>N-hydroxysuccinimide (NHS)</td><td>Sigma-Aldrich</td><td>6066-82-6</td><td>section 5.2.3</td></tr><tr><td>mercaptohexanol (MCH)</td><td>Sigma-Aldrich</td><td>CAS: 1633-78-9</td><td>section 7.7</td></tr><tr><td>Gold electrode</td><td>Shanghai Chenhua</td><td>CHI101</td><td>section 7.4. - 7.11</td></tr><tr><td>tris(2-carboxyethyl) phosphine hydrochloride (TCEP)</td><td>Sigma-Aldrich</td><td>CAS: 51805-45-9</td><td>section 7.5</td></tr><tr><td>O-(2-Mercaptoethyl)-O&#039;-methyl-hexa-(ethylene glycol)</td><td>Sigma-Aldrich</td><td>CAS: 651042-82-9</td><td>section 7.7</td></tr><tr><td>diethylhexyl phthalate (DEHP)</td><td>National Institute of Metrology, China</td><td>CAS: 117-81-7</td><td>section 7.11</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>CAS: 9005-64-5</td><td>polyoxyethy-lene(20) sorbaitan monolaurate</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>CAS: 9002-93-1</td><td>non-ionic surface active agent</td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>P5368</td><td>10 mM phosphate buffer containing 1 M NaCl, pH 7.4</td></tr></tbody></table>

phthalic acid ester-binding dna aptamer selection, characterization, and application to an electrochemical aptasensor

邻苯二甲酸酯 (PAEs) areone 主要群体的持久性有机污染物。由于同源性的快速增长, PAEs 的群特异检测非常理想。DNA 适配子已越来越多地应用于生物传感器平台上的识别元素, 但很少报道适配子对高度疏水性的小分子靶点 (如 PAEs) 的选择。这项工作描述了一种基于珠子的方法, 用于选择组特定的 DNA 适配子 PAEs。将氨基甲酸二丁酯 (DBP-NH2) 合成并固定在环氧活化的琼脂糖珠上, 使其在固定基质表面显示邻苯乙酯基团, 并因此选择组特定的粘合剂。通过定量聚合链反应和磁分离, 确定了核酸适体候选者的离解常数。适配子对其他 PAEs 的相对亲和性和选择性由竞争性化验确定, 核酸适体候选者预先限定在 DBP- NH 2 附着的磁性珠子上, 并在孵化时释放到上清被测试的 PAEs 或其他潜在的干扰物质。竞争试验的应用, 因为它提供了一个简便的亲和力比较 PAEs 之间没有功能组的表面固定化。最后, 我们演示了电化学 aptasensor 的制备方法, 并将其应用于邻苯二甲酸二 (2-己酯) 的超灵敏和选择性检测。该协议为核酸适体发现其他疏水小分子提供了见解。

我们设计并合成了氨基甲酸二丁酯 (DBP-NH2) 作为定位靶 (图 1F)。然后, 我们进行 DNA 核酸适体选择 PAEs 使用 DBP-NH 2 作为锚定目标和遵循经典的目标固定化的方法(图 2)。在每一个回合中, 使用变性页来优化 pcr 的周期数 (图 3) 进行了先导 pcr。强烈建议变性页面, 而不是本机页, 因为我们和其他?...

Watch this Scientific Journal Video about Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor at JoVE.com

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

本文提出了一种体外的协议, 用于对组特定邻苯二甲酸酯结合 DNA 适配子的筛选和表征。在电化学 aptasensor 中也包括了所选核酸适体的应用。

邻苯二甲酸酯结合 DNA 核酸适体的选择、表征及其在电化学 Aptasensor 中的应用

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

phthalic-acid-ester-binding-dna-aptamer-selection-characterization

Universidad San Sebastian

Research

JoVE Journal

Chemistry

9.8K 次观看.  Capital Normal University. 本文提出了一种体外的协议, 用于对组特定邻苯二甲酸酯结合 DNA 适配子的筛选和表征。在电化学 aptasensor 中也包括了所选核酸适体的应用。

视频: Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

映射的适体对ATP的结合部位使用MicroScale程序热泳

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

生物化学

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

电子舌生成连续的识别模式的蛋白质分析

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

使用核酸探针进行核酸活性的动力学筛选

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

引物的适体的选择，使用一个随机DNA文库

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

生物学

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

使用等温滴定量热法测定DNA适配体和四环素的热力学和动力学关联

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

电化学 DNA 生物传感器检测食源性致病菌的研究进展

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

基于分子印迹的电容生物传感器超灵敏检测生物标志物

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

免疫与感染

Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules with signal-generation capability. For the system described herein, target recognition through an aptameric domain, Stem II of a modified hammerhead ribozyme, activates the self-cleaving ribozyme by stabilizing the initially unstructured construct. The cis-cleaving RNA acts at the junction of Stem III and Stem I, creating two cleavage products. The longer cleavage product primes an isothermal exponential amplification reaction (EXPAR) of the two similar catalytically active G-quadruplexes. Those resulting amplification products catalyze peroxidase reduction, which is coupled to the reduction of a colorimetric substrate with an output that the naked eye can detect. The 3-part system described in the present study improves detection modalities such as enzyme-linked immunosorbent assays (ELISAs) by producing a visually detectable signal for indicating the presence of as low as 0.5 &#181;M theophylline in as little as 15 min.

The present protocol demonstrates the use of a fast, 3-stage, aptamer-based exponential amplification assay to detect targets. Sample preparation, signal amplification, and color development are covered to implement this system to recognize the presence of theophylline over that of caffeine.

基于适配体的靶标检测通过 3 级 G-四链体等温指数扩增反应进行

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules ...