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

We are grateful for financial support from the National Natural Science Foundation (21675112), Key project of science and technology plan of Beijing Education Commission (KZ201710028027) and Yanjing Young Scholar Program of Capital Normal University.

<ol>
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The authors declare no competing financial interest.

One outstanding benefit of aptamers is that they are identified through the in vitro method SELEX, while antibodies are generated via in vivo immunoreactions. Therefore, aptamers can be selected with desired target specificity under well-designed experimental conditions, whereas antibodies are limited to physiological conditions.
To facilitate the separation of bound sequences from free sequences, several modified SELEX have recently been reported, in which capillary electrop...

Along with rapid economic development, acceleration of industrialization, and urban construction, environmental pollution is more severe than ever. Typical environmental pollutants include heavy metal ions, toxins, antibiotics, pesticides, endocrine disruptors, and persistent organic pollutants (POPs). Besides metal ions and toxins, other pollutants are small molecules that quite often consist of a variety of congeners. For example, the most toxic POPs include polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), polybrominated biphenyl ethers (PBDEs), polychlorinated dibenzo-p-dioxin (PCDDs), polychlorinated dibenzofuran (PCDFs), and phthalic acid esters (PAEs)1,2, which all consist of many congeners. Small molecule detection has been mainly performed by chromatography/mass spectrometry-based techniques due to their diversity of applications3,4,5,6. For on-site detections, antibody-based methods have recently been developed7,8,9. However, since these methods are highly specific for a certain congener, multiple tests must be performed. What is more serious is that the novel congeners grow so fast that their antibodies can&#39;t be generated in time. Therefore, the development of group-specific biosensors to monitor the total levels of all congeners in one test may provide an invaluable metric for assessing environmental pollution status.
Recently, nucleic acid aptamers have been widely applied as recognition elements in various biosensing platforms due to their capability of recognizing a wide variety of targets, from ions and small molecules to proteins and cells10,11,12. Aptamers are identified through an in vitro method called systematic evolution of ligands by exponential enrichment (SELEX)13,14. SELEX begins with the random synthetic single strand oligonucleotide library, which contains approximately 1014-1015 sequences. The size of the random library ensures the diversity of the RNA or DNA candidate structures. The typical SELEX process consists of multiple rounds of enrichment until the library is enriched in sequences with high affinity and specificity to the target. The final enriched pool is then sequenced, and the dissociation constants (Kd) and selectivity against potential interfering substances are determined by different techniques such as filter binding, affinity chromatography, surface plasmon resonance (SPR), etc.15
Due to the extremely poor water solubility and lack of functional groups for surface immobilization, the aptamer selection of POPs is theoretically difficult. Significant advances for SELEX have speeded up the discovery of aptamers. However, the selection of group-specific aptamers for POPs has not yet been reported. So far, only PCB-binding DNA aptamers with high specificity for a certain congener have been identified16. PAEs are mainly used in polyvinyl chloride materials, changing polyvinyl chloride from a hard plastic to an elastic plastic, thus acting as a plasticizer. Some PAEs have been identified as endocrine disruptors, can cause serious damage to liver and kidney function, reduce the motility of male sperm, and may result in abnormal sperm morphology and testicular cancer17. Neither the compound- nor group-specific PAE-binding aptamers have been reported.
The goal of this work is to provide a representative protocol for selecting group-specific DNA aptamers to highly hydrophobic small molecules such as PAEs, a representative group of POPs. We also demonstrate the application of the selected aptamer for environmental pollution detection. This protocol provides guidance and insights for the aptamer discovery of other hydrophobic small molecules.

<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

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 rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

We designed and synthesized the amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target (Figure 1F). We then performed the DNA aptamer selection of PAEs using DBP-NH2 as the anchor target and following the classical target immobilization-based method (Figure 2). In each round, a pilot PCR was performed using the denatured PAGE to optimize the cycle number of PCR (Figu...

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

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

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

Research

JoVE Journal

Chemistry

9.8K Views.  Capital Normal University. A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

Video: 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.

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. ...

Bioengineering

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.

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

Biochemistry

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.

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

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

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.

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

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.

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

Biology

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.

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.

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.

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

Immunology and Infection

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.

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

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

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