Small molecule detection with aptamer based lateral flow assays: Applying aptamer-C-reactive protein cross-recognition for ampicillin detection (original) (raw)
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Antibodies have been known for more than three decades and have proved to be invaluable tool for rapid and advanced diagnostics. Although, antibodies are used routinely in most of diagnostic tests as biorecognition elements, however, their high cost of production, shorter-shelf life, animal requirement for production, stability issues, batch-to-batch variations had significant drawbacks on the field of diagnostics. Aptamers are short oligonucleotides of less 100 nucleotides that bind selectively and specifically with high affinity to their targets, ranging from small molecule like a toxin to a large cancerous cells, due to their unique 3-D conformational. Combining aptamers with different nanostructures has elevated their diagnostic capability and made them highly useful in various biosensing platforms. Some of the biosensing assays like ELASA, Colorimetry, Electrochemical, Microfluidics, Lateral flow, etc. using aptamers and their modified forms with nanostructures are expatiated i...
Kinetic Exclusion Assay of Biomolecules by Aptamer Capture
Sensors
DNA aptamers are short nucleotide oligomers selected to bind a target ligand with affinity and specificity rivaling that of antibodies. These remarkable features recommend aptamers as candidates for analytical and therapeutic applications that traditionally use antibodies as biorecognition elements. Numerous traditional and emerging analytical techniques have been proposed and successfully implemented to utilize aptamers for sensing purposes. In this work, we exploited the analytical capabilities offered by the kinetic exclusion assay technology to measure the affinity of fluorescent aptamers for their thrombin target and quantify the concentration of analyte in solution. Standard binding curves constructed by using equilibrated mixtures of aptamers titrated with thrombin were fitted with a 1:1 binding model and provided an effective Kd of the binding in the sub-nanomolar range. However, our experimental results suggest that this simple model does not satisfactorily describe the bin...
Application of aptamers in therapeutics and for small-molecule detection
Handbook of experimental pharmacology, 2006
Nucleic acids that can bind with high affinity and specificity to target molecules are called "apta mers". Aptamers recognise a large variety of different molecule classes. The main focus of this chapter is small molecules as targets. Aptamers are applied complementarily to antibody technologies and can substitute antibodies or small molecules wherever their different properties, such as biochemical nature or highly discriminating capacities, are advantageous. Examples of promising applications of these versatile molecules are discussed in the field of therapeutics and biotechnology with a special view to small-molecule detection.
Nucleic acid aptamers function as versatile sensing and targeting agents for analytical, diagnostic, therapeutic, and gene-regulatory applications, but their limited characterization and functional validation have hindered their broader implementation. We report the development of a surface plasmon resonance-based platform for rapid characterization of kinetic and equilibrium binding properties of aptamers to small molecules. Our system is label-free and scalable and enables analysis of different aptamer−target pairs and binding conditions with the same platform. This method demonstrates improved sensitivity, flexibility, and stability compared to other aptamer characterization methods. We validated our assay against previously reported aptamer affinity and kinetic measurements and further characterized a diverse panel of 12 small molecule-binding RNA and DNA aptamers. We report the first kinetic characterization for six of these aptamers and affinity characterization of two others. This work is the first example of direct comparison of in vitro selected and natural aptamers using consistent characterization conditions, thus providing insight into the influence of environmental conditions on aptamer binding kinetics and affinities, indicating different possible regulatory strategies used by natural aptamers, and identifying potential in vitro selection strategies to improve resulting binding affinities.
Aptamers are short single-stranded oligonucleotides that fold into unique three-dimensional structures, facilitating selective and high affinity binding to their cognate targets. It is not well understood how aptamer–target interactions affect regions of structure in an aptamer, particularly for small molecule targets where binding is often not accompanied by a dramatic change in structure. The DNase I footprint-ing assay is a classical molecular biology technique for studying DNA–protein interactions. The simplest application of this method permits identification of protein binding where DNase I digestion is inhibited. Here, we describe a novel variation of the classical DNase I assay to study aptamer–small molecule interactions. Given that DNase I preferentially cleaves duplex DNA over single-stranded DNA, we are able to identify regions of aptamer structure that are affected by small molecule target binding. Importantly, our method allows us to quantify these subtle effects, providing an in solution measurement of aptamer– target affinity. We applied this method to study aptamers that bind to the mycotoxin fumonisin B 1 , allowing the first identification of high affinity putative minimers for this important food contaminant. We confirmed the binding affinity of these minimers using a magnetic bead binding assay.