Aptamer‐Based Bioanalytical Assays: Amplification Strategies

Tombelli, Sara; Minunni, Maria; Mascini, Marco

doi:10.1002/9780470380772.ch8

Cited by 10 publications

(10 citation statements)

References 48 publications

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“…This combination of properties has led to growing interest in applications of aptamers in fields including therapeutics, chemical analysis, biotechnology, chemical separations and environmental diagnostics. 3 Aptamers are identified from large libraries of random nucleic acid sequences via an iterative in vitro process called SELEX (Systematic Evolution of Ligands by EXponential enrichment). 4 – 6 A typical SELEX round includes the following three steps: i) Binding: incubation of the library with the target, ii) Partitioning: separation of target-bound library sequences from unbound ones, iii) Amplification: generation of a new pool of nucleic acids by making multiple copies of the sequences that bound to the target.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Microcolumn-Based Process for Efficient Selection of RNA Aptamers

et al. 2013

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We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin—a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 108 with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.

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Section: Introductionmentioning

confidence: 99%

Multiplexed Microcolumn-Based Process for Efficient Selection of RNA Aptamers

et al. 2013

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“…At present, aptamers have been combined with various transducers, such as optical transduction [39], quartz crystal microbalance [40], surface plasmon resonance (SPR) [41,42], fluorescence [43,44], colorimetry [45,46], electrophoresis [47], atomic force microscopy [48,49], electrochemistry [50][51][52][53][54][55][56], field-effect transistor [57,58], and so on. Due to their inherent selectivity, affinity, and their multifarious advantages, aptamers are now involved in preparing all kinds of multiple biosensing systems [59,60]. These aptasensors can detect a large range of specific targets containing small inorganic and organic substances, and even proteins or cells [12][13][14].…”

Section: Aptamer-based Biosensorsmentioning

confidence: 99%

Analytical potential of gold nanoparticles in functional aptamer-based biosensors

Wang

2010

Bioanal Rev

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Gold nanoparticles (AuNPs) exhibit many predominant capabilities such as high biocompatibility, chemical stability, strong localized surface plasmon resonance absorption, and high extinction coefficient in the visible region. These properties have enabled the extensive use of AuNPs in optical and electrochemical biosensors. As a kind of functional nucleic acids, aptamers can be considered as a valid alternative to antibodies or other bio-receptors and have been widely employed to develop novel biosensors. We are summarizing here the state of the art of AuNP-based biosensors that use functional aptamers as molecular recognition elements. In many cases, AuNPs themselves can be used as a probe for detection, such as various colorimetric aptasensors and fluorescent aptasensors. They also can be used as probe vectors to enlarge detection signals and to increase the number of conceivable substrates in electrochemical aptasensors.

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“…This, combined with the ability to specifically place chemical agents, opens up a wealth of possible signal transduction schemas [12]. Electrochemical approaches using aptamers in analytical applications have become increasingly popular over the past two decades [13,14] because electrochemical techniques have high sensitivities, easy operation and low cost, and are suitable for miniaturization.…”

Section: Introductionmentioning

confidence: 99%