Article

Aptamer-Based Sensor Arrays for the Detection and Quantitation of Proteins

Department of Electrical & Computer Engineering, University of Texas at Austin, Austin, Texas, United States
Analytical Chemistry (Impact Factor: 5.64). 08/2004; 76(14):4066-75. DOI: 10.1021/ac049858n
Source: PubMed

ABSTRACT

Aptamer biosensors have been immobilized on beads, introduced into micromachined chips on the electronic tongue sensor array, and used for the detection and quantitation of proteins. Aptamer chips could detect proteins in both capture and sandwich assay formats. Unlike most protein-based arrays, the aptamer chips could be stripped and reused multiple times. The aptamer chips proved to be useful for screening aptamers from in vitro selection experiments and for sensitively quantitating the biothreat agent ricin.

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    • "Over the last 25 years, DNA aptamers have gained recognition to complement antibodies as useful affinity molecules binding to specific targets. Thus, aptamers have been used in a variety of bioanalytic assays and array devices which conventionally use antibodies (Cho et al. 2006; Deng et al. 2014; Drolet et al. 1996; Kirby et al. 2004; Li et al. 2015; Ma et al. 2015; Zhao et al. 2012). In addition to their longer shelf-life, better temperature stability , and ease of site-specific chemical modification, DNA aptamers also possess several intrinsic and unique properties that enable various signal-amplification mechanisms to facilitate detection of trace amounts of biomolecules (Li et al. 2015; Zhang et al. 2013). "
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    ABSTRACT: Aptamers of high affinity and specificity have a wide range of analytic and clinical applications. Selection of DNA or RNA aptamer molecules usually involves systematic evolution of ligands via exponential enrichment (SELEX), in which a random DNA or RNA library is incubated with a target molecule, and the oligonucleotides that bind the target are then separated from the nonbinders, PCR amplified, and used as refined libraries in the next round of selection. Conventional SELEX methodologies require the use of purified target molecules and their immobilization onto a solid support. However, purified targets from cells are not always available, and fixing the target to a support may alter its conformation. To overcome these problems, we have developed a SELEX technique using live bacterial cells in suspension as targets, for selecting DNA aptamers specific to cell-surface molecules. Through the selection of aptamers binding to Lactobacillus acidophilus and Streptococcus pyogenes, we report here optimization of this technique and show how varying selection conditions impact the characteristics of resultant aptamer pools, including the binding affinity, selectivity, and the secondary structures. We found that the use of larger starting library sequence diversity, gel purification of the subsequent pools, and the introduction of counter-selection resulted in a more efficient SELEX process and more selective aptamers. A SELEX protocol with lower starting sequence diversity, the use of heat denaturation, and the absence of counter-selection still resulted in high-affinity aptamer sequences specific to the target cell types; however, the SELEX process was inefficient, requiring 20 rounds, and the aptamers were not specific to the strain of the bacterial cells. Strikingly, two different SELEX methodologies yielded the same sequence that bound strongly to the target S. pyogenes cells, suggesting the robustness of the bacterial cell-SELEX technique.
    Full-text · Article · Nov 2015 · Journal of Molecular Evolution
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    • "3.1. Experimental principle of the aptamer-based EWA biosensor Aptamers exhibit high target-binding affinity because their binding function is largely dependent upon stable secondary structure interactions (Kirby et al., 2004; Zhou et al., 2010); however, it is challengeable to keep the restoration to the original DNA probe form after repeated uses (Liu and Tan, 1999). Inspired by the immunosensors undergoing the antibody–antigen recognition which feature with highly reusability of up to 300 times of regenerated test cycles (Rodriguez-Mozaz et al., 2005; Zhou et al., 2014), we describe here a novel aptamer-based EWA biosensing strategy as shown in Scheme 1. "
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    ABSTRACT: Although aptamer-based biosensors have attracted ever-increasing attentions and found potential applications in a wide range of areas, they usually adopted the assay protocol of immobilizing DNA probe (e.g., aptamer, aptamer-complementary oligonucleotides) on a solid sensing surface, making it critical and challengeable to keep the integration of nucleic acid surface during the regeneration and the restoration to its original DNA probe form after repeated uses. In order to address the issue, we report a novel aptamer-based biosensing strategy based on an evanescent wave all-fiber (EWA) platform. In a simple target capturing step using aptamer-functionalized magnetic microbeads, signal probes conjugated with streptavidin are released and further detected by a EWA biosensor via a facial dethiobiotin-streptavidin recognition. Apart from the inherent advantages of aptamer-based evanescent wave biosensors (e.g. target versatility, sensitivity, selectivity and portability), the proposed strategy exhibits a high stability and remarkable reusability over other aptasensors. Under the optimized conditions, the typical calibration curve obtained for Ochratoxin A has a detection limit of 3 nM with a linear response ranging from 6 nM to 500 nM. The dethiobiotin-streptavidin sensing surface instead of the traditional nucleic acid one can be reused for over 300 times without losing sensitivity.
    Full-text · Article · Apr 2015 · Biosensors & Bioelectronics
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    • "Be‐ cause of their high thermostability when compared with antibodies, aptamers have potential applications in analytical devices, including biosensors, and as therapeutic agents [38]. Assays for protein identification and quantitation were developed and ap‐ plied to ricin detection [39] [40]. A multiplex aptamer microarray was generated by print‐ ing an anti-ricin RNA aptamer onto either streptavidin (SA)-or neutravidin (NA)-coated glass slides. "

    Full-text · Chapter · Dec 2012
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