Solid‐Phase Enzyme Activity Assay Utilizing an Entrapped Fluorescence‐Signaling DNA Aptamer

Rupcich, Nicholas; Nutiu, Razvan; Li, Yingfu; Brennan, John D.

doi:10.1002/ange.200504576

Cited by 8 publications

(4 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[20,21] Aptamers, that is, DNA/RNA oligonucleotide probes that can substitute for antibodies and even overcome the drawbacks of antibodies, [22,23] have great potential for biological applications due to their ability to bind target molecules with high affinity and specificity. [24][25][26][27][28][29][30][31] For specific label-free detection of target proteins (human a-thrombin), as described above, the surface of the CPPy nanotubes was covalently functionalized with thrombin aptamers, and was treated with a blocking agent (skim milk) to prevent nonspecific adsorption of undesired species. The sensing test was performed to investigate the dependence of sensor response on the chemical functionality at the V SD (CPNT-1: À15 mV; CPNT-2: À10 mV), where both CPNT-1 and CPNT-2 had similar I SD -E GD characteristics (see Figure 10, g m, max : ca.…”

Section: Resultsmentioning

confidence: 99%

A Novel Sensor Platform Based on Aptamer‐Conjugated Polypyrrole Nanotubes for Label‐Free Electrochemical Protein Detection

et al. 2008

View full text Add to dashboard Cite

We first present a simple yet versatile strategy for the functionalization of polymer nanotubes in a controlled fashion. Carboxylic-acid-functionalized polypyrrole (CPPy) nanotubes were fabricated by using cylindrical micelle templates in a water-in-oil emulsion system, and the functional carboxyl groups were effectively incorporated into the polymer backbone during the polymerization by using pyrrole-3-carboxylic acid (P3CA) as a co-monomer without a sophisticated functionalization process. It was noteworthy that the chemical functionality of CPPy nanotubes was readily controlled in both qualitative and quantitative aspects. On the basis of the controlled functionality of CPPy nanotubes, a field-effect transistor (FET) sensor platform was constructed to detect specific biological entities by using a buffer solution as a liquid-ion gate. The CPPy nanotubes were covalently immobilized onto the microelectrode substrate to make a good electrical contact with the metal electrodes, and thrombin aptamers were bonded to the nanotube surface via covalent linkages as the molecular recognition element. The selective recognition ability of thrombin aptamers combined with the charge transport property of CPPy nanotubes enabled the direct and label-free electrical detection of thrombin proteins. Upon exposure to thrombin, the CPPy nanotube FET sensors showed a decrease in current flow, which was probably attributed to the dipole-dipole or dipole-charge interaction between thrombin proteins and the aptamer-conjugated polymer chains. Importantly, the sensor response was tuned by adjusting the chemical functionality of CPPy nanotubes. The efficacy of CPPy nanotube FET sensors was also demonstrated in human blood serum; this suggests that they may be used for practical diagnosis applications after further optimization.

show abstract

Section: Resultsmentioning

confidence: 99%

A Novel Sensor Platform Based on Aptamer‐Conjugated Polypyrrole Nanotubes for Label‐Free Electrochemical Protein Detection

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Inhibition of the enzyme allowed adenosine to penetrate through the upper layer and bind the aptamer, resulting in displacement of a quencher and an increase in fluorescence signaling. Failure to separate the enzyme and aptamer into layers resulted in an unworkable assay owing to the effects of substrate depletion upon binding the aptamer (62).…”

Section: Microwell Plate-based Solid-phase Assaysmentioning

confidence: 99%

Solid-Phase Biological Assays for Drug Discovery

Forsberg

Sicard²,

Brennan

2014

Annual Rev. Anal. Chem.

View full text Add to dashboard Cite

In the past 30 years, there has been a significant growth in the use of solid-phase assays in the area of drug discovery, with a range of new assays being used for both soluble and membrane-bound targets. In this review, we provide some basic background to typical drug targets and immobilization protocols used in solid-phase biological assays (SPBAs) for drug discovery, with emphasis on particularly labile biomolecular targets such as kinases and membrane-bound receptors, and highlight some of the more recent approaches for producing protein microarrays, bioaffinity columns, and other devices that are central to small molecule screening by SPBA. We then discuss key applications of such assays to identify drug leads, with an emphasis on the screening of mixtures. We conclude by highlighting specific advantages and potential disadvantages of SPBAs, particularly as they relate to particular assay formats.

show abstract

“…However, the sequences of duplexes in this approach should be more carefully optimized to guarantee a satisfactory sensing efficiency and high signal/noise ratio. Now, through continuous improvement, the duplex-to-complex switching approach has been used successfully in enzyme-related assays (Rupcich et al, 2006) and DNAzyme-related assays (Stojanovic et al, 2001b).…”

Section: Posfalf Modementioning

confidence: 99%

“…Several of them have been proven feasible in practical biosamples. Various techniques to get a detectable signal have been used or combined, such as optical transduction (Lee and Walt, 2000), quartz crystal microbalance (QCM) (Liss at al., 2002), surface plasmon resonance (SPR) (Tombelli et al, 2005b), fluorescence (Nutiu et al, 2004;Pavlov et al, 2005;Yang et al, 2005;Rupcich et al, 2006), colorimetry Liu, 2006, 2007), chromatography (Ravelet et al, 2006), and electrochemistry (Willner and Zayats, 2007). On the whole, among all the convenience-aimed aptasensors, label-free seems to be one of the most widely used methods with whatever detection techniques are adopted.…”

mentioning

confidence: 99%

Strategy for Use of Smart Routes to Prepare Label‐Free Aptasensors for Bioassay Using Different Techniques

Wei

Dong

2008

Aptamers in Bioanalysis

View full text Add to dashboard Cite

Biosensors, which usually need biological molecules as recognition elements, are playing a growing role in various fields, due to their effective selectivity, high sensitivity, and easy performance. Thus, even for only one useful target, it is worth large numbers of designs and improvements to develop detection that is effective enough for a variety of applications. In this way, more and more techniques and novel recognition elements are being brought into bioassays.Aptamers are screened through the systematic evolution of ligands by exponential enrichment (SELEX) process as functional oligonucleotides (Ellington and Szostak, 1990;Robertson and Joyce, 1990;Tuerk and Gold, 1990). Because of their generally impressive selectivity and affinity, they have already attracted much analyst attention and were quick to be used in analytical assays as novel recognition elements. Aptamers exhibit multifarious advantages over traditional recognition elements (Osborne and Ellington, 1997;Jayasena, 1999;Famulok et al., 2000;Tombelli et al., 2005a). For example, they are easy to synthesize in vitro, easy to modify, and flexible to design. These inherent advantages as oligonucleotides not only make target diversification but also improve detection efficiency. They can also help analysts simplify the analytical process and fabricate a sensing platform smartly and conveniently.

show abstract

Solid‐Phase Enzyme Activity Assay Utilizing an Entrapped Fluorescence‐Signaling DNA Aptamer

Cited by 8 publications

References 37 publications

A Novel Sensor Platform Based on Aptamer‐Conjugated Polypyrrole Nanotubes for Label‐Free Electrochemical Protein Detection

A Novel Sensor Platform Based on Aptamer‐Conjugated Polypyrrole Nanotubes for Label‐Free Electrochemical Protein Detection

Solid-Phase Biological Assays for Drug Discovery

Strategy for Use of Smart Routes to Prepare Label‐Free Aptasensors for Bioassay Using Different Techniques

Contact Info

Product

Resources

About