Graphene Signal Amplification for Sensitive and Real-Time Fluorescence Anisotropy Detection of Small Molecules

Liu, Jinhua; Wang, Changyao; Jiang, Ying; Hu, Yaping; Li, Jishan; Yang, Sheng; Li, Yinhui; Yang, Ronghua; Tan, Weihong; Huang, Cheng

doi:10.1021/ac3023982

Cited by 150 publications

(83 citation statements)

References 46 publications

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“…16C-TMR-A25 and 23A-TMR-A25 can be used as a pair of aptamer probes for FA sensing, showing complementary FA response to ATP. The obtained sensitivity for ATP analysis in this work is comparable to that obtained in many aptamer-based FA assays and fluorescence assays for ATP (Cui et al, 2012;Feng et al, 2014;Juskowiak, 2011;Liu et al, 2009Liu et al, , 2013Sassolas et al, 2011;Zhu et al, 2012).…”

Section: Fa Sensing Atpsupporting

confidence: 81%

“…proteins and oligonucleotides) or nanomaterials (e.g. gold nanoparticles, graphene, and silica nanoparticles) have been introduced in assay development by increasing the binding-induced molecular weight change (Cruz-Aguado and Penner, 2008;Cui et al, 2012;Huang et al, 2012;Liu et al, 2013;Ye, and Yin 2008;Yu et al, 2013;Zhu et al, 2011Zhu et al, , 2012 Recently, we have reported a simple and noncompetitive FA strategy for a small molecule, ochratoxin A, by using a TMR-labeled aptamer. (Zhao et al, 2014) It is based on the target-binding induced change of intramolecular interactions between TMR and the guanine (G) bases of the aptamer.…”

Section: Introductionmentioning

confidence: 99%

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Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides

Zhao

Wang

2015

Biosensors and Bioelectronics

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a b s t r a c tWe previously reported a fluorescence anisotropy (FA) approach for small molecules using tetramethylrhodamine (TMR) labeled aptamer. It relies on target-binding induced change of intramolecular interaction between TMR and guanine (G) base. TMR-labeling sites are crucial for this approach. Only terminal ends and thymine (T) bases could be tested for TMR labeling in our previous work, possibly causing limitation in analysis of different targets with this FA strategy. Here, taking the analysis of adenosine triphosphate (ATP) as an example, we demonstrated a success of conjugating TMR on other bases of aptamer adenine (A) or cytosine (C) bases and an achievement of full mapping various labeling sites of aptamers. We successfully constructed aptamer fluorescence anisotropy (FA) sensors for adenosine triphosphate (ATP). We conjugated single TMR on adenine (A), cytosine (C), or thymine (T) bases or terminals of a 25-mer aptamer against ATP and tested FA responses of 14 TMR-labeled aptamer to ATP. The aptamers having TMR labeled on the 16th base C or 23rd base A were screened out and exhibited significant FA-decreasing or FA-increasing responses upon ATP, respectively. These two favorable TMRlabeled aptamers enabled direct FA sensing ATP with a detection limit of 1 mM and the analysis of ATP in diluted serum. The comprehensive screening various TMR labeling sites of aptamers facilitates the successful construction of FA sensors using TMR-labeled aptamers. It will expand application of TMR-G interaction based aptamer FA strategy to a variety of targets.

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Section: Fa Sensing Atpsupporting

confidence: 81%

Section: Introductionmentioning

confidence: 99%

Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides

Zhao

Wang

2015

Biosensors and Bioelectronics

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“…31−34 The comparison in assay performance with other FA assays for OTA and aptamer-based FA assays for small molecules is summarized in Table S4 in the Supporting Information. [25][26][27][28][29][31][32][33][34]57,58 ■ CONCLUSION In summary, we demonstrated a simple fluorescence anisotropy strategy for direct analysis of a small molecule, OTA, with a TMR-labeled anti-OTA aptamer. It relied on the changes of the intramolecular interaction between TMR and guanine bases of aptamer and the subsequent local rotation of TMR upon target binding.…”

Section: ■ Results and Discussionmentioning

confidence: 90%

“…One reported FA assay for small molecules relies on the binding-induced structure switch of the aptamer with an end-labeled dye (e.g., fluorescein or Texas red) and the subsequent change of the local rotation of the fluorescent dye, but only small FA increase is observed in most of cases. 25,26 Mass amplifying strategies recently have been exploited by using DNA oligos, 27−29 DNA-binding proteins (e.g., single-stranded DNA binding (SSB) protein and thrombin), 30−32 or nanomaterials 33,34 to improve the fluorescence anisotropy response by increasing the change of the molecular size of the aptamer upon binding of small-molecule targets. In these approaches, competitive displacement or structure switching is usually involved.…”

mentioning

confidence: 99%

Identification of Allosteric Nucleotide Sites of Tetramethylrhodamine-Labeled Aptamer for Noncompetitive Aptamer-Based Fluorescence Anisotropy Detection of a Small Molecule, Ochratoxin A

Zhao

Wang

2013

Anal. Chem.

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Aptamer-based fluorescence anisotropy (FA) assay combines the advantages of affinity aptamers in good stability, easy generation, and facile labeling and the benefits of FA in homogeneous analysis, such as robustness, simplicity, and high reproducibility. By using a fluorophore-labeled aptamer, FA detection of a small molecule is not as easy as detection of protein because the binding of a small molecule cannot cause significant increase of molecular weight of the dye-labeled aptamer. The intramolecular interaction between labeled tetramethylrhodamine (TMR) and DNA aptamer bases dramatically affects the local rotation and FA of TMR. This intramolecular interaction can be altered by aptamer conformation change upon target binding, leading to a significant change of FA of TMR. Taking this unique feature of a TMR-labeled aptamer, we described a noncompetitive aptamer-based fluorescence anisotropy assay for detection of small molecules by using ochratoxin A (OTA) as a model. We successfully identified the specific TMR-labeling sites of aptamers with sensitive FA response to OTA from the 5′-end, 3′-end and the internal thymine (T) bases. The aptamer with a TMR labeled on the 10th T base exhibited a remarkable FA reduction response to OTA (Δr = 0.078), without requiring any proteins or nanomaterials as FA signal enhancers. This FA approach for OTA showed high sensitivity with a detection limit of 3 nM, a dynamic range from 3 nM to 3 μM, and good selectivity over the tested compounds with similar structures to OTA. The new strategy allowed the detection of OTA in diluted red wine and urine samples. F luorescence anisotropy (FA) is a powerful and unique analytical technique for molecular interactions study and assay developments, which relies on the rotational diffusion of fluorescent tracers or probes under polarized excitation light. 1−5Fluorescence anisotropy shows strength in real-time analysis, simplicity, robustness, and sensitivity. FA assays have been applied to the studies about protein−protein interaction and protein−DNA interaction and the immunoassays for drug discovery, diagnostics, environmental monitoring, and food analysis. 1−7The emergence of aptamers as affinity ligands makes the aptamer-based FA assay attractive due to their advantages over antibodies in ease of synthesis and labeling, great stability, good selectivity, and high binding affinity. 8−12 Target-dependent allosteric structural change is one unique feature of an aptamer, and it allows the construction of an aptamer switch for target analysis through the adaptive transition converting a binding event into a detectable signal. 8,13−17 The applications of fluorophore-labeled aptamers to direct FA analysis of proteins are relatively straightforward as aptamers are usually smaller than proteins in size. 5,8,11,12 The binding of the fluorophorelabeled aptamers to proteins can cause enhanced FA signals by restricting the rotational diffusion of the labeled fluorophore as a result of increased molecular size. 18−23 In contrast, the ...

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“…Using this method, ATP was detected down to 1 μM. Importantly, the detection could be achieved in human serum samples [77].…”

Section: Fluorescence Anisotropy Based Assaysmentioning

confidence: 97%

Fluorescent sensors using DNA-functionalized graphene oxide

et al. 2014

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In the past few years, graphene oxide (GO) has emerged as a unique platform to develop DNA-based biosensors. This takes advantage of the DNA adsorption and fluorescence quenching property of GO.The adsorbed DNA probes can be desorbed from the GO surface in the presence of target analytes, producing fluorescence signal. In addition to this initial design, many other strategies have been reported including the use of aptamers, molecular beacons, and DNAzymes as probes, label-free detection, using the intrinsic fluorescence of GO and covalently linked DNA probes. The potential applications of DNA-functionalized GO range from environmental monitoring, cell imaging and biomedical diagnosis. In this review, we first summarize the fundamental surface interactions between DNA and GO and the related fluorescence quenching mechanism. Following that, the various sensor design strategies are critically compared. Problems at the current stages are described and a few future directions are also discussed.

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Graphene Signal Amplification for Sensitive and Real-Time Fluorescence Anisotropy Detection of Small Molecules

Cited by 150 publications

References 46 publications

Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides

Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides

Identification of Allosteric Nucleotide Sites of Tetramethylrhodamine-Labeled Aptamer for Noncompetitive Aptamer-Based Fluorescence Anisotropy Detection of a Small Molecule, Ochratoxin A

Fluorescent sensors using DNA-functionalized graphene oxide

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