High Specificity, Electrochemical Sandwich Assays Based on Single Aptamer Sequences and Suitable for the Direct Detection of Small-Molecule Targets in Blood and Other Complex Matrices

Zuo, Xiaolei; Xiao, Yi; Plaxco, Kevin W.

doi:10.1021/ja901315w

Cited by 401 publications

(316 citation statements)

References 25 publications

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“…7. Complicating this analysis, however, is the fact that the aptamer is thought to contain a large number of non-WatsonCrick base pairs (38), and thus DINAMelt Mfold likely fails to accurately model its folding free energy. To overcome this, we instead used the experimentally determined folding free energy of the parent aptamer (39) to determine K close .…”

Section: Resultsmentioning

confidence: 99%

Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors

Simon

Vallée‐Bélisle

Ricci

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. Such control could likewise be of utility in artificial biotechnologies, such as biosensors, genetic logic gates, and "smart" materials, in which highly responsive behavior is of value. In nature, the control of molecular responsiveness is often achieved using "Hilltype" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. Here, we use an intrinsicdisorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. To do so, we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. The first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a preformed site. Using this approach, we have rationally introduced cooperativity into three unrelated DNA aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. The extent of cooperativity and thus the steepness of the binding transition are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy. ultrasensitivity | intrinsically disordered proteins | biosensors | synthetic biology | ribozymes

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Section: Resultsmentioning

confidence: 99%

Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors

Simon

Vallée‐Bélisle

Ricci

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…These results suggest that serum did not change the molecular recognition property of the sensor. 29,48 We next challenged the sensor with 90% serum. Pure serum was not tested because 10% of the volume was used to add adenosine.…”

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confidence: 99%

Flow Cytometry-Assisted Detection of Adenosine in Serum with an Immobilized Aptamer Sensor

Huang

Liu

2010

Anal. Chem.

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Aptamers are single-stranded nucleic acids that can selectively bind to essentially any molecule of choice. Because of their high stability, low cost, ease of modification and availability through selection, aptamers hold great promise in addressing key challenges in bioanalytical chemistry. In the past fifteen years many highly sensitive fluorescent aptamer sensors have been reported. However, few such sensors showed high performance in serum samples. Further challenges related to practical applications include detection in a very small sample volume and a low dependence of sensor performance on ionic strength.We report the immobilization of an aptamer sensor on a magnetic microparticle and the use of flow cytometry for detection. Flow cytometry allows the detection of individual particles in a capillary and can effectively reduce the light scattering effect of serum. Since DNA immobilization generated a highly negatively charged surface and caused an enrichment of counter ions, the sensor performance showed a lower salt dependence. The detection limits for adenosine are determined to be 178 and 167 M in buffer and in 30% serum, respectively. Finally, we demonstrated that the detection can be carried out in 10 L of 90% human blood serum.3

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“…This includes sensors with a pseudoknot 12 , triple strand 13 , sandwich 14 , super sandwich 15 , or triplex 16 architecture.…”

Section: Discussionmentioning

confidence: 99%

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

Rowe¹,

White²,

Bonham³

et al. 2011

JoVE

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As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this "E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes. Video LinkThe video component of this article can be found at https://www.jove.com/video/2922/ Protocol 1. Setting the Stage 1. Purchase the relevant, chemically modified probe DNA from a custom oligonucleotide synthesis company such as Biosearch Technologies (Novato, CA) or Midland Certified (Midland, TX). The probe is modified during synthesis by the addition of a C6 thiol at its 3'-end and a redoxactive methylene blue at its 5'-end. 2. Dissolve the probe DNA in phosphate buffered saline pH 7.4 to a concentration of 200 μM and verify its concentration by measuring its absorbance at 260 nm using a spectrophotometer. Due to the methylene blue moiety on the DNA probe the solution should have a visible blue tint. 3. Freshly prepare 1 mL of a 10 mM solution of tris(2-carboxyethyl)phosphine TCEP in distilled, deionized water (DI-water). In our experience, these TCEP solutions remain fresh for one week when stored in the dark at 4°C. 4. To reduce any disulfide bonds that might be present in the probe DNA solution, combine 1 μL of the probe DNA stock solution with 2 μL of the TCEP solution and mix gently with a pipette. Incubate the mixture for one hour in a dark, refrigerated container. The initially blue solution should become clear as the TCEP reversibly reduces the methylene blue. If the solution does not become clear, repeat the procedure using a fresh TCEP solution or, possibly, at room temperature. 5. After one hour dilute the reduced DNA probe solution with 1 mL of buffer; this will dilute it to a concentration of 200nM. Later on, you will incubate a set of gold disk electrodes in 200 μL portions of this diluted probe solution. 6. Freshly prepare at least 2 mL of 2mM mercaptohexanol in phosphate buffered saline. Sensor Preparation1. Combine 0.05 micron alumina powder with water on a fine polishing cloth. Polish a set of gold disk electrodes (CH Instruments, Austin, TX) by pressing the gold surface firmly into the wet cloth, and moving them in a figure eight pattern for approximately three minutes per electrode. 2. Rinse the polished electrodes with DI-water and...

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High Specificity, Electrochemical Sandwich Assays Based on Single Aptamer Sequences and Suitable for the Direct Detection of Small-Molecule Targets in Blood and Other Complex Matrices

Cited by 401 publications

References 25 publications

Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors

Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors

Flow Cytometry-Assisted Detection of Adenosine in Serum with an Immobilized Aptamer Sensor

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

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