Aptamer-Based Electrochemical Detection of Picomolar Platelet-Derived Growth Factor Directly in Blood Serum

Lai, Rebecca Y.; Plaxco, Kevin W.; Heeger, Alan J.

doi:10.1021/ac061592s

Cited by 342 publications

(296 citation statements)

References 28 publications

(49 reference statements)

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“…[13][14][15]27,41,43,44 The latter class of sensors, directly analogous to the E-DNA sensor, is based on the binding-induced folding of DNA aptamers and has been demonstrated for targets ranging from proteins 26,41,42 to small molecules 27 and inorganic ions. 43,44 The results presented here suggest that careful optimization of probe density and measurement techniques will be necessary in order to achieve maximum performance across this broad and increasingly important class of sensors.…”

Section: Discussionmentioning

confidence: 99%

Effect of Molecular Crowding on the Response of an Electrochemical DNA Sensor

et al. 2007

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E-DNA sensors, the electrochemical equivalent of molecular beacons, appear to be a promising means of detecting oligonucleotides. E-DNA sensors are comprised of a redox-modified (here, methylene blue or ferrocene) DNA stem-loop covalently attached to an interrogating electrode. Because E-DNA signaling arises due to binding-induced changes in the conformation of the stemloop probe, it is likely sensitive to the nature of the molecular packing on the electrode surface. Here we detail the effects of probe density, target length, and other aspects of molecular crowding on the signaling properties, specificity, and response time of a model E-DNA sensor. We find that the highest signal suppression is obtained at the highest probe densities investigated, and that greater suppression is observed with longer and bulkier targets. In contrast, sensor equilibration time slows monotonically with increasing probe density, and the specificity of hybridization is not significantly affected. In addition to providing insight into the optimization of electrochemical DNA sensors, these results suggest that E-DNA signaling arises due to hybridization-linked changes in the rate, and thus efficiency, with which the redox moiety collides with the electrode and transfers electrons.

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Section: Discussionmentioning

confidence: 99%

Effect of Molecular Crowding on the Response of an Electrochemical DNA Sensor

et al. 2007

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“…We presume this occurs because, at higher probe densities, the collision rate of the single-stranded probe is slow enough that electron transfer from unbound probes is also inhibited under these conditions. In support of this collision-limited signaling mechanism, we find that the rate of electron transfer slows by approximately an order of magnitude upon target binding (see Figure SI4).All of the groups responsible for the initial development of E-DNA sensors employed stem-loop DNA probes, [1][2][3][4][5][6][7][8][9]14,15 presumably due to the misconception, 1-3,9,14 shared by us, that, by analogy to molecular beacons, a specific conformational (i.e., geometric) change is required in order to support robust signaling. We have shown here, however, that bindinginduced changes in DNA dynamics are sufficient to support E-DNA signaling.…”

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confidence: 89%

“…All of the groups responsible for the initial development of E-DNA sensors employed stem-loop DNA probes, [1][2][3][4][5][6][7][8][9]14,15 presumably due to the misconception, 1-3,9,14 shared by us, that, by analogy to molecular beacons, a specific conformational (i.e., geometric) change is required in order to support robust signaling. We have shown here, however, that bindinginduced changes in DNA dynamics are sufficient to support E-DNA signaling.…”

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confidence: 99%

Linear, redox modified DNA probes as electrochemical DNA sensors

2007

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"Linear, redox modified DNA probes as electrochemical DNA sensors" (2007). Rebecca Lai Publications. 2. http://digitalcommons.unl.edu/chemistrylai/2 E-DNA sensors, which consist of a redox-tagged stemloop DNA covalently attached to an interrogating electrode, are the electrochemical equivalents of optical molecular beacons. [1][2][3][4][5][6][7][8][9] We show here, however, that unlike molecular beacons, which rely on a rigid, binding-induced conformational change (to segregate a fluorophore-quencher pair), 10-12 E-DNA signaling arises due to binding-induced changes in the dynamics of the probe DNA. We do so by demonstrating that hybridization-linked changes in the dynamics of an electrodebound linear (as opposed to stem-loop) probe DNA efficiently support E-DNA signaling. That is, whereas a large Faradaic current is observed from a redox-modified, single-stranded DNA probe, this current is reduced upon hybridization to the appropriate target DNA sequence due to changes in the rate with which the terminal redox label collides with the electrode surface ( Figure 1).We have fabricated E-DNA sensors using a 27-base linear probe sequence that, in order to facilitate direct comparison with earlier studies, is directly analogous to a previously characterized stem-loop E-DNA sensor 9,13 save that the five base sequences at the two termini are identical and thus do not form a double stranded stem. In the absence of target, the sensor gives rise to a sharp, well-defined AC voltammetry peak consistent with the ~ -0.26 V (vs. Ag/AgCl) formal potential of the methylene blue redox moiety employed (Figure 2). Upon hybridization to a fully complementary, 17-base target this current is significantly reduced. Furthermore, because the observed signal change arises due to a hybridization-specific change in DNA dynamics (as opposed to the simple adsorption of mass or charge to the sensor surface), we can readily observe this change even when the sensor is challenged with complex, multi-component sample matrices, such as targetdoped blood serum (Figure 2, right). Finally, like the original stem-loop E-DNA architecture, the linear-probe E-DNA sensor is label-free and reusable: a 30 sec wash in room temperature distilled water or (after deployment in blood serum) room temperature detergent solution is enough to regenerate >97% original sensor current (Figure 2).The signaling characteristics of linear probe E-DNA sensors are improved relative to those of the equivalent stem-loop sensor. Whereas a linear probe E-DNA sensor exhibits an 85% signal reduction at a given target concentration (Figure 2), the equivalent stem loop sensor exhibits only 71% signal suppression at this target concentration. 9 We presume this difference Published in Chemical Communications (2007), no. 36, pp. 3768-3770; doi Supplementary information follows the "References," including experimental procedures, controlling probe surface density, sensor equilibration time and specificity, the effect of target length and bulk on signaling, electron transfer rate meas...

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“…Electrochemistry has been used to detect nucleic acids with high sensitivity and without the need for PCR amplification in bacterial lysate and serum (18)(19)(20)(21)(22), but protein detection remains a challenge (23)(24)(25)(26). In fact, although protein detection from simple serum has been accomplished (27,28), to date no reported electrochemical systems have effectively detected active protein, of any kind, from crude cell lysate.…”

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confidence: 99%

Label-free electrochemical detection of human methyltransferase from tumors

Furst

Muren

Hill

et al. 2014

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

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The role of abnormal DNA methyltransferase activity in the development and progression of cancer is an essential and rapidly growing area of research, both for improved diagnosis and treatment. However, current technologies for the assessment of methyltransferase activity, particularly from crude tumor samples, limit this work because they rely on radioactivity or fluorescence and require bulky instrumentation. Here, we report an electrochemical platform that overcomes these limitations for the label-free detection of human DNA(cytosine-5)-methyltransferase1 (DNMT1) methyltransferase activity, enabling measurements from crude cultured colorectal cancer cell lysates (HCT116) and biopsied tumor tissues. Our multiplexed detection system involving patterning and detection from a secondary electrode array combines low-density DNA monolayer patterning and electrocatalytically amplified DNA charge transport chemistry to measure selectively and sensitively DNMT1 activity within these complex and congested cellular samples. Based on differences in DNMT1 activity measured with this assay, we distinguish colorectal tumor tissue from healthy adjacent tissue, illustrating the effectiveness of this two-electrode platform for clinical applications.DNA electrochemistry | methylation detection | electrocatalysis

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Aptamer-Based Electrochemical Detection of Picomolar Platelet-Derived Growth Factor Directly in Blood Serum

Cited by 342 publications

References 28 publications

Effect of Molecular Crowding on the Response of an Electrochemical DNA Sensor

Effect of Molecular Crowding on the Response of an Electrochemical DNA Sensor

Linear, redox modified DNA probes as electrochemical DNA sensors

Label-free electrochemical detection of human methyltransferase from tumors

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