Ryan J. White

Fluorescence Detection of Single-Nucleotide Polymorphisms with a Single, Self-Complementary, Triple-Stem DNA Probe†

Singled out for its singularity: In a single-step, single-component, fluorescence-based method for the detection of single-nucleotide polymorphisms at room temperature, the sensor is comprised of a single, self-complementary DNA strand that forms a triple-stem structure. The large conformational change that occurs upon binding to perfectly matched (PM) targets results in a significant increase in fluorescence (see picture; F = fluorophore, Q = quencher).

CheapStat: An Open-Source, “Do-It-Yourself” Potentiostat for Analytical and Educational Applications

Although potentiostats are the foundation of modern electrochemical research, they have seen relatively little application in resource poor settings, such as undergraduate laboratory courses and the developing world. One reason for the low penetration of potentiostats is their cost, as even the least expensive commercially available laboratory potentiostats sell for more than one thousand dollars. An inexpensive electrochemical workstation could thus prove useful in educational labs, and increase access to electrochemistry-based analytical techniques for food, drug and environmental monitoring. With these motivations in mind, we describe here the CheapStat, an inexpensive (<

Simultaneous Alternating and Direct Current Readout of Protein Ion Channel Blocking Events Using Glass Nanopore Membranes

80), open-source (software and hardware), hand-held potentiostat that can be constructed by anyone who is proficient at assembling circuits. This device supports a number of potential waveforms necessary to perform cyclic, square wave, linear sweep and anodic stripping voltammetry. As we demonstrate, it is suitable for a wide range of applications ranging from food- and drug-quality testing to environmental monitoring, rapid DNA detection, and educational exercises. The devices schematics, parts lists, circuit board layout files, sample experiments, and detailed assembly instructions are available in the supporting information and are released under an open hardware license.

Re-engineering aptamers to support reagentless, self-reporting electrochemical sensors

Alternating current (ac) phase-sensitive detection is used to measure the conductance of the ion channel alpha-hemolysin (alphaHL), while simultaneously applying a direct current (dc) bias to electrostatically control the binding affinity and kinetics of charged molecules within the protein lumen. Ion channel conductance was recorded while applying a 10-20 mV rms, 1-2 kHz bias across a single alphaHL protein inserted in a 1,2-diphytanoyl-sn-glycero-3-phosphocholine lipid bilayer that is suspended across the orifice (100-500 nm radius) of a glass nanopore membrane. Step changes in the ac ion channel conductance with a temporal response (t(10-90)) of 1.5 ms and noise amplitude of approximately 2 pA were obtained using a low-noise potentiostat and a lock-in amplifier. These conditions were used to monitor the reversible and stochastic binding of heptakis-(6-O-sulfo)-beta-cyclodextrin and a nine base pair DNA hairpin molecule to the ion channel. Alternating current methodology allows the binding kinetics and affinity between the protein ion channel and analyte to be investigated as a function of the dc bias, including ion channel conductance measurements in the absence of a dc bias.

Polarity‐Switching Electrochemical Sensor for Specific Detection of Single‐Nucleotide Mismatches

Electrochemical aptamer-based (E-AB) sensors have emerged as a promising and versatile new biosensor platform. Combining the generality and specificity of aptamer-ligand interactions with the selectivity and convenience of electrochemical readouts, this approach affords the detection of a wide variety of targets directly in complex, contaminant-ridden samples, such as whole blood, foodstuffs and crude soil extracts, without the need for exogenous reagents or washing steps. Signaling in this class of sensors is predicated on target-induced changes in the conformation of an electrode-bound probe aptamer that, in turn, changes the efficiency with which a covalently attached redox tag exchanges electrons with the interrogating electrode. Aptamer selection strategies, however, typically do not select for the conformation-switching architectures, and as such several approaches have been reported to date by which aptamers can be re-engineered such that they undergo the binding-induced switching required to support efficient E-AB signaling. Here, we systematically compare the merits of these re-engineering approaches using representative aptamers specific to the small molecule adenosine triphosphate and the protein human immunoglobulin E. We find that, while many aptamer architectures support E-AB signaling, the observed signal gain (relative change in signal upon target binding) varies by more than two orders of magnitude across the various constructs we have investigated (e.g., ranging from -10% to 200% for our ATP sensors). Optimization of the switching architecture is thus an important element in achieving maximum E-AB signal gain and we find that this optimal geometry is specific to the aptamer sequence upon which the sensor is built.

Sensitivity and signal complexity as a function of the number of ion channels in a stochastic sensor.

Single-nucleotide polymorphisms (SNPs)—genetic variations that involve only a single DNA base-pair—can directly affect transcriptional regulations and protein functions. Thus, SNP genotyping serves as an important diagnostic for genetic diseases and drug responses. To date, methods of detecting such single-nucleotide mismatches can be broadly categorized into enzyme-aided and hybridizationbased approaches. The enzyme-aided approach typically involves a two-step, multi-component assay, in which a singlenucleotide-specific enzymatic reaction, such as primer extension, ligation, or cleavage, is coupled with a downstream detection of reaction products. As such, these methods are inherently complex, and the assay specificity is limited by both the activity of the enzyme and the sensitivity of the detection technique. In contrast, hybridization-based methods utilize DNA probes and various measurement techniques to report the hybridization difference between perfectly matched (PM) and single-nucleotide mismatched (SM) targets in a single-step. However, to resolve the small difference in thermodynamic stability between the two targets, these detection methods generally require complex probe designs and the careful control of hybridization conditions such as buffer composition, washing stringency, and melting temperature. In addition to these complex requirements, both enzymeaided and hybridization-based approaches are susceptible to false-positives because they can only measure the difference in the signal amplitude between PM and SM targets—and signal amplitude measurements are prone to fluctuation in target/probe concentrations, background contaminants, and other experimental perturbations (e.g., enzyme activity, washing stringency or temperature). Thus, for robust detection of single-nucleotide mismatches, there is a need for alternative sensor architectures that are less prone to errors from fluctuations in the signal amplitude. Toward this end, we present a single-step, room-temperature electrochemical sensor that detects single-nucleotide mismatches with a “polarity-switching” response. Our “bipolar” sensor reports a decreased output signal (signal-off) when hybridized with a PM target (Figure 1a, top right) but reports an opposite, increased signal (signal-on) when hybridized with a SM target (Figure 1a, bottom right). The output signal of the sensor is generated by the redox reporter methylene blue (MB), which is covalently attached to an electrodebound DNA probe. The polarity-switching response is achieved by tuning two key parameters—the structural flexibility of the probe and its interaction with the MB tag—that control the electron transfer between the MB tag and the electrode. In this work, we describe the design principles of the bipolar sensor and demonstrate its performance in discriminating SM and PM targets under various conditions. Furthermore, we elucidate the mechanism behind the polarity-switching behavior and quantify the relative contributions of the two parameters that govern the sensor output. The change in the output Faradaic current of our sensor is caused by alterations in the rate of electron transfer to the gold interrogating electrode, which is governed by the equilibrium probability of the DNA-boundMB tag approaching the electrode surface. In our sensor design, we exploited the structural flexibility of the DNA probe and the interaction between MB and DNA (e.g., intercalation and groove binding) to achieve mismatch detection through polarity switching. Regarding the probe flexibility parameter, the higher flexibility of single-stranded DNA (ssDNA) relative to rigid double-stranded DNA (dsDNA) increases the MB electron transfer rate and yields higher Faradaic currents. In parallel, the interaction between the MB tag and dsDNA decreases the electron transfer rate, thus reducing the Faradaic current. This decreased electron transfer is presumably due to the confinement of theMB tag within the DNA duplex, which lowers the probability of the MB approaching the electrode. Of note, this MB-dsDNA interaction depends on the DNA sequence, which therefore needs to be evaluated prior to sensor design. In the present case, we have experimentally determined that the interaction between MB and poly(thymine–adenosine) (T-A) duplexes effectively slows MB electron transfer rate com[*] Dr. Y. Xiao, Prof. H. T. Soh Materials Department, Department of Mechanical Engineering University of California, Santa Barbara Santa Barbara, CA 93106 (USA) E-mail: yixiao@physics.ucsb.edu tsoh@engr.ucsb.edu

Biomimetic glass nanopores employing aptamer gates responsive to a small molecule

Alternating current, phase-sensitive stochastic detection using between 1 and 26 alpha-hemolysin ion channels reconstituted in a lipid bilayer, suspended over a 160-nm-radius orifice glass nanopore, is reported. As predicted by the binomial distribution, simultaneous analyte detection at large numbers of channels is effectively zero, independent of the number of ion channels. The results indicate that alphaHL channels are noninteracting and that significant gains in sensitivity are possible without sacrificing the simplicity of single-molecule detection strategies.

Engineering New Aptamer Geometries for Electrochemical Aptamer-Based Sensors

We report the preparation of 20 and 65 nm radii glass nanopores whose surface is modified with DNA aptamers controlling the molecular transport through the nanopores in response to small molecule binding.

Single Ion-Channel Recordings Using Glass Nanopore Membranes

Electrochemical aptamer-based sensors (E-AB sensors) represent a promising new approach to the detection of small molecules. E-AB sensors comprise an aptamer that is attached at one end to an electrode surface. The distal end of the aptamer probed is modified with an electroactive redox marker for signal transduction. Herein we report on the optimization of a cocaine-detecting E-AB sensor via optimization of the geometry of the aptamer. We explore two new aptamer architectures, one in which we concatenate three cocaine aptamers into a poly-aptamer and a second in which we divide the cocaine aptamer into pieces connected via an unstructured, 60-thymine linker. Both of these structures are designed such that the reporting redox tag will be located farther from the electrode in the unfolded, target-free conformation. Consistent with this, we find that signal gains of these two constructs are two to three times higher than that of the original E-AB architecture. Likewise all three architectures are selective enough to deploy directly in complex sample matrices, such as undiluted whole blood, with all three sensors successfully detecting the presence of cocaine. The findings in this ongoing study should be of value in future efforts to optimize the signaling of electrochemical aptamer-based sensors.