Ryan J White

University of California, Santa Barbara, Santa Barbara, California, United States

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Publications (31)176.9 Total impact

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    ABSTRACT: A sensor capable of continuously measuring specific molecules in the bloodstream in vivo would give clinicians a valuable window into patients' health and their response to therapeutics. Such technology would enable truly personalized medicine, wherein therapeutic agents could be tailored with optimal doses for each patient to maximize efficacy and minimize side effects. Unfortunately, continuous, real-time measurement is currently only possible for a handful of targets, such as glucose, lactose, and oxygen, and the few existing platforms for continuous measurement are not generalizable for the monitoring of other analytes, such as small-molecule therapeutics. In response, we have developed a real-time biosensor capable of continuously tracking a wide range of circulating drugs in living subjects. Our microfluidic electrochemical detector for in vivo continuous monitoring (MEDIC) requires no exogenous reagents, operates at room temperature, and can be reconfigured to measure different target molecules by exchanging probes in a modular manner. To demonstrate the system's versatility, we measured therapeutic in vivo concentrations of doxorubicin (a chemotherapeutic) and kanamycin (an antibiotic) in live rats and in human whole blood for several hours with high sensitivity and specificity at subminute temporal resolution. We show that MEDIC can also obtain pharmacokinetic parameters for individual animals in real time. Accordingly, just as continuous glucose monitoring technology is currently revolutionizing diabetes care, we believe that MEDIC could be a powerful enabler for personalized medicine by ensuring delivery of optimal drug doses for individual patients based on direct detection of physiological parameters.
    Science translational medicine 11/2013; 5(213):213ra165. · 10.76 Impact Factor
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    ABSTRACT: We fabricated and tested encoders and decoders based on a multiplex, DNA-based electrochemical biosensor that uses electronic (electrochemical) signals as its readout. These devices use two or more sequence-specific DNA probes, with each being modified with a distinct redox reporter. These probes, when interrogated together, serve as encoders and decoders, converting patterns that are encoded and decoded by the presence or absence of specific DNA sequences into specific electronic outputs. We demonstrated these multifunctional, bio-electrochemical devices, for example, 4-to-2 and 8-to-3 encoders and 1-to-2 and 2-to-3 decoders. Accordingly, these devices bridge the division between DNA-based devices and silicon-based electronics.
    NPG Asia Materials. 01/2012; 4(1).
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    ABSTRACT: The diagnosis, prevention, and treatment of many illnesses, including infectious and autoimmune diseases, would benefit from the ability to measure specific antibodies directly at the point of care. Thus motivated, we designed a wash-free, electrochemical method for the rapid, quantitative detection of specific antibodies directly in undiluted, unprocessed blood serum. Our approach employs short, contiguous polypeptide epitopes coupled to the distal end of an electrode-bound nucleic acid "scaffold" modified with a reporting methylene blue. The binding of the relevant antibody to the epitope reduces the efficiency with which the redox reporter approaches, and thus exchanges electrons with, the underlying sensor electrode, producing readily measurable change in current. To demonstrate the versatility of the approach, we fabricated a set of six such sensors, each aimed at the detection of a different monoclonal antibody. All six sensors are sensitive (subnanomolar detection limits), rapid (equilibration time constants ∼8 min), and specific (no appreciable cross reactivity with the targets of the other five). When deployed in a millimeter-scale, an 18-pixel array with each of the six sensors in triplicate support the simultaneous measurement of the concentrations of multiple antibodies in a single, submilliliter sample volume. The described sensor platform thus appears be a relatively general approach to the rapid and specific quantification of antibodies in clinical materials.
    Analytical Chemistry 12/2011; 84(2):1098-103. · 5.70 Impact Factor
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    Angewandte Chemie International Edition 11/2011; 50(47):11176-80. · 13.73 Impact Factor
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    ABSTRACT: 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.
    Journal of Visualized Experiments 01/2011;
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    ABSTRACT: 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 (<$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 device's 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.
    PLoS ONE 01/2011; 6(9):e23783. · 3.73 Impact Factor
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    ABSTRACT: 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.
    Chemical Communications 11/2010; 46(42):7984-6. · 6.38 Impact Factor
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    ABSTRACT: In a traditional sandwich assay, a DNA target hybridizes to a single copy of the signal probe. Here we employ a modified signal probe containing a methylene blue (a redox moiety) label and a "sticky end." When a DNA target hybridizes this signal probe, the sticky end remains free to hybridize another target leading to the creation of a supersandwich structure containing multiple labels. This leads to large signal amplification upon monitoring by voltammetry.
    Journal of the American Chemical Society 10/2010; 132(41):14346-8. · 10.68 Impact Factor
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    ABSTRACT: Electrode-bound, redox-reporter-modified oligonucleotides play roles in the functioning of a number of electrochemical biosensors, and thus the question of electron transfer through or from such molecules has proven of significant interest. In response, we have experimentally characterized the rate with which electrons are transferred between a methylene blue moiety on the distal end of a short, single-stranded polythymine DNA to a monolayer-coated gold electrode to which the other end of the DNA is site-specifically attached. We find that this rate scales with oligonucleotide length to the -1.16 ± 0.09 power. This weak, approximately inverse length dependence differs dramatically from the much stronger dependencies observed for the rates of end-to-end collisions in single-stranded DNA and through-oligonucleotide electron hopping. It instead coincides with the expected length dependence of a reaction-limited process in which the overall rate is proportional to the equilibrium probability that the end of the oligonucleotide chain approaches the surface. Studies of the ionic strength and viscosity dependencies of electron transfer further support this "chain-flexibility" mechanism, and studies of the electron transfer rate of methylene blue attached to the hexanethiol monolayer suggest that heterogeneous electron transfer through the monolayer is rate limiting. Thus, under the circumstances we have employed, the flexibility (i.e., the equilibrium statistical properties) of the oligonucleotide chain defines the rate with which an attached redox reporter transfers electrons to an underlying electrode, an observation that may be of utility in the design of new biosensor architectures.
    Journal of the American Chemical Society 10/2010; 132(45):16120-6. · 10.68 Impact Factor
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    ABSTRACT: An "XOR" gate built using label-free, dual-analyte electrochemical sensors and the activation of this logic gate via changing concentrations of cocaine and the relevant cDNA as inputs are described.
    Journal of the American Chemical Society 06/2010; 132(25):8557-9. · 10.68 Impact Factor
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    Ryan J White, Aaron A Rowe, Kevin W Plaxco
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    ABSTRACT: 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.
    The Analyst 03/2010; 135(3):589-94. · 4.23 Impact Factor
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    Ryan J White, Kevin W Plaxco
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    ABSTRACT: Electrochemical sensors employing redox-tagged, electrode-bound oligonucleotides have emerged as a promising new platform for the reagentless detection of molecular analytes. Signal generation in these sensors is linked to specific, binding-induced changes in the efficiency with which an attached redox tag approaches and exchanges electrons with the interrogating electrode. We present here a straightforward means of optimizing the signal gain of these sensors that exploits this mechanism. Specifically, using square-wave voltammetry, which is exquisitely sensitive to electrode reaction rates, we can tune the frequency of the voltammetric measurements to preferentially enhance the signal associated with either the unbound or target-bound conformations of the probe. This allows us to control not only the magnitude of the signal gain associated with target binding but also the sign of the signal change, generating "signal-on" or "signal-off" sensors. This optimization parameter appears to be quite general: we show here that tuning the square-wave frequency can significantly enhance the gain of the sensors directed against specific oligonucleotide sequences, small molecules, proteins, and protein-small molecule interactions.
    Analytical Chemistry 12/2009; 82(1):73-6. · 5.70 Impact Factor
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    ABSTRACT: Many electrochemical biosensor approaches developed in recent years utilize redox-labeled (most commonly methylene blue or ferrocene) oligonucleotide probes site-specifically attached to an interrogating electrode. Sensors in this class have been reported that employ a range of probe architectures, including single- and double-stranded DNA, more complex DNA structures, DNA and RNA aptamers, and, most recently, DNA-small molecule chimeras. Signaling in this class of sensors is generally predicated on binding-induced changes in the efficiency with which the covalently attached redox label transfers electrons with the interrogating electrode. Here we have investigated how the properties of the redox tag affect the performance of such sensors. Specifically, we compare the differences in signaling and stability of electrochemical DNA sensors (E-DNA sensors) fabricated using either ferrocene or methylene blue as the signaling redox moiety. We find that while both tags support efficient E-DNA signaling, ferrocene produces slightly improved signal gain and target affinity. These small advantages, however, come at a potentially significant price: the ferrocene-based sensors are far less stable than their methylene blue counterparts, particularly with regards to stability to long-term storage, repeated electrochemical interrogations, repeated sensing/regeneration iterations, and employment in complex sample matrices such as blood serum.
    Analytical Chemistry 10/2009; 81(21):9109-13. · 5.70 Impact Factor
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    ABSTRACT: Recent years have seen the emergence of a new class of electrochemical sensors predicated on target binding-induced folding of electrode-bound redox-modified aptamers and directed against targets ranging from small molecules to proteins. Previous studies of the relationship between gain and probe-density for these electrochemical, aptamer-based (E-AB) sensors suggest that signal transduction is linked to binding-induced changes in the efficiency with which the attached redox tag strikes the electrode. This, in turn, suggests that even well folded aptamers may support E-AB signaling if target binding sufficiently alters their flexibility. Here we investigate this using a thrombin-binding aptamer that undergoes binding-induced folding at low ionic strength but can be forced to adopt a folded conformation at higher ionic strength even in the absence of its protein target. We find that, under conditions in which the thrombin aptamer is fully folded prior to target binding, we still obtain a ca. 30% change in E-AB signal upon saturated target levels. In contrast, however, under conditions in which the aptamer is unfolded in the absence of target and thus undergoes binding-induced folding the observed signal change is twice as great. The ability of folded aptamers to support E-AB signaling, however, is not universal: a fully folded anti-IgE aptamer, for example, produces only an extremely small, ca. 2.5% signal change in the presence of target despite the larger steric bulk of this protein. Thus, while it appears that binding-induced changes in the dynamics in fully folded aptamers can support E-AB signaling, this signaling mechanism may not be general, and in order to ensure the design of high-gain sensors binding must be linked to a large-scale conformational change.
    Electroanalysis 06/2009; 21(11):1267-1271. · 2.82 Impact Factor
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    ABSTRACT: 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).
    Angewandte Chemie International Edition 06/2009; 48(24):4354-8. · 13.73 Impact Factor
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    ABSTRACT: Previous work has described several reagentless, electrochemical DNA (E-DNA) sensing architectures comprised of an electrode-immobilized, redox-tagged probe oligonucleotide. Recent studies suggest that E-DNA signaling is predicated on hybridization-linked changes in probe flexibility, which will alter the efficiency with which the terminal redox tag strikes the electrode. This, in turn, suggests that probe length, probe geometry, and redox-tag placement will affect E-DNA signaling. To test this we have characterized E-DNA sensors comprised of linear or stem-loop probes of various lengths and with redox tags placed either distal to the electrode or internally within the probe sequence (proximal). We find that linear probes produce larger signal changes upon target binding than equivalent stem-loop probes. Likewise, long probes exhibit greater signal changes than short probes provided that the redox tag is placed proximal to the electrode surface. In contrast to their improved signaling, the specificity of long probes is poorer than that of short probes, suggesting that sensor optimization represents a trade off between sensitivity and specificity. Finally, we find that sensor response time and selectivity are only minimally affected by probe geometry or length. The results of this comparative study will help guide future designs and applications of these sensors.
    Analytical Chemistry 03/2009; 81(6):2150-8. · 5.70 Impact Factor
  • Noelle Phares, Ryan J White, Kevin W Plaxco
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    ABSTRACT: Alkane thiol self-assembled monolayers (SAMs) have seen widespread utility in the fabrication of electrochemical biosensors. Their utility, however, reflects a potentially significant compromise. While shorter SAMs support efficient electron transfer, they pack poorly and are thus relatively unstable. Longer SAMs are more stable but suffer from less efficient electron transfer, thus degrading sensor performance. Here we use the electrochemical DNA (E-DNA) sensor platform to compare the signaling and stability of biosensors fabricated using a short, six-carbon monothiol with those employing either of two commercially available trihexylthiol anchors (a flexible Letsinger type and a rigid adamantane type). We find that all three anchors support efficient electron transfer and E-DNA signaling, with the gain, specificity, and selectivity of all three being effectively indistinguishable. The stabilities of the three anchors, however, vary significantly. Sensors anchored with the flexible trithiol exhibit enhanced stability, retaining 75% of their original signal and maintaining excellent signaling properties after 50 days storage in buffer. Likewise these sensors exhibit excellent temperature stability and robustness to electrochemical interrogation. The stability of sensors fabricated using the rigid trithiol anchor, by comparison, are similar to those of the monothiol, with both exhibiting significant (>60%) loss of signal upon wet storage or thermocycling. Employing a flexible trithiol anchor in the fabrication of SAM-based electrochemical biosensors may provide a means of improving sensor robustness without sacrificing electron transfer efficiency or otherwise impeding sensor performance.
    Analytical Chemistry 02/2009; 81(3):1095-100. · 5.70 Impact Factor
  • Eric N Ervin, Ryan J White, Henry S White
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    ABSTRACT: 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.
    Analytical Chemistry 02/2009; 81(2):533-7. · 5.70 Impact Factor
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    Ryan J White, Kevin W Plaxco
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    ABSTRACT: 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.
    Proceedings - Society of Photo-Optical Instrumentation Engineers 01/2009; 7321:732105.
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    ABSTRACT: Electrochemical, aptamer-based (E-AB) sensors, which are comprised of an electrode modified with surface immobilized, redox-tagged DNA aptamers, have emerged as a promising new biosensor platform. In order to further improve this technology we have systematically studied the effects of probe (aptamer) packing density, the AC frequency used to interrogate the sensor, and the nature of the self-assembled monolayer (SAM) used to passivate the electrode on the performance of representative E-AB sensors directed against the small molecule cocaine and the protein thrombin. We find that, by controlling the concentration of aptamer employed during sensor fabrication, we can control the density of probe DNA molecules on the electrode surface over an order of magnitude range. Over this range, the gain of the cocaine sensor varies from 60% to 200%, with maximum gain observed near the lowest probe densities. In contrast, over a similar range, the signal change of the thrombin sensor varies from 16% to 42% and optimal signaling is observed at intermediate densities. Above cut-offs at low hertz frequencies, neither sensor displays any significant dependence on the frequency of the alternating potential employed in their interrogation. Finally, we find that E-AB signal gain is sensitive to the nature of the alkanethiol SAM employed to passivate the interrogating electrode; while thinner SAMs lead to higher absolute sensor currents, reducing the length of the SAM from 6-carbons to 2-carbons reduces the observed signal gain of our cocaine sensor 10-fold. We demonstrate that fabrication and operational parameters can be varied to achieve optimal sensor performance and that these can serve as a basic outline for future sensor fabrication.
    Langmuir 10/2008; 24(18):10513-8. · 4.19 Impact Factor

Publication Stats

383 Citations
36 Downloads
2k Views
176.90 Total Impact Points

Institutions

  • 2008–2012
    • University of California, Santa Barbara
      • Department of Chemistry and Biochemistry
      Santa Barbara, California, United States
  • 2005–2009
    • University of Utah
      • Department of Chemistry
      Salt Lake City, UT, United States