Kuangwen Hsieh

Rapid, Sensitive, and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic Electrochemical Quantitative Loop-Mediated Isothermal Amplification†

Single-step DNA detection: a microfluidic electrochemical loop mediated isothermal amplification platform is reported for rapid, sensitive, and quantitative detection of pathogen genomic DNA at the point of care. DNA amplification was electrochemically monitored in real time within a monolithic microfluidic device, thus enabling the detection of as few as 16 copies of Salmonella genomic DNA through a single-step process in less than an hour.

Genetic Analysis of H1N1 Influenza Virus from Throat Swab Samples in a Microfluidic System for Point-of-Care Diagnostics

The ability to obtain sequence-specific genetic information about rare target organisms directly from complex biological samples at the point-of-care would transform many areas of biotechnology. Microfluidics technology offers compelling tools for integrating multiple biochemical processes in a single device, but despite significant progress, only limited examples have shown specific, genetic analysis of clinical samples within the context of a fully integrated, portable platform. Herein we present the Magnetic Integrated Microfluidic Electrochemical Detector (MIMED) that integrates sample preparation and electrochemical sensors in a monolithic disposable device to detect RNA-based virus directly from throat swab samples. By combining immunomagnetic target capture, concentration, and purification, reverse-transcriptase polymerase chain reaction (RT-PCR) and single-stranded DNA (ssDNA) generation in the sample preparation chamber, as well as sequence-specific electrochemical DNA detection in the electrochemical cell, we demonstrate the detection of influenza H1N1 in throat swab samples at loads as low as 10 TCID(50), 4 orders of magnitude below the clinical titer for this virus. Given the availability of affinity reagents for a broad range of pathogens, our system offers a general approach for multitarget diagnostics at the point-of-care.

Integrated Microfluidic Electrochemical DNA Sensor

Effective systems for rapid, sequence-specific nucleic acid detection at the point of care would be valuable for a wide variety of applications, including clinical diagnostics, food safety, forensics, and environmental monitoring. Electrochemical detection offers many advantages as a basis for such platforms, including portability and ready integration with electronics. Toward this end, we report the Integrated Microfluidic Electrochemical DNA (IMED) sensor, which combines three key biochemical functionalities--symmetric PCR, enzymatic single-stranded DNA generation, and sequence-specific electrochemical detection--in a disposable, monolithic chip. Using this platform, we demonstrate detection of genomic DNA from Salmonella enterica serovar Typhimurium LT2 with a limit of detection of <10 aM, which is approximately 2 orders of magnitude lower than that from previously reported electrochemical chip-based methods.

Real-Time, Aptamer-Based Tracking of Circulating Therapeutic Agents in Living Animals

An aptamer-based biosensor continuously measures the concentration of drug molecules in the blood of living animals and in patient samples. Tracking Drugs in Real Time You have the drug, it’s time to give to the patient. Now, what is the ideal dose? Many drugs have unwanted side effects when given at large doses; conversely, they are not efficacious at too low of a dose. Continuously monitoring a drug as it circulates throughout the body would give doctors a better grip on personalized medicine, by allowing them to then tailor the therapeutic dose and schedule for each patient. To this end, Ferguson et al. developed a biosensor that reports the concentration of a drug in real time in live animals and in patient samples. The microfluidic sensing device, which the authors named MEDIC (microfluidic electrochemical detector for in vivo continuous monitoring), consisted of an electrochemically modified aptamer—a oligonucleotide that is highly specific for a target drug—attached to a gold electrode, as well as a filter to prevent blood cells from clogging up the device. The electrodes reported the change in charge as the drug bound to the aptamer. Ferguson et al. used two different aptamers: one specific for doxorubicin (DOX; a cancer drug) and one for kanamycin (an antibiotic). The authors first demonstrated that MEDIC could detect submicromolar concentrations of DOX in human whole blood. The MEDIC was then hooked up to live rats to continuously draw blood for monitoring. Injecting the animals with a drug-free solution yielded no change in device signal. However, injecting therapeutically relevant doses of DOX or kanamycin—depending on the device configuration—quickly produced a signal that corresponded to the in vivo drug concentration. Such continuous monitoring of drugs could afford clinicians the opportunity to tailor therapeutic regimens to individual patients, thus preventing toxic side effects or dialing up the drug effect. Translating this technology to people may require tweaking the sensor for longer operation times (days to weeks, versus the hours described here), as well as safety testing. Once deemed useful and safe, the device could replace periodic and disruptive blood draws at the patient’s bedside, much like continuous glucose monitors in widespread use today for diabetes. 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.

Controlled delivery of DNA origami on patterned surfaces

Due to its capacity for programmable self-assembly, wellestablished modes of chemical synthesis, and exceptional stability, DNA serves as a powerful nanoscale structural material. In particular, the recent invention of DNA origami technology has established a paradigm in which DNA’s capacity for deterministic self-assembly into essentially any discrete two-dimensional (2D) shape can be exploited for the construction of molecular ‘‘bread boards’’. For example, previous groups have demonstrated the delivery of nanoparticles to specific positions within an origami scaffold with nanometer-scale precision and the weaving of DNA aptamers into origami for the assembly of protein arrays. However, in order to fully harness the potential of DNA as a universal nanoscale template, it is important not only to control the position of cargo material within the origami scaffold but also to accurately control the position and orientation of the

Quantification of Transcription Factor Binding in Cell Extracts Using an Electrochemical, Structure-Switching Biosensor

Transcription factor expression levels, which sensitively reflect cellular development and disease state, are typically monitored via cumbersome, reagent-intensive assays that require relatively large quantities of cells. Here, we demonstrate a simple, quantitative approach to their detection based on a simple, electrochemical sensing platform. This sensor sensitively and quantitatively detects its target transcription factor in complex media (e.g., 250 μg/mL crude nuclear extracts) in a convenient, low-reagent process requiring only 10 μL of sample. Our approach thus appears a promising means of monitoring transcription factor levels.

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

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

Electrochemical real-time nucleic acid amplification: towards point-of-care quantification of pathogens

Real-time nucleic acid amplification, whereby the amplification rate is used to quantify the initial copy number of target DNA or RNA, has proven highly effective for monitoring pathogen loads. Unfortunately, however, current optical methods are limited to centralized laboratories due to complexity, bulk and cost. In response, recent efforts aim to develop lower-cost, electrochemical real-time amplification platforms for point-of-care applications, with researchers already having developed platforms that not only perform in situ and concurrent electrochemical detection during amplification, but also deliver sensitivity and specificity potentially rivaling bench-top optical systems. This report chronicles the evolution of the different strategies, describes the current state of the art, and identifies challenges of bringing the power of real-time detection to the point-of-care.

Accurate Zygote‐Specific Discrimination of Single‐Nucleotide Polymorphisms Using Microfluidic Electrochemical DNA Melting Curves

We report the first electrochemical system for the detection of single-nucleotide polymorphisms (SNPs) that can accurately discriminate homozygous and heterozygous genotypes using microfluidics technology. To achieve this, our system performs real-time melting-curve analysis of surface-immobilized hybridization probes. As an example, we used our sensor to analyze two SNPs in the apolipoprotein E (ApoE) gene, where homozygous and heterozygous mutations greatly affect the risk of late-onset Alzheimers disease. Using probes specific for each SNP, we simultaneously acquired melting curves for probe-target duplexes at two different loci and thereby accurately distinguish all six possible ApoE allele combinations. Since the design of our device and probes can be readily adapted for targeting other loci, we believe that our method offers a modular platform for the diagnosis of SNP-based diseases and personalized medicine.