Brian Scott Ferguson

Continuous, Real-Time Monitoring of Cocaine in Undiluted Blood Serum via a Microfluidic, Electrochemical Aptamer-Based Sensor

The development of a biosensor system capable of continuous, real-time measurement of small-molecule analytes directly in complex, unprocessed aqueous samples has been a significant challenge, and successful implementation has been achieved for only a limited number of targets. Toward a general solution to this problem, we report here the Microfluidic Electrochemical Aptamer-based Sensor (MECAS) chip wherein we integrate target-specific DNA aptamers that fold, and thus generate an electrochemical signal, in response to the analyte with a microfluidic detection system. As a model, we demonstrate the continuous, real-time (approximately 1 min time resolution) detection of the small-molecule drug cocaine at near physiological, low micromolar concentrations directly in undiluted, otherwise unmodified blood serum. We believe our approach of integrating folding-based electrochemical sensors with miniaturized detection systems may lay the groundwork for the real-time, point-of-care detection of a wide variety of molecular targets.

Selection of phage-displayed peptides on live adherent cells in microfluidic channels

Affinity reagents that bind to specific molecular targets are an essential tool for both diagnostics and targeted therapeutics. There is a particular need for advanced technologies for the generation of reagents that specifically target cell-surface markers, because transmembrane proteins are notoriously difficult to express in recombinant form. We have previously shown that microfluidics offers many advantages for generating affinity reagents against purified protein targets, and we have now significantly extended this approach to achieve successful in vitro selection of T7 phage-displayed peptides that recognize markers expressed on live, adherent cells within a microfluidic channel. As a model, we have targeted neuropilin-1 (NRP-1), a membrane-bound receptor expressed at the surface of human prostate carcinoma cells that plays central roles in angiogenesis, cell migration, and invasion. We show that, compared to conventional biopanning methods, microfluidic selection enables more efficient discovery of peptides with higher affinity and specificity by providing controllable and reproducible means for applying stringent selection conditions against minimal amounts of target cells without loss. Using our microfluidic system, we isolate peptide sequences with superior binding affinity and specificity relative to the well known NRP-1-binding RPARPAR peptide. As such microfluidic systems can be used with a wide range of biocombinatorial libraries and tissue types, we believe that our method represents an effective approach toward efficient biomarker discovery from patient samples.

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.

Microfluidic Device Architecture for Electrochemical Patterning and Detection of Multiple DNA Sequences

Electrochemical biosensors pose an attractive solution for point-of-care diagnostics because they require minimal instrumentation and they are scalable and readily integrated with microelectronics. The integration of electrochemical biosensors with microscale devices has, however, proven to be challenging due to significant incompatibilities among biomolecular stability, operation conditions of electrochemical sensors, and microfabrication techniques. Toward a solution to this problem, we have demonstrated here an electrochemical array architecture that supports the following processes in situ, within a self-enclosed microfluidic device: (a) electrode cleaning and preparation, (b) electrochemical addressing, patterning, and immobilization of sensing biomolecules at selected sensor pixels, (c) sequence-specific electrochemical detection from multiple pixels, and (d) regeneration of the sensing pixels. The architecture we have developed is general, and it should be applicable to a wide range of biosensing schemes that utilize gold-thiol self-assembled monolayer chemistry. As a proof-of-principle, we demonstrate the detection and differentiation of polymerase chain reaction (PCR) amplicons diagnostic of human (H1N1) and avian (H5N1) influenza.

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

Microfluidic Chip-Based Detection and Intraspecies Strain Discrimination of Salmonella Serovars Derived from Whole Blood of Septic Mice

ABSTRACT Salmonella is a zoonotic pathogen that poses a considerable public health and economic burden in the United States and worldwide. Resultant human diseases range from enterocolitis to bacteremia to sepsis and are acutely dependent on the particular serovar of Salmonella enterica subsp. enterica, which comprises over 99% of human-pathogenic S. enterica isolates. Point-of-care methods for detection and strain discrimination of Salmonella serovars would thus have considerable benefit to medical, veterinary, and field applications that safeguard public health and reduce industry-associated losses. Here we describe a single, disposable microfluidic chip that supports isothermal amplification and sequence-specific detection and discrimination of Salmonella serovars derived from whole blood of septic mice. The integrated microfluidic electrochemical DNA (IMED) chip consists of an amplification chamber that supports loop-mediated isothermal amplification (LAMP), a rapid, single-temperature amplification method as an alternative to PCR that offers advantages in terms of sensitivity, reaction speed, and amplicon yield. The amplification chamber is connected via a microchannel to a detection chamber containing a reagentless, multiplexed (here biplex) sensing array for sequence-specific electrochemical DNA (E-DNA) detection of the LAMP products. Validation of the IMED device was assessed by the detection and discrimination of S. enterica subsp. enterica serovars Typhimurium and Choleraesuis, the causative agents of enterocolitis and sepsis in humans, respectively. IMED chips conferred rapid (under 2 h) detection and discrimination of these strains at clinically relevant levels (<1,000 CFU/ml) from whole, unprocessed blood collected from septic animals. The IMED-based chip assay shows considerable promise as a rapid, inexpensive, and portable point-of-care diagnostic platform for the detection and strain-specific discrimination of microbial pathogens.