Rebecca Y. Lai

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.

Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing

Recent years have seen the development of a number of reagentless, electrochemical sensors based on the target-induced folding or unfolding of electrode-bound oligonucleotides, with examples reported to date, including sensors for the detection of specific nucleic acids, proteins, small molecules and inorganic ions. These devices, which are often termed electrochemical DNA (E-DNA) and E-AB (electrochemical, aptamer-based) sensors, are comprised of an oligonucleotide probe modified with a redox reporter (in this protocol methylene blue) at one terminus and attached to a gold electrode via a thiol-gold bond at the other. Binding of an analyte to the oligonucleotide probe changes its structure and dynamics, which, in turn, influences the efficiency of electron transfer to the interrogating electrode. This class of sensors perform well even when challenged directly with blood serum, soil and other complex, multicomponent sample matrices. This protocol describes the fabrication of E-DNA and E-AB sensors. The protocol can be completed in 12 h.

A folding-based electrochemical aptasensor for detection of vascular endothelial growth factor in human whole blood

We herein report a folding-based electrochemical DNA aptasensor for the detection of vascular endothelial growth factor (VEGF) directly in complex biological samples, including blood serum and whole blood. The electrochemical signal generation is coupled to a large, target-induced conformational change in a methylene blue-modified and surface immobilized anti-VEGF aptamer. The sensor is sensitive, selective and essentially reagentless: we can readily detect VEGF down to 5 pM (190 pg/mL) directly in 50% blood serum. Similar to other aptasensors of this class, the VEGF sensor is also regenerable and reusable. In addition, the sensor performs comparably well even when fabricated on a gold-plated screen-printed carbon electrode and can potentially be implemented as a cost-effective, single-use biosensor for diseases diagnosis and therapy monitoring. The exceptional sensitivity, selectivity, and reusability of this electrochemical aptasensor platform suggest it may be a promising strategy for a wide variety of sensing applications.

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.

Folding-based electrochemical DNA sensor fabricated on a gold-plated screen-printed carbon electrode.

Here we report a folding-based electrochemical DNA (E-DNA) sensor fabricated on a gold-plated screen-printed carbon electrode and show that the E-DNA sensor is not required to be fabricated on a relatively flat gold surface; the sensor works comparably well when fabricated on an electrodeposited gold film with a surface roughness factor of approximately 7.

Development of an electrochemical insulin sensor based on the insulin-linked polymorphicregion

Here we report the design and fabrication of an electrochemical aptamer-based (E-AB) sensor for detection of insulin. The aptamer used in this study is the insulin-linked polymorphic region (ILPR) sequence, a G-rich sequence that presumably undergoes ligand-induced folding to form a G-quadruplex in presence of insulin. Our circular dichroism data, however, suggests that the ILPR sequence, even in absence of the target, is predominantly in a G-quadruplex-like form. Insulin binding, however, has shown to further induce the formation of the G-quadruplex. To evaluate the potential of the ILPR sequence as a biosensing element, we constructed two E-AB insulin sensors that are identical in all aspects but the location of the methylene blue (MB) redox label. We find that the sensor fabricated with internal MB-modified probes (In-IT) shows enhanced sensing behavior when compared to one fabricated using terminal-MB modified probes (In1). The improvements observed with the In-IT sensor could be attributed to the more effective obstruction of electron transfer upon insulin binding. Overall, both sensors perform well, affording a detection limit of 10 nM and 50 nM for the In-IT and In1 sensors, respectively.

Combined optical and acoustical method for determination of thickness and porosity of transparent organic layers below the ultra-thin film limit

Analysis techniques are needed to determine the quantity and structure of materials composing an organic layer that is below an ultra-thin film limit and in a liquid environment. Neither optical nor acoustical techniques can independently distinguish between thickness and porosity of ultra-thin films due to parameter correlation. A combined optical and acoustical approach yields sufficient information to determine both thickness and porosity. We describe application of the combinatorial approach to measure single or multiple organic layers when the total layer thickness is small compared to the wavelength of the probing light. The instrumental setup allows for simultaneous in situ spectroscopic ellipsometry and quartz crystal microbalance dynamic measurements, and it is combined with a multiple-inlet fluid control system for different liquid solutions to be introduced during experiments. A virtual separation approach is implemented into our analysis scheme, differentiated by whether or not the organic adsorbate and liquid ambient densities are equal. The analysis scheme requires that the film be assumed transparent and rigid (non-viscoelastic). We present and discuss applications of our approach to studies of organic surfactant adsorption, self-assembled monolayer chemisorption, and multiple-layer target DNA sensor preparation and performance testing.

Fabrication of electrochemical DNA sensors on gold-modified recessed platinum nanoelectrodes.

We report the use of gold-modified recessed platinum (Pt) nanoelectrodes in the fabrication of linear and stem-loop probe-based electrochemical DNA (E-DNA) sensors. Pt nanoelectrodes with a radius less than 10 nm were reproducibly fabricated using an optimized laser pulling technique. Prior to sensor fabrication, the nanoelectrode was electrochemically etched to create a recessed nanopore, followed by electrodeposition of gold into the nanopore using either cyclic voltammetry or constant potential amperometry. Both techniques enabled controlled deposition of gold into the nanopores, resulting in a nanostructured gold electrode with a well-defined surface area. In addition, we systematically determined the optimal experimental condition for DNA probe immobilization and target interrogation. The electron transfer rate constants of methylene blue, as determined using alternating current voltammetry, were found to be much higher than those obtained from E-DNA sensors fabricated on conventional macroscale electrodes. While this unique phenomenon requires further investigation, our results clearly show that these gold-modified nanoelectrodes can be used as substrates for this class of electrochemical biosensors.

A dual-signalling electrochemical DNA sensor based on target hybridization-induced change in DNA probe flexibility

We report a fully covalent, dual-signalling electrochemical DNA sensor that exploits competitive binding and target hybridization-induced change in probe flexibility for simple and robust detection of target DNA.

Use of thiolated oligonucleotides as anti-fouling diluents in electrochemical peptide-based sensors.

We incorporated short thiolated oligonucleotides as passivating diluents in the fabrication of electrochemical peptide-based (E-PB) sensors, with the goal of creating a negatively charged layer capable of resisting non-specific adsorption of matrix contaminants. The E-PB HIV sensors fabricated using these diluents were found to be more specific and selective, while retaining attributes similar to the sensor fabricated without these diluents. Overall, these results highlight the advantages of using oligonucleotides as anti-fouling diluents in self-assembled monolayer-based sensors.