Magnitude Improvement In Limit Research Articles

Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers.

Immobilization of nucleic acid aptamer recognition elements selected free in solution onto the surface of biosensor platforms has proven challenging. This study investigated the binding of multiple aptamer/target pairs immobilized on a commercially available microarray as a model system mimicking biosensor applications. The results indicate a minimum distance (linker length) from the surface and thymine nucleobase linker provides reproducible binding across varying conditions. An indirect labeling method, where the target was labeled with a biotin followed by a brief Cy3-streptavidin incubation, provided a higher signal-to-noise ratio and over two orders of magnitude improvement in limit of detection, compared to direct Cy3-protein labeling. We also showed that the affinities of the aptamer/target interaction can change between direct and indirect labeling and conditions to optimize for the highest fluorescence intensity will increase the sensitivity of the assay but will not change the overall affinity. Additionally, some sequences which did not initially bind demonstrated binding when conditions were optimized. These results, in combination with studies demonstrating enhanced binding in nonselection buffers, provided insights into the structure and affinity of aptamers critical for biosensor applications and allowed for generalizations in starting conditions for researchers wishing to investigate aptamers on a microarray surface.

Acceleration of Surface-Based Hybridization Reactions Using Isotachophoretic Focusing

We present a theoretical model and experimental demonstration of a novel method for acceleration of surface-based reactions using isotachophoresis (ITP). We use ITP to focus a sample of interest and deliver a high concentration target to a prefunctionalized surface, thus enabling rapid reaction at the sensor site. The concentration of the focused analyte is bound in space by the ITP interface and, upon reaction with the surface, continues electromigrating downstream, removing any contamination or reacted sample molecules from the surface. This constitutes a one-step react-and-wash assay which can be performed in a simple channel and does not require flow control elements or moving parts. We designed a novel microfluidic chip where reaction surfaces are formed by paramagnetic beads, immobilized at desired sites by an external magnetic field. Using this chip, we compared ITP-based surface hybridization to standard continuous flow-based hybridization and experimentally demonstrated a 2 orders of magnitude improvement in limit of detection (LoD) in a 3 min nucleic acid hybridization assay. The simple analytical model we present allows prediction of the rate of surface reaction under ITP and can be used to design and optimize such assays as a function of the physical properties of the system, including buffer chemistry, applied voltage, analyte mobility, analyte concentration, probe density, and surface length. The method, model, and experimental setup can be applied to various forms or surface reactions and may serve as the basis for highly genetic analysis and immunoassays.

Optofluidic Concentration: Plasmonic Nanostructure as Concentrator and Sensor

The integration of fluidics and optics, as in flow-through nanohole arrays, has enabled increased transport of analytes to sensing surfaces. Limits of detection, however, are fundamentally limited by local analyte concentration. We employ the nanohole array geometry and the conducting nature of the film to actively concentrate analyte within the sensor. We achieve 180-fold enrichment of a dye, and 100-fold enrichment and simultaneous sensing of a protein in less than 1 min. The method presents opportunities for an order of magnitude increase in sensing speed and 2 orders of magnitude improvement in limit of detection.

Poly(dimethylsiloxane) cross-linked carbon paste electrodes for microfluidic electrochemical sensing

Recently, the development of electrochemical biosensors as part of microfluidic devices has garnered a great deal of attention because of the small instrument size and portability afforded by the integration of electrochemistry in microfluidic systems. Electrode fabrication, however, has proven to be a major obstacle in the field. Here, an alternative method to create integrated, low cost, robust, patternable carbon paste electrodes (CPEs) for microfluidic devices is presented. The new CPEs are composed of graphite powder and a binder consisting of a mixture of poly(dimethylsiloxane) (PDMS) and mineral oil. The electrodes are made by filling channels molded in previously cross-linked PDMS using a method analogous to screen printing. The optimal binder composition was investigated to obtain electrodes that were physically robust and performed well electrochemically. After studying the basic electrochemistry, the PDMS-oil CPEs were modified with multi-walled carbon nanotubes (MWCNT) and cobalt phthalocyanine (CoPC) for the detection of catecholamines and thiols, respectively, to demonstrate the ease of electrode chemical modification. Significant improvement of analyte signal detection was observed from both types of modified CPEs. A nearly 2-fold improvement in the electrochemical signal for 100 μM dithiothreitol (DTT) was observed when using a CoPC modified electrode (4.0 ± 0.2 nA (n = 3) versus 2.5 ± 0.2 nA (n = 3)). The improvement in signal was even more pronounced when looking at catecholamines, namely dopamine, using MWCNT modified CPEs. In this case, an order of magnitude improvement in limit of detection was observed for dopamine when using the MWCNT modified CPEs (50 nM versus 500 nM). CoPC modified CPEs were successfully used to detect thiols in red blood cell lysate while MWCNT modified CPEs were used to monitor temporal changes in catecholamine release from PC12 cells following stimulation with potassium.