A review of the zone broadening contributions in free-flow electrophoresis.

The broadening of analyte streams, as they migrate through a free-flow electrophoresis (FFE) channel, often limits the resolving power of FFE separations. Under laminar flow conditions, such zonal spreading occurs due to analyte diffusion perpendicular to the direction of streamflow and variations in the lateral distance electrokinetically migrated by the analyte molecules. Although some of the factors that give rise to these contributions are inherent to the FFE method, others originate from non-idealities in the system, such as Joule heating, pressure-driven crossflows, and a difference between the electrical conductivities of the sample stream and background electrolyte. The injection process can further increase the stream width in FFE separations but normally influencing all analyte zones to an equal extent. Recently, several experimental and theoretical works have been reported that thoroughly investigate the various contributions to stream variance in an FFE device for better understanding, and potentially minimizing their magnitudes. In this review article, we carefully examine the findings from these studies and discuss areas in which more work is needed to advance our comprehension of the zone broadening contributions in FFE assays.

Fluorescence signal amplification by optical reflection in metal-coated nanowells.

A significant amplification in the fluorescence signal is demonstrated when measured in metal (aluminum)-coated fluidic wells with volumes on the order of a nanoliter or smaller (nanowells). Photolithographic and wet etching procedures were used to fabricate these nanowells on glass substrates followed by vapor deposition of an aluminum layer on them. The fluorescence signal recorded in these structures was enhanced due to the reflection of the incident and emitted radiation by the metal layer as well as focusing of this light by the curvature of the well surface. While the first effect amplified the background signal in the entire assay chamber, the latter one produced signal hotspots around the edges and center of the nanowell. In this work, we were able to realize over a 20-fold enhancement in the fluorescence signal upon quantitating it at the central hotspot of an aluminum-coated circular nanowell with a depth and photo-patterned diameter of 30 µm and 38 µm, respectively. More interestingly, our experiments indicate that this enhancement factor may be further improved by optimizing the curvature of the nanowell surface to merge all the signal hotspots within a smaller detection zone. Finally, quantitative assays using horseradish peroxidase samples were performed on the reported signal enhancement platform to further demonstrate its utility for making sensitive analytical measurements.

Correction: Microfluidic ELISA employing an enzyme substrate and product species with similar detection properties.

Correction for 'Microfluidic ELISA employing an enzyme substrate and product species with similar detection properties' by Basant Giri et al., Analyst, 2018, 143, 989-998, https://doi.org/10.1039/C7AN01671A.

Absorbance Detection in Multireflection Microfluidic Channels Using a Commercial Microplate Reader System.

Absorbance detection is often prohibited in microfluidic channels due to the limited optical path length available in these systems. However, this optical distance may be significantly increased by guiding the probing light beam along the channel length via multiple reflections by patterned metallic surfaces. In this work, we demonstrate enhanced absorbance detection in glass microfluidic channels using a commercial microplate reader based on this principle, yielding detection limits comparable to that measured on standard microwell plates. This improvement in detectability was realized through careful optimization of the mirror lengths and locations combined with the appropriate design of a microchip holder to suitably position the microchannels in the microplate reader. Additionally, it was determined that the angle by which our device was tilted relative to the horizontal plane played an important role in this optimization. For an optimum choice of parameters accessible with our design, the sensitivity of our absorbance measurements in a 30 µm-deep channel was improved by as much as 52-fold, raising this quantity to about 84% of the corresponding value realized for 75 µL samples placed within 7 mm i.d. standard cylindrical microwells. Quantitative ELISAs employing the absorbance detection method were demonstrated on the noted multireflection microchip device for assessing West Nile viral IgM antibody levels in human serum samples yielding analyte detection limits comparable to that measured on standard microwell plates.