A Miniature 3D Printed LED-Induced Fluorescence Detector for Capillary Electrophoresis and Dual-Detector Taylor Dispersion Analysis.

Taylor dispersion analysis (TDA) provides absolute determination of diffusion coefficients for analytes ranging from small molecules to particulate matter. TDA has seen a resurgence in recent years, as modern commercial capillary electrophoresis (CE) instrumentation is well equipped to meet the precision flow requirements of TDA. Discontinuous flow velocities, which occur during sample injection, can lead to substantial inaccuracies in single-point detection TDA. Dual-point detection allows TDA to be carried out under continuous flow in the volume between the detection points, but dual-point fluorescence detection has not previously been feasible within the confines of commercial CE instrumentation. Here, we describe a compact light-emitting diode (LED)-induced fluorescence detector designed for online, dual-point capillary detection within a commercial CE system. The three-dimensional (3D) printed detector houses an inexpensive LED excitation source, a bandpass excitation filter, an integral 3D printed pinhole collimator, and a ball lens, which collects fluorescence emission. Multivariate optimization of operating conditions yielded a detection limit of 613 ± 13 pM for CE of fluorescein disodium salt solution in borate buffer. The miniature size of the device allowed integration of two detectors within a commercial CE system without modification to the instrument, thereby enabling dual-detector assays including TDA and CE-TDA. Monitoring of the bioconjugation reaction between fluorescein isothiocyanate (FITC) and a model protein illustrates the utility of direct, calibration-free size determination, which enabled the resolution of fluorescence originating from free FITC from that of protein-bound FITC. TDA detection coupled to CE enabled the determination of peak identities without the need for standard solutions.

Assessment of aptamer-steroid binding using stacking-enhanced capillary electrophoresis.

The binding affinity of 17ß-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17ß-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 µM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.

Method for detecting and quantitating capture of organic molecules in hypervelocity impacts.

Enceladus is a prime candidate in the solar system for in-depth astrobiological studies searching for habitability and life because it has a liquid water ocean with significant organic content and ongoing cryovolcanic activity. The presence of ice plumes that jet up through fissures in the ice crust covering the sub-surface ocean, enables remote sampling and in-situ analysis via a fly-by mission. However, capture and transport of organic materials to organic analyzers presents distinctive challenges as it is unknown whether, and to what extent, organic molecules imbedded in ice particles can be captured and survive hypervelocity impacts. This manuscript provides a fluorescence microscopic method to parametrically determine the amount of an organic fluorescent tracer dye, Pacific Blue™ (PB) deposited on a metallic surface. This method can be used to measure the capture and survival outcomes of terrestrial hypervelocity impact experiments where an ice projectile labeled with Pacific Blue impacts a soft metal surface. This work is an important step in the advancement of instruments like the Enceladus Organic Analyzer for detecting biosignatures in an Enceladus plume fly-by mission. An apparatus consisting of a substrate humidification shroud coupled with an epifluorescence microscope with CCD detector is developed to measure the intensity of quantitatively deposited Pacific Blue droplets under controlled humidity. Calibration curves are produced that relate the integrated fluorescence intensity of humidified PB droplets on metal foils to the number of PB molecules deposited. To demonstrate the utility of this method, our calibrations are used to analyze and quantitate organic capture and survival (up to 11% capture efficiency) following ice particle impacts at a velocity of 1.7 km/s on an aluminum substrate.

Fabrication of high-quality glass microfluidic devices for bioanalytical and space flight applications.

Microfabricated glass microfluidic and Capillary Electrophoresis (CE) devices have been utilized in a wide variety of applications over the past thirty years. At the Berkeley Space Sciences Laboratory, we are working to further expand this technology by developing analytical instruments to chemically explore our solar system. This effort requires improving the quality and reliability of glass microfabrication through quality control procedures at every stage of design and manufacture. This manuscript provides detailed information on microfabrication technology for the production of high-quality glass microfluidic chips in compliance with industrial standards and space flight instrumentation quality control.•The methodological protocol provided in this paper includes the scope of each step of the manufacturing process, materials and technologies recommended and the specific challenges that often confront the process developer.•Types and sources of fabrication error at every stage have been identified and their solutions have been proposed and verified.•We present robust and rigorous manufacturing and quality control procedures that will assist other researchers in achieving the highest possible quality glass microdevices using the latest apparatus in a routine and reliable fashion.