Ph.D, University of Alberta, Chemistry (2017)
B.Sc, Sun Yat-sen University, Chemistry (2012)
Michael Lin, Postdoctoral Faculty Sponsor
We introduce a centrifugal microfluidic disc that accepts a small volume in (∼5 μL), performs sample cleanup on human serum samples, and delivers a small volume out, for subsequent metabolite analysis by surface assisted laser desorption/ionization (SALDI) mass spectrometry (MS) or hydrophilic interaction liquid chromatography (HILIC)-MS. The centrifugal microfluidic disc improves the MS results by removing proteins and lipids from serum. In the case of SALDI-MS, sample background electrolytes are segregated from analytes during the spotting process by the action of the SALDI-chip during drying, for further cleanup, while HILIC separates the salts in HILIC-MS. The resulting mass spectra of disc-prepared samples show a clean background and high signal-to-noise ratio for metabolite peaks. Several representative ionic metabolites from human serum samples were successfully quantified. The performances of the sample preparation disc for SALDI-MS and HILIC-MS were assessed and were comparable. Reproducibility, sample bias, and detection limits for SALDI-MS compared well to ultrafiltration sample preparation.
View details for DOI 10.1021/acs.analchem.8b05756
View details for Web of Science ID 000472682000012
View details for PubMedID 31090394
We introduce a single-phase flow microfluidic cell sorter with a two-point detection system capable of two-parameter screening to assist with directed evolution of a fluorescent protein based Ca2+ sensor expressed in bacterial cells. The new cell sorting system utilizes two fluorescence microscopes to obtain signals at two different points along a flow path in which a change in concentration of the analyte, Ca2+, is induced. The two detectors thus determine the magnitude of fluorescence change of the sensor following the reaction, along with the overall brightness of the sensor. A design for a 3D focusing flow was configured to enhance the spatial control of cells and signal pair-matching. The cell sorter screens the sensors at a moderate throughput, 10 cells per s and 105 cells per round, enriching top variants for the subsequent manual screening with higher accuracy. Our new μFACS greatly accelerates the directed evolution of genetically encoded Ca2+ sensors compared to the previous version with single point detection for brightness-based screening. Two rounds of directed evolution led to a variant, named Y-GECO2f, which exhibits a 26% increase in brightness and a greater than 300% larger Ca2+-dependent fluorescence change in vitro relative to the variant before evolution.
View details for DOI 10.1039/c9lc00779b
View details for PubMedID 31641712
We have developed a series of yellow genetically encoded Ca2+ indicators for optical imaging (Y-GECOs) with inverted responses to Ca2+ and apparent dissociation constants (Kd') ranging from 25 to 2400 nM. To demonstrate the utility of this affinity series of Ca2+ indicators, we expressed the four highest affinity variants (Kd's = 25, 63, 121, and 190 nM) in the Drosophila medulla intrinsic neuron Mi1. Hyperpolarization of Mi1 by optogenetic stimulation of the laminar monopolar neuron L1 produced a decrease in intracellular Ca2+ in layers 8-10, and a corresponding increase in Y-GECO fluorescence. These experiments revealed that lower Kd' was associated with greater increases in fluorescence, but longer delays to reach the maximum signal change due to slower off-rate kinetics.
View details for DOI 10.1038/s41598-018-30080-x
View details for Web of Science ID 000440782000045
View details for PubMedID 30082904
View details for PubMedCentralID PMC6079023
We demonstrate a simple, low cost and disposable microfluidic fluorescence activated cell sorting system (μFACS) for directed evolution of fluorescent proteins (FP) and FP-based calcium ion (Ca(2+)) indicators. The system was employed to pre-screen libraries of up to 10(6) variants of a yellow FP-based Ca(2+) indicator (Y-GECO) with throughput up to 300 cells per s. Compared to traditional manual screening of FP libraries, this system accelerated the discovery of improved variants and saved considerable time and effort during the directed evolution of Y-GECO. Y-GECO1, the final product of the μFACS-aided directed evolution, has a unique fluorescence hue that places it in the middle of the spectral gap that separates the currently available green and orange FP-based Ca(2+) indicators, exhibits bright fluorescence in the resting (Ca(2+) free) state, and gives a large response to intracellular Ca(2+) fluctuations in live cells.
View details for DOI 10.1039/c4ib00039k
View details for Web of Science ID 000338298200007
View details for PubMedID 24840546
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