Honors & Awards
School of Medicine Dean's Postdoctoral Fellowship, Stanford University (07/2021)
Ph.D, University of Alberta, Chemistry (2017)
B.Sc, Sun Yat-sen University, Chemistry (2012)
Michael Lin, Postdoctoral Faculty Sponsor
Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
2021; 22 (1)
Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca2+-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca2+ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor's functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca2+ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R0) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.
View details for DOI 10.3390/ijms22010445
View details for Web of Science ID 000606034200001
View details for PubMedID 33466257
View details for PubMedCentralID PMC7794744
High-Performance Intensiometric Direct- and Inverse-Response Genetically Encoded Biosensors for Citrate
ACS CENTRAL SCIENCE
2020; 6 (8): 1441-1450
Motivated by the growing recognition of citrate as a central metabolite in a variety of biological processes associated with healthy and diseased cellular states, we have developed a series of high-performance genetically encoded citrate biosensors suitable for imaging of citrate concentrations in mammalian cells. The design of these biosensors was guided by structural studies of the citrate-responsive sensor histidine kinase and took advantage of the same conformational changes proposed to propagate from the binding domain to the catalytic domain. Following extensive engineering based on a combination of structure guided mutagenesis and directed evolution, we produced an inverse-response biosensor (ΔF/F min ≈ 18) designated Citroff1 and a direct-response biosensor (ΔF/F min ≈ 9) designated Citron1. We report the X-ray crystal structure of Citron1 and demonstrate the utility of both biosensors for qualitative and quantitative imaging of steady-state and pharmacologically perturbed citrate concentrations in live cells.
View details for DOI 10.1021/acscentsci.0c00518
View details for Web of Science ID 000566668400024
View details for PubMedID 32875085
View details for PubMedCentralID PMC7453566
Sample Preparation in Centrifugal Microfluidic Discs for Human Serum Metabolite Analysis by Surface Assisted Laser Desorption/Ionization Mass Spectrometry
2019; 91 (12): 7570–77
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
A single-phase flow microfluidic cell sorter for multiparameter screening to assist the directed evolution of Ca2+ sensors.
Lab on a chip
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
Inverse-response Ca2+ indicators for optogenetic visualization of neuronal inhibition
2018; 8: 11758
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
Microfluidic cell sorter-aided directed evolution of a protein-based calcium ion indicator with an inverted fluorescent response
2014; 6 (7): 714–25
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
- Controllable synthesis of hierarchical ZnO nanodisks for highly photocatalytic activity CRYSTENGCOMM 2012; 14 (5): 1850–55
- Preparation and Magnetic Properties of Polycrystalline Eu2O3 Microwires JOURNAL OF THE ELECTROCHEMICAL SOCIETY 2012; 159 (4): D204–D207