2 research outputs found
Directed Evolution of Key Residues in Fluorescent Protein Inverses the Polarity of Voltage Sensitivity in the Genetically Encoded Indicator ArcLight
Genetically encoded
calcium indicators (GECIs) produce unprecedentedly
large signals that have enabled routine optical recording of single
neuron activity in vivo in rodent brain. Genetically encoded voltage
indicators (GEVIs) offer a more direct measure of neuronal electrical
status, however the signal-to-noise characteristics and signal polarity
of the probes developed to date have precluded routine use in vivo.
We applied directed evolution to target modulable areas of the fluorescent
protein in GEVI ArcLight to create the first GFP-based GEVI (Marina)
that exhibits a Δ<i>F</i>/Δ<i>V</i> with a positive slope relationship. We found that only three rounds
of site-directed mutagenesis produced a family of “brightening”
GEVIs with voltage sensitivities comparable to that seen in the parent
probe ArcLight. This shift in signal polarity is an essential first
step to producing voltage indicators with signal-to-noise characteristics
comparable to GECIs to support widespread use in vivo
Low-Copy Number Protein Detection by Electrode Nanogap-Enabled Dielectrophoretic Trapping for Surface-Enhanced Raman Spectroscopy and Electronic Measurements
We
report a versatile analysis platform, based on a set of nanogap
electrodes, for the manipulation and sensing of biomolecules, as demonstrated
here for low-copy number protein detection. An array of Ti nanogap
electrode with sub-10 nm gap size function as templates for alternating
current dielectrophoresis-based molecular trapping, hot spots for
surface-enhanced Raman spectroscopy as well as electronic measurements,
and fluorescence imaging. During molecular trapping, recorded Raman
spectra, conductance measurements across the nanogaps, and fluorescence
imaging show unambiguously the presence and characteristics of the
trapped proteins. Our platform opens up a simple way for multifunctional
low-concentration heterogeneous sample analysis without the need for
target preconcentration