An often limiting factor for studying protein folding by single-molecule fluorescence resonance energy transfer (FRET) is the ability to site-specifically introduce a photostable organic FRET donor (D) and a complementary acceptor (A) into a polypeptide chain. Using alternating-laser excitation and chymotrypsin inhibitor 2 as a model, we show that chemical labeling of a unique cysteine, followed by enzymatic modification of a reactive glutamine in an N-terminally appended substrate sequence recognition tag for transglutaminase (TGase) affords stoichiometrically D-/A-labeled protein suitable for single-molecule FRET experiments. Thermodynamic data indicate that neither the presence of the TGase tag nor D/A labeling perturbs protein stability. As the N terminus in proteins is typically solvent accessible, a TGase tag can (in principle) be appended to any protein of interest by genetic engineering. Two-step chemical/enzymatic labeling may thus represent a simple, low-cost, and widely available strategy for DIA labeling of proteins for FRET-based single-molecule protein folding studies, even for non-protein-experts laboratories.

Biomarkers from the human body can provide dynamic and powerful insight a broad spectrum of health conditions. Monitoring biomarkers in body fluids will improve and advance prediction, screening, diagnosis, and treatment of disease. At present, however, the ability to study and to track the everchanging mixtures of chemicals inside and on the human body is limited. For example, chemical communication between neurons plays central roles in information processing in the brain, yet technologies for neurochemical recordings with high chemical, spatial, and temporal resolution are limited, and for some neurotransmitters, nonexistent. For the past five years, I have focused on developing transformative biosensors towards in vivo neurotransmitter monitoring to advance our understanding of brain activity and how behavior arises from this activity.To achieve this goal, we developed ultrathin (~3-nm) In2O3 field-effect transistor (FET) biosensors, where DNA sequences (aptamers) covalently functionalized to the device surfaces enable specific, high-sensitivity molecular recognition. Building on the capabilities of these aptamer-FET biosensors, we developed multiplexed sensors that simultaneously target several important biomarkers, including dopamine, serotonin, glucose, phenylalanine, and cortisol. A high-throughput, wafer-scale, and low-cost nanolithographic approach (chemical lift-off lithography, CLL) was developed and applied for the fabrication of nanoscale FETs, which are the functional core of these sensors. We have advanced CLL by using the self-collapse nature of polymeric stamps to achieve small features (~15 nm). The capability of CLL has been expanded to pattern chemical molecules, biomolecules, metallic nanostructures, and semiconductor nanostructures. We were able to fabricate different types of sensors including plasmonic sensors and FET biosensors using micro- and nanostructured features patterned by CLL. We designed and fabricated implantable neurochemical probes and wearable bioelectronics using micro-electro-mechanical-system technologies. We performed ex vivo and in vivo experiments with implanted neural probes to monitor neurotransmitters, e.g., serotonin, in living, conscious animals. The technologies we are developing will advance our understanding of healthy brain function in relation to complex behavior, as well as corresponding dysfunction in psychiatric and neurodegenerative disorders.

We study protein and nucleic acid structure and dynamics using single-molecule FRET and alternating-laser excitation. Freely diffusing molecules are sorted into subpopulations based on single-molecule signals detected within 100 mu s to 1 ms. Distance distributions caused by fluctuations faster than 100 mu s are studied within these subpopulations by using time-correlated single-photon counting. Measured distance distributions for dsDNA can be accounted for by considering fluorophore linkers and fluorophore rotational diffusion, except that we find smaller fluctuations for internally labeled dsDNA than DNA with one of the fluorophores positioned at a terminal site. We find that the electrostatic portion of the persistence length of short single-stranded poly(dT) varies approximately as the ionic strength (l) to the -1/2 power (l(-1/2)), and that the average contribution to the contour length per base is 0.40-0.45 nm. We study unfolded chymotrypsin inhibitor 2 (02) and unfolded acyl-CoA binding protein (ACBP) even under conditions where folded and unfolded subpopulations coexist (contributions from folded proteins are excluded by using alternating-laser excitation). At lower denaturant concentrations, unfolded C12 and ACBP are more compact and display larger fluctuations than at higher denaturant concentrations where only unfolded proteins are present. The experimentally measured fluctuations are larger than the fluctuations predicted from a Gaussian chain model or a wormlike chain model. We propose that the larger fluctuations may indicate transient residual structure in the unfolded state.