Electrokinetic delivery of single fluorescent biomolecules in fluidic nanochannels

Davis, Lloyd M.; Canfield, Brian K.; Li, Xiaoxuan; Hofmeister, William; Shen, Guoqing; Lescano-Mendoza, Isaac P.; Bomar, Bruce; Wikswo, John P.; Markov, Dmitry A.; Samson, Philip C.; Daniel, Claus; Sikorski, Zbigniew; Robinson, William N.

doi:10.1117/12.798173

Cited by 11 publications

(24 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Experiments in our lab 13 have found that diffusion of fluorescent dye molecules in a nanochannel fabricated from fused silica is slowed by a factor of ϳ50, in agreement with the observations of Lyon and Nie. 15 However, it is likely that this is due to sticking of molecules to the nanochannel walls and that avoidance of sticking by surface treatment or other means will restore the free solution diffusion.…”

Section: ͑13͒supporting

confidence: 77%

“…This time-stamp ts i , or laser-pulse count at the time of detection of the i'th photon, which is stored as a 32-bit unsigned integer for compatibility with the analysis routines for the experiments of Ref. 13, is either even or odd, depending on whether the photon detection follows a laser pulse to the left or right of the center of the trap. Thus, time-gated photon detection may be achieved by sorting photons based on the value of the least significant bit of the timestamp.…”

Section: Photon Detectionmentioning

confidence: 99%

“…The simulations provide a means for developing and testing algorithms that may be implemented in software within a field-programmable gate array ͑FPGA͒, which is a part of the hardware for controlling the electrokinetic voltages of the single-molecule trap. 13 The simulation also enables the robustness of the trap to be studied under different experimental conditions. The goal is to trap a molecule in a nanochannel at detected photon count rates of ϳ10 5 s −1 and to rapidly replace it with a new one following photobleaching or escape.…”

Section: Introductionmentioning

confidence: 99%

“…16 Initial experiments on single-molecule detection with actively controlled electrokinetic transport of the solution within a nanochannel in a device fabricated from fused silica have been reported. 13 Maximum-likelihood data analysis strategies for sensing the position of a single molecule within a trap have also been described. 17 This paper discusses Monte Carlo computer simulations of the successive delivery and 1-D trapping of single molecules within a nanochannel.…”

Section: Introductionmentioning

confidence: 99%

“…For applications that can tolerate such disturbances, a 1-D nanochannel trap should be equally useful but simpler to implement and control. 13 There has been increasing interest in single-molecule trapping in solution.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Simulation of single-molecule trapping in a nanochannel

Robinson

Davis

2010

J. Biomed. Opt.

Self Cite

View full text Add to dashboard Cite

Abstract. The detection and trapping of single fluorescent molecules in solution within a nanochannel is studied using numerical simulations. As optical forces are insufficient for trapping molecules much smaller than the optical wavelength, a means for sensing a molecule's position along the nanochannel and adjusting electrokinetic motion to compensate diffusion is assessed. Fluorescence excitation is provided by two adjacently focused laser beams containing temporally interleaved laser pulses. Photon detection is time-gated, and the displacement of the molecule from the middle of the two foci alters the count rates collected in the two detection channels. An algorithm for feedback control of the electrokinetic motion in response to the timing of photons, to reposition the molecule back toward the middle for trapping and to rapidly reload the trap after a molecule photobleaches or escapes, is evaluated. While accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware, the algorithm is shown to be effective for trapping fastdiffusing single-chromophore molecules within a micron-sized confocal region. Studies show that there is an optimum laser power for which loss of molecules from the trap due to either photobleaching or shot-noise fluctuations is minimized.

show abstract

Section: ͑13͒supporting

confidence: 77%

Section: Photon Detectionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Simulation of single-molecule trapping in a nanochannel

Robinson

Davis

2010

J. Biomed. Opt.

Self Cite

View full text Add to dashboard Cite

show abstract

Optimized Two‐Color Single‐Molecule Tracking of Fast‐Diffusing Membrane Receptors

Schirripa Spagnolo,

Moscardini,

Amodeo

et al. 2023

Advanced Optical Materials

View full text Add to dashboard Cite

Single particle tracking (SPT) combined with total internal reflection fluorescence microscopy (TIRFm) is an outstanding approach to decipher mechanisms on the cell membrane at the nanoscale. Multicolor configurations, needed to investigate interactions, are still hindered by several challenges. This work systematically and quantitatively analyzes the impact of necessary elements of SPT‐TIRFm setups on the signal‐to‐noise ratio (SNR), which must be optimized especially in dynamic studies needing minimally invasive dyes for biomolecule labeling. Autofluorescence originating from commonly used optical glass results in the dominant limiting factor in TIRFm, and a cover glass material is tested yielding significant SNR improvements in multichannel TIRFm. Moreover, methodologies are optimized for reducing fluorophore photobleaching in multicolor implementations requiring simultaneous stabilization of multiple dyes. The developed strategies are applied to the fast p75NTR receptors labeled by two fluorophores on the membrane of living cells, achieving reliable, simultaneous two‐color SPT, contrary to configurations using standard cover glasses. This work highlights the importance of optical materials suitable for microscopy and with reduced autofluorescence for increasing sensitivity toward ultimate spatiotemporal resolutions. In particular, the present protocols can pave the way for multicolor super‐resolved localization and tracking of single molecules by TIRFm, greatly expanding the potential of SPT.

show abstract