On-line green fluorescent protein sensor with LED excitation.

We present an intensity based sensor designed for on-line monitoring of green fluorescent protein, a revolutionary marker of protein expression. The device consisted of a blue light emitting diode as the excitation source. A band pass excitation filter cut off light longer than 490 nm. The light was directed into a bifurcated optical fiber bundle with the common end inserted into a stainless steel housing equipped with a quartz window. The fiber bundle and stainless steel housing are steam sterilizable. The emission radiation was collected through a long wave pass filter to reject the excitation light shorter than 505 nm and was detected by a photomultiplier tube. The signal was amplified and sent to a computer for recording time course data. The sensor was tested in an Escherichia coli fermentation of JM105 transformed with pBAD-GFP. The on-line signal was compared to off-line fluorescence spectrophotometer measurements. The on-line profile closely followed the off-line. Western blot data showed that with a time shift, the sensor was able to both continuously and quantitatively monitor expression of green fluorescent protein on-line in real time.

A long-lived, highly luminescent Re(I) metal-ligand complex as a biomolecular probe.

A highly luminescent rhenium (I) metal-ligand complex [Re(bcp)(CO)3(4-COOHPy)](ClO4), where bcp is 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline and 4-COOHPy is isonicotinic acid, has been synthesized and characterized. High quantum yields (> 0.5) and long excited-state lifetimes (0.3-10 micronseconds) in fluid solutions at room temperature were found for this complex, with remarkable emission sensitivity to microenvironment. This compound also displays highly polarized emission with a maximum anisotropy near 0.3 in the absence of rotational diffusion. This Re complex was conjugated to several biomolecules, including the proteins human serum albumin and bovine immunoglobulin G, as well as an amine-containing lipid. When bound to a protein or lipid, the decay time is near 3 microseconds and the quantum yield is approximately 0.12 in aqueous oxygenated solution at room temperature. This compound's unique spectral properties along with its conjugatability allowed us to utilize it as biomolecular probe in a variety of environments.

A lifetime-based fluorescence resonance energy transfer sensor for ammonia.

A lifetime-based optical NH3 sensor based on the principle of fluorescence resonance energy transfer was developed. The sensor consisted of sulforhodamine 101 as the donor, bromocresol green as the acceptor, ethyl cellulose as the polymer support, and tributyl phosphate as the plasticizer. When the concentration of NH3 changed, it caused a change in the decay time of the SR101, which was measured by phase-modulation fluorometry. At 100 MHz, increasing the concentration of NH3 from 0 to 175 ppm resulted in a decrease in phase angle of about 31 degrees and an increase in modulation of about 18%. Oxygen and carbon dioxide did not interfere with the sensor. However, a 30% relative humidity could cause a downward shift of the response by 5 degrees, while additional increase in the relative humidity to 100% showed little further effect. For a film thickness of 40 microns, the typical response and recovery times for 90% of total signal change were 1 and 2.5 min, respectively. The phase angle measurements for the same sample were reproducible for 5 days, with no special care of the film sample.

A lifetime-based optical CO2 gas sensor with blue or red excitation and stokes or anti-stokes detection.

We describe the fabrication and characterization of an optical CO2 sensor based on the change in fluorescence lifetimes due to fluorescence resonance energy transfer from a pH-insensitive donor, sulforhodamine 101, to a pH-sensitive acceptor, either m-cresol purple or thymol blue, entrapped in an ethyl cellulose film. A phase transfer agent allows incorporation of the dyes and water into the film, while providing an initially basic environment for the acceptor. Diffusion of CO2 into the water entrapped in the film produced carbonic acid, causing a pH-dependent decrease in the spectral overlap of the acceptor absorbance with the donor emission, and decreased energy transfer, resulting in increased SR101 donor lifetimes. The lifetime changes were detected as a change in the phase of the emission, relative to the modulated excitation, and were insensitive to excitation intensities and emission signal levels. In addition to an externally modulated 442-nm light source, we excited the sensor with a directly modulated 635-nm laser diode and detected the anti-Stokes emission. The CO2 sensor is not fragile and can provide stable readings for weeks. The use of fluorescence resonance energy transfer, along with the simple entrainment procedure, allows facile change of the CO2 response range through change of the acceptor dye and the use of laser diode excitation sources.

Sensing oxygen through skin using a red diode laser and fluorescence lifetimes.

The most difficult impediments to transcutaneous optical sensing are the absorbance and scatter of light caused by skin and the lack of fluorescent sensing probes which can be excited at wavelengths over 600 nm. Furthermore, current optical sensing techniques rely on absorbance or fluorescence intensity measurements, both of which are sensitive to drifts in lamp intensity, changes in probe concentration and inner filter effects. We demonstrate oxygen sensing through a layer of skin by using red light which readily penetrates skin as diffusely scattered light. The oxygen sensitive osmium-ligand complex used in this study can be excited at 635-680 nm. In addition, we measure fluorescence lifetimes, which are inherently unaffected by factors that limit absorbance and fluorescence intensity measurements. By using phase fluorimetry and long lived fluorophores, we are able to demonstrate the potential for subdermal oxygen sensing with simple and inexpensive instrumentation. This work describes a paradigm for future non-invasive measurements of other analytes.

Solvent complexes of jet-cooled ß-phenylethylamine.

Laser-induced fluorescence spectroscopy was used to investigate ß-phenylethylamine molecules that were cooled via supersonic gas expansions. The bare molecule spectra reveal 0-0 transitions that can be attributed to four separate conformers. Upon the addition of water or alcohol solvents, a series of new peaks is induced that appear to be built off separate bare molecule transitions. This is a pattern that is markedly different from the one seen for solvent addition to tyramine, which includes an-OH in the para position. As a result, definite hypotheses can be made to explain the influence of the para-OH in affecting solvent induced conformations.