Regenerable fiber-optic-based immunosensor.

An immunosensor is described that is based on fluorescently labeled F(ab') anti-human serum albumin antibody fragments covalently immobilized to the distal end of a fiber-optic probe. When human serum albumin is present, it is bound to the sensor and shields the fluorescent label from the solvent water, and a significant increase in the label fluorescence results. The sensor can be regenerated by simply immersing the sensing tip in chaotropic media. Under these conditions the antigen-antibody complex is selectively disrupted without adversely affecting the sensor. In the present configuration, the same sensor can be recycled over 50 times before the immunosurface inactivates significantly. With proper storage the sensor can last for up to 4 months.

Effects of inaccurate reference lifetimes on interpreting frequency-domain fluorescence data.

The effects of assigning inaccurate reference lifetimes in lifetime determinations are predicted theoretically by using standard equations. This theory leads to a method to remove reference error effects using common least-squares software. This method cannot, however, be used to deconvolute data collected with isochronal references. Uncorrected data can always be exactly solved with models containing one more degree of freedom than the true model. Monoexponential decays are fit by double-exponential decays or excited-state processes. Unimodal distributed decays often appear as discrete, double-exponential decays.

Multicomponent fluorometric analysis using a fiber-optic probe.

Single-frequency phase-resolved fluorometry through single and bifurcated fiber-optic probes is used to quantify mixtures of spectrally similar fluorophores. Correlation coefficients from correlation plots are greater than 0.98, and standard errors of estimate are less than 0.13 microM for 80 binary mixtures. Quantification of fluorophores at up to 10:1 molar ratios with lifetime separations of less than 200 ps is possible. The simultaneous quantification of the individual components of ternary and quaternary synthetic mixtures is demonstrated. Application of phase resolution to ambient light rejection is reported also. In this case, the emission spectrum of 10 nM Rhodamine 6G is easily obtained in the presence of a fluctuating, unmodulated background.