Fluorescence Properties of Rhodamine 800 in Whole Blood and Plasma

• Published in: Analytical biochemistry 2000-03, Vol.279 (2), p.142-150
• Main Authors: Abugo, Omoefe O., Nair, Rajesh, Lakowicz, Joseph R.
• Format: Article
• Language: English
• Subjects: Blood Chemical Analysis - methods; Evaluation Studies as Topic; Fluorescence Polarization; Fluorescent Dyes - analysis; Humans; Plasma - chemistry; Rhodamines - blood; Spectrometry, Fluorescence; Spectrophotometry
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2000.4486

We have characterized the fluorescence spectral properties of rhodamine 800 (Rh800) in plasma and blood in order to test the possibility of making clinical fluorescence measurements in whole blood without separation steps. Rh800 was used because of its absorption at red/near-infrared wavelengths away from the absorption bands of hemoglobin. We utilized the front-face illumination and detection to minimize the effects of absorption and/or scatter during measurements. The presence of Rh800 was detected in plasma and blood using steady-state fluorescence measurements. Absorption due to hemoglobin reduced the Rh800 intensity from the blood. Fluorescence lifetime measurements in plasma and blood showed that it is possible to recover lifetime parameters of Rh800 in these media. We obtained mean lifetimes of 1.90 and 1.86 ns for Rh800 in plasma and blood, respectively. Using the recently described modulation sensing method, we quantified the concentrations of Rh800 in plasma and blood. Rh800 was detected at a concentration of as low as 2 μM in both media. High anisotropy values were obtained for Rh800 in plasma and blood using steady-state and anisotropy decay measurements, implying the tight binding of this probe to the contents of these media. This binding can be exploited to monitor the concentrations of different blood components using already existing or new red-emitting probes that will be specially designed to bind to these components with high specificity. To test this possibility of direct measurements in blood, we used Rh800 to monitor albumin in the presence of red blood cells. Increase in the polarization of Rh800 as the concentration of albumin was increased in the presence of the red cells showed the feasibility of such measurements.