New fluorescein precursors for live bacteria detection.

Area of Science:

• Microbiology
• Analytical Chemistry
• Biotechnology

Background:

• Live bacteria detection in drinking water is crucial for human safety.
• Current methods using carboxyfluorescein diacetate (CFDA) are limited by poor membrane permeation due to the probe's charge at physiological pH.
• Improving probe diffusion is key to enhancing detection sensitivity and reliability.

Purpose of the Study:

• To develop novel fluorescent probes with improved cell membrane permeability for more sensitive live bacteria detection.
• To chemically modify the carboxyl group of CFDA and related compounds to neutralize charge and enhance diffusion.
• To evaluate the efficacy of these modified probes in detecting live bacteria in aqueous samples.

Main Methods:

• Chemical synthesis of CFDA derivatives, focusing on modifying the 5-carboxyl group.
• Testing probe stability in biological media and their ability to penetrate bacterial membranes.
• Assessing the metabolic conversion of probes into fluorescent species within live bacteria.
• Comparing the sensitivity of novel probes against CFDA using fluorescence detection.
• Evaluating the effect of contrast agents (trypan blue, brilliant blue R, bromophenol blue) on signal-to-noise ratio.

Main Results:

• Ethylamido derivatives of CFDA and Oregon Green diacetate/diacetoxymethyl ester demonstrated stability, cell penetration, and conversion to fluorescent species.
• Live bacteria were successfully detected, with no labeling of non-viable cells.
• Derivatives with ester or thiourea modifications of the carboxyl group showed poor performance.
• The probe 'triembarine' (5-ethylaminocarboxy-Oregon Green, 3',6'diacetoxymethyl ester) achieved 6-10 times higher sensitivity than CFDA.
• Contrast agents like trypan blue and brilliant blue R enhanced signal-to-noise by quenching extracellular fluorescence.

Conclusions:

• Chemical modification of the carboxyl group significantly enhances the performance of fluorescent bacterial viability probes.
• Triembarine represents a superior probe for live bacteria detection, offering substantially increased sensitivity.
• Contrast agents can be utilized to optimize detection accuracy and standardize measurements in water quality monitoring.