Fluorescence modulation sensing of positively and negatively charged proteins on lipid bilayers.

BACKGROUND

Detecting ligand-receptor binding on cell membrane surfaces is required to understand their function and behavior. Detection platforms can also provide an avenue for the development of medical devices and sensor biotechnology. The use of fluorescence techniques for such purposes is highly desirable as they provide high sensitivity. Herein, we describe a technique that utilizes the sensitivity of fluorescence without directly tagging the analyte of interest to monitor ligand-receptor interactions on supported lipid bilayers. The fluorescence signal is modulated according to the charge state of the target analyte. The binding event elicits protonation or deprotonation of pH-responsive reporter dyes embedded in the lipid bilayer.

METHODS

Supported lipid membranes containing ortho-conjugated rhodamine B-POPE (1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine), which fluoresces in its protonated but not in its deprotonated form, were utilized as sensor platforms for biotin-avidin and biotin-streptavidin binding events. The membranes contained 5 mol% biotin-PE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) as a capture ligand. Supported lipid bilayers were formed in the channels of microfluidic devices and the fluorescence intensity of the dye was monitored as protein was introduced.

RESULTS

The binding of avidin, which is positively charged at pH 7.2, made the bilayer surface charge more positive, which in turn deprotonated the ortho-rhodamine B dye, reducing its fluorescence. The binding of streptavidin, which is negatively charged at pH 7.2, had the opposite effect. Reducing the ionic strength of the analyte solution by removing 150 mM NaCl from the 10 mM phosphate buffered saline (PBS) solution raised the apparent pKa of the ortho-rhodamine B titration point by about 1 pH unit. This could be exploited in conjunction with bulk solution pH changes to turn the rhodamine B-POPE dye into a sensor for streptavidin involving a decrease, rather than an increase, in the fluorescence response, at pH values below streptavidin's pI value.

CONCLUSIONS

This study demonstrates the ability to monitor ligand-receptor interactions on supported lipid bilayers through the protonation or deprotonation of reporter dyes for both negatively and positively charged analytes over a range of pH and ionic strength conditions. Specifically, the sensitivity and pH-operating range of this technique can be optimized by modulating the sensing conditions which are employed.