Label-free pH Modulation Sensor

A biosensor based on pH modulation on the lipid membrane surface has been developed [J. Am. Chem. Soc. 131 (2009) 1006-1014 pdf]. By incorporating pH sensitive fluorescent dyes into supported lipid bilayer, we can monitor the interactions between membranes and small molecules, peptides and proteins without the tedious work of labeling the analytes. The commonly used pH sensitive dyes are ortho-conjugated Texas Red-DHPE and ortho-conjugated Rhodamine B-POPE. The pH sensitive ortho isomer of Texas Red-DHPE can be turned on at lower pHs and be turned off at higher pHs, while the para isomer is not sensitive to pH changes and is usually used as a reference. The biosensor is built in microfluidic device channel with supported lipid bilayer as platform, which renders this heterogeneous assay with both high throughput and high interface specificity. The figure 1 is a schematic illustration of how this biosensor works: when the negatively charged analytes are introduced into device channels and bind at the membrane surface, local electric field gets more negative thus more hydronium ions are recruited to the membrane surface. The pH sensitive dyes located at the surface can sense the interfacial potential change, and their fluorescence will be turned on. Upon positively charged analytes binding, the mechanism is vice versa. And in this case, hydroxide ions are recruited to the surface and the fluorescent signal should be turned off.

Fig. 1 Schematic diagram illustrating the principle of pH-sensitive dye as a reporter for interfacial binding of negatively charged analytes

The above biosensor system has been successfully applied to sense the behavior of small drug molecules, antimicrobial peptides and virus proteins at lipid membrane surface [literatures]. For small drug molecules, positively charged tetracaine and negatively charged ibuprofen have been studied [Anal. Chem., 2013, 85, 10240-10248 pdf].  We were able to get binding curves for both drugs with lipid membrane. And we unraveled the details of how cholesterol, negatively charged lipids, and glycolipids affect membrane interactions with such small molecule drugs. Moreover, we found that ibuprofen showed multiple-step binding modes, and high concentration of ibuprofen can disrupt lipid membrane by forming pores [undergoing project].

For antimicrobial peptide (AMP) measurements, we compared the binding behaviors of two typical AMPs: nonalysine and nonaarginine. Although the structural make-up of Lys₉ and Arg₉ peptides is similar in many ways, they have very different modes of interaction with lipid membranes. We proved that Lys₉ bound much weaker to lipid membrane compared to Arg₉ due to the strong anti-cooperativity. As for Arg₉, the intermolecular guanidinium interactions lead to aggregation and dimer formation of Arg₉ which overcome the like-charge repulsion.

We are also collaborating with Craig Cameron’s lab in BMMB to study the interactions of poliovirus protein 3C with phosphoinositide on a supported lipid bilayer platform. Preliminary data showed that 3C protein specifically binds to phosphoinositide with high affinity. And divalent ion Mg²⁺ was found to block this interaction. Further work will be done to explore protein-lipid binding as a function of phosphoinositide concentration, membrane fluidity and how the lipid composition may change phosphoinositide dynamics and phosphoinositide-protein interactions.

Electrophoretic-Electroosmotic Focusing—A label-free Bioanalytical Platform

An electrophoretic-electroosmotic focusing (EEF) method was developed and used to separate membrane-bound proteins and charged lipids based on their charge-to-size ratio from an initially homogeneous mixture. EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands on supported lipid bilayers (SLBs). Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion (Fig. 2). The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field, and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations, and it overcame diffusive peak broadening to allow four bands to be separated simultaneously within a 380 μm wide isolated supported membrane patch. (Anal. Chem. 83 (2011) 7876-7880  pdf)

Fig. 2 Electrophoretic-Electroosmotic Focusing—A label-free Bioanalytical Platform

This EEF platform was utilized to study Phosphatidic Acid Formation in Intact Phosphatidylcholine Bilayers upon Phospholipase D Catalysis. [Anal. Chem., 2014, 86, 1753-1759 pdf ]   The production of the negatively charged lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid acid (POPA), in supported lipid bilayers via the enzymatic hydrolysis of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC), will re-position the green dye-labeled streptavidin protein due to the membrane charge gradient change  (Fig. 3). On the basis of these results, other enzymatic reactions involving the change in membrane charge could also be monitored in a similar way. This would include phosphorylation, dephosphorylation, lipid biosynthesis, and additional phospholipase reactions.

Fig.3 Monitoring Phosphatidic Acid Formation in Intact Phosphatidylcholine Bilayers upon Phospholipase D Catalysis

This method is also capable of simultaneously identifying multiple competitively binding proteins for the same ligand on supported lipid bilayers (SLBs). This strategy used unlabeled target proteins that bind to the same fluorescently-tagged, lipid-conjugated ligands within the SLB. The protein-ligand complexes were then focused under an applied potential to different locations within the SLB based upon each protein’s size and charge. Both protein identity and relative surface concentration information could be obtained, simultaneously (Fig. 4). This assay provides a simple and convenient method for simultaneously monitoring target analytes that bind to the identical ligand and may ultimately be useful in developing assays that help overcome problems associated with cross-reactivity. (Submitted to Analytical Chemistry)

Fig. 4 The Simultaneous Detection of Multiple Proteins that Bind to the Identical Ligand in Supported Lipid Bilayers