Investigating ligand-receptor interactions at bilayer surface using electronic absorption spectroscopy and fluorescence resonance energy transfer

Abstract

We investigate interactions between receptors and ligands at bilayer surface of polydiacetylene (PDA) liposomal nanoparticles using changes in electronic absorption spectroscopy and fluorescence resonance energy transfer (FRET). We study the effect of mode of linkage (covalent versus noncovalent) between the receptor and liposome bilayer. We also examine the effect of size-dependent interactions between liposome and analyte through electronic absorption and FRET responses. Glucose (receptor) molecules were either covalently or noncovalently attached at the bilayer of nanoparticles, and they provided selectivity for molecular interactions between glucose and glycoprotein ligands of E. coli. These interactions induced stress on conjugated PDA chain which resulted in changes (blue to red) in the absorption spectrum of PDA. The changes in electronic absorbance also led to changes in FRET efficiency between conjugated PDA chains (acceptor) and fluorophores (Sulphorhodamine-101) (donor) attached to the bilayer surface. Interestingly, we did not find significant differences in UV-vis and FRET responses for covalently and noncovalently bound glucose to liposomes following their interactions with E. coli. We attributed these results to close proximity of glucose receptor molecules to the liposome bilayer surface such that induced stress were similar in both the cases. We also found that PDA emission from direct excitation mechanism was ~2-10 times larger than that of the FRET-based response. These differences in emission signals were attributed to three major reasons: nonspecific interactions between E. coli and liposomes, size differences between analyte and liposomes, and a much higher PDA concentration with respect to sulforhodamine (SR-101). We have proposed a model to explain our experimental observations. Our fundamental studies reported here will help in enhancing our knowledge regarding interactions involved between soft particles at molecular levels.

Figure 1

Changes in the absorption (A) and emission (B) spectra of PDA liposomes (N) with the addition of E. Coli at different concentrations. Figure 1C and 1D shows the spectral overlap (J value) (yellow region) between SR 101 (Pink) and Blue PDA (blue) liposome solution and spectral overlap between SR 101 and Red PDA (red) liposome solution respectively. The concentration of E. Coli stock solution was 3.3 × 10⁷ E. Coli/mL while the concentration of BSA was 150 μg/mL. In Figure 1A and 1B, m stands for 10⁷ E.Coli particles. For example, 0.033m means 0.033*10⁷ of E.Coli particles. The excitation wavelength for FRET experiments was 560 nm.

Figure 2

(A) Colorimetric response (CR) of the liposomes-versus-E. coli concentration for liposomes N, (B) shows FRET efficiency for liposomes N. Minus sign in (B) denotes a decrease in the FRET efficiency after addition of E. Coli to the solution. CR and E were calculated using Eqs. 1 and 3 respectively.

Figure 3

(A) Fluorescence micrograph of SR-101tagged-GUV bound to excess amount of E. Coli. The red emission originated from SR-101. (B) DAPI-stained E. Coli bound to GUV showed blue emission. (C) Shows the composite of E. Coli-bound GUVs. (D) The fluorescence micrograph of nano-sized liposomes tagged with SR-101 bound on the surface of E. Coli (blue emission). Here the liposomes are totally covered with E. Coli surface. For these experiments, E. Coli was in excess (>20 times the liposome concentration). Please see experimental section for information on excitation and emission filters. The red emission was obtained using a 41004 Texas Red filter (exciting and emitting band widths of the filter used were 527-567 nm and 605-682 nm respectively). The blue emission was obtained using a DAPI filter (excitation and emission band widths were (349 ± 25) nm and (459 ± 25 nm).

Figure 4

The interaction of liposomes with E. Coli under our experimental conditions. The glucose receptors were close to the liposomal bilayer-aqueous interface such that it is proposed that, apart from specific glucose-glycoprotein interactions between liposomes and E. Coli, non-specific interactions are possible. The concentration of liposomes with respect to E. Coli concentration was low for these experiments.

Figure 5

(A) R_FRET for N series liposome at different E. Coli concentrations. R_FRET represents the ratio of SR-101 emission intensity (excitation wavelength was 560 nm) after addition of E. Coli of a given concentration to SR-101 emission intensity in the absence of E. Coli. (B) R_Direct at different E. Coli concentrations. R_Direct represents the ratio of PDA emission intensity (excitation wavelength was 490 nm) in the presence and absence of E. Coli. The excitation wavelengths for FRET and direct excitation were 560 nm and 490 nm respectively.

Scheme 1

All chemicals used in the preparation of liposomes.