Investigating Ligand−Receptor Interactions at Bilayer Surface UsingElectronic Absorption Spectroscopy and Fluorescence ResonanceEnergy TransferNavneet Dogra,† Xuelian Li,† and Punit Kohli*

Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois 62901, United States

*S Supporting Information

ABSTRACT: We investigate interactions between receptorsand ligands at bilayer surface of polydiacetylene (PDA)liposomal nanoparticles using changes in electronic absorptionspectroscopy and fluorescence resonance energy transfer(FRET). We study the effect of mode of linkage (covalentversus noncovalent) between the receptor and liposomebilayer. We also examine the effect of size-dependentinteractions between liposome and analyte through electronicabsorption and FRET responses. Glucose (receptor) molecules were either covalently or noncovalently attached at the bilayer ofnanoparticles, and they provided selectivity for molecular interactions between glucose and glycoprotein ligands of E. coli. Theseinteractions 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) andfluorophores (Sulphorhodamine-101) (donor) attached to the bilayer surface. Interestingly, we did not find significantdifferences in UV−vis and FRET responses for covalently and noncovalently bound glucose to liposomes following theirinteractions with E. coli. We attributed these results to close proximity of glucose receptor molecules to the liposome bilayersurface such that induced stress were similar in both the cases. We also found that PDA emission from direct excitationmechanism was ∼2−10 times larger than that of the FRET-based response. These differences in emission signals were attributedto 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 ourexperimental observations. Our fundamental studies reported here will help in enhancing our knowledge regarding interactionsinvolved between soft particles at molecular levels.

■ INTRODUCTIONMolecular recognition events involving carbohydrates are veryimportant in various biological processes such as functioning ofthe immune system1 and in the interaction of viruses andbacteria with cell.2 These recognition events usually involvecommunication between cells that is driven by specificinteractions between molecules on the cell surfaces.3 Tostudy these receptor−ligand interactions for the molecularrecognition, a wide variety of studies are reported in theliterature.4−7 The natural cell membrane, which has manyhighly specific molecular recognition receptor sites on itssurface, may be considered a completely self-containedbiosensing system where molecular recognition is directlylinked to signal transduction.6 When specific ligand bindingoccurs at these sites, the binding event is often transduced intoa cellular message which can be communicated into cell oramong many cells.7 To mimic the complex molecularchoreography of cell membrane in a simple way, manysynthetic membranes are designed and prepared.8,9 Conjugatedpolymer-based biosensor systems are a promising choice forcell membrane mimic because in these systems the signal of aspecific ligand binding to the receptor in the bilayer can betransduced along the conjugated polymer chain.6−9 The

ligand−receptor interactions occurring at bilayer surface canaffect the properties of the collective system producing largersignal amplification.5,7,10−13

Recently, polydiacetylene (PDA), a conjugated polymer, hasbeen widely utilized as transducer because of its distinctivechromatic transition property and convenient preparationmethod through self-assembly and subsequent photopolymeri-zation among diacetylene bonds.14−18 It is well-known thatPDA shows intense blue color due to light absorption by itsextended ene−yne conjugated backbone in the visible region.The torsion angle between substituent groups plays animportant role in the appearance of conjugated polydiacetylene,as nearly flat structures are proposed to appear blue whereasstrongly twisted structures appear red.19 This structural/colorimetric transformation of PDA can be generated byvarious external stimuli, such as temperature,20,21 mechanicalstress,22,23 pH,24 ionic strength,25 or biointeractions.26,27 It isdemonstrated that by incorporating biological receptors in thePDA liposome bilayer, the binding of analytes to receptors may

Received: February 19, 2012Revised: June 17, 2012Published: June 26, 2012

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change the conformation of the polymer backbone providing acolorimetric response for a variety of biological targets.28,29 Todate, PDA has been successfully employed for the developmentof colorimetric biosensors for cholera toxin,30 influenza virusand bacteria,31,32 epitopes,33 lipopolysaccharides,34 and phos-pholipase A2.

35 Recently, oligopeptide-functionalized polydia-cetylene liposomes were used as specific fluorescent sensor forbacterial lipopolysaccharides and E. coli in aqueous solution.36

The emissive properties of PDA have potential for providingincreased detection sensitivity and a lower detection limitcompared to colorimetric sensors due to an inherent highersensitivity of fluorescence. The development of fluorescence“turn on” sensors is a highly desirable and may also providemore sensitive approach for the detection of a target

molecule.37−40 In “off” state of these materials, the excitedstates propagating along the conjugated backbone are quenchedwhen encountering an energy trap at an occupied receptor sitewhich can lead to quenching of fluorescence.4,7,41 Previously,we have demonstrated that the protein sensors based onfluorescence resonance energy transfer (FRET) betweenconjugated polymers and fluorophores can provide “turn-on”sensors with high sensitivity and lower detection limit thanthose based on colorimetric change.42 We demonstrated anovel FRET-based system that utilizes changes in J value(spectral overlap) and changes in the quantum yield of theacceptors for fluorescence amplification.43,44

The receptors containing lipids can be cross-linked throughthe use of diacetylene groups which form conjugated polymer

Scheme 1. All Chemicals Used in the Preparation of Liposomes

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backbone. It is also shown that naturally driven lipophilicmolecules can be incorporated into PDA’s bilayer mem-branes.45 Several studies published in recent years havedemonstrated that PDA liposomes and films with naturallipid assemblies intercalated within the polymer matrix alsoundergoes blue−red transitions in response to interactions withvaried analytes.46−48 Glucose molecules present at the surfaceof liposomes were previously used as receptors for theglycoprotein on E. coli surface.36,32,49 E. coli, a Gram-negativebacterium, usually uses fimbrial adhesions to bind or adsorb tothe surface of host cells. For E. coli P fimbriae, recognizing thegalactose-containing receptors;50 type 1 fimbriae, binding tomannose-containing receptors;51 S fimbriae, recognizing sialyl-(α2−3)galactosides;52 G fimbriae, binding preferentially toterminal N-acetyl-D-glucosamine residues.53 In particular, thecell surface glycolipid conjugates seem to be importantmediators of adhesion of E. coli.54 In general, E. coli interactswith glucose, but these interactions are not as selective ascompared to those present in the case of antibody and antigen.In the present case, use of glucose on the liposome surfacepresents a convenient way of probing the interactions betweenliposomes and E. coli.In a recent work, we found that the interactions between

covalently bonded receptors on liposome surface and proteinligands appeared to induce a larger stress on the PDA backbone(hence, possessed a higher response) than those involved innoncovalently bonded receptors.42 The main focus of thepresent work is to investigate the effect of interactions betweenreceptor attached to PDA liposomes (covalent versus non-covalent) and ligands of E. Coli through UV-Vis and FRETresponses. To elucidate this effect, we synthesized two differentglucose-tagged lipids (3 and 4, Scheme 1). In the case of 3, theglucose was tagged to a long alkyl chain fatty acid and wasinserted non-covalently into bilayer of the liposomes (denotedby N). For liposomes prepared with 4, the glucose residue wascovalently linked to a diacetylene monomer that formed the

conjugated backbone of liposomes after photopolymerization(denoted by C). In all the experiments, sulforhodamine 101(SR-101) and PDA (blue) were served as energy donor andacceptor, respectively (Figure 1C). We observed changes in thespectral overlap (J) between SR-101 emission and PDAabsorption after addition of E. coli to a glucose containingliposome solution (Figure 1C,D and Figure 3S). The changesin spectral overlap J also caused changes in SR-101 emissionsignal (Figure 1B and Figure 1S(B)) which in turn provided aconvenient way of observing interactions between E. coli andglucose in solution.In this article, we investigate ligand−receptor interactions at

bilayer using fluorescence resonance energy transfer betweenSR-101 molecules and polydiacetylene acceptor molecules(Figure 1C and Figure 1S(C)) after binding of glucosereceptors attached to the surface of liposomes with proteinligands on E. coli. To visualize the liposome−E. coli interactions,we prepared glucose receptors containing giant unilamellarvesicles (GUVs) for fluorescence microscopic analysis (seeSupporting Information for more details). We have employedoptical spectroscopic (UV−vis, emission, and FRET) andfluorescence microscopic techniques for investigating ligand−receptor interactions at the bilayer−aqueous interface.

■ EXPERIMENTAL SECTIONChemicals and Materials. N-Boc-L-threonine (>98%), acetobro-

mo-α-D-glucose (>95%), iodine (>99.8%), potassium carbonate(anhydrous, >99%), and trifluoroacetic acid (>98%) were purchasedfrom Aldrich. Triethylamine (>98%) and acetonitrile (MeCN) werepurchased from Acros; dichloromethane (DCM), diethyl ether,chloroform (TCM), methanol (MeOH), and ethyl acetate (EtOAc)were purchased from Fisher Scientific. Solvents were purified anddried by distillation after refluxed with calcium hydride (CaH2) andstored under Ar. 2 was synthesized according to a previously reportedmethod.40,44 Scheme 1 shows the chemical structures of lipids andmonomers used in our experiments.

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

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Giant Unilamellar Liposome (GUVs).55 Besides the use ofnanoscale liposomes, we have used GUVs (∼15−60 μm) for thestudies involving the liposome−E. coli interactions using fluorescencemicroscopy. The use of GUVs provided an excellent way to visuallyprobe the interactions of liposomes with E. coli. These studies clearlyshow that the liposomes are attached to E. coli and also provided usefulinformation on E. coli distribution on the surface of GUVs. The self-assembled GUVs were prepared by using a mixture of 10,12-pentacosadiynoic acid (1), glucose-tagged lipid (4), and rhodamine-tagged DMPC (6) [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine-rhodamine-B-sulfonyl) (ammonium salt)].GUVs Were Synthesized According to the Following Proce-

dure.55,56 Briefly, a mixture containing 1, 4, and 6 in a desired ratio(4:4:2) was dissolved in chloroform in a round-bottom flask. The totalconcentration of all the monomers was 1 mM in the final solution. Thesolution was passed through a 0.45 μm nylon filter to remove the lipidaggregates. The solvent was then evaporated completely. The driedmaterial was again dissolved in a mixture of 3 mL of chloroform and 1mL of methanol. The aqueous phase (25 mL of nanopure water) wasthen added carefully along the flask walls. The organic solvent wasremoved in a rotary evaporator under reduced pressure, and anopalescent fluid was obtained. The resultant solution is cooled at 4 °Covernight to promote self-assembly of monomers.E. coli Staining with 4′,6-Diamidino-2-phenylindole (DAPI)

Fluorophore. E. coli was stained with DAPI fluorophore forfluorescence microscopic studies. DAPI is a well-known agent thatselectively stains nuclei.57 The staining of E. coli was performedaccording to a reported procedure.58 Briefly, 5 mL aliquot of E. coli wastaken from stock bacterial culture. The aliquot was transferred tocentrifuge tube to which equal amount of PBS was added. Thesolution was centrifuged for 15 min at 8000 rpm, the supernatant wasdiscarded, and an equal volume (1 μM) of DAPI was added to theremaining solution. The solution mixture was then centrifuged for 15min, and supernatant was removed. An equal amount of PBS wasadded again to the bacterial solution prior to and before centrifugation.This process was repeated three times. DAPI-tagged E. coli was storedin a refrigerator for future uses. The interactions between E. coli andliposomes were investigated using fluorescence microscopy (seeSupporting Information).Spectroscopic Measurements. PDA liposomes C (covalently

bound glucose) and N (noncovalently bound glucose) containing 5−20% glucose-lipid (3) or glucose-PDA (4), respectively, wereincubated with different concentrations (between 0.033 × 107 and3.3 × 107 cells/mL) of E. coli in deionized water for 30 min at roomtemperature. UV−vis absorption spectra of all of the samples wererecorded using a Perkin-Elmer Lambda 25 (spectral slit width 1 nm)UV−vis spectrophotometer. The color transition of the PDA liposomesolution was observed by comparing two main absorption maxima ofblue- and red-PDA forms at 540 and 640 nm, respectively. Thecolorimetric response (CR) is calculated as a percent change in blue tored color after incubated the samples with different concentration of E.coli solution as shown in eqs 1 and 2.31

=−

×CRPB PB

PB100%0

(1)

=+

×A

A APB 100%640

540 640 (2)

PB and PB0 are blue- and red-PDA ratios of a sample after and beforeaddition of E. coli, respectively. A640 and A540 represent the absorbancevalues at wavelengths of 640 and 540 nm, respectively.

The emission spectra were measured using a Photon TechnologyInternational spectrofluorometer. For all emission spectra, theexcitation wavelengths for SR-101 and PDA were set at 560 and490 nm, respectively, and the spectral slit widths (both excitation andemission) were 6 nm. Under these conditions, the contribution ofemission from PDA to total emission and that of SR-101 to totalemission was minimum when excited at 560 and 490 nm, respectively.

■ RESULTS AND DISCUSSIONAbsorption Spectroscopy of PDA Liposomes with

Addition of E. coli. Polymerized liposomes appeared blue incolor and exhibited an intense absorption maximum at ∼640nm along with less intense peaks centered at 590 nm (Figure1A and Figure 1S(A)). The peaks centered at 640 and 590 nmare attributed to 0−0′ and 0−n′ vibrational transitions in π−π*electronic transition respectively (Figure 1A).59 The blue-shifted peak centered at ∼540 nm is attributed to red form ofPDA. We did not observe this peak in our previous reports42−44

where the ligands were small and were present at a longerdistance from aqueous−bilayer interface. The low-intensitypeak at 540 nm (Figure 1A and Figure 1S(A)) resulted fromstress induced on conjugated PDA due to cross-sectional areamismatch between glucose and carboxylic acids that led tochanges in the bilayer packing density.14 It is interesting to seethat at higher concentration of E. coli the increase in theabsorbance in the 400−450 nm region was much larger thanthe rest of the spectrum (Figure 1A and Figure 1S(A)). Thislarge increase in the absorbance in 400−450 nm region iscaused by scattering of light due to binding of E. coli withliposomes because of significant increase in the particle size.60

One of the consequence of increase in absorbance is theintroduction of significant errors in the calculation of spectraloverlap (J). On the basis of our recent work,61 we would like toemphasize that the calculations of J, E, and CR provide veryuseful qualitative information, and our interpretations of thedata do not change significantly due to errors resulted fromlight scattering.With addition of E. coli to PDA solution, the blue absorption

peak was decreased and the intensities of the red absorptionpeaks (centered at 490 and 540 nm) were increased (Figure 1A

Figure 2. (A) Colorimetric response (CR) of the liposomes versus E. coli concentration for liposomes N. (B) shows FRET efficiency for liposomesN. 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.

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and Figure 1S(A)). The color of the liposome solution turnedpurple and then red with increasing concentration of E. coli inthe solution. This blue to red chromatic transition presumablyis attributed to applied stress on the PDA backbone followinginteractions between glucose and surface proteins on E. coli.These interactions led to reduced effective conjugated length ofthe PDA backbone chains which shifted the electronicabsorption band to shorter wavelengths.26,32,33 To evaluatethe effect of receptor concentration on the sensor response, wecalculated CR (%) values and emission signal for a range ofglucose receptors containing liposomes. Figure 2A and Figure2S(A) show CR versus E. coli concentration curves forliposomes with three different receptor concentrations of 3and 4, respectively, on the liposome surface. The CR values ofboth series (C and N) increased with an increase in the E. coliconcentration in the solution. At 3.3 × 107 E. coli particles/mLin aqueous solution, the CR values were ∼32 and ∼34 forliposomes C (15% of 3) and N (15% of 4), respectively. TheseCR values are much higher than CR ∼ 8.1 for controlexperiments under same E. coli concentration for the liposomesthat did not contain glucose receptors on their surface. Thesubtle nonspecific interactions (such as hydrophobic andelectrostatic interactions) between E. coli and liposomesappears to contribute to CR ∼ 8.1 in the control experiments.Further, we did not observe a significant color change to aliposome solution when a BSA protein solution containing 150μg/mL was added to C and N liposome solutions. Theseexperiments agree with our argument that the biorecognitioninteractions between glucose and E. coli dominantly contributedto larger CR values whereas the samples without specificinteractions between E. coli and receptors showed much smallerCR values. In both the cases, the incorporation of glucosereceptors covalently or noncovalently in the liposomesenhanced the colorimetric response due to specific interactionof glucose at the liposome surface with surface ligands on E. colicell wall. As the concentration of glucose (3 or 4concentration) in the liposomes increased, the CR values ofthe solution also increased upon the addition of E. coli. The CRvalues for the liposome C and N series were almost the same.Emission Spectra of Glucose-Tagged Liposomes with

Addition of E. coli. In our previous studies of biotin−streptavidin interactions using a similar PDA system, weobserved that the covalently bound receptors on the surface ofliposomes (C) showed much larger (∼2−3 times) responseafter interactions with protein ligands than for liposomescomposed with surface receptors that were noncovalently (N)linked to the bilayer.42 We hypothesized that the induced stressdue to biotin−streptavidin interactions on PDA backbonechains was higher for liposomes composed with receptormolecules covalently attached to the backbone of the PDA thanfor liposomes composed with noncovalently inserted receptorsin the bilayer of the liposomes. Thus, the enhanced stresstransport was hypothesized to result in larger changes in bothcolorimetric and FRET signals for liposomes with covalentlybound receptors to the surface of the liposomes. For biotin−streptavidin PDA liposome system, the size of streptavidin(analyte) was much smaller than that of liposomes, and a largeportion or whole surface of the liposomes is accessible tostreptavidin (Figure 4S). However, in the present studies, E. coli(analyte) is much larger than nanoscale liposomes.The emission spectra of liposomes N and C upon addition of

various concentration of E. coli are shown in Figure 1B andFigure 1S(B), respectively. The emission intensity of SR-101

increased gradually with the increasing concentration of E. coli.This is consistent with our UV−vis spectroscopic analysis andthe design of our system that the quenching of emission of SR-101 will be reduced due to decrease in the spectral overlap (J)between SR-101 and PDA after addition of E. coli to theliposome solution. A decrease in the FRET efficiency andincrease in the SR-101 emission was observed following E. coliaddition to the solution (Figure 2B and Figure 2S(B)). Weestimated FRET efficiency (E), J, and changes in J (ΔJ) usingthe equations 3, 4, and 5 respectively:62

=−

EF F

F0

0 (3)

where F and F0 is the emission intensity of donor in thepresence and absence of the acceptor (PDA). F0 here meansemission intensity of SR-101 when PDA was unpolymerized.

∫ ∫λ λ λ ε λ λ λ= =∞ ∞

‐J J( ) d PL ( ) ( ) d0 0

D corr A4

(4)

where PLD‑corr and εA represent the donor emission(normalized dimensionless spectrum) and molar absorptioncoefficient of the acceptor.

Δ =−

×JJ J

J100%0

0 (5)

where J0 and J are the donor−acceptor spectral overlap ofsample before and after addition of E. coli, respectively.

Trend of Electronic Absorption and FRET Responseswith E. coli Concentration. The present study investigatesthe changes in electronic absorption of PDA and FRETresponse due to molecular recognition between moleculespresent at the bilayer interfaces of liposomes and E. coli. Wehave used CR, ΔJ, and FRET responses to evaluate interactionsbetween liposomes and E. coli. CR is a measure of changes inthe electronic absorbance of blue-PDA after E. coli addition, ΔJprovides a measure of integrated area between the spectraloverlap between PDA absorbance and SR-101 emission curves,and it depends on changes in the PDA absorbance. The overallenergy transfer probability is effected as a consequence ofconformational changes in the conjugated PDA chains. Finally,ΔE represents changes in the FRET efficiency as a result ofspectroscopic changes in the PDA absorbance spectrum. Weobserved that all three responses (CR, ΔE, and ΔJ) showsimilar trend with increase of E. coli in the solution (Figure 2,Figures 2S and 3S). For example, there is a rapid increase inresponse followed by plateau for all three response−[E. coli]curves. These trends were reproducible for different liposomepreparations. The magnitude of these responses was also similarfor liposomes composed with 3 and 4. For example, whereas ΔJwas 18% and 24% for liposomes with 15% 3 and 4, respectively(Figure 3S), E was ∼20% for these liposomes (Figure 2B andFigure 2S(B)). The estimated values of ΔJ contains some errordue to scattering contribution in the absorption spectra (seeabove). However, for all purposes, ΔJ for both the systems (Cand N) were similar in values. The shape of the response trendis not unexpected because the underline molecular phenom-enon that contributes to these three responses is stress inducedon the PDA backbone due to interactions of E. coli withliposomes. The molecular interactions between glucose and E.coli presumably resulted in stress that is transported toconjugated polydiacetylene chain. The application of this subtlemolecular stress on the PDA backbone led to blue-shift in the

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UV−vis spectrum of PDA and a decrease in the spectral overlap(J) between the emission of SR-101 and absorption of PDA.Overall, the result of E. coli−liposome binding led to decreasein the quenching SR-101 fluorophores; that is, the emissionfrom SR-101 emission was enhanced (Figure 1B and Figure1S(B)).During the initial additions of E. coli, the liposome bilayer

was enriched with glucose receptors which were stericallyunhindered for binding with E. coli. The ratio of number ofglucose receptors to binding protein on E. coli was large in thebeginning of the experiments, and E. coli can access to glucosereceptors on the liposome surface. Because of these reasons, theresponses due to binding of liposomes with E. coli were sharp inthe initial stages of addition of E. coli to the liposome solution

(Figure 2A and Figure 2S). However, with increase in the E. coliconcentration in the solution, many protein ligands on E. colisurface were occupied, and both the number of availableglucose receptors on the liposome surface and glycol−proteinon E. coli were significantly reduced as well. This led todecrease in the probability of glucose−E. coli interactions whichresulted in slow rise of responses at later stages of theexperiments (Figures 2, 5, 3S and 5S).Interestingly, irrespective of receptor concentration in the

liposomes, the responses appear to be saturated at around 1 ×107 E. coli/mL (Figure 2 and Figure 2S). The saturation of theresponse means that further increase in the number ofinteractions between liposome and E. coli appears small. Thedimension (both length and diameter) of E. coli can depend

Figure 5. (A) RFRET for N series liposome at different E. coli concentrations. RFRET represents the ratio of SR-101 emission intensity (excitationwavelength was 560 nm) after addition of E. coli of a given concentration to SR-101 emission intensity in the absence of E. coli. (B) RDirect at differentE. coli concentrations. RDirect 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 and 490 nm, respectively.

Figure 3. (A) Fluorescence micrograph of SR-101-tagged 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 ofnanosized 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). See Experimental Section for information on excitation andemission filters. The red emission was obtained using a 41004 Texas Red filter (exciting and emitting band widths of the filter used were 527−567and 605−682 nm, respectively). The blue emission was obtained using a DAPI filter (excitation and emission band widths were 349 ± 25 and 459 ±25 nm).

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upon the strain of E. coli; E. coli was ∼2 μm long with adiameter of ∼500 nm in our experiment.58 Assuming themonodispersed distribution of liposomes with an averagediameter of about 250 nm,58 the number of liposomes were∼4.19 × 1010/mL of the solution (see Supporting Informationfor more information). This means on average total number ofmolecules and glucose in a liposome are ∼2.9 × 106 and 1.45 ×105, respectively. We estimated that each E. coli on an averageinteracted with ∼40 liposomes that contain 10 mol % glucoseconcentration on their surface. This corresponds to only ∼1%of the total surface area is covered with liposomes under ourexperiment conditions. Interestingly, the signal (both E andRFRET) in our experiments get saturated at a concentration of∼107 E. coli in the solution. Increase in CR values for similarexperiments was larger; however, as we noted earlier, due toscattering in the UV−vis spectra the interpretation of theseresults are qualitative in nature. These estimates suggest thatassuming the whole surface of E. coli was accessible toliposomes for interactions under our experimental conditions,the signal generated following E. coli−liposome interactions isless sensitive to E. coli concentration >1 × 107 particles/mL.One of the reasons for saturation in signal response isattributed to insufficient number of receptors on the liposomesurface for interactions with E. coli. This is also evident fromour experimental data: when the concentration of glucose inthe liposomes was increased from 5 to 15 mol %, the increase inboth E and RFRET responses was modest with increase in E. coliconcentration from 1 × 107 to 3.3 × 107 particles/mL ascompared to a much larger signal enhancement when E. coliconcentration was increased from 0 to 1 × 107 particles/mL.The available ligand density on E. coli surface is also not known;therefore, it is not possible to estimate with certainty how muchfurther enhancement in the signal can be achieved with increasein the glucose concentration in the liposomes.In these calculations, we have assumed that (1) E. coli are

cylindrical in shape with hemispheres at their two ends; (2) theconcentration of glucose on the liposomes are evenlydistributed and that half of them are inside of the liposomeswhich were not available for binding to E. coli; (3) liposomesare monodispersed; and (4) only half of the glucose moleculeson the outer surface of the liposomes interacted with E. colithrough specific glucose E. coli interactions. The lastassumption is valid because we have not observed E. coliaggregation in the solution under our experimental conditions.There is also a possibility that liposomes are able to interactwith multiple E. coli, but due to significantly large size of E. coliand a low concentration of liposomes in the solution, webelieve that there is large steric hindrance for two or more E.coli to bind with the liposome already attached to an E. coli.Figures 3A and 3B show the fluorescence micrograph ofliposomes tagged with SR-101 and E. coli stained with DAPI,respectively. For these experiments, the number of E. coli werein excess (>20 times) as compared to number of GUVs. Ourfluorescence microscopic data showed that whole surface ofGUVs liposomes is accessible to E. coli although some spots onliposomes appeared to have an aggregation of E. coli which isbelieved to be result of an excess of E. coli relative to liposomesin the solution (Figure 3A−C). Further, we have alsoperformed binding of E. coli with nanoscale liposomes usingfluorescence microscopic analysis (Figure 3D). Interestingly, inthe latter case, the size of liposomes was much smaller than thatof E. coli, and the liposomes (which were in excess) bound to E.coli surface. The red emission around the blue fluorescent E. coli

indicated bound liposomes. Analysis with both smaller andlarger liposomes confirmed the binding of liposomes with E.coli (Figure 3).We believe that the fourth assumption needs more

discussion. For our experiments, the glucose receptors arepresent very near to the surface of the liposomes (see chemicalstructures in Scheme 1). Although our experiments suggestedthat the presence of glucose on the liposome surface isnecessary for response, however, once E. coli is attached withliposomes, nonspecific surface interactions between E. coli andliposomes cannot be ruled out. This is because the binding ofliposomes and E. coli is very close to the surface of liposomesand that both the E. coli and liposomes are soft particles whichcan be easily deformed (Figure 4 and Figure 4S). Further, these

nonspecific interactions between E. coli and liposomes deformconjugated polymer chain in the PDA liposomes. One of theconsequences of binding of liposomes with E. coli through thesenonspecific interactions is large PDA emission signal throughdirect excitation mechanism (Figure 5) (see more discussionbelow).Finally, with increase in the glucose receptor concentration in

the liposomes, the initial response (slope between the responseand E. coli concentration at the beginning of the experiment)was larger than that for particles with a lower receptorconcentration (Figure 2A and Figure 2S(A)). This is becauselarger number of glucose receptors interacted with E. coliproducing enhanced stress on the conjugated PDA chain.

Direct versus FRET Response. We now compare theemission of the direct excitation of PDA versus FRET response.It is interesting and important to compare the emission fromSR-101 and red-PDA to the total emission intensity whenexcited at different wavelengths. This discussion also shed somelight on specific versus nonspecific interactions betweenliposomes and E. coli. For direct excitation, we meant excitationof PDA chains at 490 nm, and the emission was observed in510−700 nm region (Figure 6S). For FRET experiments, theexcitation was performed at 560 nm, and the emission wascollected in the 570−700 nm region (Figure 1B and Figure1S(B)). Previously, we have observed a smaller emissionresponse for direct excitation as compared to FRET mechanismfor a PDA system in which biotin was tagged on to liposomesurface and the response was induced from biotin−streptavidininteractions.42 In contrast to our previous results, however, inthe present study we observed a much higher emissionresponse (>10 times) for PDA direct excitation (Figure 5A,Band Figure 5S) than the emission response due to FRETmechanism (Figure 1B, Figures 5S(A) and 5S(B)). Figure 5and Figure 5S show RFRET,N and RFRET,C, which represents ratioof SR-101 emission in the presence and absence of E. coli. In

Figure 4. Interaction of liposomes with E. coli under our experimentalconditions. The glucose receptors were close to the liposomal bilayer−aqueous interface such that it is proposed that, apart from specificglucose−glycoprotein interactions between liposomes and E. coli,nonspecific interactions are possible. The concentration of liposomeswith respect to E. coli concentration was low for these experiments.

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both of these cases, the maximum FRET ratio for N and Cseries was ∼2. That is, the maximum increase in the FRETemission of SR-101 after addition of E. coli to the liposomesolution was ∼2. However, under the same conditions, themaximum increase in RDirect of PDA was >10 for liposomesprepared with 3 and 4 after E. coli was added to the liposomesolution (Figure 5B).We would like to emphasize E value of ∼34% (for 20 mol %

of glucose receptors) was calculated using eq 3 where F0corresponds to emission intensity of SR-101 for theunpolymerized PDA liposomes (in the absence of acceptor),and F is the emission intensity of liposomes after addition of E.coli in the solution. RFRET represents a different measure ofdecrease in FRET in our experiments. It is calculated bydividing emission intensity of SR-101 in the presence andabsence of E. coli, but in all of RFRET calculations the FRETmechanism was present. The major difference between E andRFRET is that the F0 value in E calculations does not possessFRET component whereas the emission intensity in all RFRETcalculations contains a FRET component.In general, the emission intensity for direct excitation

depends on concentration (C) of PDA, its excitation coefficient(ε), and quantum yield (Qy). The FRET emission response, onthe other hand, depends upon the FRET efficiency (E)between SR-101 and PDA along with quantum yield (Qy) ofthe emitting species (SR-101). In our case, the Qy and ε of SR-101 are ∼3−4 and 2−3 orders of magnitude higher than redPDA, whereas E ranges between 10 and 34% (Figure 2B andFigure 2S(B)). PDA concentration is about 106 times largerthan that of SR-101 concentration. A much larger directemission response compared to FRET response in our studiesis attributed to two major contributions: First, although both Qyand ε of SR-101 are significantly larger than that of PDA, theoverall PDA concentration in liposomes is 3 orders ofmagnitude larger than that of SR-101. This large concentrationdifference between PDA and SR-101 compensated for lower Qyand ε of PDA. E. coli is much larger than liposomes; thus, abouthalf of the total glucose receptors can interact with surfaceproteins of the E. coli under our experimental conditions. Thismeans that FRET response in glucose−E. coli system isobtained from approximately half of the available glucosereceptors. Second, our calculations suggested that only ∼1% ofE. coli surface is occupied by the liposomes, and rest of thesurface is available for specific and/or nonspecific interactions.These nonspecific interactions presumably through inducedstress on PDA backbone leading to conformation changes inthe conjugated PDA chain without affecting FRET responsefrom other half of the liposome surface which is not in contactwith E. coli (Figure 4 and Figure 4S). We believe that thesenonspecific interactions are a major contributor to the PDAemission signal from direct excitation, but they do notcontribute significantly to the FRET response. We also notethat for this to happen the first step is specific binding ofglucose with E. coli. Without this step, the possibility ofproposed nonspecific interactions is small. This is observed inour control experiments that the emission response is negligiblein the absence of glucose receptors on the liposome surface(Figure 1B and Figure 1S(B)), Thus, the size differencesbetween analyte and liposomes, a much higher PDAconcentration with respect to SR-101, and nonspecificinteractions between E. coli and liposomes contributed to amuch larger direct emission signal as compared to the FRETresponse.

■ CONCLUSIONSIn summary, we have investigated interactions between E. coliand glucose-tagged liposomal nanoparticles through changes inelectronic absorption spectroscopy and FRET response. Theglucose groups on the liposome surface provided selectivitythrough interactions between carbohydrate and E. coli. We didnot find significant differences in UV−vis and FRET responsesbetween covalently and noncovalently bound glucose toliposomes following their interactions with E. coli. Weattributed these results to close proximity of glucose receptormolecules to liposome bilayer surface such that induced stressdue to receptor−ligand interactions were similar in both thecases. We also found that PDA emission from direct excitationmechanism was ∼5 times larger than the FRET-based response.We attributed these differences in emission signals to threemajor reasons: nonspecific interactions between E. coli andliposomes, size differences between analyte and liposomes, anda much higher PDA concentration with respect to SR-101. Thissystem has implications in several biosensing applicationswhere low cost and sensitivity are the top priorities.

■ ASSOCIATED CONTENT*S Supporting InformationFluorescence microscopic analysis, synthesis of monomers 3and 4, procedure for the synthesis of nanoscale liposomes,Figures 1S−6S, estimation of number of liposomes/E. coli. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: pkohli@chem.siu.edu.Author Contributions†Equal contributions.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe acknowledge financial support from the National ScienceFoundation (CAREER award) and the National Institute ofHealth (GM 8071101). Drs. John Bozzola and Steve Schmittand Ms. Hillary Gates (IMAGE center at SIUC) helped us inthe electron microscopy analysis. We acknowledge Mr. KunalChatterjee and Prof. Ramesh Gupta for providing E. coli. Ms.Julia Reyes helped us in liposome preparation. We would like toacknowledge positive critics of three anonymous reviewerswhich made the manuscript stronger after revisions.

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