Investigating Molecular Interactions in Biosensors Based on

Mar 22, 2010 - This article references 22 other publications. 1. (a) McQuade , D. T.; Pullen , A. E.; Swager , T. M. Chem. Rev. 2000, 100, 2537– 257...
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J. Phys. Chem. C 2010, 114, 6255–6264

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Investigating Molecular Interactions in Biosensors Based on Fluorescence Resonance Energy Transfer Xuelian Li and Punit Kohli* Department of Chemistry and Biochemistry, Southern Illinois UniVersity at Carbondale, Carbondale, Illinois 62901 ReceiVed: December 6, 2009; ReVised Manuscript ReceiVed: February 18, 2010

We describe here molecular sensors based on fluorescence resonance energy transfer (FRET) between sulforhodamine 101 (donor) and polydiacetylene (PDA, acceptor) for selective detection of biomolecules (streptavidin) in solution. These novel FRET-based systems primarily utilize changes in J values (the spectral overlap between the emission of the donor and absorption of the acceptor) for the modulation of FRET efficiency between donors and acceptors. The biotin-streptavidin interactions were used as a sensing model system to test our sensor response. In this paper, four different biotin-tagged lipids were used as receptors to investigate the effect of interactions between ligand-receptors on the FRET efficiency. The biotin was covalently linked to the liposome surface when using biotin-tagged diacetylene; whereas the biotin-tagged lipids with hydrophobic chains but without diacetylene functionalities provided noncovalently inserted lipids in liposomes. The polymerized liposomes, consisting of sulforhodamine-tagged-diacetylene and receptors linked to lipids in different molar ratios, were investigated using UV-vis and steady-state emission spectroscopy. The liposome solution yielded a weak emission from the donor after photopolymerization of diacetylene monomers due to energy transfer from the donor to PDA backbone chains (acceptors). The addition of streptavidin resulted in increase of the donor emission, which was due to a decrease in the FRET efficiency from donor to PDA. These studies are intended to enhance our basic understanding of the interactions at the molecular level and will guide us for the fabrication of highly sensitive and selective biosensors. Introduction Presently, there is a large interest in conjugated polymers due to the substantial π-electron delocalization along their backbones which gives rise to very interesting linear and nonlinear optical1,2 and electrical properties.3 These polymers have been found to have a wide range of applications including solar and photovoltaic cells, light-emitting devices, optoelectronics, actuators, sensor information storage, and optical signal processing, substitutes for batteries.4 Due to their special responsive properties, conjugated polymers have also been investigated for the development of sensing applications.5 Polydiacetylene (PDA) and polythiophene (PTP) have been used for the sensing of ions, proteins, bacteria, and viruses,1,5–7 because they exhibit special colorimetric changes following stress application. A majority of the reported PDA and PTP sensors are based on its colorimetric changes.5 Excellent reviews and tutorials are published on polydiacetylene-based materials by Kim’s group.7i,j Recently, changes in emission and fluorescence resonance energy transfer (FRET) efficiency after application of external stress (such as thermal heat, mechanical stress, biomolecular recognition between receptors and analytes) on PDA have also been reported.7 Sensors based on FRET between conjugated polymers and fluorophores can be more sensitive than those based on colorimetric changes because of the possibility of signal amplification in response to external stimulation.8 Many excellent books1c,9 and reviews1a,b,7h on sensing applications using conjugated polymers are readily available. Using PTPs, Leclerc and co-workers sensed ions such as I- and Na+, proteins, and nucleic acids.6 Groups led by Heeger and Bazan * To whom chem.siu.edu.

correspondence

should

be

addressed,

pkohli@

have used FRET between organic fluorophores and fluorenederivative conjugative polymers for highly sensitive detection of nucleic acids and peptides.8a–f Whitten’s group used conjugated phenylene-vinylene polymers for reversible FRET-based highly sensitive protein sensing.3a,10, FRET based sensing using semiconducting quantum dots have also been extensively employed for sensing applications.11 Reppy and co-workers have detected E. coli using a PDAbased FRET assay.7d Similarly, Cheng7b,c and Kim7e,8g have also used FRET for the sensing of amines in solution. Recently, we7f,g have shown that the FRET efficiency between dansyl (donor) and PDA (acceptor) can be modulated through thermal treatment of the liposome solution. We have found that the monomer ratio of acceptor to donor (Rad) and the length (L) of linkers (the functional part that connected dansyl fluorophores to the diacetylene group in the monomer) strongly affected the FRET efficiency following heating of the liposome solutions.7f,g For Rad ) 10000, PDA emission intensity was amplified by >18 times when the temperature of the liposome solution was raised from 298 to 338 K. Decreasing Rad resulted in diminished acceptor emission amplification which was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background emission signal. We also observed that the dansyl-attached liposomes with shorter and more rigid linkers exhibited much lower PDA emission amplification than those with longer and more flexible ethylene glycol linkers.7g The stress transport efficiency to PDA backbone depends upon factors such as the mode of receptor-ligand interactions, the linker that connects receptors and PDA backbone, and intermolecular interactions of the lipids within bilayers. Thus, investigations involving these factors may help us in optimizing

10.1021/jp911573g  2010 American Chemical Society Published on Web 03/22/2010

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Figure 1. Schematic presentation of the effect of receptor-sensor interactions on the FRET sensor response. The weaker receptor-sensor interactions provides smaller response (left-most figure) than stronger receptor-sensor interactions (right-most figure).

and increasing the PDA sensor response. In this paper, we investigate the effect of molecular interactions between receptors (attached to the liposome surface) and PDA backbone on FRET response following receptor-analytes binding. We used conjugated PDA backbone chain as a transducing element of the sensor in which the molecular-recognition (MR) interactions between receptor and analytes are converted into an observable emission signal due to changes in the FRET signal from sulforhodamine (acting as donor denoted by SR-101) to PDA. Here, we show that the strength of molecular interactions, i.e., strong-Versus-weak coupling between the PDA backbone and biotin can affect the sensor response (Figure 1). We hypothesize that the transport of the induced stress due to biotin-streptavidin MR interactions to PDA backbone chains will be higher for liposomes composed with covalently attached biotin (referred as liposome solution A) than those that consist of noncovalently bound receptors (Figure 1). Thus, the enhanced stress transport is hypothesized to result in larger PDA spectral colorimetric changes and FRET signal changes in solution A than in solution B. We have systematically performed a detailed analysis for studying the effect of molecular interactions between biotin and conjugated PDA chains on FRET sensor response. We demonstrate that the liposomes that contained covalently bound biotin showed two to three times larger emission response than those made up of noncovalently bound biotin. Thus, the present studies are aimed at increasing our fundamental knowledge of interactions at molecular levels for PDA liposome-based sensors. Materials and Methods All chemicals were purchased from Fisher Scientific and were used as received unless noted otherwise. Different monomers and lipids used in our studies are shown in Figure 2. The synthesis procedures of 2, 3, and 6 are shown in Schemes 1, 2, and 3 respectively. Synthesis of Monomer Sulforhodamine 101-Tagged Pentacosadiynoic Acid (2) (Scheme 1). Synthesis of 2a. The sulforhodamine acid chloride was synthesized according to published literature.13 Laser grade sulforhodamine 101 (0.5 g, 0.814 mmol) was dried under vacuum at 100 °C overnight and was then treated with 3 mL of phosphorus oxychloride. After the mixture was stirred for approximately 12 h, the dark solution was poured onto ice. After 15 min the product was extracted into chloroform, washed several times with cold water, and dried over MgSO4. The chloroform-soluble sulforhodamine 101 acid chloride was dried by evaporation and then redissolved in 25 mL of dichloromethane (CH2Cl2). Triethylamine (0.1 g) and sulforhodamine 101 acid chloride were added to a solution of 1,2-diaminoethane (0.9 g, 15 mmol) in 50 mL of dry CH2Cl2. After the mixture was stirred overnight, the solvent was removed by rotary evaporation, and the residue was subjected to silica

Figure 2. The chemical structures of materials were used to prepare liposome with covalent and noncovalent receptors.

gel column chromatography (ethanol:methanol:chloroform, 3:3: 2) to give the highly fluorescent intermediate 2a (yield: 42%). 1 H NMR (300 MHz, DMSO), δ (ppm): 1.792 (m, 4H), 1.962 (m, 4H), 2.64 (t, 2H), 2.84 (m, 8H), 3.11-3.13 (m, 10H), 6.93 (s, 2H), 7.16 (d, 1H), 7.96 (d, 1H), 8.26 (d, 1H). Synthesis of 2b. The PDA-NHS was synthesized according to a method we described before.7g To a solution of 10,12pentacosadiynoic acid (0.267 g, 0.713 mmol) in dry CH2Cl2 (10 mL), N-hydroxysuccinimide ((NHS), 0.0914 g, 0.786 mmol) was added followed by 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride ((EDC), 0.144 g, 0.713 mmol). The solution was stirred at room temperature for 2 h followed by rotary evaporation of the CH2Cl2. The residue was extracted with diethyl ether and washed with water three times. The organic layer was dried with MgSO4 for half an hour and then filtered. The solvent was removed by rotary evaporation to give a white solid powder 2b (yield: 91%). 1H NMR (300 MHz, CDCl3), δ (ppm): 0.87 (t, 3H), 1.10-1.34 (m, 26H), 1.52(m, 4H), 1.74(m, 2H), 2.23(t, 4H), 2.58 (t, 2H), 2.82 (t, 4H). Synthesis of 2. A solution of 2b in 20 mL of dry CH2Cl2 was added to a solution of fluorescent intermediate 2a dissolved in CH2Cl2. After being allowed to stir overnight at room

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SCHEME 1: Synthetic Route of SR-101-Tagged Diacetylene 2

SCHEME 2: Synthetic Route of Biotin-Tagged Diacetylene 3

temperature, the mixture was concentrated by rotary evaporation, and the residue was purified using silica gel column chromatography (ethyl acetate:chloroform:ethanol 3:3:1 is changed to chloroform:methanol 3:1) to yield 0.165 g of 2 (45%). 1H NMR (400 MHz, CDCl3), δ (ppm): 0.84 (t, 6H), 1.13-1.55 (m, 68H), 1.80-2.19 (m, 20H), 2.67(m, 2H), 2.75 (m, 2H), 2.94 (m, 8H), 3.13 (m, 2H), 3.24 (m, 2H), 3.35-3.40 (m, 8H), 6.8 (s, 2H), 7.11-7.13 (m, 1H), 7.92 (d, 1H), 8.68 (d, 1H). Synthesis of Biotinylated-PDA (3) (Scheme 2). The biotinylated lipid was synthesized according to literature via a fourstep route outlined in Scheme 2.14 The white solid 3a which is same as 2b (see above) was synthesized according to the procedure as described above.

Synthesis of 3b. A solution of 3a (1.67 g, 3.39 mmol) in 20 mL of dry CH2Cl2 was added to tetra(ethylene glycol) diamine (3.24 g, 17 mmol) in 25 mL of CH2Cl2 dropwise with vigorous stirring. Three drops of triethylamine were then added. The progress of the reaction was monitored by TLC. After a few hours, the solvent and the excess residue amine were evaporated. The residue was extracted with ethyl acetate and then washed with water twice. The organic layer was dried with MgSO4 for half an hour, the solution was filtered, and the solvent was removed by rotary evaporation. The product was purified by silica gel chromatography (19:1 CHCl3: MeOH) to give 1.0 g of 3b as a white solid (54% yield). 1H NMR (300 MHz, CDCl3), δ (ppm): 0.87 (t, 3H), 1.10-1.32 (m, 26H), 1.34-1.50 (m, 4H),

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SCHEME 3: Synthetic Route of Biotin-Tagged Arachidonic Acid 6

1.58-1.64(m, 2H), 2.08-2.15 (t, 2H), 2.18-2.22(t, 4H), 3.40-3.60 (m, 16H), 6.35 (b, 1H). Synthesis of 3c. 3c was synthesized according to ref 15. To a solution of D-biotin (0.80 g, 3.27 mmol) in dimethylformamide (DMF) N-hydroxysuccinimide (NHS) (0.38 g, 3.3 mmol) was added followed by EDC (0.67 g, 3.3 mmol). The reaction was stirred overnight, and the solvent DMF was evaporated in vacuo. The residue was washed with water and MeOH several times to get a white solid 3c (63% yield). 1H NMR (300 MHz, DMSO), δ (ppm): 1.40-1.46 (m, 4H), 1.67 (m, 2H), 2.52(d, 1H), 2.67 (t, 2H), 2.81 (t, 4H), 2.84 (d, 1H), 3.11 (m, 1H), 4.15 (t, 1H), 4.30 (t, 1H), 6.36 (d, 1H), 6.42 (d, 1H). Synthesis of 3. A 0.5 g (0.98 mmol) portion of 3b was dissolved in a mixture of 20 mL of DMF and 15 mL of CH2Cl2. Four drops of triethylamine and 0.33 g (0.98 mmol) of 3c were dissolved in 10 mL of dry DMF. This mixture was added dropwise to the solution of 3b. The mixture was stirred overnight. The progress of the reaction was monitored by TLC. The product 3 was isolated after precipitation washed several times with DMF and diethyl ether with a 65% yield. The product was used without further purification. 1H NMR (300 MHz, CDCl3), δ (ppm): 0.87 (t, 3H), 1.10-1.34 (m, 28H), 1.34-1.72 (m, 10H), 2.12-2.22 (m, 8H), 2.87 (m, 1H), 2.93(m, 1H), 3.16 (m, 1H), 3.43-3.50 (m, 4H), 3.54-3.62 (m, 12H), 4.32 (m,1H), 4.53(m,1H), 5.2 (s,1H), 5.9 (s, 1H), 6.28-6.42 (m, 2H). Synthesis of 6 (Scheme 3). The biotinylated arachidonic acid was synthesized according to modified literature methods via a four-step route outlined in Scheme 3.14 The essential steps of the synthetic pathway were adapted. The white solid 6c which is same as 3c (see above) was synthesized according to a procedure as described above. Synthesis of 6a. To a solution of arachidonic acid (0.69 g, 2.27 mmol) in 20 mL of dry methylene chloride (CH2Cl2), NHS (0.268 g, 2.3 mmol) was added followed by addition of EDC (0.465 g, 2.3 mmol). The solution was stirred at room temperature overnight, and the solvent was evaporated by rotary evaporation under reduced pressure. The residue was extracted with chloroform (CHCl3) and water for three times. The organic layer was dried with MgSO4 for half an hour, the solution was filtered, and the solvent was removed by rotary evaporation to give 0.9 g of 6a (97% yield). 1H NMR (300 MHz, CDCl3), δ (ppm): 0.89 (t, 3H), 1.30-1.38 (m, 6H), 1.70 (m, 2H),

2.03-2.19 (m, 4H), 2.31 (m, 2H), 2.6-2.83 (m, 10H), 5.34-5.46 (m, 8H). Synthesis of 6b. A solution of 6a (0.9 g, 2.24 mmol) in 10 mL of CH2Cl2 was added to triethyleneglycol diamine (3.871 g, 20.2 mmol) in 15 mL of CH2Cl2 dropwise while stirring. This was followed by addition of three drops of triethylamine. The solution was stirred at room temperature overnight, and the progress of the reaction was monitored by TLC. The solvent and the amine were then evaporated. The product was purified by silica gel chromatography (10:1 CHCl3: MeOH) to give 0.56 g of 6b as a light yellow liquid (yield: 52%). 1H NMR (300 MHz, CDCl3), δ (ppm): 0.88 (t, 3H), 1.30-1.38 (m, 6H), 1.70 (m, 2H), 2.03-2.19 (m, 4H), 2.31 (m, 2H), 2.53-2.89 (m, 6H), 3.40-3.79 (m, 16H), 5.34-5.45 (m, 8H), 6.50 (b, 1H). Synthesis of 6. A 0.56 g (1.16 mmol) portion of 6b was dissolved in a mixture of 15 mL of dry dimethylformamide (DMF) and 10 mL of CH2Cl2. Four drops of triethylamine and 0.44 g (1.3 mmol) of 6c in 10 mL of DMF were added to the solution of 6b. The mixture was stirred overnight, and then the solvent was evaporated. The product was purified by silica gel chromatography (19:1 CHCl3: MeOH) to give 0.33 g of 6 (40% yield). 1H NMR (300 MHz, CDCl3), δ (ppm): 0.88 (t, 3H), 1.25-1.37 (m, 8H), 1.57-1.63 (m, 4H), 1.72 (m, 2H), 2.03-2.19 (m, 6H), 2.34 (m, 2H), 2.62-2.76 (m, 6H), 2.87 (m, 1H), 2.96 (m, 1H), 3.37-3.79 (m, 17H), 4.60 (m, 2H), 5.2-5.41 (m, 8H), and 8.02 (b, 2H). Liposome Preparation. The liposomes were synthesized according to a published literature procedure.5i,7f,g We have used four different biotin-containing monomers/lipids: 3, 4, 5, and 6. 3 contained diacetylene functionality and was covalently bound to liposomes, but 4, 5, and 6 were noncovalently inserted into bilayers. For all the experiments, the ratios of 1, 2, 7 and biotin-monomer/lipid were x:1:160:y. The total concentration [1 + 2 + 7 + biotin-monomer/lipid] of the monomers was 1 mM for all the experiments. The liposome solutions with different biotin monomer (3) or biotin lipids (4, 5, or 6) were synthesized such that x and y were varied. The concentrations of 2 and 7 were kept constant (1 and 160 µM). In this paper, the concentrations of biotin ligands (%), x, were varied from 1% to 20%. The concentration of 1 can be easily modulated by noting that total concentration of all the lipids was 1 mM.

Molecular Interactions in Biosensors Briefly, mixtures containing 1, 2, 7, and biotin-containing monomers/lipids (3, 4, 5, or 6) were mixed in a desired ratio in chloroform. The solvent was evaporated completely, and the deionized water or PBS (0.1 mM, pH 7.4) was added to make liposome solution with the desired concentration. The resultant suspension was probe sonicated at 76 °C for ∼15 min. The solution then was passed through a 0.8 µm nylon filter to remove the lipid aggregates and was cooled at 4 °C overnight. The resultant solution was optically clear. Polymerized diacetylene liposomes were prepared by UV irradiation with 254 nm for 1-2 min using a Pen Ray UV source (4.5 mW/cm2). The resulting blue liposome solution was stored in the dark at 4 °C. Dialysis of the liposome was carried out with a membrane (Mw, 10000 cutoff) against deionized water. Our dialysis experiment indicated that >95% of SR-101 remained incorporated in the liposomes after polymerization. Four series (liposome A, B, C, D) were prepared. Depending upon the biotin-tagged lipid used, receptors were either covalently (liposome A when 3 was used) or noncovalently (liposome B, C, or D when 4, 5, or 6 was used) bound to the PDA backbone. Estimation of Biotin Concentration in the Liposomes. The level of biotin in liposome was determined using a colorimetric HABA dye displacement assay according to the protocol recommended by Pierce Inc.16 The procedure was described briefly here. HABA (4′-hydroxyazobenzene-2-carboxylic acid) and avidin were obtained from Pierce Inc. The phosphatebuffered saline (PBS) (100 mM sodium phosphate, 150 mM sodium chloride, pH 7.2) was prepared in the laboratory. A 24.2 mg portion of HABA was added to 9.9 mL of ultrapure water, and then 0.1 mL of 1 N NaOH was added. If the HABA did not completely dissolve, another 0.1 mL of 1 N NaOH solution was added, and the solution was filtered and stored at 4 °C before use. Ten milligrams of avidin and 600 µL of the HABA solution were added to 19.4 mL of PBS. The solution can be stored at 4 °C for up to 2 weeks. A 900 µL portion of HABA/ avidin solution was added into a 1 mL cuvette, and A500,initial was measured. According to manufacturer’s recommendation, A500,initial of the solution should be 0.9-1.3. A 100 µL portion of liposome solution containing biotinylated lipid was then added to HABA/avidin solution, and the solution was stirred thoroughly. UV-vis absorbance of this mixture (A500,final) was measured. The calculations of biotin concentration are based on the Beer-Lambert law: A500 ) ε500bc, where A500 is the absorbance of the sample at 500 nm, ε is the extinction coefficient of HABA/avidin complex (at 500 nm and pH ) 7.0) with a value of 34000 M-1 cm-1,16 b is the cell path length expressed in centimeters (cm), and c is the concentration of the HABA expressed in molarity. The concentrations of biotin lipid in liposome A and liposome B (were determined) before and after photopolymerization were determined using HABA/avidin analysis. Spectroscopy Measurements. The synthesized liposomes were incubated with concentrations ranging from 1 to 200 µg/ mL streptavidin in PBS buffer (pH ) 7.4) solution at room temperature. UV absorption spectra of all of the samples were recorded with a Perkin-Elmer Lambda 25 UV-vis spectrophotometer (scanning speed 480 nm/min). The emission spectra were measured with a Photon Technology International spectrofluorometer (with scanning speed 300 nm/min). For all emission spectra, the excitation wavelengths of PDA and SR-101 were 470 and 560 nm, respectively. The slit widths (both excitation and emission) were 6 nm for all the experiments. The spectral overlap integral, J, defined as in eq 117

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J)

∫0∞ J(λ) dλ ) ∫0∞ PLD-corr(λ)εA(λ)λ4 dλ

(1)

which is a quantitative measure of the donor-acceptor spectral overlap over all wavelengths (λ), where PLD-corr represents the donor emission (normalized dimensionless spectrum) and εA represents the extinction coefficient of acceptor which was calculated from the absorption spectrum of the acceptor after scattering background correction. The scattering from the absorption spectra was corrected by subtracting the absorption of control samples (nonpolymerized samples incubated with same amount of streptavidin) from absorption of the liposome A (or B, C, or D) series. Thus, the only difference between control samples and liposome A (or B, C, or D) was that liposomes A (or B, C, or D) were polymerized for 1-2 min at 254 nm light and the control samples were unpolymerized. Results and Discussion In this paper, we investigate the effect of molecular interactions between biotin receptors and transducing elements (conjugated PDA backbone) on the sensor response. PDA has been used as selective sensors for ions, peptides, proteins, and nucleic acids.6,7 For example, a color change from blue to red was observed after an externally applied stress on the PDA backbone.5 This stress presumably originates from the interactions between a receptor attached to PDA and ligands in solution which results in changes of effective conjugation length marked by color change of the liposome solution. We demonstrate in this report that FRET efficiency between donor and acceptor is dependent upon the mode of linkage between biotin and PDA backbone. We also show that the FRET sensor response for biotin which was covalently bound to conjugated PDA chain was much larger than that which was noncovalently inserted in liposomes. In general, the rate of FRET from donor to acceptor is given by eq 218

kET ) kκ2QJ/r6

(2)

where k is a constant, κ2 is the orientation factor, Q is the emission quantum yield of donor, J is the spectral overlap function, and r is donor-acceptor distance. The change in FRET efficiency (E) in the present studies was due to changes in the spectraloverlap(J),andinturnFRET(eq2)afterstreptavidin-biotin interactions. To study this, we prepared PDA liposomes that contained SR-101 (donor), PDA (acceptor), and biotin (receptor). We chose a biotin-streptavidin system as a sensing model to test our liposome-based FRET sensor, because the interactions between streptavidin and biotin are regarded as strongest noncovalent bonds found in a biological system (dissociation constant, Kd ∼ 4 × 10-14 M-1).12 Biotin-streptavidin chemistry is well-studied in literature, and it has been used for many biomedical applications because of their extremely strong interactions and commercial availability of many biotin- and streptavidin-tagged probes.12b Figure 2 shows the chemical structures of different molecules used in this study. 4, 5, and 6 are expected to be noncovalently inserted into the bilayer even though 5 and 6 contains double bonds. This is because the polymerization of diacetylene requires special monomer packing conditions such that the distance between two diacetylene units should be 4.9 Å and a molecular orientation angle φ ∼ 45° (Figure 1S in Supporting Information). 3 is expected to be covalently bound to the conjugated PDA backbone after self-

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Figure 3. The changes in the spectral overlap (J) between SR-101 (donor) emission and PDA (acceptor) absorption after addition of different concentration of streptaVidin to biotin-containing liposome solutions.

Figure 4. The changes in J values of liposome system with covalent and noncovalent bonded biotin. ∆J was calculated by the equation J ) (J - J0)/J0 × 100%, where J0 and J are the donor-acceptor spectral overlap of sample before and after addition of streptaVidin, respectively. Here we plotted “-∆J” since ∆J is a negative value because J values were decreased after addition of streptavidin.

assembly and photopolymerization of diacetylene functionality in the bilayers. Effect of Addition of Streptavidin to Biotin-Tagged Liposome Solution on J Values. Figure 3 shows SR-101 (donor) emission and PDA absorption with increasing amount of streptavidin to a liposome solution composed with 15 mol % of 3. There was decrease in the PDA absorption peaks at 640 and 590 nm and an increase in the absorbance of 540 nm peak after an addition of streptavidin to liposome solution. This is attributed to the transfer of stress following binding of streptavidin to biotin to a more stable “red-PDA” form that had a lower effective conjugation length. We also observed similar electronic absorbance changes with the addition of streptavidin to noncovalently bound biotin liposomes, as shown in Figure 2S (Supporting Information). There was a decrease in the J values with the addition of streptavidin to the biotin-containing liposome solution as shown in Figure 3. The covalently bound biotin liposomes (liposomes A) showed a much larger decrease in J values (-∆J∼18%) compared to noncovalently inserted (NC) biotin samples (∼4%) following the addition of 150 µg/ mL streptavidin to liposome solution, as showed in Figure 4. (J was calculated using eq 117). ∆J was calculated by the equation:

∆J )

(J - J0) × 100% J0

where J0 and J are the donor-acceptor spectral overlap of samples before and after addition of streptaVidin, respectively.

Li and Kohli Here we compared “-∆J” since ∆J is a negative value because J values were decreased after addition of streptavidin to the solution. We consistently observed lower -∆J for the liposome B (or C, D) solution synthesized with varying amounts of NC lipids 4, 5, or 6) as compared to liposome A solution. These observations indicate that the stress transfer to PDA backbone in A solution was much larger than that in B solution. Larger -∆J also resulted in larger changes in FRET efficiency for A than B which was the basis of sensing in our studies (see below). Now, we consider why covalently functionalization of liposomes with biotin showed significantly higher response for streptavidin detection than those composed with NC-tagged biotin. The transport of stress from biotin-streptavidin interaction sites to PDA conjugated chain primarily depends upon interlipid interactions within bilayers, lateral lipid packing, and mode of bonding between biotin and PDA chains. The lipid packing in membranes, in general, depends upon many factors including the degree of unsaturation of lipids, the number and position of double bonds in the lipids, the length of lipids, etc.19 For example, the saturated lipids with alkyl portion pack more closely and will be more stabilized due to van der Waals interactions with their neighbors in the bilayers than those for unsaturated lipids.19 It can be envisioned that the stress transport efficiency from stress-generating points to PDA conjugated chains can be lowered in liposomes composed with unsaturated lipids and noncovalently bound receptors (with 5 or 6) with liposomes because the induced stress can be dissipated easily through vibrations and rotations of bonds, lateral diffusion, trans-cis transformations, and/or a combination of these and any other mechanisms that can aid in the damping of stress in bilayers. We also note that the FRET sensor response is similar for liposomes synthesized with different NCs (4, 5, and 6) even though the chemical structures of the biotin-tagged lipids used in our studies were very different. 4, 5, and 6 contained none, one, and four carbon double bonds, respectively, in them. These experiments suggest that the contribution from covalently bound receptors to the stress transport efficiency was a dominate factor for sensing response and that the stress efficiency decreased drastically for NC liposomes irrespective of degree of unsaturation present in the biotin-lipids. Finally, at this point, it is not clear if biotin-containing nonpolymerizable lipids can form domains within bilayers of the liposomes. This domain formation can affect stress-transport efficiency and FRET response in our system. Biotin Concentration in the Liposome Solution and the Stability of Liposomes. To accurately gauge FRET sensor response, the biotin concentration in different liposome solutions must be same. This is because the biotin concentration in the liposomes has a significant impact on changes in J and FRET efficiency (E) after its reaction with streptavidin. We measured the biotin concentration in liposome solutions using a HABA (chromophore) displacement assay in which biotin displaces HABA from avidin because of its higher affinity for avidin than that of HABA for avidin. HABA displacement from avidin binding resulted in changes in HABA UV-vis electronic absorbance.16 The changes in HABA absorbance at 500 nm (A500) is proportional to the number of displaced HABA and, hence, to biotin concentration in the solution.16 The biotin concentration present in liposome solution was evaluated in a single cuvette by measuring the absorbance of the HABA-avidin solution before and after addition of the biotin-containing liposome solution. As shown in Figure 3S (Supporting Information), the measured biotin concentration in both liposome systems (A and

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Figure 5. Normalized emission response of SR-101 for biotin functional PDA liposome solutions upon addition of streptavidin: (A) liposome solution (A series) composed of 3, 2, 1, and 7 monomers in the ratio of 150:1:1000:160; (B) liposome solution (B series) composed of 4, 2, 1, and 7 monomers in the ratio of 150:1:1000:160; (C) liposome solution (C series) composed of 5, 2, 1, and 7 monomers in the ratio of 150:1:1000:160; (D) liposome solution (D series) composed of 6, 2, 1, and 7 monomers in the ratio of 150:1:1000:160. “1” in the legends means polymerized PDA liposomes in buffer solution. A different concentration of streptavidin was added to the liposomes in buffer solution (“1”) and incubated for half an hour. (E) The relative ratio of SR-101 emission for biotin-functionalized PDA liposome solution with noncovalent binding receptor 4 (red dots) or with covalently attached biotin receptor 3 (blue dots) upon addition of streptavidin. For all emission spectra, the excitation wavelength was 560 nm, which is the excitation wavelength of SR-101.The emission was normalized to the highest peak as 1.

B) were ∼15 mol % of the total monomer solution. The measured biotin concentration values were in agreement with the biotin concentration added to the solution for liposome preparation. These experiments clearly indicated that the selfassembling of lipids with different biotin-containing lipids was efficient. These experiments also demonstrated that the effect of headgroup size on the orientation of diacetylene functional groups and on photopolymerization was not significant. This is probably due to the long linker that joined biotin and the hydrophobic part of the bilayer which provided sufficient mobility without affecting self-assembly and packing of the lipids in the bilayers. The mechanical stability of biotinylated liposomes A and B was investigated by using dialysis experiments before and after the addition of 5% (v/v) Triton X-100 to solutions. The addition of surfactant such as Triton X-100 is known to destroy the bilayer structure of liposomes.20a The lipophilic species also can be removed from polymerized PDA liposomes through simple collisional exchange with unpolymerized liposomes.20b Using a membrane (molecular weight cutoff 12000 Da) against deionized water, our dialysis liposome solutions were analyzed by using HABA-avidin assay. The results indicated that >95%

of 3 and 4 remained incorporated in the liposomes after photopolymerization while before Triton X-100 addition (Figure 3S, Supporting Information), and it was also observed that the photodegradation of 2 was minimal (