Article pubs.acs.org/Langmuir
Targeted Binding of the M13 Bacteriophage to Thiamethoxam Organic Crystals Whirang Cho,† Jeffrey D. Fowler,‡ and Eric M. Furst*,† †
Department of Chemical and Biomolecular Engineering and Center for Molecular and Engineering Thermodynamics, University of Delaware, 150 Academy Street, Newark, Delaware 19716, United States ‡ Syngenta Crop Protection, 410 Swing Road, Greensboro, North Carolina 27409, United States S Supporting Information *
ABSTRACT: Phage display screening with a combinatorial library was used to identify M13-type bacteriophages that express peptides with selective binding to organic crystals of thiamethoxam. The six most strongly binding phages exhibit at least 1000 times the binding affinity of wild-type M13 and express heptapeptide sequences that are rich in hydrophobic, hydrogen-bonding amino acids and proline. Among the peptide sequences identified, M13 displaying the pIII domain heptapeptide ASTLPKA exhibits the strongest binding to thiamethoxam in competitive binding assays. Electron and confocal microscopy confirm the specific binding affinity of ASTLPKA to thiamethoxam. Using atomic force microscope (AFM) probes functionalized with ASTLPKA expressing phage, we found that the average adhesion force between the bacteriophage and a thiamethoxam surface is 1.47 ± 0.80 nN whereas the adhesion force of wild-type M13KE phage is 0.18 ± 0.07 nN. Such a strongly binding bacteriophage could be used to modify the surface chemistry of thiamethoxam crystals and other organic solids with a high degree of specificity.
1. INTRODUCTION Thiamethoxam (TMX) is an organic solid. This neonicotinoid (insecticide) compound has a relatively high water solubility, 4 g/L at 25 °C, and is therefore prone to problems with crystal growth and colloidal stability, including the formation of large single crystals and fused agglomerates. These in turn contribute to the sedimentation of TMX colloidal suspensions and unsatisfactory performance in agricultural applications. Maintaining the colloidal stability of TMX crystals is essential to achieving a desirable shelf life and good performance. Typically, TMX is dispersed using combinations of conventional surfactants with molecular weights of up to around 1 kDa and polymeric dispersants with molecular weights of several tens of kilodaltons; however, environmental considerations and the difficulty of optimizing these complex product formulations motivate alternative strategies for controlling the interfacial properties of these organic solids. © 2012 American Chemical Society
Inspired by their highly specific molecular recognition capacities in biology, peptides, proteins, and nucleic acids have received significant attention in recent years in materials chemistry and engineering.1−6 In the search for highly specific biomimetic interactions, phage display technology7−9 has been used because of its ability to identify binding peptide motifs rapidly for a given target material, including both organic10−12 and inorganic compounds,13−21 cell receptors,22−24 and organic compounds in solution.25 Bacteriophages are viruses consisting of capsid proteins that enclose a copy of their genome.26 In particular, the filamentous M13 bacteriophage, which infects Escherichia coli has been used as a platform for presenting and selecting peptide sequences against binding targets. M13 is Received: February 3, 2012 Revised: March 8, 2012 Published: March 9, 2012 6013
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880 nm in length and 6.6 nm in diameter and has five coat proteins: pVIII, pIII, pVI, pVII, and pIX. Of these five proteins, pIII is the longest. Its sequence consists of 406 amino acids (except a leader sequence) attached to the virus particle by pVI. The amino terminus of pIII proteins protrudes away from the phage surface, where foreign polypeptides can be inserted. Although M13 has found widespread use for directing the assembly of inorganic nanoparticles,13−21 its use as a surface modifier remains relatively unstudied. Previously, Fraden and co-workers reported the pair interaction of colloidal stars formed by grafting biotinylated M13 viruses to a streptavidin-coated polystyrene latex.27 They attributed the excess repulsive potential between the virus-grafted colloids to the rodlike excluded volume of M13 viruses, suggesting that such viruses could be used to control colloidal stability and dispersion when those viruses are irreversibly and nonspecifically grafted to polymeric colloids. With this potential in mind, the goals of this study were to identify the M13 bacteriophage that binds selectively to organic crystalline surfaces and to demonstrate that they alter the colloidal properties of organic crystalline particles. In this study, combinatorial phage display libraries with randomized 7-mer peptides displayed on M13 from the N-terminus of the pIII capsid proteins are used to identify peptide sequences with selective and specific binding affinities to TMX organic crystals. Starting with randomized DNA sequences in the phage genome locations coding for end protein pIII, a diverse library of peptides (up to ∼1011 random sequences) can be displayed on the outer M13 bacteriophage.28−30 From the initial library, we identified six phages that exhibit selective TMX binding using titer count analysis, fluorescence, and atomic force microscopy (AFM). This new, straightforward method of utilizing genetically engineered viruses to identify strongly binding peptides could be used as a targeted dispersant material to enhance the colloidal stability or other applications that benefit from targeted tailoring of the surface chemistry of solids.
Figure 1. Schematic illustration of the phage display method used to screen the selective TMX binding phage. were eluted from the TMX surface by incubating with 100 μL of 0.2 M glycine-HCl (pH 2.2) and 1 M BSA solution, which was then neutralized with 15 μL of 1 M Tris base (pH 9.0). The eluted phage was amplified with 20 mL of a 1:100 diluted overnight culture of E. coli (ER 2738) that was grown in LB media at 37 °C for 4.5 h and purified through poly(ethylene glycol) precipitation. These panning procedures were repeated three times using an increasing tween-20 concentration in each round (0.3, 0.5% v/v) to increase the stringency of binding to the TMX target. The final eluted phage solutions were serially diluted and quantified by plating on lawns of E. coli (ER 2738) grown on agar containing 5-bromo-4-chloro-3indolyl-β-D-galactopyranoside (X-Gal) and isopropylβ-D-thiogalactopyranoside (IPTG) (LB/IPTG/X-Gal plates), which show blue plaques after incubation at 37 °C overnight. Individual blue plaques were selected and separately amplified in 1 mL of a 1:100 diluted overnight culture of E. coli in LB media at 37 °C for 4.5 h. From these amplified phage stocks, phages were separately precipitated and the single-stranded DNA was isolated by adding 100 μL of an iodide buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 4 M NaI) and 250 μL of ethanol. Finally, a phage sequencing analysis was performed to identify the pIII heptapeptide sequences that bind strongly to TMX (Genewiz, South Plainfield, NJ). We determined the strongest and most selective TMX-binding phages using both single-phage binding assays and competitive binding assays. For single-phage TMX-binding assays,31 the six strongest TMX-binding phage sequences were individually screened against TMX. A single round of phage screening was separately performed against 200 μL of TMX in TBS buffer using each 100 μL of phage containing different peptide inserts. The bound phages were eluted from TMX by 0.2 M glycine-HCl (pH 2.2) and a 1 M BSA solution, which was then neutralized with 1 M Tris base (pH 9.0). The bound phages were quantified by the titration method described above. We evaluated the ratio of the amount of phage after one washing step to the amount of phage initially introduced. For competitive binding assays,31 selected phages were individually picked, separately amplified, and diluted into a single mini-library at a concentration of 4 × 1012 pfu/mL. A single binding step was performed against the TMX in TBS solution. Here, all of the TMX-binding phages were simultaneously
2. EXPERIMENTAL SECTION 2.1. Materials. A commercial phage library (Ph.D.-7, New England Biolabs, Ipswich, MA) was used in these studies. Other reagents, including poly(ethylene glycol) (Mw = 8000 g/mol), Tris-HCl, glycineHCl (Trizma hydrochloride, T6666), NaCl (BioReagent ≥99.5%, S5886), sodium iodide (ACS reagent ≥99.5%, 383112), tween-20 (P9416), and ethanol (ACS reagent ≥99.5%, 459844) were obtained from Sigma-Aldrich and used as received (St. Louis, MO). 5-Bromo-4chloro-3indolyl-β-D-galactopyranoside (X-gal, AC327241000) was purchased from Acros Organics (Morris Plains, NJ). Isopropyl-β-Dthiogalactopyranoside (IPTG, BP1755) and bovine serum albumin (BSA, BP1600) were purchased from Fisher Scientific (Pittsburgh, PA). TMX air-milled to less than 10 μm was provided by Syngenta Crop Protection (Greensboro, NC). 2.2. Phage Display Screening for the Selection of TMXBinding Peptide Motifs. Panning using the phage library was performed against TMX. The initial phage library solution was diluted by adding 10 μL of the library (∼1 × 1013 plaque-forming units/mL or pfu/mL) to 1 mL of 0.1% TBST buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% v/v tween-20). TMX was treated with a blocking solution (0.1 M NaHCO3 pH 8.6, 5 mg/mL BSA) and washed several times with 0.1% TBST and dispersed into 200 μL of TBS to a final concentration of 10 mg/mL. One hundred microliters of the diluted phage library was then introduced into the TMX suspension and incubated for 1 h with gentle rocking. The sample was washed several times with TMX-saturated 0.1% TBST buffer and transferred to new centrifuge tubes. This step removed nonspecific binding phage or any phage with a strong affinity to the polypropylene centrifuge tubes. Next, to measure the amount of phage adsorbed to the TMX, phage 6014
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Figure 2. Phage display screening results. (a) Amino acid sequences from the third round of phage display screening with plaque titer abundance. (b) Compositional distribution of the six most abundant peptide sequences displayed from pIII capsid proteins of the TMX-binding M13 bacteriophage. The inset shows the total amino acid frequency among the six phages. exposed to the TMX solution. After this competitive binding, the remaining strongly bound phages were eluted and quantified using titration. A total of 23 plaques were randomly picked and sequenced to identify the most strongly binding peptide sequences. 2.3. Characterization of TMX−Phage Complex. Atomic force microscopy (AFM) images of the M13 bacteriophage on a mica substrate were obtained using multimode AFM in tapping mode under ambient conditions (Nanoscope III, Bruker Inc., Santa Barbara, CA). First, an M13 bacteriophage solution was placed onto a cleaved mica substrate. After being incubated for 30 min, the mica surface was washed with distilled water, air dried, and then analyzed by AFM. Height, amplitude, and phase signals were simultaneously recorded. The morphology of the M13 bacteriophage on the mica substrate was imaged using the tapping mode at a 1.5 Hz scan rate. AFM microscopy
(Bruker Inc., Bioscope II mounted with a Zeiss Axiovert 200 inverted light microscope) was also performed to measure the unbinding forces between the M13 bacteriophage and TMX. To attach the M13 bacteriophage, the silicon nitride AFM tips (DNP-10, Bruker Inc., Santa Barbara, CA) were first cleaned with piranha solution (70% H2SO4/ 30% H2O2 v/v). Following washing, a silanizing agent, 4% 3-aminopropyltriethoxysilane (APTES, 440140, Sigma-Aldrich) in ethanol was used to attach amines covalently to the tip by incubation for 1 h at room temperature. Silanized tips were subsequently treated with 2.5% glutaraldehyde (GA, Ted Pella) for 10 min to facilitate phage immobilization. The surface was then rinsed with pure deionized water and incubated with the M13 bacteriophage for 1 h. The TMX sample was affixed to a glass substrate using an epoxy resin. The prepared samples were mounted in the AFM liquid flow cell. Experiments were 6015
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performed in TBS buffer saturated in TMX at room temperature. A Bruker tip with a 0.12 N/m spring constant was used to measure the interaction between the M13 bacteriophage and TMX. The measurements were performed at a 2.06 μm/s retraction rate. Force−distance curves were collected at three randomly chosen points on the TMX surface. At least 1000 force measurements were made at each location. The entire experimental procedure was repeated and yielded consistent results. We characterized the TMX-M13 bacteriophage complex with electron microscopy. To image the bound phage, samples suspended in TBS solution were applied to a poly-L-lysine-coated glass coverslip and then completely air dried in a fume hood for approximately 2 to 3 h. To prevent electron charging, Au was sputtered on the sample. Fieldemission scanning electron microscopy (FE-SEM) images were taken using a scanning electron microscope (Hitachi 4700 FE-SEM) at a 5 kV accelerating voltage. For transmission electron microscopy (TEM), a drop of the TMX-M13 complex suspension was added to poly-L-lysine-coated 200 mesh Formvar-carbon-coated copper grids. The grids were rinsed three times with water and then negatively stained with 0.5% aqueous uranyl acetate. TEM images of the TMX− phage complex were obtained with a transmission electron microscope (Zeiss LIBRA 120) at a 120 kV accelerating voltage. The TMX-M13 complex was characterized with confocal laser scanning microscopy (5 live duo, Zeiss) using the 488 nm vacuum wavelength emission line of an argon ion laser. To confirm that the TMX surfaces were bound to the M13 bacteriophage, TMX-M13 complexes were incubated for 1 h with Alexa Fluor 488-tagged anti M13 monoclonal antibody and centrifuged for 30 min to remove free dye molecules. The fluorescent image was taken using emission between 505 and 550 nm. We measured the surface charge of TMX by characterizing the zeta potential (ZetaPALS, Brookehaven Instruments). For each sample measured, 10 mg of TMX was suspended in 1 mL of Trisbuffered saline (TBS) solution at pH 7.5.
ent facets and defects, and hence nonconserved sequences are expected. Nonetheless, there is a significant overabundance of proline, and using the results of Rodi et al.32 to estimate the likelihood of finding proline in any single clone within this phage library, we calculate that the probability of the observed proline frequency within the 44 plaques is approximately 1 in 580, suggesting that proline is a significant factor in binding. We assigned six peptide sequences found in more than one plaque as candidates for highly specific and strongly binding peptide sequences for TMX: His-Thr-Pro-Pro-Val-Thr-Ser (HTPPVTS), Ala-Ser-Thr-Leu-Pro-Lys-Ala (ASTLPKA), GlnPro-Gln-Val-Pro-Asp-Ala (QPQVPDA), Ala-Leu-Thr-Pro-ThrPro-Pro (ALTPTPP), Ile-Leu-Gly-Val-Gly-Leu-Pro (ILGVGLP), and Ser-Ile-Leu-Pro-Tyr-Pro-Tyr (SILPYPY). From here on, we refer to the phage identified through the panning protocol by its pIII heptapeptide sequence. The amino acid distribution of these six peptides is shown in Figure 2b. There is a strong presence of hydrophobic amino acids (>50%, A, V, L, and I) and hydroxyl-containing amino acids (S, T, and Y). This suggests that the binding between the TMX and peptide binding motif occurs via hydrophobic and some hydrogen bonding interactions. Previous work has shown that the SILPYPY peptide binds to hydrophobic octyltrimethoxysilane molecular inks.33 Interestingly, all six peptides contain proline (P) residues adjacent to hydrophobic amino acids (A, V, L, and I) with the exception of one peptide sequence (ALTPTPP). Although the measured ζ potential of TMX dispersed in TBS Table 1. Results of Binding to TMX with Subsequent Washing Stepsa
3. RESULTS AND DISCUSSION 3.1. Identification of the TMX Binding Phage. The combinatorial library of random peptides, which displays a linear heptapeptide at the pIII protein of the M13 bacteriophage, provides approximately 1011 different peptide sequences that were screened against TMX crystals. Figure 1 schematically depicts a phage display selection for the selective binding phage with TMX. The amino acid sequences of each heptapeptide are summarized in Figure 2a. From a total of 44 plaques, 33 different sequences were found. This demonstrates that it is unlikely to identify a single consensus peptide sequence of the pIII coat proteins because of the complex surface structure of TMX. Organic crystals are not homogeneous; they have differ-
phage sequence
input phage, a (pfu/mL) × 1012
bound phage, b (pfu/mL) × 109
binding ratio, c
relative degree of binding efficiency
ASTLPKA ALTPTPP ILGVGLP SILPYPY HTPPVTS QPQVPDA M13KE
7 17 12 10 4 19 5
13 24 8.8 6 1.8 6.6 1.3 × 10−3
0.18 0.14 0.07 0.06 0.045 0.039 2.6 × 10−5
6900 5400 2300 2300 1700 1500 1
a
The binding ratio represents the ratio of the amount of phage present after one round of panning to the amount of phage initially introduced (c = b/a).
Figure 3. Binding comparison showing the (a) relative binding affinity against TMX of different heptapeptides displayed by the M13 bacteriophage and (b) competitive binding assay results that show that the strongest TMX-binding phage is ASTLPKA. 6016
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Figure 5. Confocal fluorescence microscopy of an ASTLPKA bacteriophage bound to TMX crystals under wet conditions. The samples are dyed using a fluorescently tagged anti-M13 monoclonal antibody. (a, b) Confocal fluorescence and transmission optical microscopy of the TMX-binding phage containing heptapeptide (ASTLPKA) bound to TMX. (c, d) Control experiments that were not preincubated with phage before incubating with the antibody.
competitive binding of all six phages.31 In the first assay, the relative binding affinity of each phage was evaluated on the basis of the ratio of the initial number of phages introduced to the number retained after a washing step (Table 1). The ratio of the initial and retained phage concentrations is the binding ratio. As presented in Figure 3a, each subset of the TMXbinding phage selected in the panning experiment binds to TMX with greater selectivity than does the wild-type M13KE phage, which shows a negligible binding affinity with TMX. The binding ratio for M13KE is on the order of 10−5. When normalized by this value, the selected phage exhibits binding that is 1000−6900 times stronger than that of the wild-type phage. Competitive assays identified the sequence with the strongest binding affinity to TMX. The six TMX-binding phages were separately amplified and diluted to a concentration of 4 × 1012 pfu/mL to create a mini-library. After a single binding step, the bound phages were eluted with acidic buffer, amplified, and titered on LB/IPTG/Xgal plates. Figure 3b shows that the ASTLPKA phage has the strongest binding affinity to TMX on the basis of its high abundance (∼50%) among 23 randomly chosen plaques. Note that HTPPVTS, which had the highest frequency in our initial screening, does not show the highest specificity or strength, which suggests that HTPPVTS readily tends to amplify with E. coli (ER2738).8 3.2. Microscopy and Zeta Potential of the Phage− TMX Complex. The binding of the ASTLPKA phage to TMX is confirmed by FE-SEM and TEM imaging. Amplified phages displaying the ASTLPKA peptide were incubated with a dispersion of TMX and then washed to remove nonspecifically bound phages. Figure 4a shows an FE-SEM image of an ASTLPKA bacteriophage binding to the surface of TMX. Figure 4b shows a phage on the TMX surface at a higher magnification. The
Figure 4. (a, b) Field-emission scanning electron microscopy (FE-SEM) and (c) transmission electron microscopy (TEM) images of an ASTLPKA bacteriophage attached to TMX crystal surfaces.
buffer is −3.5 ± 0.7 mV under the panning conditions (pH 7.5), there are relatively few positively charged amino acids represented in the peptide sequences (only histidine, H, and lysine, K), implying that electrostatic interactions are relatively unimportant for phage binding. Next, we characterized the six phages described above using two TMX-binding assays: the first assay protocol measured the affinity of a single phage. The second assay measured the 6017
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Figure 6. Measurement of the unbinding force between the M13 bacteriophage and the TMX immobilized onto the AFM tip and glass substrate, respectively. Histograms of unbinding forces obtained by (a) the M13 bacteriophage containing heptapeptide (ASTLPKA) and (b) M13KE against TMX. (Inset) representative force−distance curves (retraction) of the phage with the TMX surface.
phages have a filamentous shape that is around 880 nm long and a diameter of 6.6 nm corroborated by the reported dimensions of M13.34 The phages appear to be lying on the surface, but this may be an artifact of the SEM sample preparation. Because we expect the washing steps to reduce or possibly eliminate weaker nonspecific binding, for instance, via the pVIII phage proteins,35 the phages likely anchored by their tips then come into contact during sample drying. Evidence for binding at the tip is presented in Figure 4c, which shows a TEM image of the bound phage. The image clearly shows the extension of the phage from the TMX surface, creating an open, brushlike layer on the crystal. Imaging by confocal microscopy after fluorescence labeling provides access to samples under hydrated conditions in which the virus particles remain functional and intact. The fluorescence confocal image in Figure 5a shows phage -coated TMX incubated with Alexa Fluor 488-tagged anti-M13 monoclonal antibodies. After incubation, the samples were washed to eliminate nonspecifically bound anti-M13 monoclonal antibodies. Considerable fluorescence emanates from the surfaces of the ASTLPKA bacteriophage−TMX complex but not from the
control (Figure 5c) in which TMX was incubated with the antiM13 antibody. Lastly, the zeta potential of TMX crystals in the presence of the bacteriophage provides additional evidence for binding. The surface of the M13 bacteriophage has an acidic pI of close to 4.218,36,37 and is thus negatively charged at pH 7.5 The ζ potentials of the phage itself and the TMX-ASTLPKA peptide complex are −12.4 and −6.1 ± 2.3 mV, respectively (Supporting Information, Figure S1). The overall ζ potential of the TMX-phage complex is more than that of the TMX itself, which can be attributed to an increase in the negative charge by the presence of associated phages on the TMX. In all, electron and confocal microscopy confirm that the M13 bacteriophage can be selected on the basis of the pIII peptide sequence used to bind to TMX crystal surfaces. The zeta potential measurements confirm that the surface chemistry is modified by these bound phage particles. 3.3. Adhesion Force of Bound Phage. AFM was used to investigate the interaction forces between the M13 bacteriophage and the TMX. Force curves are obtained by monitoring the cantilever deflection (d) as a function of the vertical displacement of the piezoelectric scanner.34 The unbinding force 6018
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nize the organic crystal surfaces of TMX. In general, such specific binding of M13 to solid surfaces could lead to the targeted manipulation of surface chemistry in colloidal suspensions in potentially complex mixtures. Applications of this phenomenon include tailoring their interactions, including using the phage as a specific steric stabilizer. In addition, binding experiments showed that the charge density of the crystals could be altered.
between the M13 bacteriophage and the TMX was measured by obtaining force−distance curves under a constant loading rate of 247.2 pN/s ( f = ksvc), where ks is the effective spring constant (ks = 0.12 N/m) and vc is the retraction velocity (vc = 2.06 μm/s).38,39 The cantilever deflection was measured as it approached and retracted from the TMX surface at least 1000 individual times at each of three randomly chosen positions on the crystal surface. The difference between these two curves can be attributed to the interaction between the TMX and the phage-coated AFM tip. The maximum adhesion force is thus known as the unbinding force and appears as a discontinuous jump out of contact (representative force−displacement curves are shown in the insets of Figure 6).40 In the context of binding specificity and strength, we compared the adhesion force between the ASTLPKA bacteriophage and wild-type M13KE. As shown in Figure 6, histograms of binding dissociation forces were tabulated from the retraction portion of individual force−displacement curves. The most probable unbinding force is determined by fitting a Gaussian to the histogram of the force distribution, and the resultant median value of the Gaussian curve is taken as the most probable unbinding force (Fm).41 To resolve and accurately fit each distribution, the histograms were fitted with two Gaussian funtions.42 The solid black curves in each image show the estimated mixture model. In the case of the TMX-binding phage displaying the ASTLPKA heptapeptide, a major Gaussian (Fm = 1.47 ± 0.80 nN) and a minor Gaussian (Fm = 8.00 ± 5.63 nN) were observed at frequencies of 82 and 18% of the measurements, respectively (Figure 6a). We assigned the mean force corresponding to the dominant interaction (major Gaussian in the force histogram) as the most probable adhesion force. In contrast, the unbinding events of Gaussian curves corresponding to wild-type M13 were observed at frequencies of 91 and 9%, of which the mean unbinding forces are 0.18 ± 0.07 and 0.50 ± 0.31 nN, respectively (Figure 6b). The maximum in the unbinding force distribution of the TMX-binding phage expressing the ASTLPKA heptapeptide from the TMX surface exhibits approximately 8-fold statistically higher values compared to wild-type M13KE. In addition, we performed control experiments in the absence of the TMX-binding phage (i.e., using a bare AFM tip modified with only 3-amino-propyltriethoxysilane (APTES) and glutaraldehyde (GA)) on the TMX substrate to ensure that the binding forces correspond to the specific phage and the TMX interaction, not to other nonspecific sources.10,43−45 In the absence of phage immobilization, force curves showed almost no hysteresis between the approach and retraction traces under identical experimental conditions. Furthermore, the adhesion force shows statistically significantly different values (p ≈ 0) of the unbinding force, indicating that the interaction between the controls and TMX is negligible (Supporting Information, Figure S3). These results confirm that the M13 bacteriophage expressing the ASTLPKA heptapeptide binds to TMX with significant strength. 3.4. Phage Amplification. As a final note, the potential applications of bacteriophages in surface and colloid chemistry are limited by the cost effectiveness of producing the virus. Notably, in our studies, we found that the yield of M13 from E. coli is quite high, with biomass conversion to the phage approaching 14.5% of the bacterial culture mass.
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ASSOCIATED CONTENT
S Supporting Information *
Transmission electron microscopy and atomic force microscopy images of the thiamethoxam (TMX)-binding phage displayed with the heptapeptide (ASTLPKA) at the tip end of the pIII proteins. Comparison of zeta potentials of pristine TMX, the M13 bacteriophage, and thiamethoxam (TMX) coated with the M13 bacteriophage expressing the ASTLPKA peptide. Histograms of unbinding force values. t-test results with the phage-expressing heptapeptide and no inserted peptide. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1-302-831-0102. Fax: +1-302-831-1048. E-mail: furst@ udel.edu. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
Financial support for this work by Syngenta Crop Protection is gratefully acknowledged. We thank our colleagues A. S. Robinson, N. George, W.-J. Chung, Z. Britton, and Y. S. Nam for fruitful discussions and E. Adams for assistance with AFM force measurements.
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4. CONCLUSIONS This work identified the M13 bacteriophage and the corresponding peptide-binding motifs that specifically recog6019
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dx.doi.org/10.1021/la300522g | Langmuir 2012, 28, 6013−6020
