ARTICLE

https://doi.org/10.1038/s42004-019-0198-0 OPEN Probing molecular mechanisms of M13 adhesion

Chanoong Lim 1,3, Jina Ko 1,3, Dasom Jeon2, Yoojung Song 1, Jinwoo Park 1, Jungki Ryu 2 & Dong Woog Lee 1 1234567890():,;

M13 can provide a versatile platform for nanobiotechnology because of their unique biological and physicochemical properties. Polypeptides on their surfaces can be finely tuned on demand through genetic engineering, enabling tailored assembly of multiple functional components through specific interactions. Their versatility has been demonstrated by synthesizing various unprecedented hybrid materials for energy storage, biosensing, and catalysis. Here we select a specific type of genetically engineered M13 bacteriophage (DSPH) to investigate the origin of interactions. The interaction forces between the phage-coated surface and five different functionalized self-assembled monolayers are directly measured using a surface forces apparatus. We confirm that the phages have strong adhesion energies in acidic environments due to π-π stacking and hydrophobic interactions, while hydrogen bonding interactions remain relatively weak. These results provide quantitative and qualita- tive information of the molecular interaction mechanisms of DSPH phages, which can be utilized as a database of the bacteriophage interactions.

1 Department of Chemical Engineering, School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), 50 UNIST- gil, Ulsan 44919, Republic of Korea. 2 Department of Energy Engineering, School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), 50 UNIST-gil, Ulsan 44919, Republic of Korea. 3These authors contributed equally: Chanoong Lim, Jina Ko. Correspondence and requests for materials should be addressed to J.R. (email: [email protected]) or to D.W.L. (email: [email protected])

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fi he discovery and identi cation of biomolecular interac- hydroxyl (OH), amine (NH2), methyl (CH3), and phenyl (Ph) tions have improved our understanding of the unique groups. Direct and precise measurement of the force versus dis- T fi functions of biological systems and signi cantly con- tance curves with the SFA under various conditions allow the tributed to the development of nanobiotechnology. Due to the identification of different types of interaction forces between the polyfunctionality of biomolecules1, they often employ unique M13 bacteriophages and functional groups. These provide clues combinations of individual interactions (e.g., hydrogen bonding, to the molecular origin of its CNT-binding ability. Our results steric hydration, electric double layer, van der Waals, cation–π, suggest that histidine and proline moieties play critical roles in and hydrophobic interactions) and, as a result, can form strong the binding of the phages to CNTs and aggregate formation in ensemble interactions with specificmoleculesormatter(so aqueous solutions, respectively. These results are further con- called specific interaction or molecular recognition)2–4.These firmed by the pH-dependent behaviors of the phage complexation specific biomolecular interactions have been successfully with the CNTs, indicating that aggregation and precipitation of employed to develop target-specific drug delivery, bioimaging, the complexes can be tuned by pH. We believe that this study can and biosensing systems5–8. Furthermore, various unprecedented provide a versatile platform to characterize various specific bio- functional materials have been developed using these interac- molecular interactions and enable better understanding and uti- tions9–12. The M13 bacteriophage (or ) is a representative lization of biomolecules. example of a toolkit for such applications13–18.Duetoits inherent nanostructure, abundant polypeptides present on its surface, and modification flexibility through genetic engineering, Results the M13 bacteriophage has been successfully utilized to assemble Experimental setup and contact angle measurements. For SFA and fabricate various functional nanomaterials. Examples analysis (Fig. 1), we prepared a monolayer of M13 bacteriophages include hybrid materials of carbon nanotubes (CNTs) and TiO2 and SAMs with various functional groups. Mica substrates, which for photovoltaics15, CNTs and inorganic materials for are frequently used for SFA analysis because of their atomically 19 fl rechargeable batteries , and porphyrin and IrOx for solar water at nature, were readily coated with DSPH phages after replacing splitting20. surface K+ ions with Mg2+ ions and exposing the substrate to a However, the polyfunctionality of biomolecules also poses a phage solution (see the Methods section for details). Atomic force significant challenge for experimentally exploring the funda- microscopy (AFM) confirmed the monolayer deposition of fila- mental origins of these interactions. The unidentified origins of mentous DSPH phages with uniform lengths and diameters of biomolecular interactions lead to difficulties in rationally 880 and 6.6 nm (Supplementary Fig. 1), as reported previously14. designing new biomolecules with desired binding affinities for Their deposition density was easily controlled by varying the unexplored target molecules/matter. In the case of M13 bacter- concentration of the DSPH solution. For the SFA analysis, the iophages, randomly mutated bacteriophage libraries were number density was controlled to be ~30 per μm2, because this repeatedly exposed to target materials to screen specific biomo- led to the highest coverage (82%) of the mica by the DSPH phages lecular interactions ()21–24. There have also been without the formation of phage multilayers and aggregates, which several attempts to rationally design biomolecules with molecular can hinder the precise measurement of the interaction forces. In recognition capabilities rather than relying on serendipitous the case of the SAMs, each of the end-functionalized alkanethiols findings (e.g., phage display)9. However, most conventional stu- was deposited on a molecularly smooth gold surface (Fig. 1, see dies have relied on empirical design rules because of the lack of the Methods section for details). We prepared SAMs with five proper experimental analysis and our understanding on these different end-functional groups: positively chargeable NH2, biomolecular interactions. negatively chargeable COOH, polar OH, aliphatic and nonpolar In this work, we first report the precise measurement of various CH3, and aromatic and nonpolar Ph groups. interaction forces between the M13 bacteriophage and common Prior to the measurement of the interaction forces, the wetting functional groups using a surface forces apparatus (SFA) to properties of the DSPH and SAMs were investigated as a function understand the origin of its molecular recognition capability. In of the pH and/or waiting time (t) because of their pH-dependent particular, we measure the interaction forces of the M13 virus physicochemical properties (Fig. 2; see the Methods section for fi with CNT-binding polypeptides (DSPH) along its lamentous more details). Considering the pKa values of the tested functional capsid—an sequence of DSPHTELP on pVIII coat groups (Supplementary Table 1) and side-chain functional groups —with various functional groups. Among various types of the DSPH phage (Supplementary Table 2), as well as the of phages, we choose the DSPH phage for the following reasons: isoelectric point (pI) value of the latter (~5.3)15, contact angles (1) it has a specific binding affinity toward CNTs, which can find were measured at pH 3.0 and 8.5, respectively. Figure 2 shows the diverse applications in various fields, such as catalysis13, biome- contact angles of the SAM-coated and DSPH-coated surfaces at dical engineering16, materials science15, and energy conversion these pHs. There was not a significant difference between the and storage19. In addition, the molecular structure of CNTs are contact angles measured at pH 3.0 and 8.5 for most of the tested very similar to that of other carbon materials, such as fullerene, SAMs (Fig. 2a). The Ph-/CH3-SAM and OH-/NH2-SAM graphene, and graphite. Thus, our study can provide valuable remained hydrophobic and hydrophilic, respectively, regardless insights to a broader range of researchers. (2) Because the CNT- of the pHs. On the contrary, the COOH–SAM showed significant binding sequences of the DSPH phage were abundantly displayed differences between the contact angles at pH 3.0 (θ = 44.6 ± 1.3°) fi θ = 25 (~2700 copies) along its lamentous structure, it can provide and 8.5 ( 19.9 ± 1.4°). Due to its pKa value of ~5.5 , it could more reliable measurement for SFA analysis than other types of be deprotonated and become more hydrophilic at pH 8.5. It is fi – phages with the CNT-binding sequences on pIII (~ ve copies). In noteworthy that the NH2 SAM should also be pH sensitive (pKa addition, a high aspect ratio and flexibility of the M13 bacter- ~7.5), but it exhibited a negligible difference in the contact angles iophage make it difficult to study the interaction forces for pIII upon pH changes26. The observed difference in the pH- – – proteins. (3) Practically, the DSPH phage can be more readily dependent wetting properties of the COOH and NH2 SAMs prepared in a large scale compared with other types of phage due can be attributed to the differences in their molecular conforma- fi – to its high ampli cation rate in E. coli. Five different types of tions. According to the literature, the NH2 SAM can form functional groups are prepared using self-assembled monolayers hydrogen bonding (H-bonding) networks with surrounding water (SAMs) bearing different terminal groups: carboxylic (COOH), molecules, regardless of its protonation state27,28. However, the

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a b c Disc HS n Glue layer Gold layer COOH s s s s s s s s s s s s s s s s s s s s OH

NH2 pH 3.0 / pH 8.5 CH3 (–Ph) KNO3 solution

D d e

ssDNA pVI pVII pIX R

pIII pVIII Sequence : DSPHTELP

Fig. 1 Experimental scheme. a Surface forces apparatus setup for measuring interaction forces between the functionalized SAM layer (top surface) and M13 bacteriophage deposited on mica (bottom surface). b Funtionalized SAM layer on gold surface. c Molecular structure of five different alkanethiols for the formation of the functionalized SAM layers. d AFM image of the M13 bacteriophage on mica. Scale bar, 1 µm. e Structure of the M13 bacteriophage

(t = 0s)to θ = 27.1 ± 0.6° (t = 10 min). The contact angle at pH a –COOH –OH –NH –CH –Ph 2 3 3.0 was relatively insensitive to the waiting time, showing a slight 44.6 ± 1.6° 43.1 ± 2.3° 39.6 ± 3.3° 101.0 ± 0.8° 73.8 ± 1.6° decrease from θ = 66.3 ± 1.7° (t = 0s) to θ = 60.4 ± 1.1° (t = pH 3.0 10 min). Considering its pI value (~5.3), the positively charged DSPH phages at pH 3.0 could interact more strongly with the 19.9 ± 1.4° 39.3 ± 1.1° 39.4 ± 2.4° 102.1 ± 0.2° 73.2 ± 2.1° negatively charged mica surface than with water molecules, pH 8.5 resulting in a slightly high contact angle and a negligible change over time. However, when exposed to pH 8.5, significant amounts of negatively charged amino acids (e.g., aspartic and glutamic b M13 bacteriophage (DSPH) deposited surface acids) in the DSPH could reorient and become exposed to the 70 DSPH–water interface, increasing the hydrophilicity with time. Moreover, swelling of the DSPH (which was confirmed by SFA, 60 as presented in a later section) could increase the rotational degrees of freedom, which may have accelerated the rearrange- 50

(°) 31,32

ment process .   40 Interaction force measurements. In addition to evaluating the fi

30 hydrophobicity/hydrophilicity of DSPH phage and the ve dif- ferent functional terminated SAMs, we measured the interaction

Contact angle, 20 forces between the phage and the five different SAMs using SFA (Figs. 3, 4). Force versus distance profiles were measured upon pH 3.0 KNO 10 3 approach and detachment of the DSPH- and SAM-modified pH 8.5 KNO3 surfaces to determine the adhesion force and energy (Wad) under 0 various conditions: pHs of 3.0 and 8.5 and contact times (tc)of5s 0246810and 1 h. Waiting time, t (min) We first measured the interaction forces at pH 3.0. The Fig. 2 The wetting properties of the functionalized SAMs and DSPH. adhesion energy between the COOH–SAM and DSPH increased = −2 = = Contact angle values of (a) functionalized SAMs and (b) DSPH-coated from Wad 0.23 ± 0.02 mJ m at tc 5s to Wad 2.32 ± −2 = – surface as a function of pH and/or waiting time. The error bars represent 0.40 mJ m at tc 1 h (Fig. 3a). The OH SAM increased = −2 = = the s.e.m. (standard error of mean), where n ≥ 7 from Wad 3.08 ± 0.75 mJ m at tc 5s to Wad 5.32 ± −2 = 0.26 mJ m at tc 1 h (Fig. 3b). The similar contact angles of the COOH– (θ = 44.6 ± 1.6°) and OH–SAMs (θ = 43.1 ± 2.3°) at COOH–SAM can form intermolecular H-bonding between end- pH 3.0 suggest that they should have similar magnitudes of functional groups only when they are protonated, leading to the hydration repulsion or hydrophobic attraction. Interestingly, decrease in the hydrophilicity with the pH29,30. despite the expected similarity in the origin of adhesion for both The wettability of the DSPH-coated surface was measured as a SAMs (i.e., H-bonding), the measured adhesion energy of the function of the pH and waiting time (t) after contact with the COOH–SAM was ~30–50% smaller than that of the OH–SAM. solution to determine the rearrangement/reorientation of the coat This quantitative discrepancy is attributed to the differences in proteins of the bacteriophage on the mica surface (Fig. 2b). The the molecular conformation of their terminal functional groups contact angle at pH 8.5 gradually decreased from θ = 59.1 ± 3.8° in aqueous solutions. As explained earlier, the protonated

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a 20 b 20 ) ) –2 2 –2 2 Out ) ) In –1 –1 (mJ m 0 0 (mJ m 0 0 R R  –2  Wad : 0.23 ± 0.02 mJ m (mN m (mN m F/2 Jump out F/2 –2 –2 = = –2 F/R F/R W : 2.32 ± 0.41 mJ m –2 W ad W W : 3.08 ± 0.75 mJ m –20 Contact time, tc –20 ad 5 s –4 –4 –2 1 h Wad : 5.32 ± 0.26 mJ m

–COOH SAM . –6 –OH SAM . . –6 –40 . . . . . –40 . . . . Force/radius, Force/radius, pH 3.0 KNO solution 3 pH 3.0 KNO3 solution DSPH layer ++ ++++ –8 DSPH layer ++ + +++ –8 Interaction energy, –60 Interaction energy, –60 010203040 0 10203040

c 20 d

) 20 ) –2 2 2 –2 ) ) –1 (mJ m –1 0 0 (mJ m R 0 0 R   W : 3.71 ± 0.33 mJ m–2 (mN m (mN m F/2 ad –2 –2 F/2 = –2 = F/R F/R W : 5.54 ± 0.48 mJ m W –20 –20 ad W –2 –4 –4 Wad : 4.30 ± 0.29 mJ m

W : 7.00 ± 0.10 mJ m–2 –6 ad –6 –NH SAM + –CH SAM . . –40 2 + + –40 3 . . + + + . . Force/radius, Force/radius, pH 3.0 KNO solution pH 3.0 KNO3 solution 3 DSPH layer ++++++ –8 DSPH layer + + + + + + –8 Interaction energy, –60 –60 Interaction energy, 010203040 010203040 Separation distance, D (nm) ef20 ) 14 –2 2 pH 3.0, tc = 5 s ) )

–2 12 pH 3.0, tc = 1 h –1 0 0 (mJ m R

 10 (mJ m

(mN m F/2 –2 ad = W 8 F/R –20 W –4 6 –2 Wad : 8.08 ± 0.82 mJ m –Ph SAM . . –6 4 –40 . . . . Force/radius, pH 3.0 KNO3 solution DSPH layer ++++++ 2

–8 Adhesion energy, W : 12.24 ± 0.50 mJ m–2

ad Interaction energy, –60 0 0 10203040 –COOH –OH –NH2 –CH3 –Ph Separation distance, D (nm) Terminal functional group of SAMs

Fig. 3 Force versus distance profiles measured at pH 3.0. The interactions force of the DSPH-coated surface was measured against SAMs with five different terminal functional groups: a –COOH, b –OH, c –NH2, d –CH3, and e –Ph. The empty and solid arrows correspond to the approach and detachment of the two surfaces, respectively. The orange and sky-blue curves correspond to the force-profiles at contact times (tc) of 5 s and 1 h, respectively. f Bar graph showing the overall adhesion energy (Wad) of different SAMs at pH 3.0 as a function of contact time. The error bars represent the s.e.m., where n = 5 in each group

– – carboxylic head group ( COOH) at pH 3.0 can partially form a the NH2 SAM and DSPH, as both are rich in H-bonding donors H-bonding with a neighboring group (–Hwith–O–)oradimer and acceptors. In addition to H-bonding, it was expected that (–Hwith= O)29. This suggests that the DSPH bacteriophages additional interactions would be present because the measured may have fewer chances to form H-bonding with the protonated force versus distance profile indicated that electrostatic repulsion COOH–SAM than with the OH–SAM at pH 3.0, which between them was negligible compared with the adhesion forces. also coincides with a molecular dynamics simulation study by It is well known that protonated primary amines can have cation– Yang et al.33. π interactions as a cationic donor with aromatic moieties, such – To our surprise, despite the positive charges of the NH2 SAM as histidine of the pVIII . Assuming that half of the and the DPSH bacteriophage at pH 3.0, the adhesion energy pVIII proteins from the deposited phages participated in these = −2 = between them (Wad 4.30 ± 0.29 mJ m at tc 1 h) was com- interactions, the adhesion force per pVIII protein would be parable with that between the OH–SAM and the bacteriophage ~15.3 kcal mol−1 (~26 kT). This suggests that there could (Fig. 3c). It was expected that H-bonding would occur between be multiple cation–π interactions per pVIII protein because the

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a 20 b 20 ) –2 )

Out 2 –2 2 )

) In (mJ m –1 –1 0 0 R

(mJ m 0 0 

–2 R –2

W : 0.21 ± 0.02 mJ m W : 0.24 ± 0.01 mJ m /2 ad  ad F –2 /2 –2 (mN m F (mN m W : 0.22 ± 0.01 mJ m –2 Wad : 0.22 ± 0.01 mJ m –2 ad = = W F/R F/R

–20 W –20 Contact time, tc 5 s –4 –4 1 h –6 –6 –40 –COOH SAM –40 –OH SAM

Force/radius, pH 8.5 KNO3 solution pH 8.5 KNO3 solution DSPH layer –8 DSPH layer –8 Interaction energy, –60 –60 Interaction energy, 0 20406080 010203040

c 20 d 20 ) –2 )

2 –2 2 ) ) Force/radius, (mJ m –1 –1 (mJ m 0 0 0 0 R  –2 R 

Wad : 0.25 ± 0.01 mJ m /2 /2 F

–2 F –2 (mN m W : 0.26 ± 0.01 mJ m –2 (mN m Wad : 0.24 ± 0.04 mJ m –2 ad = = F/R

–2 W F/R –20 W –20 Wad : 1.00 ± 0.27 mJ m –4 –4

–NH SAM –6 –CH SAM –6 –40 2 –40 3 Force/radius, pH 8.5 KNO solution Force/radius, pH 8.5 KNO solution DSPH layer 3 3 –8 DSPH layer –8 Interaction energy, –60 –60 Interaction energy, 010203040 010203040 Separation distance, D (nm)

e 20 f 6 )

2 –2 pH 8.5, tc = 5 s ) ) –2 pH 8.5, t = 1 h

–1 c

0 0 (mJ m R  (mJ m

/2 4 F ad (mN m –2 W = F/R –20 W –2 Wad : 2.64 ± 0.70 mJ m –4

–2 Wad : 5.23 ± 0.55 mJ m 2 –6 –40 –Ph SAM Force/radius, pH 8.5 KNO3 solution DSPH layer –8 Adhesion energy, Interaction energy, –60 0 010203040 –COOH –OH –NH2 –CH3 –Ph Separation distance, D (nm) Terminal functional group of SAMs

Fig. 4 Force versus distance profiles measured at pH 8.5. The interactions force of the DSPH-coated surface was measured against SAMs with five different terminal functional groups: a –COOH, b –OH, c –NH2, d –CH3, and e -Ph. The empty and solid arrows correspond to the approach and detachment of the two surfaces, respectively. The red and blue curves correspond to the force profiles at contact times (tc) of 5 s and 1 h, respectively. f Bar graph showing the overall adhesion energy (Wad) of different SAMs at pH 8.5 as a function of contact time. The error bars represent the s.e.m., where n = 5in each group strength of the cation–π interaction was estimated to be curve indicates that any types of repulsive forces were completely ~5.5 kcal mol−1 in water34. overwhelmed by the strong adhesion until the surfaces became – 35 The adhesion energy between the CH3 SAM and DSPH layer closer than their steric wall distance, Dsw (Fig. 3d) . A simple fi = −2 – – was signi cantly higher (Wad 7.00 ± 0.10 mJ m ) compared DSPH adsorption experiment on the CH3 and COOH SAM – – – with those of the COOH ,OH , and NH2 SAMs (Fig. 3d). The perfectly corresponded with the SFA result, which showed the – fi – origin of the strong attraction between the DSPH and CH3 SAM deposition of a signi cantly denser DSPH layer on the CH3 SAM was most likely due to the strong hydrophobic interactions, as compared with the COOH–SAM (Supplementary Fig. 2). evidenced by the contact angle measurement, in which the Among all the tested SAMs, the Ph–SAM exhibited the highest – θ = CH3 SAM was very hydrophobic ( 101.0 ± 0.8°). The approach adhesion energy against the DSPH bacteriophage at pH 3.0

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= −2 = = – −16 = (Wad 8.08 ± 0.82 mJ m at tc 5 s and Wad 12.24. ± 0.50 mJ the Ph SAM was ~4.2 × 10 J (at pH 3.0 and tc 1 h), which −2 = 5 m at tc 1 h) (Fig. 3e). If the strong adhesion between the corresponds to ~10 kT at room temperature. Considering the phage and Ph–SAM was solely caused by hydrophobic interac- number of pVIII proteins per M13 phage (~2700)41, with the tions, the adhesion energy between them should be lower assumption that about 50% of the pVIII proteins were involved in compared with that between the phage and CH3–SAM, as the the adhesion, the adhesion energy per pVIII protein was ~74 kT. – fi CH3 SAM was more hydrophobic, as shown in Fig. 2. The strong It is noteworthy that this value is signi cantly higher than the interactions between the DSPH phage and Ph–SAM can be adhesion energy of well-known mussel foot proteins; the adhesion attributed to the π–π stacking (as hypothesized from other work), energy between a 25-mer-long mussel foot protein-3s and the cation–π, and hydrophobic interactions. The high proportions of hydrophobic surface was estimated to be ~34.7 kT by replica- histidine (H) and proline (P) in the surface-exposed portion of exchange molecular dynamics (REMD) simulations42. Using the the pVIII protein (DSPHTELP) for the DSPH phage (~12.5 and same assumption, the adhesion energy of one pVIII against the ~25%, respectively) were closely correlated to these results. It was Ph–SAM was calculated to be ~32 kT at pH 8.5. reported that even though proline is not a π-conjugated system at pH 3.0, it can favorably interact with the π–electron-rich phenyl aromatic face and induce the aforementioned interactions, Complexation and dispersion of SWCNT using DSPH phage. minimizing the steric penalty36. Based on the finding that DSPH phage strongly interacts via π–π Previous studies showed that the magnitude of the π–π interaction, we investigated the pH-dependent properties of stacking energy mediated by protonated histidine is stronger than single-walled carbon nanotubes (SWCNT) and DSPH-phage that of deprotonated histidine37. In biological systems, the complexes. The DSPH bacteriophage was reported to strongly positively charged histidine has been known to be an important bind with SWCNTs and maintain a stable dispersion without re- cationic source in cation–π interactions for regulating protein bundling15,16, enabling diverse applications including in vitro and folding and reactivity38. Considering that histidine is always in vivo bioimaging and synthesis of CNT-based hybrid materials protonated under acidic conditions (pH 3.0), cation–π interac- in aqueous solutions. However, little is known about the under- tions would be one of the major contributors to the interactions lying mechanism for their complexation and dispersion stability, with the Ph–SAM. Furthermore, all the amino acids’ α protons especially at high CNT concentrations. The complexation was could interact with the Ph–SAM via CH/π interactions, regardless conducted through the dialysis of a surfactant-assisted SWCNT of the pH conditions36. dispersion in the presence of DSPH bacteriophages at three dif- All the adhesion energies measured at pH 3.0 were higher at ferent pHs (pH 3.0, 5.0, and 8.5). Possible interactions between = = tc 1 h compared with those at tc 5 s. The increase in adhesion the DSPH bacteriophages and SWCNTs are listed in Fig. 5aasa π–π with tc is a typical sign of structural rearrangements or function of pH. It was anticipated that stacking and hydro- reorientation of biological macromolecules39,40, indicating that phobic interactions were always present, regardless of the pH the adhesive bonds (physical interactions, including van der values. At a low pH, cation–π and electrostatic interactions may Waals, H-bonding, and hydrophobic attraction in this study) be additionally present. Due to the presence of only attractive developed favorably while the functional groups and DSPH were interactions at pH 3.0 and 5.0, the dialysis of the complexing in contact. The decrease in Dsw during contact also suggests that solution resulted in the formation of hazy aggregates (Fig. 5b). molecular rearrangement occurred (Supplementary Fig. 3). This seems to be contrary to previous studies that found the In general, the adhesion energies at pH 8.5 were much lower formation of stable SWCNT–DSPH complexes under similar than those in acidic conditions, regardless of the opposing conditions15,16. Note that we have used a much higher con- functional group (Fig. 4). The increase in the hydrophilicity of the centration of DSPH and SWCNTs for their complexation than DSPH-coated surface (as shown in the contact angle measure- previous studies; their high concentrations can decrease the ments) and the larger Dsw between the two opposing layers separation distance and help overcome (repulsive) kinetic barriers indicate the existence of strong steric- and hydration–repulsion to achieve thermodynamic equilibrium, allowing the facile caused by the swelling of the bacteriophages on the mica surface. acquisition of the (aggregated) global minimum state. However, Consequently, for the COOH–,OH–, and NH2–SAMs, we could not observe the formation of any aggregates for the repulsive forces dominated over all the other adhesive forces, sample prepared at pH 8.5. The resulting SWCNT–DSPH com- yielding purely repulsive force versus distance profile. On the plex solution remained stable over 6 months without any – – contrary, for the CH3 and Ph SAM, the adhesive forces were noticeable precipitation or change. The observed high-dispersion still stronger than the repulsive counterparts, even though stability of the SWCNT–DSPH complexes was attributed to the significant decreases in the adhesion energies were observed at presence of additional electrostatic repulsive interactions between pH 8.5. The adhesion energies between the Ph–SAM and DSPH the complexes. The repulsive interactions can rise from negatively −2 = −2 layer (~2.64 ± 0.70 mJ m at tc 5 s and ~5.23 ± 0.55 mJ m at charged amino acids on the DSPH phage (e.g., glutamic acid and = tc 1 h) were still the strongest (Fig. 4e), indicating the aspartic acid) and dominate over other short-range attractive dominance of π–π interactions due to the histidine and CH/π interactions (e.g., π–π stacking and hydrophobic interactions). interactions induced by proline in the DSPH phage36. The plateau The complex mixtures were analyzed by AFM to observe the before the “jump-out” upon the detachment of the Ph–SAM morphology of the SWCNT–DSPH complex in detail (Fig. 5c). (Fig. 4e) at pH 8.5, which was absent at pH 3.0, may indicate that AFM imaging and cross-section analyses showed severe aggre- highly hydrated residues of the DSPH phage were “peeled-off”’ gation of the DSPH phages and SWCNTs at pH 3.0. These results before detachment (Supplementary Fig. 4). suggest that π–π stacking and hydrophobic interactions were the The adhesion energy per virus can be calculated based on the main driving forces for the binding of the DSPH phages with the number density of DSPH (calculated from AFM images) and the SWCNTs, which correlated well with the SFA results. At higher measured Wad. Compared with the known dimension of DSPH pHs, the phages became negatively charged due to the deproto- (diameter ~6.5 nm)16, adsorbed DSPH in AFM image (Fig. 1) nation of glutamic acids and aspartic acids in the pVIII protein, seems to be in a “compressed cylinder” shape (lateral thickness increasing the electrostatic repulsion between the phages and ~20 nm) rather than a normal cylinder. Thus, 50% is a reasonable phage-bound SWCNTs. Hence, the degree of repulsion was a upper bound of the pVIII protein fraction directly involved in major factor that affected the dispersion stabilities of the adhesion. In particular, the adhesion energy per DSPH phage on SWCNT–DSPH phage complexes. Therefore, the aggregate

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a pH 3.0 pH 5.0 pH 8.0

C O C O C O

R CH R CH R CH NH NH NH

- stacking, - stacking Repulsion, Cation-, and - stacking, Hydrophobic, Hydrophobic Hydrophobic Electrostatic interaction interaction interaction

b

pH 3.0 pH 5.0 pH 8.0

c

pH 3.0 pH 5.0 pH 8.0

5 4 1.5 0 0 0.5 –5 –2 0 nm nm nm

Fig. 5 Dispersion stability of SWCNT–DSPH complex at different pHs. a Graphical illustration showing a list of potential interactions between the DSPH phages and SWCNTs upon their complexation. b Photographs of SWCNT–DSPH phage complexes prepared at different pHs: 3.0, 5.0, and 8.5. Schematic illustration explaining the observed difference in their dispersion stabilities. c The corresponding AFM images of SWCNT–DSPH phage complexes. Scale bar, 100 nm formation and dispersion of SWCNTs can be simply tuned by the Using a model biomolecule (i.e., pVIII peptides on M13 bac- pH conditions when using DSPH phages as a dispersant. teriophage with a specific sequence of DSPHTELP) and SAMs with various terminal functional groups, we could identify pos- sible interaction forces and their strengths precisely. We found Discussion that histidine and proline moieties may play a critical role in the Despite the highly consistent and reliable results, there remain a molecular recognition of CNTs by the DSPH phages through few issues that should be addressed in the future studies. First, we their engagement in attractive π interactions, as expected pre- cannot completely exclude the possibility that another portion of viously15, and additionally through hydrophobic interactions. the bacteriophage (e.g., pIII or pVII) other than the exposed The measured strength of each identified interaction was com- pVIII sequences (i.e., DSPHTELP in this study) participated in parable with previously reported values. It is thought that a much the interactions with the SAMs. Second, it would be useful to higher abundance of pVIII at the phage surface compared with measure the interaction forces of various point-mutated bacter- other proteins allowed the SFA analysis with adequate reliability. iophages (e.g., DSAHTELA) for a more straightforward and We also experimentally demonstrated that DSPH–SWCNT systematic investigation of the origin of the molecular recognition complexes can be stabilized at a high pH through electrostatic capabilities. More ideally, one can utilize the solid-phase- and hydration repulsion. synthesized short peptides rather than the whole phage. Never- The interaction origin of M13 bacteriophages (DSPH phages) theless, we believe that this study can provide insights and solid was investigated by directly measuring interaction forces against – – – – – foundations for studies on specific interactions of biomolecules. functionalized SAMs ( COOH, OH, NH2, CH3, and Ph).

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The overall results indicated that the DSPH phage exhibited the coverage of the M13 bacteriophages on the mica substrate were analyzed using the highest and lowest adhesion energy with the phenyl and car- freeware, ImageJ. boxylic acid groups, respectively, indicating that the phages strongly interacted via π–π stacking and hydrophobic interac- Contact angle measurements. Wetting properties of the M13 bacteriophages and alkanethiol compounds were investigated using a DSA-100B-basic contact angle tions, while the H-bonding interactions remained relatively weak. μ fi analyzer (KRÜSS GmbH, Germany). First, 4 L of 3 mM KNO3 solution (pH 3.0 Moreover, we con rmed that DSPH phages interacted strongly and pH 8.5) was dropped on each SAM surface. The contact angle values were with the SWCNTs in acidic conditions via the aforementioned obtained in tangent 1 fitting mode. In the case of the M13 bacteriophage-coated physical interactions. Hence, pH-responsive tuning of the M13 surface, a sample was placed in an acryl box with wetted tissues and left for 10 min μ bacteriophage–SWCNT complexes is possible. The obtained to achieve air saturation, after which a 4 L droplet of 3 mM KNO3 (pH 3.0 and pH 8.5) was placed on the phage surface. The measurements were recorded as a video results can be used as a fundamental database in bacteriophage- clip for 10 min to investigate the contact angle profile over time. Every measure- based applications to enhance the performance of future phage- ment was repeated at least four times. based templates. Furthermore, the utilized measurement protocol, using functional SAMs as opposing surfaces, can be applied to Measurement of interaction forces using an SFA. Molecular interactions and the study the molecular interaction mechanisms of various bio and absolute separation distances between two surfaces were measured in an asymmetric synthetic materials. system with the Surface Forces Apparatus 2000 (Surforce LLC, Santa Barbara, CA, USA). A freshly cleaved back-silvered mica and gold surface were glued onto each cylindrical glass disc (Radius R, ~2 cm) using an optical adhesive, Norland optical Methods adhesive 81 (Norland Products, Inc. Cranbury, NJ, USA). The SAMs deposited on Materials. HiPCo SWCNTs were purchased from Unidym. Unless stated other- the gold layers were placed onto the upper discs. The DSPH phage layer on the wise, all chemicals, including 10-carboxy-1-decanethiol (95%), 11-amino-1-unde- back-slivered mica was placed on the lower disc. The two opposing surfaces were canethiol, hydrochloride (99%), 11-hydroxy-1-undecanethiol (97%), 1- arranged with a cross-cylindrical geometry in the SFA chamber, and 50 μLofa undecanethiol (98%), and 2-phenylethanethiol (98%), were purchased from Sigma- 3 mM KNO3 solution (pH 3.0 and 8.5) was injected between the surfaces. The SFA Aldrich. chamber was thoroughly sealed and maintained for 30 min after the injection of each solution to achieve equilibration. The contact time (tc) effects were also fi = = investigated by taking measurements rst at tc 5 s and followed by at tc 1 h. All Preparation of SAMs with different end-functional groups. Each alkanethiol measurements were conducted at room temperature (T = 23 °C). was prepared on atomically smooth gold surfaces. The smooth gold surfaces Force versus distance profiles were measured at a constant rate of 5 nm s−1. The (thickness: 45 nm) were prepared on cleaved clean muscovite mica (Grade #1, S&J normal force F was calculated as a function of the absolute surface separation Trading. Floral Park, NY, USA) through electron beam evaporation. The gold Δ = Δ −Δ distances D as follows: F(D) k ( Dapplied Dmeans), where k is the double- layers were attached to the curved surfaces of cylindrical glass discs (Radius, R cantilever spring constant of 1225.8 N m−1. The distances D were confirmed from ~2 cm) by painting them with an optical adhesive, Norland optical adhesive 81 fringes of equal chromatic order (FECO) by using multiple-bean interferometry (Norland Products, Inc. Cranbury, NJ, USA), and subsequently UV treating the (MBI) in real time. The forces were normalized by their radii (R ~2 cm) as samples for 40–60 min. The discs with gold-coated top surfaces were immersed Fcurved(D) R for the cylindrical discs. The normalized force F/R was converted to into 1 mM alkanethiol-ethanol solution for the formation of SAM structure via the interaction energy per unit area W between thep twoffiffiffiffiffiffiffiffiffiffiflat surfaces based on the gold-sulfur bonds on the gold (111) surfaces43. After 16–18 h, the discs were = π = π Derjaguin approximation, Wflat(D) Fcurved(D)/2 R1R2 Fcurved(D)/2 R, with sonicated for 30 s, washed with ethanol, and dried by nitrogen gas to remove = – – R1 R2. The adhesion energy was obtained using the Johnson Kendall Roberts physical impurities and excess SAM molecules from the surfaces. (JKR) model, which is useful for soft materials with large deformations, Wad = π 39 2Fad/3 R . Each experiment was repeated, at least three times at the same contact M13 bacteriophage cultivation. The DSPH phage was obtained from Prof. Bel- point to investigate hysteresis effect, and at least at three different contact points cher’s group at Massachusetts Institute of Technology. Cultivation of M13 bac- under the same conditions for reproducibility and repeatability. teriophages was performed using strain ER2738 from New England Biolab. First, 10 mL of the Luria Bertani (LB) medium was mixed with 10 μLof SWCNT–DSPH phage complexation. Before complexation with the DSPH pha- TET (antibiotic) and a single colony of ER2738 cells, and incubated overnight. For ges, SWCNT (HiPCoTM, diameter: 0.8−1.2 nm, length: 100−1000 nm) dispersions large-scale amplification, 1 mL of TET, 10 mL of the overnight culture, and ~1011 were prepared using a 2 wt% sodium cholate surfactant based on a previous pfu of the DSPH phage were added to a freshly prepared LB medium (25 g of LB report15. The DSPH phage and SWCNTs were mixed at a molar ratio of 5:1 14 medium and 1 g of MgCl2.6H2O in 1 L of distilled water). Thus, around 10 pfu fi 12 − (SWCNTs:DSPH phage) with a nal concentration of DSPH phages of 1.0 × 10 mL 1 of bacteriophages were produced. Finally, the culture was incubated at 37 °C pfu mL−1. The SWCNT–DSPH solution mixture was first dialyzed against water in a shaker for 225–250 rpm for about 6 h. (10 mM NaCl) at pHs of 3.0, 5.0, and 8.5 for complexation. Dialysis was conducted overnight with frequent solution changes. Bacteriophage purification. First, 500 mL of the culture was poured into each large centrifuge tube and centrifuged at 7800 rpm for 30 min. After centrifugation, Data availability 70 mL of PEG/NaCl (200 g of PEG and 146 g of NaCl in 1 L deionized water) was The authors declare that all data supporting the findings of this study are available within added to a 420 mL of supernatant, and the mixture was left overnight at 4 °C. The the article and its supplementary information files. Additional data related to this study solution was centrifuged again at 8140 rpm for 30 min, the supernatant was dis- can be requested from the corresponding author upon reasonable request. carded, and the solution was centrifuged at 8140 rpm for another 5 min. The white phage pellets in the centrifuged solution were dissolved completely in a 30 mL of TBS solution. The solution was centrifuged at 10,000 rpm for 5 min to remove Received: 29 April 2019 Accepted: 29 July 2019 residual impurities, after which 5 mL of PEG/NaCl solution was added to the solution and mixed until the solution became homogeneous. Finally, the phage solution was centrifuged at 11,000 rpm for 30 min, the supernatant was discarded, and the solution was centrifuged again at 11,000 rpm for 5 min. The resulting phage solution concentration was determined by the amount of the white phage pellets that were obtained just before centrifuging to dissolve the 1 mL TBS solution References (pH 7.2). 1. Beveridge, D. 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