Potassium Triggers a Reversible Specific Stiffness Transition of

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Potassium Triggers a Reversible Specific Stiffness Transition of Poly-ethylene Glycol Laura Tüting, Weixiang Ye, Giovanni Settanni, Friederike Schmid, Bernhard Anton Wolf, Rubén Ahijado-Guzmán, and Carsten Sönnichsen J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b08987 • Publication Date (Web): 14 Sep 2017 Downloaded from http://pubs.acs.org on September 15, 2017

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The Journal of Physical Chemistry

Potassium Triggers a Reversible Specific Stiffness Transition of Poly-ethylene Glycol Laura Tüting1, 2‡, Weixiang Ye1, 2‡, Giovanni Settanni3, Friederike Schmid3, Bernhard A. Wolf1, Rubén Ahijado–Guzmán1*, and Carsten Sönnichsen1* 1

Institute of Physical Chemistry, University of Mainz, Duesbergweg 10–14, D–55128 Mainz, Germany 2

Graduate School Materials Science in Mainz, Staudinger Weg 9, D–55128 Mainz, Germany

3

Institute of Physics, University of Mainz, Staudinger Weg 7–9, D–55128 Mainz, Germany

KEYWORDS Plasmon Rulers, Polyethylene glycol (PEG), Plasmonic nanosensors, macromolecular transitions,

gold

nanoparticle

ACS Paragon Plus Environment

dimers.


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ABSTRACT We use plasmon rulers made from two connected gold nanoparticles to monitor the conformation and stiffness of single PEG molecules and their response to cations. By observing equilibrium fluctuations of the interparticle distance, we obtain the spring constants or stiffness of the connecting single molecule tether with pico–Newton sensitivity. We observe a transition of the PEG molecules’ extension and stiffness above about 1.2 mM K+ ion concentration which is specific to potassium ions. Molecular dynamics simulations reveal the formation of crown–like structures as the most likely molecular mechanism responsible for this specific effect.


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INTRODUCTION Polyethylene glycol (PEG) is a commonly used polymer in medicine, pharmacy, and cosmetics due to its water solubility and its exceptionally high biocompatibility.

1–3

PEG molecules tend to organize

around some specific metal cations, e.g. potassium, in helical conformations, so called ‘in–chain’ pseudo–crown structures.3–5 Polyelectrolytes, on the other hand, react mainly un–specifically to ions with a transition between compact and expanded states depending mostly on the ionic strength of the solvent.6 However, it is not completely clear how PEG reacts towards cations, whether it reacts specifically to certain ions or un–specifically, especially with regards to chain stiffness, conformation and hydrodynamic radius. In this work, we monitor the cation–triggered conformational response and stiffness of PEG molecules using plasmon rulers. Plasmon rulers spectroscopically resolve the end–to– end distance of a single PEG molecule sandwiched between two plasmonic gold nanoparticles.7,8 Our results show that PEG stiffness sharply responds to potassium but not to sodium, suggesting a specific PEG–cation interaction. These single–molecule results are supported by generalized intrinsic viscosity measurements. Additional molecular dynamics simulations reveal that indeed ‘in–chain’ crown–like structures forming around potassium ions (but not around sodium ions) are the most likely mechanism responsible for the observed response to cations. This specific response towards potassium ions provides an attractive possibility to create ‘smart polymers’ responding to external environmental stimuli, for example in the context of electro– or opto–mechanical systems, tissue engineering, drug delivery and diagnosis.9,10 More generally, the observed stimuli–responsive behavior of PEG could be used as model system, foldamer–like, for other more complex macromolecular transitions including conformational changes and folding of proteins.11,12 The method we use here, single–molecule plasmon rulers, is an attractive tool to study such systems that has so far not been used to the full extent possible. Single– molecule plasmon rulers could directly resolve transient intermediate states, possible alternative folding pathways, and characterize complex conformational dynamics of proteins.



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Plasmon rulers emerged as potential substitute of classic single–molecule techniques like fluorescence resonance energy transfer (FRET), atomic force microscopy (AFM) or optical tweezers to measure molecular distances.7,8 FRET suffers from limited observation bandwidth (time resolution and total monitoring time) due to photo–bleaching and the maximum useful distance is limited to approximately 10 nm.13,14 AFM and optical tweezers induce a force on the molecule of interest, thus extrapolation to zero–force is necessary to extract equilibrium values. In AFM the cantilever stiffness limits the applicable force range. In the strong light fields used for optical tweezers, the samples may suffer from photo–damage and heating.15,16 Plasmon rulers potentially overcome these drawbacks due to the absence of bleaching or blinking in plasmonic light scattering allowing, in principle, indefinite observation times. In a plasmon ruler, the plasmons of two adjacent nanoparticles are coupled. With decreasing inter–particle distance, the plasmon resonance wavelength of the coupled system shifts to lower frequencies (‘red shift’) and the peak scattering efficiency increases. The coupling strength of plasmonic particles decreases approximately exponentially with distance with a typical decay length corresponding to around 0.2 times the particle diameter.17 If the particle pair is connected by a single macromolecule tether, the plasmon resonance frequency follows the molecular dynamics. In principle, plasmon resonances occur in all metal nanoparticles, but high chemical stability, a resonance frequency in the visible, and the comparatively low damping make gold the material of choice for the plasmonic particles in plasmon rulers.

RESULTS AND DISCUSSION Plasmon rulers require spherical gold nanoparticles of a diameter tailored to the expected molecular distances under investigation. Since the plasmon resonance of individual spherical nanoparticles is also dependent on the particle diameter, a low polydispersity among the nanospheres is required for an accurate conversion of the plasmon resonance frequency into molecular distance. For our study, we used therefore a nanosphere synthesis method known18 to result in low polydispersity batches of gold spheres ACS Paragon Plus Environment

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(see supporting information Figure S1). Our batch had a mean particle diameter = 52 nm with a size variation of σD = 2 nm. This size variation of about 4% of the mean (polydispersity index PDI = 1+σ2/2 = 1.0015) is lower than most nanoparticle batches where the diameter typically varies around 10% (or worse). In addition, our sample had a small shape anisotropy (aspect ratio variation < 0.1). Both size variation and shape anisotropy resulted in a remarkably small variation in the single particle plasmon resonance wavelengths λres of only 9 nm (standard deviation) around the median value of λres = 561 nm. A plasmon ruler experiment needs a dimer of gold nanopheres connected by a single macromolecule tether, with one of the spheres attached to a transparent substrate and the other sphere free to move in a solvent. We chose to prepare the dimers step–by–step in a microscope flow cell, which allows to follow the assembly process ‘life’, avoids purification steps and simplifies the selective attachment of only the first sphere to the surface. To form dimers in the flow cell, we deposited nanospheres from a first batch of particles on the glass surface (Figure 1a). After washing out unreacted excess of particles and blocking the remaining substrate surface, we added a second batch of particles into the flowcell, functionalized to react specifically to the first particles. We monitored the formation of dimers in the microscope, which is readily observable by a strong color change, and removed unreacted particles once a sufficiently high number of dimers had formed. Later, the flowcell allowed exchanging the liquid environment around the dimers. The two batches of nanospheres used to create the dimers were taken from the same stock of particles as mentioned above. The first batch was functionalized with thiol–PEG–biotin, the second batch with the protein streptavidin (see Supporting Information).19 The strong affinity of streptavidin to biotin was used to capture a streptavidin coated particle from solution by the immobilized biotin–PEG coated particle on the substrate. In order to ensure that only a single PEG molecule connects the two particles, we attached a mixture of biotin-functionalized PEG and unreactive methoxy–PEG to the first batch of



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particles, typically with a ratio of about 1:19. The biotin–PEG was slightly longer (3 vs 2 kDa) than the unreactive PEG to ensure the availability of the biotin group. Steric hindrance due to the curvature of the spheres is an important factor to prevent multiple linker formation (see sketch drawn to scale in Figure S2 as schematic indication).20 This deposition strategy allowed us to monitor the stretching/shrinking response of individual PEG macromolecule attached to both nanoparticles as a function of potassium and sodium concentration in the liquid environment.

Figure 1. Dimer formation and principle of measurement. a. Simplified step–by–step schematic illustration of the deposition strategy for the dimer formation in the flow–cell (cf. Figure S2 for details). b. Measured single particle spectra of dimers with a single PEG tether (inset) in a stretched (violet) and compressed (pink) configuration. Compared to the compressed configuration, the stretched configuration shows a red–shifted plasmon resonance of slightly higher intensity. The intensity change (ρ) at wavelength λ2 (normalized and corrected for the value at wavelength λ1) is approximately proportional to the change in interparticle distance ∆x = x2 – x1 (cf. also Figure S3 and S4).


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To study the stiffness of the PEG molecule linking the plasmon ruler (inset, Figure 1b), we need to record the plasmon resonance wavelength as a function of time and convert it to distance changes ∆x(t). The distance fluctuations are then directly connected to the stiffness of the linker. Typical light scattering spectra of a single dimer in both stretched (inset left) and shrunk (inset right) configurations of PEG are shown in Figure 1b. As discussed before, a shorter linker leads to a red–shifted spectrum with higher maximum scattering efficiency. To measure distance changes quickly, we determined the entire spectrum shown in Figure 1b only at the beginning of the experiment and then monitored the intensities I1(t) and I2(t) at the two wavelengths λ1 and λ2 as indicated in Figure 1b. The shorter wavelength (λ1) corresponds to the plasmon resonance in the transverse direction of the particle pair. The transverse plasmon mode does not react (much) to distance changes, thus serves as an internal reference for the intensity. We normalized the intensity at λ1 and λ2 to their initial values Inorm(t) = I(t) / I(t=0) and follow the ratio ρ(t) = I2,norm(t) / I1,norm(t). The normalized intensity changes

ρ(t) can be converted to interparticle distance changes ∆x(t) using a theoretical model similar to the one previously described17 (see supporting information Figure S3). For small fluctuations in interparticle distances ∆x, the conversion can be simplified by a linear conversion factor L = ∆ρ / ∆x that is approximately L ≈ 0.03 / nm in our system. The hyperspectral imaging setup we used to perform the above mentioned measurements is shown schematically in Figure S4. This setup, based on a white light laser connected to an acousto–optical tunable filter (AOTF), allows switching between two modes: The complete spectra of all the particles in the field of view was recorded at the beginning of the experiment by cycling through all possible wavelength. Fast timetraces were then recorded in ‘dual–line mode’ by rapidly switching the illumination between two wavelengths λ1 and λ2. With this setup, we recorded the interparticle distance of up to 70 dimers simultaneously with a time–resolution of 50 ms continuously for many hours.



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An example of such a timetrace of a single plasmon ruler connected with presumably a single PEG molecule is shown in Figure 2a (purple-grey-pink line). As is evident from the graph, the interparticle distance fluctuates around an equilibrium value by as much as +/– 3 nm. Such timetraces are simultaneously recorded on dozens of dimers – some of which were not fluctuating much because, presumably, both nanospheres were immobilized on the glass surface. These ‘control dimers’ allowed us to determine our instrumental noise because no molecular fluctuations could take place. The noise is much smaller (10x) than the fluctuations observed on the freely moving dimers (see dark blue timetrace in Figure 2a). As an additional control, we changed the salt concentration after 30 min from 0.1 mM to 10 mM. At the higher salt concentration, the end–to–end distance of the PEG molecule decreased and, at the same time, the fluctuation amplitude decreased by a factor of about 2. Both observations are consistent with a shrinking PEG molecule with higher stiffness and not consistent with alternative explanation of fluctuations, for example instrument noise or particle surface coverage fluctuations.


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Figure 2. Molecular stiffness from equilibrium fluctuations a. Timetrace (purple, grey and pink line) of the normalized intensity ratio ρ (left axis) and corresponding interparticle distance change ∆x (right axis) at two different K2SO4 concentrations (shaded areas) for a dimer connected with a single PEG tether. The dark blue line corresponds to the values measured on a ‘control dimer’ where both particles are immobilized on the substrate. b. Interparticle potential ϕ as a function of fluctuation (extension) around the equilibrium value. The values extracted from the probability functions for the particle separation ∆x shown in Figure 2a are shown as dots (color corresponds to the time sequences shown in Figure 2a). The measured values (dots) clearly follow two different trends as indicated by the fitted parabolas (lines in the corresponding colors).

To convert distance fluctuations quantitatively to polymer stiffness, we assume that the probability distribution P(x) of the interparticle distances follows a Boltzmann distribution given by a molecular potential φ(x) (we neglect here contributions from noise).21,22 We extract the probability distribution P(x) from our timetraces and convert it to a potential φ(x) by φ(x)/kBT = –ln(P(x)). In Figure 2b those potentials are shown for the two different salt concentrations used before (0.1 mM, violet, and 10 mM, pink). Assuming the potential is caused by the stiffness of the molecular linker, a parabolic fit to φ(x) yields the spring constant k. We show the parabolic fits as solid lines in Figure 2b together with the corresponding spring constants. The calculation confirms the qualitative picture presented before: a lower spring constant in the lower salt environment, consistent with a more flexible, extended polymer chain.



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Figure 3. Conformational transitions as function of salt concentration. a. Example of a timetrace of the intensity ratio ρ(t) (left axis) and interparticle distance ∆x(t) (right axis) of a single plasmon ruler (pink line). The salt (K2SO4) concentration was changed every 30 min (shaded areas) to the values shown on top. Between every salt titration step we reverted back to 0.1 mM for a short time to test for the reversibility of the PEG shrinking process. We recorded about 70 such timetraces in parallel in each experiment. b. The spring constants (left axis) calculated from pieces of timetraces shown in ‘a’ for different salt concentrations are show as small open circles. Their mean value for every salt concentration is shown as solid blue dot with error bars corresponding to the error of the mean. A sigmoidal function fitted to the data (solid blue line) shows a transition at 0.6 ± 0.2 mM (1.2 mM K+ ion concentration). c. For comparison, the intrinsic viscosity measured on free PEG molecules in solution. The orange line, to guide the eye, correspond to the expected intrinsic viscosity behavior. The dotted orange line shows approximately the transition in the intrinsic viscosity measurements at a concentration of around 0.5 mM which corroborates our single–molecule data. The red line indicates the intrinsic viscosity value in total absence of salt (pure water) and the dotted red lines correspond to the error of that measurement. d. Same data as in Figure 3b but from an experiment with Na2SO4. At the end of the experiment, K2SO4 was added at 30 mM resulting in the same stiffness as shown before (panel above).


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We performed some more control experiments to show that the observed fluctuations are governed by the properties of a single linker molecule. First, we determined that the estimated spring constants are not influenced by the gold nanoparticles size and shape (as determined from the observed scattering intensity and wavelength) (Figure S5). Second, we reduced the amount of thiol–PEG–Biotin on the nanoparticle from 5 to 0.5 %. Whereas this reduction dramatically reduces, as expected, dimerization efficiency, the resulting spring constants are not affected (see Figure S6). Taken together, these controls (and previous work on plasmon rulers)7,8,21 make a single PEG linker the most likely scenario. However, the system consisting of a single molecule sandwiched between two solid nanoparticles is more complex in many aspects than we are able to treat in this work (electrochemical changes at the particle surface, forces between particles, optically induced effects). As in previous work, we assumed those aspects of minor relevance compared to

the observed changes. After we validated the method, we determined the stiffness of single PEG molecules as a function of potassium and sodium ion concentration. First, we titrated K2SO4 step–by–step from 0.1 mM to 300 mM while recording the normalized intensities ρi(t) of all 70 dimers in the field of view. Figure 3a shows one of the 70 time traces ρi(t) for different K2SO4 concentrations as indicated by the shaded areas. Between every titration step, we reverted briefly to the initial conditions of 0.1 mM K2SO4 to ensure the reversibility of the shrinking process, i.e. the absence of irreversible aggregation. Indeed, all timetraces recovered the original signal level after each concentration step, usually within seconds (indicating that the transition occurs and equilibrium is reached faster than our solvent exchange time). We tested for complete reversibility also in another experiment, cf. Figure S7, and repeated the titration experiment several times with different salt concentration steps. The dimers were surprisingly stable against irreversible


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aggregation, indicating that the strategy of shorter polymeric spacer molecules passivating the remaining particle surface seems to be working well. For each of the K2SO4 concentrations, we obtained the linker stiffness for every dimer by performing an analysis analogous to Figure 2. Figure 3b shows these stiffness values as a function of K2SO4 concentration (small pink open circles). The mean values (averaged over all dimers) at each concentration, indicated by blue symbols with error bars, follow a clear sigmoidal trend with a transition to a stiffer, less extended state at higher salt concentrations. A fit with a sigmoidal function (blue line) gives a critical transition concentration of 0.6 ± 0.2 mM (K+ concentration 1.2 ± 0.4 mM) and two conformations or ‘states’ with stiffness of 23.2 ± 0.2 pN/nm and 8.0 ± 0.5 pN/nm. To test if these results are reasonable, we compared the results with literature and performed an ensemble measurement (Figure 3c) to verify the transition concentration. In the literature, we found a value of around 10 pN/nm, obtained by single–molecule AFM, which agrees with the low–salt state value stated above.23 For the ensemble measurement showed in Figure 3c (see also Figure S8), we measured the generalized intrinsic viscosity {η} (hydrodynamic specific volume) as a function of the K2SO4 concentration as described by Xiong et al.6 These ensemble data corroborate the results obtained in our single–molecule plasmon rulers. The specific hydrodynamic volumes decreased with increasing the salt concentration within the investigated region and reached saturation at concentrations around 0.5 mM, in excellent agreement with the value obtained above. This situation is also evident from the Huggins constant (see Figure S8) which varies accordingly with the potassium concentration. To find out whether the observed stiffness transition in PEG is specific to potassium ions or if it is an effect of the ionic strength of the solvent, we compared K+ and Na+ by repeating the


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fluctuation experiment by titrating with Na2SO4 from low to high concentrations (Figure 3d). Within the range of concentrations studied, we did not observe a shrinking of the PEG molecule or a transition of stiffness. Effectively, nothing happened at all up to 30 mM Na2SO4 (60 mM Na+). To make sure that the plasmon rulers were still intact and connected, most likely, with single molecules of PEG as before, we exchanged the final Na2SO4 solution with a K2SO4 solution of the same concentration. Indeed, we observed the shrinking of the PEG and reduced fluctuation with a higher spring constant immediately after exchanging the cations. These experimental results therefore suggest a specific conformational transition above about 1.2 mM K+.

Figure 4. Results from Molecular Dynamics simulations. a. The radial distribution functions of potassium (blue line) and sodium (green line) cations as function of distance to the oxygen atoms of the PEG chain. The strong peak at about 2.5 Å observed for potassium but not sodium suggests a much stronger coordination to PEG. b.


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Coordination of potassium ion in three representative snapshots from the simulations shows the pseudo–crown pattern around the potassium cations.

To find out more about the possible molecular mechanism responsible for this suggested specific potassium–induced PEG shrinking, we performed atomistic classical molecular dynamics simulations. For these simulations, we studied PEG in pure water, in the presence of 150 mM of potassium salt, and in the presence of 150 mM of sodium salt. To exclude polymer chain length effects, these simulations were repeated for two different length of polymers (PEG18 and PEG36) resulting in the same behavior. The simulations confirm the size reduction of PEG in the presence of potassium ions but not for sodium ions (see the radius of gyration values in Table S1). Structurally, the simulations suggest a strong coordination of the potassium cations to the oxygen atoms in the PEG chain, which is absent for sodium (Figure 4a). The strong coordination is associated with the formation of in–chain pseudo–crown–like patterns in PEG molecules around the potassium cations (see snapshots in Figure 4b). Sodium may also induce such patterns occasionally, but much less frequently. Quantitatively, we observed that the probability to find more than 3 PEG oxygen atoms around a cation is 100 times larger in the simulations at 150 mM K+ than in the simulations at 150 mM Na+ (12 % vs. 0.12 % of the trajectories). The net effect of the formation of pseudo–crown patterns is the reduction of the effective polymer length. The different behavior of the two cations is probably related to the solvation free energy of Na+, which is higher than the one of K+.24 Thus, removing the first hydration shell of Na+ in order to interact with the oxygen atoms in the PEG molecule is notably more difficult than for potassium atoms. In addition, crowning around the larger potassium involves a lower degree of bending than around sodium, which should be energetically more favorable .3,25 We do not


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consider the contraction observed in the simulations as a compelling demonstration of the one observed experimentally, although it is qualitatively in agreement with it. More importantly, the simulations show the significantly larger propensity of K+ ions to induce crown-like structures in PEG. This phenomenon is extremely pronounced and clearly sets a difference between sodium and potassium ions. When scaled to the length of experimentally tested PEG molecules, it provides a compelling ex-planation to the origin of the observed contractions.

CONCLUSIONS Our single–molecule study of the shrinking and stiffness of polyethylene glycol (PEG) in different salt concentrations demonstrates pico–Newton sensitivity of plasmon rulers, comparable to single molecule AFM or optical tweezers.

15,16

Compared to Förster resonant

energy transfer (FRET), plasmon rulers work at much farther separations13,14 and monitoring, in principle, for indefinite time – without bleaching, blinking, and rotation. We carefully validated the method and performed controls that strongly suggest single molecule connections and the absence of nanoparticle induced artefacts on the polymer equilibrium conformation. We observed a potassium-triggered specific transition by using plasmon rulers and molecular dynamics simulations reveal the formation of crown–like structures as molecular mechanism responsible for this specific effect. This work will establish plasmon rulers as tool to study the structural dynamics of single macromolecules in polymer, soft–matter, biophysical, biochemical, and biomedical science.

METHODS


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If not specified, all the chemicals and reagents were purchased with analytical grade from Sigma–Aldrich or Merck. Deionized water from a Millipore system (> 18 MΩ, Milli Q) was used in all experiments. Nanoparticle synthesis: We performed the gold nanoparticle (AuNP) preparation as described in reference 18 with minor modifications. The process has four steps: first seeds are prepared, then grown to a size of 10 nm, then further grown to a larger size, and finally etched down to the desired size. Gold seeds: We added 50 µL of a 0.05 M HAuCl4 solution to 5 mL of a 0.1 M CTAC solution, then injected 200 µL of a freshly prepared 0.02 M NaBH4 solution under vigorous stirring. After 5 min, we diluted the mixture 10 times with 100 mM CTAC. To grow AuNPs with 10 nm diameter, we added 900 µL of the gold seeds (prepared as described above) and 40 µL of 0.1 M ascorbic acid to 10 mL of 25 mM CTAC solution. Then, we injected 50 µL of 0.05 M HAuCl4 under vigorous stirring. AuNP growth: We mixed 200 µL of 10 nm AuNPs and 400 µL of 0.1 M ascorbic acid with 100 mL of a 25 mM CTAC solution. Then, we added 50 µL of a 0.05 M HAuCl4 solution under stirring. This growth solution was left undisturbed for 1 hour. Oxidative etching: We used oxidative etching to smooth the nanoparticle surface, make them more spherical and decrease the degree of polydispersity.18 We injected 100 µL of a dilute NaClO solution (1.5 wt % of available chlorine) under stirring and then left undisturbed for 30 minutes. The 10 nm AuNP were grown into the final nanospheres as described in reference 18 using the growth and etching solutions described above. Nanoparticle Functionalization: PEG-Biotin coated gold nanospheres (AuNP@PEG– Biotin):We centrifuged 500 µL AuNPs (6000 g, 5 min), removed the supernatant and resuspended the pellet in 100 µL freshly prepared 2 mM PEG solution (mix of Thiol–PEG– Biotin with 3317 DA and Thiol-PEG-OMe with 1981 Da (5:95 molar ratio), Iris Biotech GmbH).


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We incubated the mixture for 2 hours at room temperature under stirring. We repeated the process and incubated overnight at 4°C. To remove excess of Thiol–PEG–Biotin, we washed the AuNP@PEG–Biotin twice with 400 µL of Milli–Q water (by centrifugation and resuspension as described above) and stored the particles at 4°C until use. Streptavidin coated gold nanospheres (AuNP@Streptavidin): 500 µL of AuNPs were centrifuged (6000 g, 5 min) and the supernatant removed. We prepared a solution containing 50 µl

of

1 mg/mL

streptavidin

and

50 µL

of

0.3 mM

DTSSP

(3,3´–

Dithiobis(sulfosuccinimidylpropionate)) in phosphate saline buffer (PBS). After 30 min of incubation, we desalted the solution in a desalting column and added to the AuNPs pellet. This solution was mixed incubated overnight at 4°C. To remove the excess of streptavidin, we washed the AuNPs twice with 400 µL of Milli–Q water (by centrifugation as described above) and stored at 4°C until use. Dimer formation: The microfluidic flow cell was first washed with ‘Hellmanex II special cleaning concentrate’ and then rinsed with 1 mL of Milli–Q water. We flushed AuNP@PEG– Biotin through the flow cell for 3–4 minutes. For their immobilization on the flow cell surface, we used a 1 M NaCl solution. Then, we passivated the glass–surface for 10 min with a mixture of ‘superblock’ (Pierce) and conjugation buffer (2.5 mM Tris–HCl pH 7.5, 0.25 mM EDTA, 0.5 M NaCl, 0.025% Tween 20) in a 60:40 ratio. After the passivation, we injected a solution containing AuNP@Streptavidin in a 60:40 mixture of superblock and conjugation buffer into the flow cell and incubated for 10 minutes. We removed unbound particles by rinsing 10 minutes with a 60:40 mixture of superblock and conjugation buffer. Molecular dynamics simulations: We carried out all the simulations using the program NAMD26 and the charmm27 force field27,28 with the extension for PEG.29 We used Tip3p30 as


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model for the explicit treatment of water. The CHARMM force field shows very good agreement with experimental data on the solvation free energies and structure of the hydration shell for potassium and sodium ions in combination with the TIP3P model.31,32 The parameters for PEG were used successfully in combination with NaCl solutions including proteins, for example in reference 33. We note that, although, potassium ions have not been used in combination with PEG, its parameters (VdW radius and depth of the VdW potential) have been extracted in the same way as those of sodium. So it is reasonable to believe that they will pro-vide similar accuracy. We used an integration time step of 1 fs across the simulations. We carried out the simulations using periodic boundary conditions. We maintained the pressure and temperature constant at 1atm and 300K, respectively, during the simulations using the Langevin piston algorithm and Langevin thermostat.34,35 We used a cutoff of 1.2 nm for the non-bonded interactions with a switch function. We treated long range electrostatic interactions using the smooth particle mesh Ewald (PME)36 method with a grid spacing of about 0.1 nm. We prepared the systems by placing a PEG molecule (H-[O-CH2-CH2]n-OH, with n either 18 or 36) in a 7.2 nm-long cubic box of water molecules. For each PEG length we prepared three systems: one with no ions, one including 0.15 M NaCl and one including 0.15 M KCl. We then minimized the complete systems using the steepest descent algorithm for 10000 steps. Then we equilibrate the systems at room temperature and pressure for 1.0 ns. The equilibrated systems were then subjected to replica exchange molecular dynamics with solute tempering (REST2),37,38 using 10 replicas where the temperature of PEG (the solute), ranged from 300 to 650 K with replica swap attempts every 1 ps. Each replica ran for 100 ns. We used the replica at the lowest temperature for analysis. This approach allows for an efficient sampling of the conformational space of PEG. In these simulations we observed replica swap acceptance rates of 25 to 50% for PEG18 and


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around 20% for PEG36, leading to a uniform distribution of the 10 walkers over the 10 temperatures, which is expected at convergence. We analyzed the simulations using the program VMD and WORDOM.39,40 We used both programs to measure the radius of gyration, the radial distribution function of the oxygen atoms of PEG and the ions, the persistence length and the number of contacts between ions an PEG.

ASSOCIATED CONTENT Supporting Information Available. Detailed information on nanoparticle characterization, BEM simulations, setup improvements, MD simulations and additional control experiments. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

Author Contributions ‡L.T. and ‡W.Y. contributed equally. Experimental single particle data was measured by L.T., W.Y. and R.A.–G. on a setup developed by W.Y. in the laboratory of C.S. and with guidance of R.A.–G. and C.S. The simulations were performed and analyzed by G.S. and F.S. B.A.W. analyzed the viscosity measurements. All authors contributed to the analysis, discussion of


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results and its implications. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources This work was financially supported by the ERC grant 259640 (“SingleSens”). L.T. and W.Y. are recipients of DFG fellowships through the Excellence Initiative by the Graduate School Materials Science in Mainz (GSC 266). G.S. acknowledges support from the Max Planck Graduate Center (MPGC). We gratefully acknowledge support with computing time from HPC facility Mogon at the University of Mainz, and the High Performance Computing Center Stuttgart (ACID 12911). This work was partly supported by the DFG/SFB1066 (‘Nanodimensionale polymere Therapeutika fuer die Tumortherapie’, project Q1). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Anja Eckelt and Dr. John Eckelt (WEE–Solve GmbH, Mainz, Germany) for the intrinsic viscosity measurements. We acknowledge excellent support from Dr. Arpad Jakab who initially developed the hyperspectral imaging setup including software, and Dipl.-Chem. Sirin Celiksoy and Dipl.-Ing. Karl Wandner for significant setup improvements. We thank Mathias Schmitt for help with transmission electron microscopy. REFERENCES


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1. J. M. Harris. Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; J. M. Harris, Ed.; Plenum Press, New York, 1992; Chapter 1, pp 1–14. 2. S. Zalipsky; J. M. Harris. Poly (ethylene glycol) Chemistry and Biological Applications, J. M. Harris and S. Zalipsky, Eds.; ACS Symposium Series 680, American Chemical Society, Washington, DC, 1997; Chapter 1, pp 1–13. 3. Chen, J.; Spear, S. K.; Huddleston, J. G.; Rogers R. D. Polyethylene Glycol and Solutions of Polyethylene Glycol as Green Reaction Media. Green. Chem., 2005, 7, 64–82. 4. Rogers, R. D,; Bond, A. H.; Aguinaga, S.; Reyes, A. Complexation Chemistry of Bismuth (III) Halides with Crown Ethers and Polyethylene Glycols. Structural Manifestations of a Stereochemically Active Lone Pair. J. Am. Chem. Soc. 1992, 114, 2967–2977. 5. Terashima, T.; Kawabe, M.; Miyabara, Y.; Yoda, H.; Sawamoto, M. Polymeric Pseudo-Crown Ether for Cation Recognition Via Cation Template-Assisted Cyclopolymerization. Nat. Commun. 2013, 4, 2321. 6. Xiong, X.; Wolf, B. A. Intrinsic Viscosities of Polyelectrolytes: Specific Salt Effects and Viscometric Master Curves. Soft Matter 2014, 10, 2124–2131. 7. Sönnichsen, C.; Reinhard, B. M.; Liphardt, J.; Alivisatos, A. P. A Molecular Ruler Based on Plasmon Coupling of Single Gold and Silver Nanoparticles. Nat. Biotechnol. 2005, 23, 741–745. 8. Reinhard, B. M.; Siu, M.; Agarwal, H.; Alivisatos, A. P.; Liphardt, J. Calibration of Dynamic Molecular Rulers Based on Plasmon Coupling between Gold Nanoparticles. Nano Lett. 2005, 5, 2246–2252. 9. Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Materials, 2013, 12, 991–1003. 10. Stuart, M. A. C.; Huck, W. T. S.; Genzer, J.; Mueller, M.; Ober, C.; Stamm, M.; Sukhorukov, G. B.; Szleifer, I.; Tsukruk, V. V.; Urban, M.; Winnik, F.; Zauscher, S.; Luzinov, I.; Minko, S. Emerging Applications of Stimuli-Responsive Polymer Materials. Nat. Materials, 2010, 9, 101–113. 11. Goodman, C. M.; Choi, S.; Shandler, S.; DeGrado, W. F. Foldamers as Versatile Frameworks for the Design and Evolution of Function. Nat. Chem. Biol. 2007, 3, 252– 262. 12. Gellman, S. H. Foldamers: A Manifesto. Acc. Chem. Res. 1998, 31, 173–180. 13. Weiss, S. Fluorescence Spectroscopy of Single Biomolecules. Science 1999, 283, 1676––1683.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 24

14. Roy, R.; Hohng, S.; Ha, T. A Practical Guide to Single-Molecule FRET. Nat. Methods 2008, 5, 507–516. 15. Neuman, K. C.; Nagy, A. Single-Molecule Force Spectroscopy: Optical Tweezers, Magnetic Tweezers and Atomic Force Microscopy. Nat. Methods 2008, 5, 491–505. 16. Viani, M. B.; Schäffer, T. E.; Chand, A.; Rief, M.; Gaub, H. E.; Hansma, P. K. Small Cantilevers for Force Spectroscopy of Single Molecules. J. App. Phys. 1999, 86, 2258. 17. Jain, P.; Huang, W.; El–Sayed, M. A. On the Universal Scaling Behavior of the Distance Decay of Plasmon Coupling in Metal Nanoparticle Pairs: A Plasmon Ruler Equation. Nano Lett. 2007, 7, 2080–2088. 18. Hanske, C.; González–Rubio, G.; Hamon, C.; Formentín, P.; Modin, E.; Chuvilin, A.; Guerrero–Martínez, A.; Marsal, L. F.; Liz–Marzán, L. M. Large-Scale Plasmonic Pyramidal Supercrystals via Templated Self-Assembly of Monodisperse Gold Nanospheres. J. Phys. Chem. C 2017, 121, 10899–10906. 19. Diamandis, E. P.; Christopoulos, T. K. The Biotin-(Strept)avidin System: Principles and Applications in Biotechnology. Clin. Chem. 1991, 37, 625–636. 20. Pierrat, S.; Zins, I.; Breivogel, A.; Sönnichsen, C. Self-Assembly of Small Gold Colloids with Functionalized Gold Nanorods. Nano Lett. 2007, 7, 259–263. 21. Chen, T.; Hong, Y.; Reinhard, B. M. Probing DNA Stiffness through Optical Fluctuation Analysis of Plasmon Rulers. Nano Lett. 2015, 15, 5349–5357. 22. Dill, K. A.; Bromberg, S. Molecular driving forces: statistical thermodynamics in chemistry, physics, biology, and nanoscience, 2nd ed.; Garland Science: New York, 2011. 23. Sulchek, T. A.; Friddle, R. W.; Langry, K.; Lau, E. Y.; Albrecht, H.; Ratto, T. V.; DeNardo, S. J.; Colvin, M. E.; Nox, A. Dynamic Force Spectroscopy of Parallel Individual Mucin1-Antibody Bonds. Proc. Natl. Acad. Sci.USA 2005, 102, 16638– 16643. 24. Yizhak, M. Thermodynamics of Solvation of Ions. Part 5.—Gibbs Free Energy of Hydration at 298.15 K J. Chem. Soc. Faraday Trans., 1991, 87, 2995–2999. 25. Pedersen, C. J. Cyclic Polyethers and Their Complexes with Metal Salts. J. Am. Chem. Soc. 1967, 89, 7010–7036. 26. Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E; Chipot, C.; Skeel, R. D.; Kalé, L.; Schulten, K. Scalable Molecular Dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781–1802.


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Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


27. MacKerell, A. D.; Bellott, M.; Dunbrack, R. L.; Evanseck, J. D.; et al. All-atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins. J. Phys. Chem. B 1998, 102, 3586–3616. 28. MacKerell, A. D.; Feig, M.; Brooks, C. L. Extending the Treatment of Backbone Energetics in Protein Force Fields: Limitations of Gas-phase Quantum Mechanics in Reproducing Protein Conformational Distributions in Molecular Dynamics Simulations. J. Comput. Chem. 2004, 25, 1400–1415. 29. Lee, H.; Venable, R. M.; Mackerell, A. D.; Pastor, R. W. Molecular Dynamics Studies of Polyethylene Oxide and Polyethylene Glycol: Hydrodynamic Radius and Shape Anisotropy. Biophys. J. 2008, 95, 1590–1599. 30. Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926–935. 31. Beglov, D.; Roux, B. Finite representation of an infinite bulk system: Solvent Boundary Potential for Computer Simulations. J. Chem. Phys. 1994, 100, 9050–9063. 32. Joung, I. S.; Cheatman III, T. E. Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations. J. Phys. Chem. B, 2008, 112, 9020–9041. 33. Chao, S-H.; Matthews, S. S.; Paxman, R.; Aksimentiev, A.; Gruebele, M.; Price, J. L. Two Structural Scenarios for Protein Stabilization by PEG. J. Phys. Chem. B, 2014, 118, 8388–8395. 34. Martyna, G. J.; Tobias, D. J.; Klein, M. L. Constant Pressure Molecular Dynamics Algorithms. J. Chem. Phys. 1994, 101, 4177–4189. 35. Feller, S. E.; Zhang, Y.; Pastor, R. W.; Brooks, B. R. Constant Pressure Molecular Dynamics Simulation: The Langevin Piston Method. J. Chem. Phys. 1995, 103, 4613– 4621. 36. Essmann, U.; Perera, L.; Berkowitz, M. L. A Smooth Particle Mesh Ewald Method. J. Chem. Phys. 1995, 103, 8577–8593. 37. Wang, L.; Friesner, R. A.; Berne, B. J. Replica Exchange with Solute Scaling: A More Efficient Version of Replica Exchange with Solute Tempering (REST2). J. Phys. Chem. B 2011, 115, 9431–9438. 38. Jo, S.; Jiang, W. A Generic Implementation of Replica Exchange with Solute Tempering (REST2) Algorithm in NAMD for Complex Biophysical Simulations. Computer Physics Communications 2015, 197, 304–311. 39. Seeber, M.; Cecchini, M.; Rao, F.; Settanni, G.; Caflisch, A. Wordom: a Program for Efficient Analysis of Molecular Dynamics Simulations. Bioinformatics 2007, 23, 2625– 2627.


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40. Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graphics 1996, 14, 33.

TOC GRAPHICS


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Potassium Triggers a Reversible Specific Stiffness Transition of

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