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Cite This: Nano Lett. 2017, 17, 6598-6605

An Optical Tweezers Platform for Single Molecule Force Spectroscopy in Organic Solvents Jacob W. Black, Maria Kamenetska, and Ziad Ganim* Department of Chemistry, Yale University, 350 Edwards St., New Haven, Connecticut 06520, United States S Supporting Information *

ABSTRACT: Observation at the single molecule level has been a revolutionary tool for molecular biophysics and materials science, but single molecule studies of solutionphase chemistry are less widespread. In this work we develop an experimental platform for solution-phase single molecule force spectroscopy in organic solvents. This optical-tweezerbased platform was designed for broad chemical applicability and utilizes optically trapped core−shell microspheres, synthetic polymer tethers, and click chemistry linkages formed in situ. We have observed stable optical trapping of the core− shell microspheres in ten different solvents, and single molecule link formation in four different solvents. These experiments demonstrate how to use optical tweezers for single molecule force application in the study of solution-phase chemistry. KEYWORDS: Force spectroscopy, poly(methyl methacrylate), polymers, core−shell nanoparticles, click chemistry, atom transfer radical polymerization

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provide a mechanical probe that is much softer than AFM (stiffness 0.0002−0.4 vs 10−60 pN/nm) and is therefore wellsuited to investigate the 50 nm from the optically trapped bead surface and trapping laser with no sign of surfaceinduced heterogeneity.26−28 Immobilizing single molecules with OT provides distinct advantages that complement single molecule spectroscopies such as fluorescence7−9 or Raman,29 in particular: ∼nanometer sample immobilization, little out-offocus background, and straightforward overlap of beam paths. However, a single molecule study using OT has been predominantly limited to biomolecules in water. The limited literature on optical trapping in organic solvents arises from the study of colloids and utilizes surfactant-stabilized acrylic microspheres that cannot be directly transferred across solvents.30−33 Challenges in developing a direct solventuniversal analogy of the biophysical dumbbell assay are primarily due to a lack of commercially available microspheres

or chemistry, single molecule spectroscopy has the potential to structurally and kinetically characterize reactive intermediates in complex mixtures. Yet it has been difficult for chemists to study mechanisms with single molecule tools because there remains a practical gap between techniques developed for molecular physics (low temperature, solid state) and the methods employed in molecular biophysics (aqueous, pH 7). In particular the biophysical optical tweezers have enabled exquisite single molecule mechanistic studies due to their ability to apply pN forces,1−3 measure forces down to 10 fN at room temperature,4−6 and immobilize a single molecule in a spectroscopically clear background7−10 over tens of minutes/hours.11−16 In this work, we demonstrate an extension of the optical tweezers platform to immobilize and study small molecules in polar and nonpolar solvents with the aim of accelerating the applications of single molecule spectroscopy in solution-phase chemistry. To realize the complete potential of a single molecule (SM) investigation, the molecule must be probed over a sufficiently broad set of time scales to sample its entire distribution of structures. Namely, this requires sufficient time resolution to sample the fastest events while maintaining a long enough observation period to observe the slowest events. Dual trap optical tweezers provide a method for immobilizing a single molecule in solution, applying pN-scale forces, and measuring its contour length. A key feature is that all-optical immobilization mitigates mechanical drift, which yields outstanding force stability and makes SM observation possible over the course of hours.11−16 In addition, optical tweezers (OT) © 2017 American Chemical Society

Received: June 7, 2017 Revised: August 30, 2017 Published: October 3, 2017 6598

DOI: 10.1021/acs.nanolett.7b02413 Nano Lett. 2017, 17, 6598−6605

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Figure 1. A solvent-universal platform for single molecule optical tweezers. Polystyrene@silica core−shell microspheres are stably optically trapped in a wide variety of solvents. Poly(methyl methacrylate) polymers are grafted to the microsphere surface serve as linkers. Click-chemistry end functionality of the linkers and molecule of interest allow for single molecule tethers to be formed in situ. The mechanical and optical properties of the immobilized molecule at the center can be measured in solution when the construct is extended 500−1000 nm to avoid surface-specific chemistry. (The star represents the in situ link formation via inverse electron demand Diels−Alder click chemistry.)

that can be trapped in organic solvents, (e.g., silica lacks refractive index contrast, polystyrene rapidly swells and dissolves) the complete insolubility of the DNA handles, and the incompatibility of the antibody−antigen linkages critical to this construct. To overcome these obstacles limiting applications of optical tweezers, we developed a solvent-universal design that utilizes core−shell microspheres, synthetic polymers, and click chemistry (Figure 1). With broad chemical applicability as the driving design factor, this system must: display compatibility with most common organic solvents, allow for steric, electronic, and length tunability of the polymer tethers, and utilize robust, selective and orthogonal chemistry for tether link formation. This system will avoid the chemical heterogeneity induced by direct surface attachment, as the molecules under investigation are 500−1000 nm away from the trapped microspheres’ surface when they are probed. We anticipate this new OT construct will allow for SM study of many waterincompatible molecules. Additionally, the geometry of this dual optical tweezers platform will allow for the facile coupling of an orthogonal optical probe, because the OT intrinsically provides ∼ nm localization of the probed molecule with a spectroscopically clear background. Microspheres for Solvent-Independent Optical Trapping. At the foundation of the proposed OT construct are microspheres that can be stably trapped in many different organic solvents. The design criteria for these microspheres are refractive index contrast relative to common organic solvents,34 facile chemical modification, and a monodisperse diameter on the order of the trapping laser wavelength, 1064 nm. Our first strategy was to modify the widely used Stöber process35 for SiO2 microsphere synthesis with either TiO2 dopants36 or core−shell37,38 architectures, which in our hands proved unsuccessful in reproducibly creating monodisperse and optically trappable microspheres (see SI for further information). Instead, a successful approach was to synthesize 850 nm polystyrene core−shell microspheres (PS@SiO2) as reported by Lu et al.39 Briefly, this modified Stöber process used 750 nm cationic, amine-coated PS cores dispersed in isopropanol− water as condensation seeds for tetraethylorthosilicate, resulting in a 50 nm SiO2 shell. This method was reproducible on the milligram scale across multiple batches, and both the SiO2 coating depth and microsphere size were confirmed with scanning electron microscopy (Figure 2). The resulting microspheres had a diameter of 850 nm ±35 nm, though

Figure 2. Scanning electron micrograph of the synthesized polystyrene@silica core−shell microspheres. 750 nm polystyrene cores serve as condensation seeds for tetraethylorthosilicate, which yields 850 nm microspheres that are optically trappable and stable in organic solvents. Panel a shows detail from panel b.

nucleation products of approximately 220 nm were observed with every synthetic attempt, even after employing the suggested reaction modifications from Lu et al.39 These secondary nucleations comprised 0.05% by mass and did not affect further experimental steps due to a lack of refractive index contrast. Optical trapping experiments were conducted with PS@SiO2 microspheres (referred to as simply “beads” from here on) in a 6599


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probed. Moreover, the fitted diffusion constants correlate with those predicted for 850 nm spheres from Debye−Stokes− Einstein theory (see SI for further information). Table 1

variety of solvents using a home-built OT instrument (ZELDA). Stable trapping was observed and characterized by measuring the power spectrum of trapped bead position fluctuations in response to an oscillatory driving of the sample chambera hallmark calibration procedure for the stiffness and sensitivity of optical tweezers40as shown in Figure 3. These

Table 1. Summary of Solvent Parameters, Observed Optical Trapping Behavior of the PS@SiO2 Microspheres, and Polymer−Microsphere/Polymer−Polymer Link Formation Behaviora solvent

dielectric constant

eluent strength

refractive index at 1064 nm

trap stiffness (pN/nm)

links observed

hexane toluene chloroform EtOAC THF isopropanol acetone ethanol DMF acetonitrile water

1.9 2.4 4.8 6 7.5 19 21 24 37 37.5 80.1

0.01 0.29 N/A 0.58 0.57 0.82 0.56 0.88 N/A 0.65 ≫1

1.3685 1.4812 1.4354 1.3700 1.4100 1.3763 1.3525 1.3536 1.4300 1.3357 1.3239

N/A 0.24 0.08 0.16 0.16 0.14 0.16 0.13 0.15 0.15 0.12

N/Ab yesc yes yes yes no no no no no no

a

The microspheres were trappable for all solvents in which they were soluble (eluent strength > 0.1). PMMA tether links were observed in solvents with dielectric constant 35 pN arises from nonlinearity of the trapping potential.

protein of interest is attached to the beads. A general experiment to probe for tether formation between two optically trapped beads in a given solvent was to approach the beads at 2 μm/s until they reached close proximity (0−300 nm), wait for 5 s to allow tethers to form, and increase the trap separation at 700 nm/s to probe for entropic elasticity typical of a polymer. This process was repeated three times with a bead pair before they were marked as “no interaction.” As control experiments, tether formation was probed in all combinations of non6602


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extension experiments. It was found that tether formation was correlated with solvent dielectric constant more than any other property; tethers were not observed to form in polar solvents with dielectric constants ε > 10. (Table 1) As an independent concern, bead solubility was correlated with the eluent strength (ES, a measure of solvent absorption energy on silica, with pentane defined as 0); polar solvents with an ES > 0.3, such as ethyl acetate, CHCl3, and THF, readily solubilized the beads, less polar solvents, such as toluene (ES = 0.29), required surfactant, and nonpolar solvents, such as hexane (ES = 0.01) will require modification of the bead surface chemistry. Thus, the platform described here is most applicable to the range limited by ES > 0.25 and ε < 10. Stable trapping with an average stiffness ∼0.15 pN/nm was observed in all solvents, with a weak correlation between stiffness and refractive index of the solvent.34 Changes in the diffusive behavior of the beads were clearly visible in brightfield microscopy and were correlated to predictions based on the solvent viscosity and bead diameter with a systematic offset (see SI for further information). It is likely that a more accurate measurement of the temperature near the optical trap and treatment of the surface hydrodynamics will result in better agreement here.61 The single molecule force extension experiments described here are the first observations focused on the mechanical properties of synthetic polymers in the low pN force regime.55,56 The observed contour lengths match well with the lengths obtained from GPC given the polydispersity of the high MW polymers. The persistence lengths obtained from WLC fits were 0.4−1.7 nm, greater than the 0.3 nm length expected from a tetrahedral C−C−C bond, and without a clear solvent-dependent trend. This observation warrants further study and may result from the formation of tertiary structures around the lower critical solution temperature of PMMA,57,58 knot formation,62 and modification of the effective persistence length due to attachment geometry to the optically trapped bead.1 Design Modularity. This new platform is particularly powerful given its modular synthesis with three primary avenues for synthetic manipulation. First, though the PS@ SiO2 beads described here were readily soluble in most organic solvents, surfactant or surface modification with nonpolar alkyl trichlorosilanes can expand compatibility to the lowest dielectric solvents. Second, a variety of polymers are accessible with this platform given the diverse monomer choice inherent to ATRP (Figure 5a).41,42 With the correlation observed between PMMA link formation and solvent polarity, we anticipate that different synthetic polymers may be used to form SM linkages in a broader collection of solvents, by tuning the monomer polarity. Our platform could stand as an alternative to the antibody/antigen/DNA construct used in water,68,69 or other high dielectric solvents, by using acrylic acid or acrylamide ATRP polymers or even non-ATRP polymers such as poly(ethylene glycol) and polyproline with the simple application of peptide couplings. Finally, the use of click chemistry enables any molecule that can be modified with the widely available copper-free click moieties, including N3, TCO, DBCO, and cyclopropene, to be placed into this new OT construct. Figure 5b highlights three possible bead pairing options from this work, though many more can be imagined, and Figure 5c shows how the design modularity could incorporate a diverse set of molecules, such as a force-sensitive polymer,63−65 a force-activated isomerizable system,66,67 or a transition metal complexes for purely spectrocopic investiga-

persistence lengths of 0.4−1.3 nm in THF, ethyl acetate, toluene, and CHCl3 with no clear solvent dependence. The occurrence of a single-step rupture event definitively established that a SM tether was formed and provided an empirical point of comparison for tethers that did not rupture. Interestingly, in both polymer-bead and polymer−polymer tether constructs, linking behavior was only observed in THF, toluene, ethyl acetate, and CHCl3. We hypothesize that the lack of tether formation in more polar solvents arises from a slow rate of encounter of the polymer termini (which is strongly influenced by the solubility of PMMA) rather than degradation of the end groups.51 In more polar solvents (bottom 6 rows of Table 1), the predominant behavior (95%) was no interaction between bead pairs. No SM tether formation was observed, and even the stuck bead phenotype was extremely rare. This experiment represents the OT design we anticipate employing in single molecule chemistry experiments going forward by functionalizing the molecule of interest with N3, TCO, DBCO, or TET (Figure 1 and Figure 5b and c). As the trapped SM will be

Figure 5. Highlights of design modularity in the new optical tweezers construct. (a) Polymers accessible using ATRP for use in different solvents. (b) Building on the proof of principle demonstrated herein, other easily accessible solvent-robust linkages may be formed in situ. (c) Attachment of click chemistry handles could allow for mechanical properties of polymeric or force-activated systems to be probed with this optical tweezers assay, or the tweezer handles could allow for immobilization of single molecules for spectroscopic study. Molecules in panel c pair with the described PMMA-N3 and PMMA-TET beads.

immobilized in solution, ∼750 nm away from any surface, this construct alleviates surface effects and allows the molecule to be probed by force application and potentially optical spectroscopy. Solvent Effects on OT Experiments. With many new solvents accessible to the OT platform for the first time, we were particularly interested in solvent effects on the force 6603


Nano Letters tion. We are excited by the variety of solution-phase chemistry that can be investigated mechanistically and with structural resolution on the single molecule level. Summary. We have successfully demonstrated optical trapping and single molecule tether formation in organic solvent. The core−shell beads we have synthesized demonstrate stable trapping in a wide variety of organic solvents, opening the door to SM experiments in a broad set of conditions relevant for solution-phase chemistry. While the PS@SiO2 beads described here were found to be readily soluble in most organic solvents, in less polar solvents we have demonstrated that they may still be optically trapped and used to form linkages with the addition of surfactant. Single molecule tethers of PMMA were formed in situ and characterized with force−extension measurements. These data demonstrate all the hallmarks of single tether force spectroscopy established in aqueous solutions; namely, agreement with the worm-like chain model is observed with clear one-step rupture events. Furthermore, the fit-derived contour length of the PMMA polymers is consistent with the GPC analysis and increases by a factor of 2 upon forming an end-to-end polymer linkage. These measurements establish an optical-tweezer dumbbell platform for single molecule force-spectroscopy in organic solvents and lay the groundwork for probing chemical reactions on the single molecule level. As the technical challenges for combining single molecule optical tweezers and fluorescence have been addressed,8,9,70,71 this platform is primed to incorporate single molecule fluorescence as an orthogonal probe to the force measurements and possibly provides an exciting new way to probe catalysis via fluorescent substrates28,72−74 or structurespectral correlations in fluorescent polymers.63−65

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ABBREVIATIONS

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REFERENCES

OT, optical tweezers; SM, single molecule; PMMA, poly(methyl methacrylate); AFM, atomic force microscopy; PS, polystyrene; TEOS, triethylorthosilicate; ATRP, atom transfer radical polymerization; SPAAC, strain-promoted azide−alkyne cycloaddition; iEDDA, inverse electron demand Diels−Alder; TCO, trans-cyclooctenol; TET, tetrazine; APTES, (3aminopropyl)triethoxysilane; DBCO, dibenzocyclooctyne acid; NHC, N-heterocyclic carbene

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.7b02413. Instrument description, all synthesis procedures, SEM images of microspheres, HPLC chromatogram of PMMA polymers, images of colorimetric microsphere controls, sample chamber design, click chemistry kinetics, trends in optical trapping fit parameters, table of solvent properties and optical trapping behavior (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (203) 436-9370. ORCID

Ziad Ganim: 0000-0002-8275-269X Present Address

M.K.: Boston University, Department of Chemistry, Department of Physics, Boston, MA 02215, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE1122492 to J.B. 6604


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