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An Optical Tweezers Platform for Single Molecule Force Spectroscopy in Organic Solvents Jacob Black, Maria Kamenetska, and Ziad Ganim Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b02413 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 4, 2017
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An Optical Tweezers Platform for Single Molecule Force Spectroscopy in Organic Solvents Jacob W. Black†, Maria Kamenetska†‡, Ziad Ganim*† †Yale University, Department of Chemistry, 350 Edwards St., New Haven CT 06520 ‡Current Address: Boston University, Department of Chemistry, Department of Physics, Boston, MA 02215 KEYWORDS: force spectroscopy, poly(methyl methacrylate), polymers, core-shell nanoparticles, click chemistry, atom transfer radical polymerization. ABSTRACT: Observation at the single molecule level has been a revolutionary tool for molecular biophysics and materials science, but single molecule studies of solution-phase chemistry are less widespread. In this work we develop an experimental platform for solution-phase single molecule force spectroscopy in organic solvents. This optical-tweezer-based 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.
For 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 minuteshours.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 timescales 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) provide a mechanical probe that is much softer than AFM (stiffness 0.0002-0.4 vs 10-60 pN/nm), and is therefore well-suited to investigate the 50nm from the optically trapped bead surface and trapping laser with no sign of surface-induced heterogeneity.26-28 Immobilizing single molecules with OT provides distinct advantages that complement single molecule spectroscopies such as fluorescence7-9 or Raman29, in particular:~nm sample immobilization, little out-of-focus background, and straightforward overlap of beam paths. However, 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 solvent-universal analogy of the biophysical dumbbell assay are primarily due to a lack of commercially available microspheres 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.
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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. Clickchemistry 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.)
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 water-incompatible 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 SolventSolvent-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 info). 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 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 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 Fig. 3. These power spectra, I(f), are typical of an overdamped harmonic oscillator, and were well-fit by two parameters in the Lorentzian form, D I(f) I K K , (1) π (fL + f K ) in which the corner frequency (fc) and diffusion constant of the particle (D) are related to the harmonic force constant of the optical trap, κ, by,40 2πk N T κI fL . (2) D Here, kB is Boltzmann’s constant and T is the absolute temperature. These power spectra demonstrate stable optical trapping. The optical trapping force constants are comparable to those for analogous beads in water, ~ 0.2 pN/nm at 1 W of laser power,1, 8, 15 and roughly scale with the solvent refractive index, which indicates that a similar force regime may be probed. Moreover, the fitted diffusion constants correlate with those predicted for 850 nm spheres from Debye-StokesEinstein theory (see SI for further info). Table 1 summarizes properties of the solvents, observed force constants, and eventual link formation with PMMA tethers. Stable optical trapping of PS@SiO2 beads in ten different organic solvents was observed in contrast to three separate controls: PS beads, SiO2 beads, and PS@SiO2 beads whose PS core was removed by cal-
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cination at 500 °C for 2 hours. Trapping in these solvents was indefinite, excepting toluene, tetrahydrofuran, and CHCl3 where etching of the bead core was observed after a few days, as previously reported by Lu et al.39 Toluene required 0.1 mM tetraoctylammonium bromide as a surfactant to fully solubilize the beads, and all attempts to solubilize the beads in hexanes were unsuccessful, even with many different surfactants at high concentrations. This work represents the first widely applicable optical trapping solution for a variety of different solvents ranging in polarity, hydrogen bonding character, and halogenation.
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 (b).
Synthetic Polymer Tethers. In a similar fashion to the microspheres, the polymers employed as tethers must meet specific design criteria including: solubility, monodispersity, and control over chemical functionality at both ends. Living polymers generated from atom transfer radical polymerization (ATRP)41-44 met the above criteria. ATRP allows for a variety of monomer choices, with reactivity at the polymer head determined by the choice of radical initiator, and a broadly reactive alkyl halide at the tail. Drawing inspiration from the work of Ohno et al,45 we synthesized high MW poly(methyl methylacrylate) (PMMA) polymers using a triethylorthosilicate (TEOS) initiator to facilitate SiO2 attachment. (Scheme 1a) Following a twostep synthesis, the silicate polymerization initiator (1 1) was used in the Cu(I)-catalyzed ATRP method of Ohno et al,45 which was modified to use a 1:1 ratio of anisole:methacrylate as the solvent to accommodate the highly viscous reaction mixture observed as the polymerization neared completion. The TEOS-PMMA-Br polymer (2 2) was characterized with 1H and 13C NMR spectroscopy as well as Gel Permeation Chromatography (GPC), which indicated formation of PMMA polymers with a number average molecular weight, MN, of 249 kDa and a polydispersity index of 1.2. The predicted average polymer contour length was therefore 766 nm (I249 kDa ÷ 100 Da / monomer x 0.308 nm C-C-C backbone contour length) (see Scheme 1a and SI for further info). These TEOS-PMMA-Br polymers were then attached to the surface of the beads by vigorous stirring in heterogenous mixture of 1.2 mM ammonia in toluene, and finally converted from PMMA-Br coated beads (3 3) to PMMA-N3 coated beads (4 4). 46, 47 (Scheme 1b) The success of this final conversion and integrity of the end-functionality was confirmed by formation of single molecule tethers. Click Chemistry Linkers. The strategically installed azide at the terminus of the PMMA polymer provides several avenues for attaching a SM of interest using click chemistry.48 In particular, copper-free click chemistry49, 50 is an ideal companion to this new assay because the reactions are solvent-robust and can be performed rapidly in situ without the presence of a catalyst.49, 51-53 Additionally, click reactions are selective, orthogonal, and compatible with most ATRP-synthesized polymers. (It ACS Paragon Plus Environment
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is important to note that the mechanism of the click-reaction is not under investigation in this paper, but rather serves as facile linking method with the potential to incorporate a diverse set of molecules of interest in the future.) We measured the kinetics of two common copper free click reactions - strain promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels Alder (iEDDA) - to verify their orthogonality and characterize their rates in toluene. The reaction of dibenzocyclooctyne-acid (DBCO, 5) and benzyl azide had a rate constant of 4.2 M-1s-1, while the reaction of (E)-cyclooct-4enol (TCO, 7) and the simple symmetric dipyridinyl-s-tetrazine had a rate constant of 360 M-1s-1 (see SI for further info). The reaction of dipyridinyl-s-tetrazine with DBCO did not occur, while only trace amounts of TCO and benzyl azide reactivity were observed over the course of hours. With these rapid and selective results, we were optimistic about the implementation of SPAAC and iEDDA in the final OT construct.
Figure 3. Power spectra of optically trapped beads in a variety of solvents demonstrate stable optical trapping and data used for calibration. The spectra were acquired by Fourier transforming the bead position signal acquired using back focal plane interferometry at 1 W laser
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trapping power, and averaged for 10-30s as described by Tolić-Nørrelykke et al.40 The spectra are fit to obtain the stiffness and sensitivity. Noise at harmonics of 60 Hz is evident in some traces.
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Table 1. Summary of solvent parameters, observed optical trapping behavior of the PS@SiO2 microspheres, and polymermicrosphere/polymer-polymer link formation behavior. dielectric eluent constant strength
Solvent
trap refractive links index stiffness at1064nm (pN/nm) observed
Hexane
1.9
0.01
1.3685
N/A
N/A†
Toluene
2.4
0.29
1.4812
0.24
Yes‡
Chloroform
4.8
N/A
1.4354
0.08
Yes
EtOAC
6
0.58
1.3700
0.16
Yes
THF
7.5
0.57
1.4100
0.16
Yes
Isopropanol
19
0.82
1.3763
0.14
No
Acetone
21
0.56
1.3525
0.16
No
Ethanol
24
0.88
1.3536
0.13
No
DMF
37
N/A
1.4300
0.15
No
Acetonitrile
37.5
0.65
1.3357
0.15
No
Water
80.1
>>1
1.3239
0.12
No
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 < 10. †Beads were not soluble. ‡Beads solubilized with surfactant. Laser power was 1W for all stiffness measurements.
We developed the azide terminus chemistry to allow both polymer-microsphere (Figure 4a) and polymer-polymer tether formation (Figure 4b). Polymer-polymer linkages are formed using iEDDA chemistry by functionalizing the PMMA termini on complementary beads with trans-cyclooctenol (TCO) and tetrazine (TET). For this construct, PMMA-N3 coated beads were first mixed with DBCO, to convert the azide functionality to a carboxylic acid (6 6), and split into two batches which were subsequently ester coupled with TCO (7 7) to yield PMMA-TCO-coated beads (8 8) or peptide-coupled with 6-methyltetrazine-amine (9 9) to yield PMMA-TET-coated beads (10 10) 10 (See Scheme 1c). As an alternative construct, SPAAC chemistry was used to form polymer-bead linkages by synthesizing DBCO surface-coated beads to bind the PMMA-N3 coated beads (4 4). This two-step synthesis first required the coating of beads with amines using (3-aminopropyl) triethoxysilane (APTES, 11) 11 followed by the attachment of DBCO via peptide coupling (Scheme 1d). These bead preparations were verified by the successful formation of SM tethers, with contour lengths in agreement with the molecular weight shown in GPC chromatograms. Additionally, the surface coated DBCO beads (13 13) 13 were confirmed with an observable color change when mixed with N3-sulforhodamine B dye relative to the APTES coated control (12 12) 12 (see SI for further info). Using these four different bead preparations, we demonstrate both SPAAC and iEDDA click chemistry approaches for making single molecule linkages with the optical trap. These two can approaches extend optical trapping-based single molecule spectroscopy to a wide variety of chemical reactions by drawing on the creative and rapidly proliferating applications of copper-free click chemistry. PolymerPolymer- Bead Tether Formation. Single molecule force-extension experiments were performed on the (simpler) polymer-bead construct as an intermediate check and proof of principle. (Figure 4a) This polymer-bead linkage construct operates in analogy to some OT experiments,24 in which the 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 noncomplimentary beads (two bare beads, bare + PMMA-N3 coated beads, bare + DBCO surface-coated beads, two PMMA-N3 coated beads, and two DBCO surface-coated beads). In the control experiments, only two phenotypes were observed: no bead interaction and surface-surface attached beads ("stuck"). Next, tether formation was attempted in a sample with complimentarily functionalized (PMMA-N3 coated + DBCO coated) beads and a third phenotype emerged, namely forceextension curves consistent with a SM polymer linkage (Figure 4a). These curves fit well to the worm-like chain model,54 x WK 1 x kN T 1 S T1 − V − + X, (3) F(x) I p 4 L 4 L where x is the experimentally controlled extension, L is the contour length (end-to-end length of the polymer at infinite force), and p is the persistence length (a quantification of stiffness; higher is more rigid). Polymer contour lengths spanning 600-800 nm and persistence lengths of 0.7-1.7 nm were observed in THF, ethyl acetate, toluene, and CHCl3 with no clear solvent dependence (see SI for further info). This range of contour lengths is well beyond the 1 nm measurement error, and
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reflects the dispersity of polymer lengths as a result of the ATRP synthesis and observed in the GPC analysis (see SI for further info). Previous AFM measurements of PMMA and structurally similar polymers55, 56 have shown a persistence length of 0.3 nm, about five times less stiff than we typically observe. This inconsistency may be due to a helix-coil transition known to occur in PMMA57 that would affect the apparent stiffness,58 and is reminiscent of the spread in persistence lengths measured for DNA in OT.1, 25 Figure 4a also shows a rupture event, which when observed, provided definitive proof that a single molecule tether was formed. We attribute a rupture in this force range to oxidative damage to the tether59 rather than mechanical disruption of a covalent bond.60 To our knowledge this represents the first force-extension experiment with optical tweezers in a solvent other than water. While this construct does not provide a means for immobilizing a molecule of interest away from the bead surface, it does provide a synthetically efficient means for probing the elasticity of polymers and other macrostructures where edge effects due to surface conjugation are not of great concern. PolymerPolymer-Polymer Tether Formation. Single molecule force-extension experiments were performed on a construct where two polymer ends are linked in situ; this experiment represents the OT construct designed at the outset (Figure 1, Figure 4b) and is what we currently use to immobilize single molecules for study in solution. The experiment to probe for tether formation was identical to that for the polymer-bead tethers described in the previous section. Control experiments were conducted by probing for tether formation in all combinations of non-complimentary beads (two bare beads, bare + PMMATCO coated beads, bare + PMMA-TET coated beads, two PMMA-TET coated beads, and two PMMA-TCO coated beads). In the control experiments, again only two phenotypes were observed: no bead interaction and surface-surface “stuck” beads. When a sample of the complimentary PMMA- TET coated + PMMA-TCO coated beads was investigated, force-extension behavior that was well-described by the WLC polymer model was observed (Figure 4b). Here the fits yielded contour lengths of 1300-1500 nm and 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 & Figure 5b & c). As the trapped SM will be 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.
Scheme
1.
Synthesis
of
PMMA
and
Click
Chemistry
End-Functionalization.
a) Procedure for synthesizing PMMA polymers using ATRP; (b) attaching PMMA polymers to the beads and azide derivatization; and adapting the azide terminus for (c) polymer-polymer link formation using iEDDA or (d) polymer-bead link formation using SPAAC.
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Figure 4. 4. Force-extension curves of PMMA tethers obtained using the optical trap. The data show in situ click chemistry linkages formed between (a) polymer-bead and (b) polymer-polymer. In each panel, five cycles are shown (extend, relax, extend…) that demonstrate force increase as the polymers are extended due to their entropic elasticity, and rupture in a single step (indicated by blue arrow). The dashed line indicates a worm-like chain fit (Eq. 3) with average pI1.75 nm, LI720 nm (a) or pI0.39 nm, LI1417 nm (b). Solvents were THF and toluene, respectively. Traces are horizontally offset for clarity. Deviation from the fit at >35 pN arises from non-linearity of the trapping potential. 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 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 (ESI0.29), required surfactant, and non-polar solvents, such as hexane (ESI0.01) 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
