Inserting and Manipulating DNA in a Nanopore with Optical Tweezers

The translocation of long-chain molecules, such as DNA or peptides, through membranes is an integral process for the function of living cells. During the translocation process, a number of interactions of electrostatic or hydrophobic nature govern the translocation velocity. Most of these interactions remain largely unexplored on the single-molecule level due to a lack of suitable instrumentation. We have shown that a combination of optical tweezers, single solid-state nanopores, and electrophysiological ionic current detection can provide further insight into the behavior of polymers in confinement. Here, we describe the experimental procedures necessary for manipulation of single biopolymers in a single nanopore not only by electrical fields, but also through mechanical forces using optical tweezers.

While the translocation of linear polymers through nanopores is well-understood, the underlying mechanism of transport of branched polymers through nanopores is yet to be fully developed. In this general premise, we have investigated the translocation of multiarm star-like polyethylene glycols (PEGs) through single solid-state nanopores, using single-molecule electrophoresis. Our experiments reveal the conformational trajectories of multiarm-PEGs during their sojourn inside the nanopore in exquisite detail. We quantify these pathways in terms of the number of leading arms (fin) and the number of lagging arms (fout), which depend on the pore diameter (d) and the total number of arms (f). We have measured the average translocation time (τ), polymer capture rate (Rc), and polymer conformations during translocation in terms of d, f, and applied voltage (Vm). We find a direct proportionality between Rc and fVm, and between τ and f/Vm. Interestingly, star polymers with more arms inside the nanopore (fin) than outside (fout) also translocate successfully, in contrast with previous suggestions of fin < fout. As the pore size increases, the optimal fin shifts from 0.25f to 0.5f. In addition to gaining insight into the mechanism of translocation of star-like polymers, the present experimental strategy opens new opportunities to characterize and separate polymers with different branching architectures.

We use solid-state nanopores to study the dynamics of single electrically charged colloids through nanopores as a function of applied voltage. We show that the presence of a single colloid inside of the pore changes the pore resistance, in agreement with theory. The normalized ionic current blockade increases with the applied voltage and remains constant when the electrical force increases even more. We observe short and long events of current blockades. Their durations are associated, respectively, with low and high current variation. The ratio of long events increases with the electrical force. The events frequency increases exponentially as a function of applied voltage and saturates at high voltage. The dwelling time decreases exponentially at low and medium voltages when the electrical force increases. At large voltages, this time decreases inversely proportionally to the applied voltage. The long events are associated with translocation events. We show that the dynamics of colloids through the nanopore is governed mainly by two mechanisms, by the free-energy barrier at relatively low and medium voltages and by the electrophoresis mechanism at high voltage.

Selective permeability is a key feature of biological membranes. It is controlled by the physico-chemical properties of the lipid bilayer and by channel-forming membrane proteins. Here we focus on the permeation of small molecules across channel-forming proteins in Gram-negative bacteria called porins and present a new approach based on artificial amino acids. We introduced Hco, a fluorescent amino acid with characteristic excitation and emission wavelengths, into OmpF and measured FRET from Hco to dissolved Bocillin FL using solubilized OmpF porins. We examined four variants of OmpF and by doing so, we were able to show that small molecules, like Bocillin FL, are remaining long enough in the porin in order to undergo FRET and produce a reproducible fluorescence signal. This finding opens the way to quantify translocation in the future by the simultaneous detection of resistive pulses and differential fluorescence with FRET as an example.

Single-molecule mechanics of synthetic aromatic amide helices: Ultrafast and robust non-dissipative winding

The spin‐orbit interaction (SOI) of light generated by tight focusing in optical tweezers is regularly employed in generating angular momentum ‐ both spin and orbital ‐ the effects being extensively observed in trapped mesoscopic particles. Specifically, the transverse spin angular momentum (TSAM), which arises due to the longitudinal component of the electromagnetic field generated by tight focusing is of special interest, both in terms of fundamental studies and associated applications. This study provides an effective and optimal strategy for generating TSAM in optical tweezers by tightly focusing first‐order radially and azimuthally polarized vector beams with no intrinsic angular momentum (AM) into a refractive index stratified medium. The choice of such input fields ensures that the longitudinal spin angular momentum (LSAM) arising from the electric (magnetic) field for the radial (azimuthal) polarization is zero. As a result, the effects of the electric and magnetic TSAM are exclusively observed separately in the case of input first‐order radially and azimuthally polarized vector beams on single optically trapped birefringent particles. This research opens up new and simple avenues for exotic and complex particle manipulation in optical tweezers.

Single-molecule mechanochemical sensing (SMMS) is a novel biosensing technique using mechanical force as a signal transduction mechanism. In the mechanochemical sensing, the chemical binding of an analyte molecule to a sensing template is converted to mechanical signals, such as tensile force, of the template. Since mechanical force can be conveniently monitored by single-molecule tools, such as optical tweezers, magnetic tweezers, or Atomic Force Microscopy, mechanochemical sensing is often carried out at the single molecule level. In traditional format of ensemble sensing, sensitivity can be achieved via chemical or electrical amplifications, which are materials intensive and time-consuming. To address these problems, in 2011, we used the principle of mechanochemical coupling in a single molecular template to detect single nucleotide polymorphism (SNP) in DNA fragments. The single-molecule sensitivity in such SMMS strategy allows to removing complex amplification steps, drastically conserving materials and increasing temporal resolution in the sensing. By placing many probing units throughout a single-molecule sensing template, SMMS can have orders of magnitude better efficiency in the materials usage (i.e., high Atom Economy) with respect to the ensemble biosensing. The SMMS sensing probes also enable topochemical arrangement of different sensing units. By placing these units in a spatiotemporally addressable fashion, single-molecule topochemical sensors have been demonstrated in our lab to detect an expandable set of microRNA targets. Because of the stochastic behavior of single-molecule binding, however, it is challenging for the SMMS to accurately report analyte concentrations in a fixed time window. While multivariate analysis has been shown to rectify background noise due to stochastic nature of single-molecule probes, a template containing an array of sensing units has shown ensemble average behaviors to address the same problem. In this so-called ensemble single-molecule sensing, collective mechanical transitions of many sensing units occur in the SMMS sensing probes, which allows accurate quantification of analytes. For the SMMS to function as a viable sensing approach readily adopted by biosensing communities, the future of the SMMS technique relies on the reduction in the complexity and cost of instrumentation to report mechanical signals. In this account, we first explain the mechanism and main features of the SMMS. We then specify basic elements employed in SMMS. Using DNA as an exemplary SMMS template, we further summarize different types of SMMS which present multiplexing capability and increased throughput. Finally, recent efforts to develop simple and affordable high throughput methods for force generation and measurement are discussed in this Account for potential usage in the mechanochemical sensing.

The dynamic back-action caused by electromagnetic forces (radiation pressure) in optical and microwave cavities is of growing interest. Back-action cooling, for example, is being pursued as a means of achieving the quantum ground state of macroscopic mechanical oscillators. Work in the optical domain has revolved around millimetre- or micrometre-scale structures using the radiation pressure force. By comparison, in microwave devices, low-loss superconducting structures have been used for gradient-force-mediated coupling to a nanomechanical oscillator of picogram mass. Here we describe measurements of an optical system consisting of a pair of specially patterned nanoscale beams in which optical and mechanical energies are simultaneously localized to a cubic-micron-scale volume, and for which large per-photon optical gradient forces are realized. The resulting scale of the per-photon force and the mass of the structure enable the exploration of cavity optomechanical regimes in which, for example, the mechanical rigidity of the structure is dominantly provided by the internal light field itself. In addition to precision measurement and sensitive force detection, nano-optomechanics may find application in reconfigurable and tunable photonic systems, light-based radio-frequency communication and the generation of giant optical nonlinearities for wavelength conversion and optical buffering.

Solid-State Nanopore Translocation of Idealized Helical Repeat Proteins

Cells sense mechanical signals and forces to probe the external environment and adapt to tissue morphogenesis, external mechanical stresses and a wide range of diverse mechanical cues. Here, we propose a combination of optical tools to manipulate single cells and measure the propagation of mechanical and biochemical signals inside them. Optical tweezers are used to trap microbeads that are used as handles to manipulate the cell plasma membrane; genetically encoded FRET-based force sensors inserted in F-actin and alpha-actinin are used to measure the propagation of mechanical signals to the cell cytoskeleton, while fluorescence microscopy with single-molecule sensitivity can be used with a huge array of biochemical and genetic sensors. We describe the details of the setup implementation, the calibration of the basic components and preliminary characterization of actin and alpha-actinin FRET-based force sensors.

Complete handling of single DNA molecules and their enzymatic restriction are described. For that purpose a microsphere was bound to a DNA molecule and trapped by optical tweezers. It could be moved in any direction. For stretching or rotating the molecule an electrical field was applied while the bead was fixed by the optical tweezers. In a near-equilibrium state of the resulting forces, the DNA remained stretched. Subsequently, a restriction endonuclease was activated by liberating Mg<SUP>2+</SUP> from a caged compound. The enzymatic reaction could be directly observed in the light microscope. While the bead remained in the focus of the laser trap the other restricted part of the DNA molecule is pulled away in the electrical field. The reaction was directly monitored and recorded on videotape.

We demonstrate that tight focusing of a circularly polarized Gaussian beam in optical tweezers leads to spin-momentum locking—with the transverse momentum density (Poynting vector) being helicity-dependent, while the transverse spin angular momentum density becomes independent of helicity. We further use a stratified medium in the path of the trapping beam in our optical tweezers setup to enhance the magnitude of the transverse momentum and the electric field intensity in the radial direction with respect to the beam axis and cause them to significantly overlap. This overlap allows us to experimentally observe the circular motion of a birefringent particle, trapped off-axis, in response to an input circularly polarized fundamental Gaussian beam carrying no intrinsic orbital angular momentum (OAM). The circular motion is dependent on the helicity of the input beam so that we can identify it as the signature of the elusive Belinfante spin in propagating light beams obtained in our optical tweezers configuration. Our results can be extended to beams carrying intrinsic OAM leading to simple routes for achieving complex manipulation of micro-machines or other mesoscopic matter using optical tweezers.

This thesis is focused on the potential applications of ion track-etched nanopores in polymeric membranes, such as molecular separation, gating and biosensing. The nanopores are fabricated in polyethylene terephthalate (PET) membranes by heavy ion track-etching technique. Ion track technology provides the feasibility to produce membranes with nanopores of desired diameters and geometries. The characterizations of the nanopores are performed by field emission scanning electron microscopy, replica technique and conductometry measurements. Heavy ion irradiation and subsequent track-etching lead to the generation of carboxylic groups (–COOH) on the inner pore walls. At neutral pH, nanopores are cation-selective due to the presence of ionized carboxylate groups (–COO–) on the pore surface. The chemical modification of –COOH groups with amino-terminated molecules switches the surface polarity from negative to positive which in turn flipped the nanopore perm-selective behaviour. Polymeric membranes containing cylindrical nanopores are employed for the transport of ionic organic (bio)molecules through the nanopores. The nanopores have the ability to selectively transport and discriminate the molecules based on their size and charge. For the transport studies main focus is the modulation of pore density, pore diameter, ionic strength of buffer solution and electric field which affect the ionic selectivity and flux of the membrane. Moreover, the polyethyleneimine (PEI) modified pores are also applicable as gate to control the ionic/molecular flux across the membrane. Finally, single (conical and cylindrical) nanopores modified with boronic acid receptor are studied for the molecular recognition of monosaccharides and glycoprotein inside confined geometries. The recognition events are observed by measuring the current – voltage (I–V) curves in an electrolyte solution in the presence of glucose, fructose and glycoprotein separately.

Single solid-state nanopores modified with poly-N-isopropylacrylamide (NIPAM) brushes display thermally controlled gating properties. Below the lower critical solubility temperature (LCST) NIPAM brushes are swollen and, consequently, dramatically reduce the effective cross section of the nanopores (see image). Conversely, above the LCST the brushes dehydrate and suffer a transition into a collapsed state, which promotes the widening of the nanopore and enables a substantial flow of ions.

The Unfolding Behavior of RNase H Under Force