Nano- and micro-fabrication for single-molecule biological studies

Heterogeneity is a general feature in biological system. In order to avoid possible misleading effects of ensemble averaging, and to ensure a correct understanding of the biological system, it is very important to look into individuals, such as a single bio-molecule or a single cell, for details. The size of a single bio-molecule/cell typically ranges from nanometer to micrometer scale. Therefore, the tools for study single-molecule/cell often consist of nano- and micro-features. The power of nanotechnology is such that it is possible to fabricate a wide variety of nano- and micro-scale structures and devices, which find more and more frequent application as powerful tools for biophysical studies of single-molecule/cell. This thesis reports several nano- and micro-fabricated structures that I, together with my colleagues, have developed for biophysical studies at single-molecule/cell level: (1) Nanofabricated zero-mode waveguides for single-molecule fluorescence experiments at biologically-relevant high concentrations; (2) Microfabricated mirrors for three-dimensional fluorescence imaging and tracking of single molecule / particle; (3) Microfabricated polydimethylsiloxane-based microfluidics device for studying submicron-scale bacteria (4) Microfabricated birefringent cylinders for use in optical torque wrench to study torsional properties of single biomolecules. Our main interest in zero-mode waveguide lies in its powerful potential to study single telomerase at work by visualizing individual incorporation of dye-labeled nucleotides. We have successfully developed several key fundamental elements towards single-molecule fluorescence studies of telomerase in ZMW: we have designed a biotin-labeled oligonucleotide specifically for human telomerase assay in ZMW - it can be immobilized on the ZMW floor via its biotin group, and it has the highest affinity for base-pairing with telomerase. We have also designed and acquired two types of special modified nucleotides for this assay: fluorescently phospholinked nucleotides (TMR-?-dATP and Atto532-dG6P), and we have demonstrated that both phospholinked nucleotides can be incorporated processively by human telomerase. We have constructed an optical setup specifically for single-molecule fluorescence studies in ZMWs. This setup can operates in two different modes, namely, massive parallel detection mode (using wide-field illumination and EMCCD detection), and high-speed single spot mode (using focused illumination spot and APD for high speed detection). We have successfully developed methods for nano-fabrication of ZMWs. We have also performed extensive characterizations on our ZMW devices using SEM (device geometric profiles), and FCS (detection volume, fluorophore working concentration). Our characterization results show that we are able to controlledly fabricate ZMWs with a suitable size (ca. 80nm in diameter) for single-molecule fluorescent studies at biologically relevant concentration (> 1?M), which is very important for meaningful studies of telomerase kinetics. Finally, we have demonstrated a method for successful surface treatment of ZMWs, by which the DNA substrates can be tethered specifically onto the glass floor of ZMWs, and more importantly, the non-specific transient adsorption of labeled nucleotides on ZMW surfaces has been reduced to a sufficient low level (one order of magnitude lower than the typical rate of nucleotide incorporation). Microfabricated mirror is one of the most promising tools for high-precision 3D imaging and particle tracking. We have developed a method based on electron beam lithography and wet etching of single-crystal silicon for the fabrication of V-groove micromirrors. 54.7°-symmetric V-groove micromirror was fabricated using regular (100) silicon wafer. To fabricate a mirror facet 45° relative to wafer surface, an off-axis cut silicon wafer ( off 9.7° to ) was used. We have demonstrated that our V-groove micromirrors could be assembled into flow cell structures for imaging single fluorescent particles. We have also been developing a novel algorithm based on maximum likelihood estimation (MLE) for 3D tracking of single molecule/particle using micromirrors. Our simulation results demonstrated that our MLE tracking method outperformed center-of-mass tracking method as developed by Berglund et al. The ability to restrict the movement of cells in a controlled manner using microfluidics, allows one to study individual cells and gain added insight into aspects of their physiology and behaviour that can potentially be hidden in ensemble averaging experiments. We have developed a novel protocol based on electron beam lithography together with specific dry etching procedures for the fabrication of a microfluidic device suited to study submicron-sized bacteria. In comparison to approaches based on conventional optical lithography, our method provides greater versatility and control of the dimensions of the growth channels while satisfying the rapid-prototyping needs in a research environment. The widths of the submicron growth channels allow for the potential immobilization and study of different size bacteria with widths ranging from 0.3 ?m to 0.8 ?m. We verified by means of SEM that these structures are successfully transferred from Si into polydimethylsiloxane (PDMS) as well as from PDMS into PDMS. As a proof-of-principle, we demonstrated that the bacterium L. lactis can successfully be loaded and imaged over a number of generations in this device. Similar devices could potentially be used to study other submicron-sized organisms under conditions where the height and shape of the growth channels are crucial to the experimental design. The Optical Torque Wrench (OTW) is a special type of optical tweezers (OT) that uses birefringent dielectric particles, and has proved to be one of the most promising tools for torsional manipulation and torque measurement of single biomolecules. The main difference between OTW and conventional OT is that OTW uses a birefringent dielectric particle, which can be rotated by controlling the polarization of trapping laser, and therefore is able to apply and measure torque on the biomolecule attaching to the particle. We describe the use of electron beam lithography for the design, fabrication and functionalization of micron-scale birefringtent quartz cylinders. We demonstrate excellent control of the cylinders’ geometry, fabricating cylinders with heights of 0.5–2 ?m and diameters of 200–500 nm with high precision while maintaining control of their side-wall angle. The flexible fabrication allows cylinders to be selectively shaped into conical structures or to include centered protrusions for the selective attachment of biomolecules. The latter is facilitated by straightforward functionalization targeted either to a cylinder’s face or to the centered protrusion alone. The fabricated quartz cylinders are characterized in an optical torque wrench, permitting correlation of their geometrical properties to measured torques. In addition, we tether individual DNA molecules to the functionalized cylinders and demonstrate the translation and rotational control required for single-molecule studies. By using micron-scale birefringent particles, OTW has the ability to measure torque of the order of kBT (~4 pN\u95nm), which is especially important in the study of biophysical systems at the molecular and cellular level. Quantitative torque measurements rely on an accurate calibration of the instrument. We have described and performed various methods of OTW calibration, some with direct OT analog and others developed specifically for the angular variables. Overall, the different methods lead to close results, which also agree with the theoretical prediction for the particle drag coefficient. However, the absolute values of the variables measured by the instrument should be expected to depend on the details of calibration method chosen. Motivated by the potential of the OTW to access the fast rotational dynamics of biological systems, a result of its all-optical manipulation and detection, we focus on the angular dynamics of the trapped birefringent particle, demonstrating its excitability in the vicinity of a critical point. This links the optical torque wrench to nonlinear dynamical systems such as neuronal and cardiovascular tissues, nonlinear optics and chemical reactions, all of which display an excitable binary (‘all-or-none’) response to input perturbations. On the basis of this dynamical feature, we devise and implement a conceptually new sensing technique capable of detecting single perturbation events with high signal-to-noise ratio and continuously adjustable sensitivity. Last but not least, we describe our efforts towards the study of single bacterial flagellar motor in OTW, which is one of our main interests in developing OTW technology. Bacterial flagellar motor is one of the most interesting and most complex molecular machines. Torque generation plays a crucial role in its functionality. Our progresses towards the study of torque generation in flagellar motor using OTW include: (1) A controlled functionalization of quartz cylinders has been developed for attaching a cylinder to a spinning flagellum, and importantly with the flagellum tethered to the cylinder’s center to avoid precession; (2) A theoretical framework has been developed to describe the rotational kinetics of a flagellum-tethered cylinder in the OTW. (3) A novel fabrication approach has been developed for nano-fabrication of birefringent particles using TiO2 rutile, which has a birefringence 32 times larger than quartz. This will enlarge the range of rotational frequency in which the flagellar motor can be studied in OTW; (4) A possible alternative construction of OTW based on circular polarized light for producing constant torque has been considered, and a method for calibration of such construction is also been discussed theoretically.Kavli Institute of NanoscienceApplied Science