Molecular diagnostics is a fast growing field with new technologies being developed constantly. There is a demand for more sophisticated molecular tools able to detect a multitude of molecules on a single molecule level with high specificity, able to distinguish them from other similar molecules. This becomes very important for infectious diagnostics with the increasing antibiotic resistant viruses and bacteria, in gene based diagnostics and for early detection and more targeted treatments of cancer. For increased sensitivity, simplicity, speed and user friendliness, novel readouts are emerging, taking advantage of new technologies being discovered in the field of nanotechnology.  This thesis, based upon four papers, examines two novel electrical readouts for amplified single molecule detection. Target probing is based upon the highly specific amplification technique rolling circle amplification (RCA). RCA enables localized amplification resulting in a long single stranded DNA molecule containing tandem repeats of the probing sequence as product. Paper I demonstrates sensitive detection of bacterial genomic DNA using a magnetic nanoparticles-based substrate-free method where as few as 50 bacteria can be detected. Paper II illustrates a new sensor concept based on the formation of conducting molecular nanowires forming a low resistance circuit. The rolling circle products are stretched to bridge an electrode gap and upon metallization the resistance drops by several orders of magnitude, resulting in an extremely high signal to noise ratio. Paper III explores a novel metallization technique, demonstrating the efficient incorporation of boranephosphonate modified nucleotides during RCA.  In the presence of a silver ion solution, defined metal nanoparticles are formed along the DNA molecule with high spatial specificity. Paper IV demonstrates the ability to manipulate rolling circle products by dielectrophoresis. In the presence of a high AC electric field the rolling circle products stretch to bridge a 10 µm electrode gap.

Nanopore sensors are emerging as a promising technology for single molecule analysis and polymer sequencing. Traditionally, measurements are taken by monitoring the ionic current through the nanopore, which gives information (e.g. size, shape, charge) about a molecule of interest while it is in the confined geometry of the nanopore. The dynamics of the molecule before the arrival to the nanopore, such as the capture dynamics, or molecular conformation prior to translocation, as well as clogging mechanisms and features of anomalous translocation events, are not assessed by the electrical measurements alone. To study the whole process of nanopore diffusion, capture and passage it is necessary to complement the electrical signal with another detection mode. Particularly, optical visualization of the molecules as they translocate through the nanopore has great potential. In this Thesis I present the design, construction, optimization and testing of a nanopore--‐based optofluidic instrument, which uses fluorescence microscopy to visualize individual fluorescently stained DNA molecules as they translocate a solid--‐state nanopore, while in parallel record the ionic current signal through the pore. The following challenges were overcome to achieve the integration of the optical and electrical systems: (i) the electrical detection system must account for the physical constrains of a wide field fluorescence microscope, and the optical system should in turn not affect the low--‐noise electrical detection of individual DNA molecules. The design of the instrument included a microfluidic device, so to position the nanopore within the working distance (<170--‐μm) of the microscope objective (Chapter 2). (ii) Electrical noise was optimized to a level that is indistinguishable from a standard (with no optics) nanopore system (Chapter 3). The custom instrument was used to demonstrate: 1) Electrical detection of DNA translocations with a laser light illuminating the nanopore; 2) Optical tracking of DNA capture and translocation dynamics; 3) Synchronization of the optical and electrical signals in preparation for simultaneous detection. In the process of noise optimization, a strong noise coupling between the illumination source and the ionic current was found, characterized and eliminated. Consequently, the noise performance of the custom instrument is the lowest of any other nanopore--‐based optofluidic systems described in the literature to date. This opens up the way to many new and exciting investigations of polymer translocation dynamics through nanoconfined geometries. Lastly, during the development of this custom instrument, a method to localize the fabrication of a nanopore by controlled dielectric breakdown on a membrane, with a focused laser beam, was discovered.