Single-Molecule Detection and Optical Scanning in Miniaturized Formats

• Main Author: Melin, Jonas
• Format: Doctoral Thesis
• Language: English
• Published: Uppsala universitet, Institutionen för teknikvetenskaper 2006
• Subjects: Biotechnology; microfluidics; single-molecule detection; MOEMS; μTAS; lab-on-a-chip; RCA; thermoplastics; injection molding; hot embossing; Bioteknik
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7268; http://nbn-resolving.de/urn:isbn:91-554-6715-6

In later years polymer replication techniques have become a frequently employed fabrication method for microfluidic and micro-optical devices. This thesis describes applications and further developments of microstructures replicated in polymer materials. A novel method for homogenous amplified single-molecule detection utilizing a microfluidic readout format is presented. The method enables enumeration of single biomolecules by transforming specific molecular recognition events at nanometer dimensions to micrometer-sized DNA macromolecules. This transformation process is mediated by target specific padlock probe ligation, followed by rolling circle amplification (RCA) resulting in the creation of one rolling circle product (RCP) for each recognized target. Throughout this transformation the discrete nature of the molecular population is preserved. By hybridizing a fluorescence-labeled DNA detection oligonucleotide to each repeated sequence of the RCP, a confined cluster of fluorophores is generated, which makes optical detection and quantification possible. Spectral multiplexing is also possible since the spectral profile of each RCP can be analyzed separately. The microfluidic data acquisition process is characterized in detail and conditions that allow for quantification limited only by Poisson sampling statistics is established. The molecular characteristics of RCPs in solution are also investigated. Furthermore a novel thermoplastic microfluidic platform is described. The platform allows for observation of the microchannels using high magnification optics and also offers the possibility of on-chip cell culture and the integration of mechanical actuators. A novel fabrication process for the integration of polymer micro-optical elements on silicon is presented. The process is used for fabrication of a micro-optical system consisting of a laser and a movable microlens making beam steering possible. Such a micro-scanning system could potentially be used for miniaturized biochemical analysis.