Jonas Melin

Digital quantification using amplified single-molecule detection.

We describe a scheme for biomolecule enumeration by converting nanometer-scale specific molecular recognition events mediated by rolling-circle amplification to fluorescent micrometer-sized DNA molecules amenable to discrete optical detection. Our amplified single-molecule detection (SMD) approach preserves the discrete nature of the molecular population, allowing multiplex detection and highly precise quantification of molecules over a dynamic range of seven orders of magnitude. We apply the method for sensitive detection and quantification of the bacterial pathogen Vibrio cholerae.

Ligation-based molecular tools for lab-on-a-chip devices

Molecular diagnostics can offer early detection of disease, improved diagnostic accuracy, and qualified follow-up. Moreover, the use of microfluidic devices can in principle render these analyses quickly and user-friendly, placing them within the reach of the general practitioner and maybe even in households. However, the progress launching such devices has been limited so far. We propose that an important limiting factor has been the difficulty of establishing molecular assays suitable for microfabricated formats. The assays should be capable of monitoring a wide range of molecules, including genomic DNA, RNA and proteins with secondary modifications and interaction partners, and they must exhibit excellent sensitivity and specificity. We discuss these problems and describe a series of molecular tools that may present new opportunities for lab-on-a-chip devices at the point-of-care.

Microreplication in a silicon processing compatible polymer material

We present a novel fabrication process for the integration of polymer micro-optical elements on silicon. The process relies on a reverse order protocol based on embossing in an amorphous fluorocarbon polymer, Cytoptrade

Replication of continuous-profiled micro-optical elements for silicon integration

A novel scheme for the integration of diffractive optical elements onto silicon is presented. The processing is made in reverse order, meaning that the process of structuring the optical elements on the wafer precedes the silicon microstructuring. The first processing step on the wafer is the hot embossing of the optical microstructures into an amorphous fluorocarbon polymer spin coated on the wafer. The cured polymer forms a highly stable material with excellent optical properties. The remaining silicon processing is thus performed with the diffractive optical elements already in place. Two different diffractive structures were used in the development of the method-a (Fresnel) lens with a rather low f-number and a diffractive element producing a fan-out of a large number of paraxial beams.

Confocal data acquisition for digital quantification using amplified single molecule detection

We have recently presented a method that enables single molecule enumeration 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. The transformation makes optical detection and quantification possible by counting the number of generated RCPs using standard epi-fluorescence or confocal fluorescence microscopes. We have characterized the performance of the epi-fluorescence and the confocal readout formats. Both formats exhibit a linear response of the number of counted objects as a function of starting circles, and the dynamic range is three orders of magnitude employing epi-fluorescence readout and four when using confocal. In the epi-fluorescence format flow rate has to be below 1 μl/min and flow variations are likely to be the limiting factor for precision. If the flow rate is above 3 μl/min the precision of the confocal readout format is limited only by Poisson counting statistics, due to the accurate volume definition of the confocal optics. The limit of detection in the confocal format was reduced by a factor of three by increasing the data acquisition rate by a factor of ten.