The goal of the present work was to develop a microchip for amplification and detection of mRNA by employing nucleic acid sequence-based amplification (NASBA) technology. The technology platform should in principle be adaptable for any clinical analysis using mRNA or ssDNA as a target. To demonstrate the microchip functionality, identification of human papillomavirus (HPV) type 16, the etiological agent for cervical cancer has been used.
The work shows for the first time successful real-time amplification and detection employing NASBA in microsystem formats using custom-made instruments. The first silicon-glass chips contained reaction chambers of 10 nl and 50 nl, which decreased the NASBA reaction volume by a factor of 2000 and 400, respectively. Further, experiments employing cyclic olefin copolymer (COC) microchips for simultaneous amplification and detection, automatically distributed the sample into 10 parallel reaction channels with detection volumes of 80 nl. In order to detect the simultaneous amplification in the reaction channels, a second custom-made optical detection system with increased sensitivity, heat regulation and an automatic non-contact pumping mechanism, was made. Dilution series of both artificial HPV 16 oligonucleotides and SiHa cell lines showed that the detection limits for the microchips were comparable to those obtained for experiments performed in conventional routine-based laboratory-systems. For experiments related to the development of a self-contained microchip for NASBA, the detection volume was increased to 500 nl due to the advantage of an increased fluorescence signal.
For the NASBA reaction, biocompatible surfaces are critical. It was not possible to amplify any target in microchips with native silicon or COC surfaces. Adsorption measurements indicated clearly that fluorescently labelled mouse IgG bound non-specifically to the hydrophobic native COC surfaces, while PEG coated COC surfaces showed adequate protein resistance. Of the coatings tested for the COC microchips, surfaces modified with PEG showed the best biocompatibility. Successful amplification was obtained with silicon microchips when the surfaces were modified with either SigmaCote™ or SiO2.
In order to integrate the NASBA reagents on chip, a thorough evaluation of the reagents to be spotted and dried was performed. Because of the limited number of microchipsavailable, it was necessary to map the most critical parameters on macroscale prior to transfer to the microscale. The DMSO and sorbitol enclosed in the standard NASBA reaction mixture were difficult to dry, and therefore it was necessary to add these compounds to the oligonucleotides or the sample of extracted nucleic acids before the sample was applied on the amplification chip. The standard NASBA reagents consist of the two main solutions, mastermix and enzymes, in addition to the sample. Both the mastermix and the enzymes were stable only when spotted and dried separately. Protectants, such as PEG and trehalose were essential for recovery of enzymatic activity after drying on macroscale. The times for diffusion of modified molecular beacons in dried mastermix and of fluorescently labelled mouse IgG in the dried enzyme solution were ~60 seconds and ~10 minutes, respectively. So far, only dried enzymes with 0.05% PEG protectant have been successfully amplified on chip. Successful amplification using a rehydrated mastermix on microchip still remains.
Optimal design and fabrication methods of the microchips were found to be crucial for chip performance. Rough surfaces do not only create background noise for the optical measurements, but it also contributes to generation of bubbles and problems related to manipulation of the sample within the channel network. The silicon microchips were manufactured with optically smooth surfaces. However, low surface roughness was not easily obtained for the COC microchips. Of the fabrication methods evaluated, it was the injection moulded chips which showed the smoothest surfaces, closely followed by the hot embossed chips. Milled and laser ablated chips produced the roughest surfaces.
A novel non-contact pumping mechanism based on on-chip flexible COC membranes, combined with actuation pins in the surrounding instrument, was tested and evaluated. The mechanism enabled metering, isolation and movement of nanoliter sized sample plugs in parallel reaction channels. The COC chips with integrated pumps were able to simultaneously move parallel sample plugs along the reaction channels in four different positions. Each reaction channel contained a set of 4 actuation chambers in order to obtain metering, isolation and movement of the sample plug into the detection area. The pump accuracy depended on the evaporation of sample and the deformation of the COC membranes.
The results presented in this work are promising with regard to the development of a complete integrated and self-contained mRNA amplification microchip for multi-parallel target testing of clinical samples.
Anja Gulliksen, Lars Solli, Frank Karlsen, Henrik Rogne, Eivind Hovig, Trine Nordstrøm and Reidun Sirevåg, Real-time Nucleic Acid Sequence-Based Amplification in Nanoliter Volumes, Analytical Chemistry, 2004, 76, 9 – 14
Anja Gulliksen, Lars Solli, Klaus Stefan Drese, Olaf Sörensen, Frank Karlsen, Henrik Rogne, Eivind Hovig and Reidun Sirevåg, Parallel Nanoliter Detection of Cancer Markers using Polymer Microchips, Lab on a Chip, 2005, 5, 416 – 420
Lars A. Solli, Anja Gulliksen, Olaf Sörensen, Frank Karlsen, Lars R. Sætran, Liv Furuberg, Henrik Rogne and Klaus S. Drese, A novel non-contact pump mechanism for parallel movement of nanoliter sized liquid plugs using flexible diaphragms, manuscript
Anja Gulliksen, Michal Mielnik, Bente F. Hoaas, Eivind Hovig, Frank Karlsen, Henrik Rogne and Reidun Sirevåg, Aspects towards the development of an isothermal amplification microchip, manuscript