Anja Gulliksen

Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

Microchip for the Diagnosis of Cervical Cancer

: Cancer affects more people than any other disease. About one-third of the worlds population is likely to get this diagnosis during their lifetime. Currently, the diagnostic methods for cancer detection are based on visual inspection. The lack of high analytical and clinical specificity and sensitivity makes these methods in many cases inferior to recently developed molecular methods. The increased clinical specificity and sensitivity of these new molecular approaches have great benefits, such as the possibility of implementing the molecular methods in miniaturized systems and enabling easier and faster point-of-care or bedside diagnostics. This chapter provides an introduction to performing clinical trials, screening, and molecular diagnostics against cancer-related markers. In addition, an example of molecular diagnosis of cervical cancer within a microsystem concept will be presented.

The MicroActive project : automatic detection of disease-related molecular cell activity

The aim of the MicroActive project is to develop an instrument for molecular diagnostics. The instrument will first be tested for patient screening for a group of viruses causing cervical cancer. Two disposable polymer chips with reagents stored on-chip will be inserted into the instrument for each patient sample. The first chip performs sample preparation of the epithelial cervical cells while mRNA amplification and fluorescent detection takes place in the second chip. More than 10 different virus markers will be analysed in one chip. We report results on sub-functions of the amplification chip. The sample is split into smaller droplets, and the droplets move in parallel channels containing different dried reagents for the different analyses. We report experimental results on parallel droplet movement control using one external pump only, combined with hydrophobic valves. Valve burst pressures are controlled by geometry. We show droplet control using valves with burst pressures between 800 and 4500 Pa. We also monitored the re-hydration times for two necessary dried reagents. After sample insertion, uniform concentration of the reagents in the droplet was reached after respectively 60 s and 10 min. These times are acceptable for successful amplification. Finally we have shown positive amplification of HPV type 16 using dried enzymes stored in micro chambers.

Detection of Multiple Real-Time Nasba in Nanoliter Volume

Real-time Nucleic Acid Sequence Based Amplification (NASBA) of artificial Human Papillomavirus (HPV) 16 oligonucleotides have been simultaneously detected in 25 parallel reaction chambers at the volume of 20 nl and 60 nl. A plastic chip incorporating supply channels, reaction chambers, and microfluidic actuation systems, was designed and processed by injection molding of cycloolefm copolymer (COC). An instrument with thermal control, an optical detection and microfluidic actuation system was built.