Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA. Fluorescent molecular beacon probes enable real-time monitoring of the amplification process. Successful identification, utilizing the real-time NASBA technology, was performed on a microchip with oligonucleotides at a concentration of 1.0 and 0.1 microM, in 10- and 50-nL reaction chambers, respectively. The microchip was developed in a silicon-glass structure. An instrument providing thermal control and an optical detection system was built for amplification readout. Experimental results demonstrate distinct amplification processes. Miniaturized real-time NASBA in microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.
A general multipurpose microchip technology platform for point-of-care diagnostics has been developed. Real-time nucleic acid sequence-based amplification (NASBA) for detection of artificial human papilloma virus (HPV) 16 sequences and SiHa cell line samples was successfully performed in cyclic olefin copolymer (COC) microchips, incorporating supply channels and parallel reaction channels. Samples were distributed into 10 parallel reaction channels, and signals were simultaneously detected in 80 nl volumes. With a custom-made optical detection unit, the system reached a sensitivity limit of 10(-6) microM for artificial HPV 16 sequences, and 20 cells microl(-1) for the SiHa cell line. This is comparable to the detection limit of conventional readers, and clinical testing of biological samples in polymer microchips using NASBA is therefore possible.
A Lab-On-Chip system with an instrument is presented which is capable of performing total sample preparation and automated extraction of nucleic acid from human cell samples fixed in a methanol based solution. The target application is extraction of mRNA from cervical liquid based cytology specimens for detection of transformed HPV-infections. The device accepts 3 ml of sample and performs the extraction in a disposable polymer chip of credit card size. All necessary reagents for cell lysis, washing, and elution are stored on-chip and the extraction is performed in two filter stages; one for cell pre-concentration and the other for nucleic acid capture. Tests performed using cancer cell lines and cervical liquid based cytology specimens confirm the extraction of HPV-mRNA by the system.
We present results from the MicroActive project which develops an instrument for molecular diagnostics. The instrument is first tested for patient screening for a group of viruses causing cervical cancer. Two disposable polymer chips with reagents stored on-chip are developed and will be inserted into the instrument for each patient sample analysis. The first chip will perform nucleic acid extraction from patient epithelial cervical cells, while mRNA amplification and fluorescent detection takes place in the second chip. This paper reports results on the amplification chip. Purified sample is inserted into the chip and split into ten smaller droplets for simultaneous amplification and detection of ten viruses. The droplets move in parallel channels, each with two chamber extensions containing dried reagents. Experimental results on parallel droplet movement using one external pump combined with hydrophobic restrictions show that the parallel droplet positions can be controlled. There are four valves with increasing burst pressures between 800 and 4,500 Pa in each parallel channel, positioning the droplets in metering zones and reaction chambers. The re-hydration times for the dried reagents in micro chambers have been monitored. 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 show positive amplification of HPV type 16 viruses in a micro chamber.
We provide a method for the selective surface patterning of microfluidic chips with hydrophobic fluoropolymers which is demonstrated by the fabrication of hydrophobic valves via dispensing. It enables efficient optical quality control for the surface patterning thus permitting the low-cost production of highly reproducible hydrophobic valves. Specifically, different dyes for fluoropolymers enabling visual quality control (QC) are investigated, and two fluoropolymer-solvent-dye solutions based on fluorescent quantum dots (QD) and carbon black (CB) are presented in detail. The latter creates superhydrophobic surfaces on arbitrary substrates, e.g. chips made from cyclic olefin copolymer (COC, water contact angle = 157.9 •), provides good visibility for the visual QC in polymer labs-on-a-chip and increases the burst pressures of the hydrophobic valves. Finally, an application is presented which aims at the on-chip amplification of mRNA based on defined flow control by hydrophobic valves is presented. Here, the optimization based on QC in combination with the Teflon-CB coating improves the burst pressure reproducibility from 14.5% down to 6.1% compared to Teflon-coated valves.
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.
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.
Cancer affects more people than any other disease. About one-third of the world's 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.
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