The aim of the MicroActive project is to develop an instrument for molecular diagnostics. The ins... more 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.
Microsystem Technologies-micro-and Nanosystems-information Storage and Processing Systems, 2008
We present results from the MicroActive project which develops an instrument for molecular diagno... more 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.
Determination of the number of malaria parasites by routine or even expert microscopy is not alwa... more Determination of the number of malaria parasites by routine or even expert microscopy is not always sufficiently sensitive for detailed quantitative studies on the population dynamics of Plasmodium falciparum, such as intervention or vaccine trials. To circumvent this problem, two more sensitive assays, real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) and real-time quantitative PCR (QT-PCR) were compared for quantification of P. falciparum parasites. QT-NASBA was adapted to molecular beacon real-time detection technology, which enables a reduction of the time of analysis and of contamination risk while retaining the specificity and sensitivity of the original assay. Both QT-NASBA and QT-PCR have a sensitivity of 20 parasites/ml of blood, but QT-PCR requires a complicated DNA extraction procedure and the use of 500 l of venous blood to achieve this sensitivity, compared to 50 l of finger prick blood for real-time QT-NASBA. Both techniques show a significant correlation to microscopic parasite counts, and the quantification results of the two real-time assays are significantly correlated for in vitro as well as in vivo samples. However, in comparison to real-time QT-PCR, the results of real-time QT-NASBA can be obtained 12 h earlier, with relatively easy RNA extraction and use of finger prick blood samples. The prospective development of multiplex QT-NASBA for detection of various P. falciparum developmental stages increases the value of QT-NASBA for malaria studies. Therefore, for studies requiring sensitive and accurate detection of P. falciparum parasites in large numbers of samples, the use of real-time QT-NASBA is preferred over that of real-time QT-PCR.
Hands-free sample preparation platform for nucleic acid analysis
Lab on A Chip, 2009
A Lab-On-Chip system with an instrument is presented which is capable of performing total sample ... more 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.
The aim of the MicroActive project is to develop an instrument for molecular diagnostics. The ins... more 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.
Microsystem Technologies-micro-and Nanosystems-information Storage and Processing Systems, 2008
We present results from the MicroActive project which develops an instrument for molecular diagno... more 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.
Determination of the number of malaria parasites by routine or even expert microscopy is not alwa... more Determination of the number of malaria parasites by routine or even expert microscopy is not always sufficiently sensitive for detailed quantitative studies on the population dynamics of Plasmodium falciparum, such as intervention or vaccine trials. To circumvent this problem, two more sensitive assays, real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) and real-time quantitative PCR (QT-PCR) were compared for quantification of P. falciparum parasites. QT-NASBA was adapted to molecular beacon real-time detection technology, which enables a reduction of the time of analysis and of contamination risk while retaining the specificity and sensitivity of the original assay. Both QT-NASBA and QT-PCR have a sensitivity of 20 parasites/ml of blood, but QT-PCR requires a complicated DNA extraction procedure and the use of 500 l of venous blood to achieve this sensitivity, compared to 50 l of finger prick blood for real-time QT-NASBA. Both techniques show a significant correlation to microscopic parasite counts, and the quantification results of the two real-time assays are significantly correlated for in vitro as well as in vivo samples. However, in comparison to real-time QT-PCR, the results of real-time QT-NASBA can be obtained 12 h earlier, with relatively easy RNA extraction and use of finger prick blood samples. The prospective development of multiplex QT-NASBA for detection of various P. falciparum developmental stages increases the value of QT-NASBA for malaria studies. Therefore, for studies requiring sensitive and accurate detection of P. falciparum parasites in large numbers of samples, the use of real-time QT-NASBA is preferred over that of real-time QT-PCR.
Hands-free sample preparation platform for nucleic acid analysis
Lab on A Chip, 2009
A Lab-On-Chip system with an instrument is presented which is capable of performing total sample ... more 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.
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