Dynamic DNA hybridization on a chip using paramagnetic beads.

Dynamic DNA hybridization is presented as an approach to perform gene expression analysis. The method is advantageous because of its dynamic supplies of both DNA samples and probes. The approach was demonstrated on a microfluidic platform by incorporating paramagnetic beads as a transportable solid support. A glass chip was fabricated to allow simultaneous interrogation of eight DNA target samples by DNA probes. DNA targets were immobilized on beads via streptavidin-biotin conjugation or base pairing between oligonucleotide residues. The DNA/bead complex was introduced into the device in which hybridization took place with a complementary probe. The hybridized probe was then removed by heat denaturation to allow the DNA sample to be interrogated again by another probe with a different sequence of interest. A pneumatic pumping apparatus was constructed to transport DNA probes and other reagents into the microfluidic device while hydrostatic pumping was used for the introduction of paramagnetic beads with samples. After investigating three types of paramagnetic beads, we found Dynabeads Oligo(dT)25 best suited this application. Targets on the beads could be sequentially interrogated by probes for 12 times, and the hybridization signal was maintained within experimental variation. Demonstration of specific hybridization reactions in an array format was achieved using four synthesized DNA targets in duplicate and five probes in sequence, indicating the potential application of this approach to gene expression analysis.

Chemiluminescence detection in integrated post-separation reactors for microchip-based capillary electrophoresis and affinity electrophoresis.

Chemiluminescence (CL) detection based on the horseradish peroxidase (HRP) catalyzed reaction of luminol with peroxide was investigated as a post-separation detection scheme for microchip-based capillary electrophoresis. An integrated injector, separator and post-separation reactor was fabricated on planar glass wafers. The fluorescein conjugate of HRP (HRP-F1) was used as a sample for optimization of the CL detector response. In devices etched 10 microm deep, with an aluminum mirror integrated onto the backside of the detection zone to enhance collection efficiency, the detection limit, estimated at 3 standard deviations (SD) above background noise, for 1 nL injected sample plugs was 35 nM in HRP-F1. In devices etched 40 microm deep, 8 nL plugs gave a detection limit of 7 nM. Separation and CL detection of the products of an immunological reaction of a F(ab')2 fragment of the HRP conjugate of goat anti-mouse immunoglobulin G (IgG) with mouse IgG was performed on-chip. A linear calibration curve was obtained for the decrease in peak height of the HRP conjugate (53 microg/mL) with increasing mouse IgG (0-60 microg/mL). When microperoxidase was used as an internal standard, the R2 value of a linear least-squares fit was 0.9867, and the relative errors in the slope and intercept were +/- 5.8 and +/- 1.3 %, respectively.

Microchip-based capillary electrophoresis of human serum proteins.

The separation and relative quantitation of human serum proteins is important to the clinical diagnosis of various states of disease. Microchip-based capillary electrophoresis (CE) of human serum proteins offers several advantages over sodium dodecyl sulfate-poly(acrylamide) gel electrophoresis and conventional CE methods, including decreased sample consumption and analysis time and the possibility of on-chip sample manipulation (dilution, labelling, etc.). The microchip used in these studies was designed to allow for on-chip, post-separation labelling of the proteins and subsequent laser-induced fluorescence detection. 2-Toluidinonaphthalene-6-sulfonate (TNS) is a virtually non-fluorescent reagent which, upon non-covalent association with the protein and excitation at 325 nm, produces a fluorescent product with an emission maximum near 450 nm. After optimization of buffer conditions (100 mM borate with 2 mM lactate, pH 10.5), individual serum proteins (IgG to mimic the gamma zone, transferrin the beta zone, alpha-1-antitrypsin the alpha 1 zone and albumin its own zone) were successfully resolved on-chip, as was a "synthetic" serum solution composed of a mixture of all four of the previously mentioned proteins. Analysis of all five protein zones in a true human serum sample, however, has not yet been achieved on-chip due to the poor sensitivity of the TNS label for several of the serum proteins.

Evaluation of methods for the laboratory diagnosis of malaria in Guyana.

When 297 blood samples taken from patients attending a fever clinic in Georgetown Public Hospital were examined microscopically, after thick and thin blood films had been stained with Giemsa, one hundred and forty-two (47.8%) were microscopically positive for malaria. After processing the patient's serum, samples by the Indirect Fluorescent Antibody (IFA) technique, specific IgG and IgM antibodies were detected in 239 (81.3%) and 179 (60.1%), respectively, of the sera. Based on the microscopical findings, the IFAT gave positive predictive and negative values of 54.4% and 81.8% (IgG), and 57.5% and 67.8% (IgM), suggesting that the IgM would be more useful than the IgG in the diagnosis of current malaria. An odds ratio analysis showed that the presence of symptoms, IgG or IgM antibodies, as well as visits to endemic regions, could be good indicators of current malaria. Age and occupation were not. The microscopical method will continue to be the gold standard-the best available criterion for the validation of our tests-for diagnosis of acute malaria.

Evaluation of methods for the laboratory diagnosis of malaria in Guyana

When 297 blood samples taken from patients attending a fever clinic in Georgetowm Public Hospital were examined microscopically, after thick and thin blood films had been stained with Giemsa, one hundred and forty-two (47.8 percent) were microscopically positive for malaria. After processing the patients' serum samples by the Indirect Fluourescent Antibody (IFA) technique, specific IgG and IgM antibodies were detected in 239 (81.3 percent) and 179 (60.1 percent), respectively, of the sera. Based on the microscopical findings, the IFAT gave positive and negative values of 54.4 percent and 81.8 percent (IgG), and 57.5 percent and 67.8 percent (IgM), suggesting that the IgM would be more useful than the IgG in the diagnosis of current malaria. An odds ratio analysis showed that the presence of symptoms, IgG or IgM antibodies, as well as visits to endemic regions, could be good indicators of current malaria. Age and occupation are not. The microscopical method will continue to be the gold standard - the best available criterion for the validation of our tests - for our diagnosis of acute malaria. (AU)

Evaluation of methods for the laboratory diagnosis of malaria in Guyana

When 297 blood samples taken from patients attending a fever clinic in Georgetowm Public Hospital were examined microscopically, after thick and thin blood films had been stained with Giemsa, one hundred and forty-two (47.8 per cent ) were microscopically positive for malaria. After processing the patients' serum samples by the Indirect Fluourescent Antibody (IFA) technique, specific IgG and IgM antibodies were detected in 239 (81.3 per cent ) and 179 (60.1 per cent ), respectively, of the sera. Based on the microscopical findings, the IFAT gave positive and negative values of 54.4 per cent and 81.8 per cent (IgG), and 57.5 per cent and 67.8 per cent (IgM), suggesting that the IgM would be more useful than the IgG in the diagnosis of current malaria. An odds ratio analysis showed that the presence of symptoms, IgG or IgM antibodies, as well as visits to endemic regions, could be good indicators of current malaria. Age and occupation are not. The microscopical method will continue to be the gold standard - the best available criterion for the validation of our tests - for our diagnosis of acute malaria.

The comparative value of various diagnostic tests for malaria - abstract

When 300 blood samples taken from patients attending a fever clinic in Georgetown Public Hospital were examined microscopically, 47.6 percentwere found positive for malaria. IgG antibodies stored in filter paper or liquid serum gave 68 percent and 81 percent positive rate, repsectively. Sixty-one per cent (61 percent) of the patients had positive levels of IgG antibodies. Elevation of the cut-off point of IgG antibody titres from 1:256 to 1:1,024 help to reduced false positives of the indirect flourescent antibody test. Clinical symptoms, visits to endemic areas, occupation and its life styles, as well as age could assist in making a diagnosis. The microscopic method will continue to be the gold standard (AU)