Scott O. Sundberg

Spinning disk platform for microfluidic digital polymerase chain reaction.

An inexpensive plastic disk disposable was designed for digital polymerase chain reaction (PCR) applications with a microfluidic architecture that passively compartmentalizes a sample into 1000 nanoliter-sized wells by centrifugation. Well volumes of 33 nL were attained with a 16% volume coefficient of variation (CV). A rapid air thermocycler with aggregate real-time fluorescence detection was used, achieving PCR cycle times of 33 s and 94% PCR efficiency, with a melting curve to validate product specificity. A CCD camera acquired a fluorescent image of the disk following PCR, and the well intensity frequency distribution and Poisson distribution statistics were used to count the positive wells on the disk to determine the number of template molecules amplified. A 300 bp plasmid DNA product was amplified within the disk and analyzed in 50 min with 58-1000 wells containing plasmid template. Target concentrations measured by the spinning disk platform were 3 times less than that predicted by absorbance measurements. The spinning disk platform reduces disposable cost, instrument complexity, and thermocycling time compared to other current digital PCR platforms.

Comparison of glass etching to xurography prototyping of microfluidic channels for DNA melting analysis

Two microchannel manufacturing methods—xurography of double-sided tape and glass etching (lithography and wet etching)—were compared using DNA melting analysis. A heterozygous mutation (3 base-pair deletion) was distinguished from wild type (normal) DNA in 10 nL (xurography and glass etching) and 1 nL (xurography) volumes. The results of the 10 nL and 1 nL melting curves were compared to results using commercial high-resolution instrumentation with 10 µL volumes. These 1000-fold and 10 000-fold volume reductions reduced the signal-to-noise ratio (SNR) only 29-fold and 40-fold for xurography (10 nL and 1 nL, respectively,) and 39-fold for 10 nL glass etched microchannels, still providing adequate discrimination for mutation detection. The reduced SNR of the glass etched microchannels compared to the tape microchannels was due to the in-house bonding process which gave poor optical quality on the surface of the microchip. Xurography of double-sided tape reduces the cost by 20 fold and is four times faster to manufacture than glass etching. Microchips created using the rapid prototyping technique of xurography are a reasonable prototyping alternative to channels created using traditional glass etching for DNA mutation detection.

Microfluidic Genotyping by Rapid Serial PCR and High-Speed Melting Analysis

BACKGROUND Clinical molecular testing typically batches samples to minimize costs or uses multiplex lab-on-a-chip disposables to analyze a few targets. In genetics, multiple variants need to be analyzed, and different work flows that rapidly analyze multiple loci in a few targets are attractive. METHODS We used a microfluidic platform tailored to rapid serial PCR and high-speed melting (HSM) to genotype 4 single nucleotide variants. A contiguous stream of master mix with sample DNA was pulsed with each primer pair for serial PCR and melting. Two study sites each analyzed 100 samples for F2 (c.*97G>A), F5 (c.1601G>A), and MTHFR (c.665C>T and c.1286A>C) after blinding for genotype and genotype proportions. Internal temperature controls improved melting curve precision. The platforms liquid-handling system automated PCR and HSM. RESULTS PCR and HSM were completed in a total of 12.5 min. Melting was performed at 0.5 °C/s. As expected, homozygous variants were separated by melting temperature, and heterozygotes were identified by curve shape. All samples were correctly genotyped by the instrument. Follow-up testing was required on 1.38% of the assays for a definitive genotype. CONCLUSIONS We demonstrate genotyping accuracy on a novel microfluidic platform with rapid serial PCR and HSM. The platform targets short turnaround times for multiple genetic variants in up to 8 samples. It is also designed to allow automatic and immediate reflexive or repeat testing depending on results from the streaming DNA. Rapid serial PCR provides a flexible genetic work flow and is nicely matched to HSM analysis.

Low-Cost MEMS Technologies

Many researchers and companies are seeking methods to overcome the high cost of traditional microfabrication methods. This chapter presents various methods for performing common microfabrication tasks using relatively low-cost methods. The chapter discusses these low-cost methods in four sections. Section 1.12.2 discusses inexpensive techniques for use with photolithography such as low-cost mask fabrication techniques and the use of photopatternable polymers. Section 1.12.3 discusses replacements for photolithography such as xurography, lamination, screen printing, and stamping. Section 1.12.4 focuses on the low-cost methods for rapid prototyping such as soft lithography and powder blasting. Section 1.12.5 discusses methods for low-cost manufacturing of microelectromechanical systems (MEMS) devices with a focus on molding and embossing methods. Overall, low-cost methods are found to compete well with traditional MEMS methods, especially in biomedical and microfluidic applications.