On-Chip Biomarker Extraction
“While nucleic acid biomarkers are useful for determining the type and stage of cancers, their quantification requires a series of isolation and amplification steps that can take considerable time,” says Adam Woolley, Ph.D., professor of chemistry and biochemistry at Brigham Young University. “To overcome these drawbacks, we have explored the integration of sample-processing steps on microfluidic systems.”
It is generally accepted that cancer screening that takes advantage of the simultaneous quantitation of multiple biomarkers will yield a more sensitive and accurate profiling of malignancies and a concomitant improvement in cancer mortality and morbidity statistics.
For example, while the prostate specific antigen (PSA) is perhaps the most widely adopted test for the early detection of cancer, as a single marker it has a high rate of both false positives and false negatives. Moreover, the PSA test provides no information on the aggressiveness of a tumor, so the result may be that the patient is subjected to costly and unnecessary therapies that offer no benefits, while potentially compromising quality of life.
Most biomarkers are detected using ELISAs, whose performance could be enhanced through extensive validation and quality control. But this would require a multiplexing system, and Dr. Woolley argues that microfluidic format could provide higher speed and lower reagent consumption. “Many process steps, including sample desalting, labeling, and extraction have been successfully performed in microchip systems. “The simultaneous monitoring of multiple biomarkers has the potential to greatly improve the quality of the diagnosis.”
Dr. Woolley believes that although the 96-well immunoassay technology is effective for analyzing large numbers of samples, it is an inefficient approach to smaller volumes, which can be monitored much more economically using the microfluidics approach.
His team has constructed microfluidic devices employing poly(methyl methacrylate) with monolithic columns for immunoaffinity extraction. Using a laser-induced fluorescence detection system, they have quantified α feto protein, a biomarker for liver cancer, down to 1 ng/mL levels in serum samples.
They have now applied the solid-phase extractors for sequence-specific nucleic acid biomarker determination. Monolithic columns were made using glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate. The reactive GMA epoxy groups were employed for the functionalization of the monolith with amine-terminated DNA probes complementary to the target nucleic acid fragment.
Using these columns, they extracted and eluted fluorescently labeled 20-mer target single-stranded DNA fragments in as little as 15 minutes, according to Dr. Woolley. “We tested a number of approaches for eluting the fragments,” he continues, “and settled on dilute (1.5–7.0 molar) urea as the best option. We are extending our investigations to urine as a noninvasive approach to cancer biomarker detection.”