The gold standard for pathogen detection is a phenotypic approach whereby the samples are cultured in growth media with or without different antibiotics. Although specific, the test takes 48–96 hours to complete, potentially missing the narrow treatment window. According to Vincent Gau, Ph.D., co-founder, CEO, president, and CTO of Genefluidics, his company is approaching clinical validation of a biosensor system that would enable it to shorten diagnosis time to one to two hours.
Genefluidics’ systems use crude, unseparated samples that reduce the processing time and increase specificity. However, detection of unamplified targets present at low concentrations requires highly sensitive detection methods.
“Optical detection methods measure optical density, but crude samples are often cloudy. We utilize all commonly used ELISA reagents not in optical but in electrochemical detection,” explains Dr. Gau.
A detector probe (protein or nucleic acid) is conjugated with horseradish peroxidase (HRP), which is oxidized by hydrogen peroxide and reacts with one molecule of the TMB substrate, a commonly used ELISA substrate. A working electrode supplies an electron to reduce one TMB molecule, and the cycle starts over again.
HRP cycles through 10,000 Redox steps per second generating strong electrical signals in a relatively short time span. Validation with urine samples of patients with suspected urinary tract infections confirmed 100% accuracy of the HRP electrochemical assay compared to clinical laboratory results.
“The sensor chips are designed to detect RNA and proteins simultaneously, as well as internal controls,” adds Dr. Gau. “Internal controls are critical to monitor reagent quality and time sequence of reagent delivery. Our systems continuously measure electrical impedance for internal quality control.”
Genefluidics has a POC system with 6 sensors per cartridge and a lab system with 16 sensors per cartridge. According to the company, the genetic assay is able to detect femtomolar quantities of nucleic acid without target amplification.
Gen-Probe was the first company to have an FDA-cleared nucleic acid test (NAT) in the ‘80s, says Michael Watts, vp of investor relations. “We are now the world’s largest stand-alone molecular diagnostics company. The cornerstone of our success is integration of several essential technologies with fully automated instrumentation.”
Gen-Probe’s transcription-mediated amplification (TMA) technology involves multiple rounds of synthesis of RNA and DNA copies of the target. Each of the newly synthesized molecules serves as a template for a new round of replication, leading to exponential expansion of the amplicon. This expansion can result in the production of billions of amplicons in less than one hour.
“Our technology is well positioned in areas where specificity and sensitivity requirements are paramount,” continues Watts. “For example, our blood screening test for West Nile virus has an analytical sensitivity of 9.8 RNA copies per mL of blood, with 99.91 percent specificity.”
Watts also reports that Gen-Probe’s high-throughput system, Tigris, is able to process about 500 patient samples within one eight-hour shift.
The company’s new Panther instrument preserves the automation of Tigris and is geared to lower volume hospital labs. Panther, which is expected to be launched in Europe around year-end, reportedly processes 275 samples in 8 hours, with up to 120 tubes being loaded at the same time.
Panther is able to run both qualitative tests (such as Chlamydia and gonorrhea assays) and quantitative tests.