NEW YORK (GenomeWeb) – Breaking into markets for nucleic acid testing is not an easy proposition for any startup, but Electronucleics, a company recently founded by University of California, Los Angeles researchers, believes that a device it is developing could satisfy unmet needs for use of an inexpensive, fast, and high-sensitivity device at the point of care.
The nucleic acid diagnostic test they are developing doesn't require complex amplification or expensive optics and, according to the researchers, it could be available as a manufacturing prototype in about a year.
The prototype detects bacteria spiked in human urine samples by observing a change in current when a peptide nucleic acid associated with the bacteria blocks a nanopore.
"The device is very simple and that's the power of this technique," Harold Monbouquette, a professor of chemical and biomolecular engineering at UCLA and cofounder of Electronuceics, said in an interview.
Nucleic acid testing technologies generally rely on amplifying an RNA or DNA target and on a subsequent binding event in which a probe hybridizes selectively to the target for detection. To achieve this, the devices require primers, polymerase, nucleotides, and tightly controlled reaction conditions that contribute to complexity and cost. Further, fluorescence detection methods require the incorporation of additional reagents and optics, which adds more cost and bulk.
The Electronucleics device eliminates the need to amplify targets or use optics and has potential to provide a test result within 15 minutes, which could be important to clinicians looking to apply highly sensitive tests at the point of care, the researchers said. The detection method uses simple electronics and has minimal reagent requirements, adding to the device's potential for adoption if its developers are able to build a commercial platform for clinical diagnostic applications, Monbouquette said.
He and his colleagues recently described their prototype in the journal Lab on a Chip and its identification of E. coli at low limits of detection — 10 CFU mL−1 in a 1 mL sample — against a millionfold background of P. putida, a gram-negative soil bacterium. No false positive signals were observed with P. putida RNA when the device was configured to detect E. coli 16S rRNA, and the limit of detection is sufficient to suggest that the new device has potential in clinical diagnostic applications, Monbouquette said.
Importantly, the UCLA team is now developing the platform to detect gonorrhea and chlamydia, an area of testing that could see broader adoption through inexpensive and convenient testing in a doctor's office, he said.
The developer and his colleague Jacob Schmidt, a professor of bioengineering at UCLA, launched Electronucleics to move the device closer to commercialization and look into additional indications for which the platform might be suitable.
In around a year, if they can demonstrate that current low limits of detection translate to high enough values of sensitivity and specificity through testing on larger numbers of clinical samples, the researchers anticipate being ready to collaborate with a diagnostic company interested in taking the platform to market or purchasing the technology outright.
They will seek a collaboration with a company interested in taking the platform through clinical trials in preparation for a submission for marketing clearance to the US Food and Drug Administration, Monbouquette said.
The researchers anticipate developing a platform that eventually can be inexpensively manufactured and suitable for highly sensitive nucleic acid testing for a range of pathogens.
It should be possible for the researchers to build on this work to produce a low cost, easy to use, and highly sensitive diagnostic device, said Alexander Vadas, a managing director and partner at LEK Consulting, who has a focus on diagnostics, research tools, and personalized medicine. Vadas is familiar with the UCLA team's prototype and was once a student taught by Monbouquette. However, he is not involved in the development of the diagnostic device or being paid to evaluate it.
The main point is that the platform "doesn't need to do amplification, which is unique provided that the kinetics of the detection event happen quickly," he said. That would overcome an issue in diagnostics that is being worked on to improve turnaround times, he added.
Most commercially available molecular infectious disease platforms cleared for point-of-care testing in specific applications, including flu and strep as well as sexually transmitted infections, provide far quicker turnaround than laboratory tests. Still, even faster turnarounds would benefit both providers and patients, Vadas said.
"There are public health benefits associated with getting high-risk STI patients quickly on the correct treatments," Vadas said. A platform that can provide a turnaround time of a few minutes versus one hour provides important benefits for patient workflow in a clinic, he said.
In the STI testing market, all new entrants would have to compete against existing tests, including Cepheid's point-of-care GeneXpert platform, which enables detection of chlamydia and gonorrhea within 90 minutes using RT-PCR, and conducts additional types of STI tests, such as for Trichomonas vaginalis, and non-STI tests, such as flu, MRSA, and strep A, among others
To operate the UCLA test, an electric current flows through a thin glass membrane and comes in contact with polystyrene beads that are 800 nanometers in diameter. Peptide nucleic acid (PNA) coatings are covalently bonded to the surface of the beads. Although PNA has the same backbone as proteins, it also has the same bases as RNA and DNA and therefore can be designed to hybridize to specific RNA and DNA targets.
"PNA-coated beads have little charge so they don't move much in an electric field until a target nucleic acid binds to them," Monbouquette said. "We have focused on 16S ribosomal RNA targets, which have sequences unique to specific bacterial pathogens and are about 1,500 bases with a lot of negative charge. Even if one of these 16S ribosomal RNA bases binds to PNA on a bead, the complex has a sufficient charge to move in an electric field."
A hole milled in the glass membrane using a focused ion beam is about 500 nanometers in diameter — too small for a bead to slip through it. Nonetheless, because of an increase in electric charge and the presence of electrodes on either side of the glass membrane, the beads move toward the pore and when they get closer they partly block the flow of ions and therefore the flow of current. That results in a signal that the device needs in order to detect the presence of the bead and its hybridized 16S ribosomal RNA with a sequence specific to a given pathogen.
If a doctor is interested in whether a patient has chlamydia, for instance, the PNA binds only to a stretch of RNA that’s unique to chlamydia. As a result, the platform can identify whether a given pathogen is present and whether a patient has been infected.
Among the most important outcomes of the method is that in testing so far, there have been no false positive results. "It turns out that the electric field at the pore is strong enough that non-target proteins and molecules, or junk, that would normally interfere with the platform's performance are pulled away," while target RNA remains bound to beads, Monbouquette said.
With the support of a Phase I National Institutes of Health Small Business Technology Transfer grant of $150,000, the researchers are further developing the platform into a fully automated, easy-to-operate prototype that can be inexpensively manufactured in large volumes. Electronucleics anticipates doing additional testing on around 100 clinical samples to establish the device's sensitivity and specificity, and anticipates reaching a level of performance that is robust enough to satisfy the FDA and in vitro diagnostic companies that may be interested in taking the platform to commercialization. So far, they have worked with mock clinical samples consisting of bacteria spiked in pooled human urine.
The potential to compete with existing tests on price is a motivating factor for the UCLA group. The reusable base platform could probably be sold for less than $100, and the disposable test cartridges for less than $40, "so that they can compete with traditional nucleic acid tests," Monbouquette said. Given the simplicity of its design compared to that of its commercial counterparts, those prices should be achievable, he said.
"We believe this can be done, but of course we haven't done it yet," Monbouquette added.