NEW YORK (GenomeWeb) – Biosensors hold the potential to rapidly detect DNA or RNA without the need for nucleic acid amplification. Researchers at Purdue University have now developed a new biosensing method using impedimetric sensors and have applied it to mosquito-borne viruses in proof-of-principle studies.
The ultimate goal is to combine the technique with a rapid, paper-based sample prep method employing thermally actuated wax valves in order to create a point-of-care assay system to detect viruses such as Zika and dengue in human and mosquito vector samples.
Lia Stanciu, a materials engineer at Purdue who is leading the project, said that the biosensor technology recently received a US patent. The method uses graphene-wrapped silicon particles that are also coated with complementary RNA. These create an impedimetric sensor, such that, "When coupling happens, the surface resistance of the particles increases ... and you register a signal," Stanciu said.
Her team has now designed particles to recognize Zika and dengue RNA, and tested these on infected mosquito samples. They also tested the particles to confirm there is no cross-reactivity with other flaviviruses, like West Nile virus.
Biosensor-based devices have not yet lived up to their potential as commercial point-of-care tests. As a review from 2014 noted, "Fully integrated systems that bring together the components of sample preparation and analyte detection remain a critical challenge for technology transfer from laboratories to the clinical market."
To tackle this challenge, Stanciu is collaborating with Jacqueline Linnes, a biomedical engineer at Purdue, to combine the biosensing method with rapid, paper-based sample prep.
"Sample preparation and low-cost automation are big bottlenecks to commercialization," Linnes noted in an email. "Most detection methods require moving the sample from one place to the next and incubating samples over time in order to perform detection, and this requires either expensive and highly calibrated instruments, or novel fluidic controls," she said.
The Linnes lab's paper-based sample prep technique takes advantage of capillary action in paper "to pull fluids along and separate the virus from other components, [such as] red blood cells," she said. It also has incorporated controlled heating areas to lyse the virus.
Interestingly, the method also uses the heated areas to open and close wax valves that can start and stop the fluid flow in the paper.
As reported in Lab on a Chip last year, the lab can print bands of wax-ink onto strips of nitrocellulose and cellulose membranes, and embed a thin film heater in the paper device. In the study, the lab used the device to convert a traditional lateral flow immunoassay into a multi-step, semi-autonomous assay, and showed 6-fold detection enhancement of E. coli signal intensity compared to a standard lateral flow immunoassay.
The Linnes lab is now also using the wax-valve method, which is funded in part by the Bill and Melinda Gates Foundation, to develop diagnostics for detecting sepsis-causing pathogens and HIV in blood, and cholera in water samples in Haiti, Linnes said.
Linnes was previously a member of the Klapperich lab at Boston University, a group known for pioneering paper-based diagnostics methods. She helped develop a method to detect Bordetella pertussis that used a slider to pop reagent-filled blisters in a paper device and push sample along.
But, "The need for the user to perform multiple timed steps over a period of 30 to 60 minutes does not easily fit into the clinical workflow," Linnes said, adding that the new wax-based method automates these steps, and the lab is now "able to make a device that can truly become point-of-care."
Stanciu's biosensor, meanwhile, has been shown in the lab to detect very low levels of viral RNA. It can pick up femtomolar concentrations if left to incubate over a few hours, but, importantly for a POC test, it can detect picomolar concentrations in only 30 minutes, she said.
With collaborators Ernesto Marinero and Richard Kuhn, a Purdue materials engineer and biologist, respectively, Stanciu is also working on an autonomous device that can be used for outbreak monitoring in remote areas. That device will use a portable impedance reader and operate through a low-power wireless network powered by thin-film rechargeable batteries and photovoltaics.
The group is currently seeking additional funding to develop the platform for flaviruses, and is working to make a prototype. Once a prototype is ready, Stanciu said her group will tackle the manufacturing bottleneck. She is part of a consortium at Purdue that works on scaling up production by combining advances in roll-to-roll systems, functional printing, and scalable nanomanufacturing. In the meantime, the graphene microparticle technology is also available for licensing from Purdue.Â