Chip-based technology enables reliable direct detection of Ebola virus

This hybrid device integrates a microfluidic chip for sample preparation and an optofluidic chip for optical detection of individual molecules of viral RNA. (Photo by Joshua Parks)

A team led by researchers at UC Santa Cruz has developed chip-based technology for reliable detection of Ebola virus and other viral pathogens. The system uses direct optical detection of viral molecules and can be integrated into a simple, portable instrument for use in field situations where rapid, accurate detection of Ebola infections is needed to control outbreaks.

Laboratory tests using preparations of Ebola virus and other hemorrhagic fever viruses showed that the system has the sensitivity and specificity needed to provide a viable clinical assay. The team reported their results in a paper published September 25 in Nature Scientific Reports.

An outbreak of Ebola virus in West Africa has killed more than 11,000 people since 2014, with new cases occurring recently in Guinea and Sierra Leone. The current gold standard for Ebola virus detection relies on a method called polymerase chain reaction (PCR) to amplify the virus's genetic material for detection. Because PCR works on DNA molecules and Ebola is an RNA virus, the reverse transcriptase enzyme is used to make DNA copies of the viral RNA prior to PCR amplification and detection.

"Compared to our system, PCR detection is more complex and requires a laboratory setting," said senior author Holger Schmidt, the Kapany Professor of Optoelectronics at UC Santa Cruz. "We're detecting the nucleic acids directly, and we achieve a comparable limit of detection to PCR and excellent specificity."

Sensitivity and specificity

In laboratory tests, the system provided sensitive detection of Ebola virus while giving no positive counts in tests with two related viruses, Sudan virus and Marburg virus. Testing with different concentrations of Ebola virus demonstrated accurate quantification of the virus over six orders of magnitude. Adding a "preconcentration" step during sample processing on the microfluidic chip extended the limit of detection well beyond that achieved by other chip-based approaches, covering a range comparable to PCR analysis.

"The measurements were taken at clinical concentrations covering the entire range of what would be seen in an infected person," Schmidt said.

Schmidt's lab at UC Santa Cruz worked with researchers at Brigham Young University and UC Berkeley to develop the system. Virologists at Texas Biomedical Research Institute in San Antonio prepared the viral samples for testing.

The system combines two small chips, a microfluidic chip for sample preparation and an optofluidic chip for optical detection. For over a decade, Schmidt and his collaborators have been developing optofluidic chip technology for optical analysis of single molecules as they pass through a tiny fluid-filled channel on the chip. The microfluidic chip for sample processing can be integrated as a second layer next to or on top of the optofluidic chip.

Sample preparation

Schmidt's lab designed and built the microfluidic chip in collaboration with coauthor Richard Mathies at UC Berkeley who pioneered this technology. It is made of a silicon-based polymer, polydimethylsiloxane (PDMS), and has microvalves and fluidic channels to transport the sample between nodes for various sample preparation steps. The targeted molecules--in this case, Ebola virus RNA--are isolated by binding to a matching sequence of synthetic DNA (called an oligonucleotide) attached to magnetic microbeads. The microbeads are collected with a magnet, nontarget biomolecules are washed off, and the bound targets are then released by heating, labeled with fluorescent markers, and transferred to the optofluidic chip for optical detection.

Schmidt noted that the team has not yet been able to test the system starting with raw blood samples. That will require additional sample preparation steps, and it will also have to be done in a biosafety level 4 facility.

"We are now building a prototype to bring to the Texas facility so that we can start with a blood sample and do a complete front-to-back analysis," Schmidt said. "We are also working to use the same system for detecting less dangerous pathogens and do the complete analysis here at UC Santa Cruz."

The lead authors of the paper are postdoctoral researcher Hong Cai and graduate student Joshua Parks, both in Schmidt's lab at UC Santa Cruz. A team led by Aaron Hawkins at BYU fabricated the silicon-based optofluidic chips. Virologist Jean Patterson led the team at Texas Biomedical Research Institute that prepared viral samples for testing. This research was supported by the W. M. Keck Center for Nanoscale Optofluidics at UC Santa Cruz and grants from the National Institutes of Health and the National Science Foundation.

UC Santa Cruz

Read More on Nanotechnology World Association

Nanotechnology produces cheaper and portable measuring instruments

Researching, creating and applying nanoparticles go hand in hand in bionanotechnology. However, it is high time for a periodic table of nanoparticles. “Scientists working in bionanotechnology find themselves in the same position as the alchemists of the early Renaissance,” says Professor Aldrik Velders in his inaugural lecture as Professor of Bionanotechnology at Wageningen University on 17 September.

Let's start by correcting one misunderstanding: nanoparticles are not extremely small. In fact, they vary in size - from a couple of atoms to millions of atoms or thousands of molecules - and make up an intermediate form between loose molecules and relatively large grains. Proteins are also a type of nanoparticle. “In light of this, chemists see nanoparticles as actually being quite large,” explains Professor Velders in his inaugural speech entitled 'Much ado about nano'. “Nanoparticles form a whole new world, complete with its own peculiarities. With this in mind, it is important that all nanoparticles which form spontaneously or are formed by human intervention are properly catalogued. This is why we are working on technology which will allow us to catalogue them effectively, adopting an optical approach via absorption and fluorescence and using magnetic resonance spectroscopy such as the technology used in MRI scans. We also do not have a periodic table of nanoparticles like the one we have for all chemical elements. We currently only know a few classes of particles, and we have very few predictive values. We also have very little understanding of how nanoparticles behave in biological systems - this despite the fact that the basis of life is found within the interaction of molecules and that nanoparticles could hold a range of useful applications.”

Creating nanoparticles

 

Aside from carrying out pure research, Velders is also active in creating therapeutic and diagnostic nanoparticles. “We can create nanoparticles which change colour when they are in the vicinity of other molecules. We are currently working on this project together with Leiden University Medical Center. Amongst other things, we aim to create applications for robot-assisted surgery. It is also possible to make hard and soft nanoparticles, as well as to insert extra molecules into the soft nanoparticles. We are currently researching how we can insert metal complexes into a large nanoparticle, or a 'micelle’, which can then be implemented as a sort of Trojan horse. This technique can be used in the first instance for diagnostic purposes; later, it could be used to administer medication into a cell. To give you an idea of the scale of this: if an atom were a large as a football, the micelle would be as large as the Main Auditorium of the university, and the cell would be a city as large as Wageningen. Alongside this, we are studying the coming together and break up of nanoparticles under the influence of biomarkers in blood samples and other areas. Changes in light absorption or fluorescence indicate that somebody has a certain disease.”

A lab on a chip

 

Velders is also involved in researching how nanotechnology can be applied. This mostly involves the development of diagnostic sensors, as he explains. “By developing miniature instruments, we can produce cheaper and portable devices that can be used everywhere. We can create a lab on a chip. This is useful for analyses of ditchwater on the campus, for instance, or for monitoring malaria infections in the field in Sub-Saharan Africa.” A significant development in this respect is that Velders and his group are now able to create microchannels in small blocks that are made of polydimethylsiloxane (PDMS), a type of rubber. They use the same plastic as Lego bricks to do this. By creating a flow of substances - in some cases cooled, heated, and/or illuminated - through the small channels, you can trigger reactions in the PDMS blocks or carry out measurements without the need for large and expensive apparatus.

NMR antennae

 

“We are also in the process of developing very small NMR antennae. Using NMR, we can observe energy in the form of radio frequencies. These are specific to an element or atom, so this can also tell us how atoms will look later in the same molecule or nanoparticle. Every element has its own specific frequency. Our nanospools can listen to all frequencies simultaneously instead of just one, which is usually the case. Our nanospools are also a lot cheaper. A normal spool can easily cost ten thousand euros, whereas our most recently development antennae cost less than one euro.”

Finally, Velders will soon become involved with the catching and removing of antibiotic-resistant bacteria from the waste water of hospitals in order to prevent these bacteria from spreading. “We are developing technology that will allow us to attach these cells to nanoplates. We can convert expensive hospital technology to purify waste water; so from nanotechnology used in refined medicines to nanotechnology used in mud.”

Wageningen University

Nanotechnology World Association

Diagnostic Devices the Size of a Credit Card Are Now a Possibility

A microfluidic bioreactors consists of two chambers separated by
a nanoporous silicon membrane. It allows for flow-based assays
using minimal amounts of reagent. The ultra-thin silicon membrane
provides an excellent mimic of biological barrier properties.
NOTE: This image combines two exposures in order to capture the
brighter and darker parts of the scene, which exceed the dynamic
range of the camera sensor. The resulting composite is truer to what the
eye actually sees.

The ability to shrink laboratory-scale processes to automated chip-sized systems would revolutionize biotechnology and medicine. 

For example, inexpensive and highly portable devices that process blood samples to detect biological agents such as anthrax are needed by the U.S. military and for homeland security efforts. One of the challenges of "lab-on-a-chip" technology is the need for miniaturized pumps to move solutions through micro-channels. Electroosmotic pumps (EOPs), devices in which fluids appear to magically move through porous media in the presence of an electric field, are ideal because they can be readily miniaturized. EOPs however, require bulky, external power sources, which defeats the concept of portability. But a super-thin silicon membrane developed at the University of Rochester could now make it possible to drastically shrink the power source, paving the way for diagnostic devices the size of a credit card.

"Up until now, electroosmotic pumps have had to operate at a very high voltage—about 10 kilovolts," said James McGrath, associate professor of biomedical engineering. "Our device works in the range of one-quarter of a volt, which means it can be integrated into devices and powered with small batteries."

McGrath's research paper is being published this week by the journal Proceedings of the National Academy of Sciences.

McGrath and his team use porous nanocrystalline silicon (pnc-Si) membranes that are microscopically thin—it takes more than one thousand stacked on top of each other to equal the width of a human hair. And that's what allows for a low-voltage system.

A porous membrane needs to be placed between two electrodes in order to create what's known as electroosmotic flow, which occurs when an electric field interacts with ions on a charged surface, causing fluids to move through channels. The membranes previously used in EOPs have resulted in a significant voltage drop between the electrodes, forcing engineers to begin with bulky, high-voltage power sources. The thin pnc Si membranes allow the electrodes to be placed much closer to each other, creating a much stronger electric field with a much smaller drop in voltage. As a result, a smaller power source is needed.

"Up until now, not everything associated with miniature pumps was miniaturized," said McGrath. "Our device opens the door for a tremendous number of applications."

Along with medical applications, it's been suggested that EOPs could be used to cool electronic devices. As electronic devices get smaller, components are packed more tightly, making it easier for the devices to overheat. With miniature power supplies, it may be possible to use EOPs to help cool laptops and other portable electronic devices.

McGrath said there's one other benefit to the silicon membranes. "Due to scalable fabrication methods, the nanocrystalline silicon membranes are inexpensive to make and can be easily integrated on silicon or silica-based microfluid chips."

Source: http://www.rochester.edu/news/show.php?id=7592