Researchers from the State University of New York at Buffalo have developed a novel microfabricated biochip that combines an electrical sensor and microfluidics to conduct non-invasive cellular screening based on changes in cell volume.
The researchers, whose work was published online Jan. 22 as an Analytical Chemistry “ASAP” article, now hope to commercialize the technology through SUNY Buffalo’s technology-transfer office for possible applications in high-throughput drug screening, basic laboratory research, clinical testing, and environmental biosensing.
If commercialized, the product may fill a need for an inexpensive, disposable, and miniaturized cell-based assay method for conducting high-throughput screening of small-molecule compounds.
The technology is based on a concept called cell-volume cytometry. As the researchers describe in their paper, a wide body of research has provided evidence that changes in a cell’s volume reflect its response to a variety of perturbations, including excitability, metabolism, apoptosis, necrosis, neurotransmitters, toxins, and cell division and growth.
According to Susan Hua, corresponding author on the paper and an assistant professor of mechanical and aerospace engineering at SUNY-Buffalo, one cellular characteristic that has a particular correlation with cell volume is ion channel activity, which gives the chip potential as a drug-screening tool.
Hua said that a cell typically uses ion channels — including those for calcium, potassium, and sodium — to regulate its volume. Therefore, if scientists can measure minute changes in cell volume, it can be correlated to ion channels opening and closing.
Ion channels, particularly those that regulate changes in cellular calcium levels, are believed to be one of the most important drug targets in pharmaceutical science. Many methods exist to monitor changes in cellular calcium levels, but most of them require fluorescent dyes and optical-based methods to measure them. Perhaps the most well-known example of this is Molecular Devices’ FLIPR assay, which is widely regarded as a “go-to” assay for measuring calcium flux.
Researchers have known for a while that cell volume can act as an indicator of ion channel activity, but according to the authors, no one has really devised a simple, straightforward method to measure changes in cell volume, such that it could be a useful tool for drug screening.
“Maybe people have tried to use cell volume to screen cells — we don’t know,” Hua said. “People have typically used a calcium indicator, but the reason they haven’t used cell volume is because they haven’t found a convenient way to do so.”
The SUNY-Buffalo researchers devised a way to measure cell volume changes by monitoring changes in electrical impedance. The closest thing on the market is Beckman Coulter’s Coulter counter. This technology — which was not created as a drug-screening tool — uses electrical impedance to measure the sizes of cells in flow. In addition, simple cell sorters have been used to quantify cell size, but are not robust enough to be used as screening tools.
“I only know of the Coulter counter and scattering from cell sorters as screening tools [using this method],” said Frederick Sachs, a co-author on the paper, and a professor of physiology and biophysics at the SUNY-Buffalo School of Medicine and Biomedical Sciences. “The Coulter counter requires suspended cells and is slow. The cell sorter is not very precise or fast, and doesn’t provide kinetic information.”
The SUNY-Buffalo researchers believe that their device — which, if commercialized, would be the first of its kind on the market — has three major advantages over fluorescent-based ion-channel assays or Coulter counter assays: It is inexpensive and disposable because the chip is composed of only silicon, electrodes, and a simple glass cover slip; it is completely non-invasive, since nothing needs to be added to the cell to make measurements; and it can be used for adherent cells or cells in suspension.
The experimental chip used by the researchers was fabricated on silicon with a microfluidic channel 15 microns deep and 1.5 millimeters wide, and connected to a fluid inlet and an outlet port. This channel contained two chambers, one the same height as the channel and used to measure cell volume, and the other deeper and used as a calibrating chamber.
Thin-film platinum electrodes in each chamber form a four-point probe for measuring the chamber resistance, the paper states. For adherent cells, the cells were grown on a glass coverslip, and then inverted on top of the measuring chip so the cells faced the chamber. For suspended cells, the cell suspension was simply added to the channels, and measurements were taken when the flow of cells stopped.
An active current source provided a microampere to the two outer electrodes, and the two inner electrodes were measured using a homemade instrumentation amplifier. Then, by adding various test compounds to the microfluidics channels, the researchers were able to detect how they affected cell volume.
“The cell’s membrane is not conductive,” Hua explained. “And the cells are put in the chamber with conductive saline solution. Since the cell takes up part of the area in the chamber, when it swells or shrinks the conductivity between the two electrodes changes.”
The researchers used the chip to detect volume changes in a monolayer of astrocytes responding to an anisotonic stimulus; the sensitivity of suspended E. coli strains to antibiotics; and the changes in volume of various cells in response to a natural peptide found in spider venom. All of their measurements were made in 15 to 20 minutes, and the researchers believe that by tweaking certain parameters, it can be done in about five minutes.
The researchers envision the chip being used in high-throughput screening, and have begun to manufacture chips with multiple channels for highly parallel studies, Hua said.
“High-throughput screening would involve parallel sensors with different compounds added to each channel,” Sachs added. “The assay would not need other cell-based assays, but later in the screen, more specific assays, such as electrophysiology, would be useful to remove some of the other feedback within the cell. By using hetero-expression systems with overexpression of the target, the volume assay can be made more specific.”
Because the chip can be used with bacterial cells as well as mammalian cells, the researchers think that it may also be useful as an environmental biosensor, or in biodefense applications. Hua said that they have already contacted the US Department of Defense to gauge its interest.
A further extension of the chips’ ability to measure volume changes in bacterial cells might be on-the-spot clinical antibiotic screening, the researchers said.
Hua said that patent on the chip is pending, and that the researchers are working with the SUNY-Buffalo tech-transfer office to commercialize the chip. She said that several pharmaceutical companies have contacted them about the technology, although she declined to say who they are. According to Hua and Sachs, the group will likely seek to license the technology out, as opposed to spinning off a company from the university.