Integrated microfluidic platform for oral diagnostics

Abstract

While many point-of-care (POC) diagnostic methods have been developed for blood-borne analytes, development of saliva-based POC diagnostics is in its infancy. We have developed a portable microfluidic device for detection of potential biomarkers of periodontal disease in saliva. The device performs rapid microfluidic chip-based immunoassays (<3-10 min) with low sample volume requirements (10 microL) and appreciable sensitivity (nM-pM). Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. The microfluidic chip has been integrated with miniaturized electronics, optical elements, such as diode lasers, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device and methods are being tested by detecting potential biomarkers in saliva samples from patients diagnosed with periodontal disease. Our microchip-based analysis can readily be extended to detection of biomarkers of other diseases, both oral and systemic, in saliva and other oral fluids.

FIGURE 1

Image of early generation IMPOD with hardware components labeled. Inset shows glass microdevice used in performing immunoassays.

FIGURE 2

Microchip-based electrophoretic immunoassays. (left) Protocol schematic: Binding/Preconcentration: The sample is mixed with a known amount of fluorescently labeled antibody. The mixing is done off-chip or on-chip. During initial methods development, we carry out the mixing outside the chip (requires ~15 min). Upon optimization of immunoassay conditions, the sample and the labeled antibody are added to different reservoirs in the sample manifold and are mixed (in <3 min) electrokinetically on-chip. Separation: Mixed sample is electrophoretically injected into a separation channel. The antibody-bound antigen and unbound antibody are separated based on size and charge by native polyacrylamide gel electrophoresis. Detection & Analysis: A laser-induced fluorescence detector detects the unbound antibody and bound antibody (i.e., immune complex) peaks. Migration times are recorded and peak areas are calculated. The immunoassay is repeated with samples containing known amounts of analyte to generate a multipoint calibration curve. The calibration curve is used to estimate the concentration of analyte in the unknown sample. (right) Image of glass microdevice.

FIGURE 3

IMPOD analysis is quantitative and completes in less than four minutes. (Top) Electropherograms for exogenous TNF-α analysis in whole saliva. Companion dose-response curve acquired in model buffer system. (Bottom) IL-6 measurements from IMPOD analysis of spiked whole saliva. Companion dose-response curve from spiked saliva.

FIGURE 4

On-chip sample enrichment extends the C-reactive protein dynamic range and increases the sensitivity of IMPOD. Buffer samples spiked with known concentrations of C-RP were analyzed with (solid squares) and without (hollow circles) on-chip sample enrichment. Measurement of C-RP in spiked whole saliva, without enrichment, is shown for comparison (solid circles).