Integrated, DC voltage-driven nucleic acid diagnostic platform for real sample analysis: Detection of oral cancer

Abstract

We present an integrated and low-cost microfluidic platform capable of extraction of nucleic acids from real biological samples. We demonstrate the application of this platform in pathogen detection and cancer screening. The integrated platform consists of three units including a pretreatment unit for separation of nucleic acids from lysates, a preconcentration unit for concentration of isolated nucleic acids and a sensing unit localized at a designated position on the chip for specific detection of the target nucleic acid. The platform is based on various electrokinetic phenomena exhibited by ion exchange membranes in a DC electrical field that allow them to serve as molecular filters, analyte preconcentrators and sensors. In this manuscript, we describe each unit of the integrated chip separately and show specific detection of a microRNA (miRNA 146a) biomarker associated with oral cancer as a proof-of-concept experiment. This platform technology can easily be extended to other targets of interest by optimizing the properties of the ion exchange membranes and the specific probes functionalized onto the sensors.

Keywords: Electrokinetics; Integrated platform technology; Ion exchange membranes; Oral cancer; Point-of-care diagnostics; Sample-to-answer detection.

Figure 1

Schematics of a (a) pretreatment unit, (b) preconcentration unit (c) flow-through sensing unit, (d) integrated platform with all electrical connections. (e) Top view of an actual integrated device.

Figure 2

(a) Picture of the actual pretreatment unit. (b) Separation of a fluorescently labeled ssDNA from an oral cancer cell lysate,

Figure 3

(a)Experimentally obtained voltage profile that develops between the depleting and concentrating membrane at the applied voltage of 100 V and the flow rate of 5 μl/min and b) the corresponding electric field profile (absolute values) numerically calculated from the voltage profile.

Figure 4

a) Dynamics of the preconcentration of a fluorescently labeled ssDNA, (b) Experimental study showing the effect of the voltage and the flow rate on the position of the preconcentration slug, (c) Graph showing the positions of the preconcentration slug related to the flow rate and voltage applied

Figure 5

(a) Picture of an actual flow-through sensing unit and (b) Representative experiment showing specific detection of a DNA with a sequence corresponding to miRNA146a.

Figure 6

(a) Control experiment performed with a sensor that was not functionalized with a probe, (b) Control experiment performed with a sensor that was carboxylated but not functionalized with a probe, (c) Specific detection of ssDNA from clean samples, and (d) Detection of miRNA146a from oral cancer cell lysate.