(549i) A Dielectrophoretic/Electrophoretic Device for in-Situ DNA Isolation and PCR Analysis

AIChE 2012 Topical 3: 2012Annual Meeting of the American Electrophoresis Society “T3007 DNA Analysis in Microfluidic and Nanofluidic Devices” A Dielectrophoretic/Electrophoretic Device for In-situ DNA Isolation and PCR Analysis Michael J. Heller1,2, Avery Sonneberg2, and Raj Krishnan3University of California San Diego, Departments of Nanoengineering1 and Bioengineering2, Atkinson Hall Rm 2312, La Jolla, CA, 92093-0448, and Biological Dynamics3, 9393 Towne Centre Dr., San Diego, CA 92121 Abstract – A sample to answer AC/DC dielectrophoretic/electrophoretic device has been developed that allows the isolation of cell free circulating (cfc) DNA biomarkers and bacteria/virus, with subsequent DNA processing and in-situ PCR reactions, all carried out in the same device chamber. In our initial work, an earlier generation of DEP microarray devices were used to isolate DNA from blood samples, including cell free circulating (cfc) from Chronic Lymphocytic Leukemia (CLL) and other cancer patient whole blood samples. While the DNA could be isolated from blood samples in less than twenty minutes, the PCR genotyping had to be carried out in a separate device (PCR tube). We have now developed AC/DC electrokinetic devices and procedures that allow all steps, including the PCR reactions, to be carried out in the same chamber (in-situ). This represents a major advancement in “sample to answer” diagnostics in that the isolated DNA/RNA does not have be removed or transported into a different component device. We have also been able to carry out the in-situ (in the same device chamber) isolation and preparation of DNA from bacteria for subsequent DNA sequencing, this included lyses of the cells, proteinase treatment, recapture of the extracted DNA and sequencing related tagmentation reactions, all done in-situ. Introduction - AC electrokinetic methods like dielectrophoresis (DEP) are well-known techniques for achieving effective separations of cells, nanoparticles and biomolecules. Until recently, DEP technology had remained impractical for general use with complex biological samples (undiluted blood) and in high conductance solutions (>0.1 S/m). For example, very early DEP work on separating bacteria from blood (~7-9 mS/cm) required a 50-fold dilution of the blood sample before the DEP separation process could be carried out. Many other DEP separations, including cells, virus, polystyrene nanoparticles, DNA and proteins also required low-conductance conditions. More recently, some progress has been made by several groups on carrying out DEP under high conductance conditions. We for one, have developed new DEP devices and methods that allow both high molecular weight (hmw) DNA and nanoparticles to be isolated and detected in high conductance buffers (Krishnan R., Sullivan B.D., Mifflin R.L., Esener S.C., & Heller M.J., AC electrokinetic separation & detection of DNA nanoparticles in high-conductance solutions, Electrophoresis 29, 1765-1774, 2008; Krishnan R. & Heller M.J., AC electrokinetic method for enhanced detection of DNA nanoparticles, J. Biophotonics 2, 253-261, 2009; Krishnan R., Dehlinger D.A., Gemmen G.J., Mifflin R.L., Esener S.C., & Heller M.J., Interaction of nanoparticles at the DEP microelectrode interface, Electrochem. Comm. 11, 1661-1666, 2009). Using these special microarrays with robust platinum microelectrodes over-coated with a thin hydrogel layer, DEP separations were also carried out in undiluted whole blood, where hmw-DNA and nanoparticles could be concentrated into the DEP high-field regions over the microelectrodes and the blood (red and white) cells concentrated into the DEP low-field regions between the microelectrodes. A simple fluidic wash then removes the blood cells while the DNA and nanoparticles remain concentrated in the DEP high-field regions where they can be detected by fluorescence. Hmw-DNA could be detected at 260 ng/ml, which is a detection level suitable for cell free circulating (cfc) DNA biomarker analysis. Fluorescent 40 nm nanoparticles could be detected at 9.5 x 109 particles/ml, which is a level suitable for monitoring drug delivery nanoparticles. These DEP devices were next used to isolate cell free circulating (cfc) DNA from Chronic Lymphocytic Leukemia (CLL) and other cancer patient whole blood samples. While the cfc-DNA could be isolated from blood samples in less than twenty minutes, the PCR genotyping was carried out in a separate device (tube). We now have developed AC/DC electrokinetic devices and procedures that allow all steps, including the PCR reactions, to be carried out in the same chamber (in-situ). Results: Figure 1 (below) shows the earlier generation electric field microarray device used for the initial DEP separation work. The expanded view shows how the DEP high-field (red) regions and DEP low-field regions (green) are created when and AC electric field is applied to nine microelectrodes. Figure 1 - DEP electric field microarray device and the DEP high-field and low-field regions Original DC/AC electric field microarray devices were obtained from Nanogen and have one hundred 80um platinum microelectrodes overcoated with a thin hydrogel layer. Newer AC/DC dielectrophoretic/electrophoretic microarray (chip) devices specifically designed for the isolation of cfc-DNA from high conductance buffer and blood samples, as well as in-situ PCR, were obtained from Biological Dynamics. In general, DEP experiments were carried out by adding about 20 ul of blood or buffer sample to the DEP microarray device. The DEP field was applied at 10 kHz and 20 Vp-p for 15 minutes to the nine microelectrodes. The microarray was then washed three times with 0.5x PBS with the DEP field on, and finally examined by epifluorescent microscopy. Figure 2 (below) shows the results for the DEP separation of cfc-DNA from Chronic Lymphocytic Leukemia (CLL) cancer patient whole blood samples and a normal human blood sample. All images are in green fluorescence. The DEP AC voltage was applied to the nine electrodes in columns 2, 3, 4, while no voltage was applied to the three electrodes in column 1, which serve as negative controls. Figure 2 (a) shows the results for a 20 µl sample of normal human whole blood containing 200 ng/µl SYBR green fluorescent stain, where DEP was carried out at 10 kHz and 20 Vp-p for 14 minutes, and then the microarray washed three times with 0.5x PBS. No green fluorescent rings are seen around the edges of the nine activated microelectrodes indicating no cfc-DNA is present in the normal blood sample. (Note that no fluorescence appears around the three control microelectrodes which were not activated). Figure 2 (b)-(f) shows the results for five different CLL cancer patient blood samples at different disease stages. For these CLL samples, 20 µl of undiluted whole blood containing 200 ng/µl of SYBR Green I Dye stain where added to the microarray device and DEP was carried out at 10 kHz and 20 Vp-p for 14 minutes, and then the array was washed three times with 0.5x PBS. For many of the CLL samples, green fluorescent rings can now be clearly seen around the edges of the activated microelectrodes indicating that cfc-DNA has concentrated in the DEP high-field regions. The intensity of the green fluorescent suggests that some of these CLL blood samples contain relatively large amounts of cfc-DNA. (Again note that no fluorescence appears around the three control microelectrodes.) Figure 2 – (a) Normal blood after DEP (dotted white square indicates microelectrodes to which DEP was applied). (b) First CLL patient blood after DEP, yellow arrows indicate SYBR Green stained materials which are believed to be cfc-DNA. (c) Second CLL patient blood after DEP. (d) Third CLL patient blood after DEP. (e) Fourth CLL patient blood after DEP. (f) Fifth CLL patient blood after DEP. While cfc-DNA could be isolated from patient whole blood samples in less than twenty minutes, the cfc-DNA had to be extracted from these microarray devices and the PCR genotyping was carried out in a separate tube. We now have developed new AC/DC electrokinetic devices and procedures that allow all steps, including the PCR reactions, to be carried out in the same chamber (in-situ). Figure 3 below shows results for carrying PCR in-situ in the new AC/DC DEP/Electrophoresis microarray (chip) devices. The results show a comparison for PCR amplification of lambda phage DNA in both conventional PCR tubes and in the new AC/DC DEP microarray devices. Figure 3 – PCR results for amplification of lambda phage DNA in normal PCR reaction tubes and in the new AC/DC DEP microarray (chip) devices. Discussion and Conclusions: We have now developed AC/DC electrokinetic devices and procedures that allow all steps, including the PCR reactions, to be carried out in the same chamber (in-situ). This represents a major advancement in “sample to answer” diagnostics in that the isolated DNA/RNA does not have be removed or transported into a different component device. We have also been able to carry out the in-situ (in the same device chamber) isolation and preparation of DNA from bacteria for subsequent DNA sequencing, this included lyses of the cells, proteinase treatment, recapture of the extracted DNA and sequencing related tagmentation reactions, all done in-situ. The limitations for efficient DNA/RNA sample preparation have long plagued miniaturized lab on a chip devices and Point of Care (POC) diagnostic technologies, and significantly limited the overall assay sensitivity for many detection technologies. The ability to now rapidly isolated and detect disease related DNA biomarkers and bacteria/virus from undiluted whole blood will benefit many diagnostic applications by significantly reducing sample preparation time, cost and complexity.