Page 40 - ASME_NEMB_2016_Program

Technical Program TRACK 3

9:30am DNA assays leveraging ion concentration shock waves the paper-based microfluidic device offers a simple, rapid, and cost-effective
sensing platform which provides a relevant limit of detection. Generally, this
Keynote. NEMB2016-5918 paper-based microfluidic technique could be applied to other biomarkers
such as cardiac panel, cytokines, and liver panels, which requires much high
sensitivity.
Juan Santiago, Stanford University, Stanford, CA, United States

We use on-chip isotachophoresis (ITP) to create electric-field-driven shock 10:20am Counter-Flow DNA Preconcentration Without Applied
waves of ion concentration. These waves are formed at the interface be- Electric Fields
tween a high mobility leading electrolyte (LE) and a low mobility trailing
electrolyte (TE). Ionic species with mobilities bracketed by these electrolyte Technical Presentation. NEMB2016-6042
species focus at the LE-to-TE interface. For trace sample concentrations,
multiple species mix and co-focus inside a single, order 10 µm wide zone. Sarah M. Friedrich, Johns Hopkins University, Baltimore, MD, Unit-
Multiple reactants can be mixed and then pre-concentrated by more than ed States, Jeffrey M. Burke, Circulomics, Inc., Baltimore, MD, Unit-
50,000x in a few minutes to accelerate chemical reactions. We apply this ed States, Kelvin Liu, Circulomics Inc., Baltimore, MD, United States,
technique to extract and purify DNA or RNA targets from complex biological Tza-huei Wang, Johns Hopkins University, Baltimore, MD, United
samples and to immediately co-focus these with synthetic DNA probes that
we design. We can complete in 30 sec chemical reactions which would nor- States
mally take 4 days. Presented will be recent work on applying this technique
to heterogenous reactions between macromolecule targets in solution and We report a method to spontaneously induce DNA migration and concen-
synthetic DNA probes immobilized on a surface. Quantitation of the reaction tration without the use of externally applied electric fields. Electric fields are
product provides a sequence-specific detection scheme, and so the tech- commonly used to manipulate DNA molecules because they have a high
nique has applications to medical diagnostics and basic biological studies. negative charge. For example, gel and capillary electrophoresis techniques
Current work also includes integrating ITP-based extraction and focusing to exploit differences in electrokinetic mobility to separate DNA molecules
fractionate and analyze nuclear versus cytoplasmic RNA from single cells. by size. Similarly, concentration techniques such as large volume sample
stacking and isotachophoresis utilize changes in electrokinetic velocity to
concentrate DNA molecules at buffer interfaces. However, employment of
10:00am Chemically Patterned Paper-Based Microfluidic Device
for Bioassay electric fields in microfluidic devices necessitates careful design and place-
ment of electrodes, additional microfabrication steps, consideration for gas
bubble generation and pH shifts, and the use of expensive power supplies.
Technical Presentation. NEMB2016-6062 Alternatively, pressure-driven flow can be used to manipulate DNA mole-
cules without the use of electric fields. Indeed, pressure-driven flow through
TRINH LAM, Jungkyu Kim, Texas Tech University, Lubbock, TX, long microchannels has been used to separate DNA molecules by size
United States analogously to capillary electrophoresis, but without a gel matrix or applied
electric field. Using Cylindrical Illumination Confocal Spectroscopy (CICS) as
Paper-based microfluidics has been a promising technology that has ac- the detection method for single molecule hydrodynamic separations (SML-
complished many achievements in biological and chemical analysis. This FSHS) increased the limit of detection to tens of molecules, but the small
technique offers a number of useful capabilities: the ability to minimize pL injection volume restricts the concentration range of DNA molecules to
consumption of samples and reagents for various bioassays, cost effective the mg/mL (or pM) level. In-line preconcentration methods could enable
fabrication, and relatively rapid analysis. However, current fabrication meth- analysis of circulating cell-free DNA (ng/mL), an emerging biomarker for can-
ods creating hydrophobic barriers such as photolithography, wax printing, cer and prenatal testing, without sample amplification or adjustment of the
and ink jet printing, still require a complex fabrication technique, expensive microfluidic handling and injection volume. In this work, we demonstrate that
equipment, and thermal sensitive property. In this study, we introduce a pressure-driven flow can also be used to concentrate DNA without the use
simple procedure to create hydrophobic barriers using printing cutter and of electric fields in a method we call Counter-Flow Concentration (CFC). We
trichlorosilane (TCS) coating through chemical vapor deposition (CVD). Using also demonstrate the ease with which this concentration method could be
this Chemically Patterned Paper-based Microfluidic (cPMD) technique, we integrated with downstream separation and sizing analysis by coupling CFC
are able to create a micro scale patterns on various paper matrices which to Single Molecule Free Solution Hydrodynamic Separation (SML-FSHS).
are independent of temperature changes. As opposed to current electrokinetic concentration methods, counter-flow
concentration (CFC) is very simple to assemble and operate. A high con-
To create a hydrophilic fluidic pattern, a part of chromatographic paper was centration salt solution (25 mM Tris-HCl) is driven via pressure-driven flow
masked by using patterned vinyl tape, and TCS was then vaporized onto the through a microcapillary (5 micrometer diameter, commercially purchased
masked chromatographic paper. After a certain settling time inside vacuum from Polymicro Technologies) to empty into a large reservoir containing a
for CVD, the vinyl tape was removed to fabricate a paper-based microfluidic low ionic strength solution (water) and dilute DNA molecules. Diffusion of
device with desired shaped channels. The cPMD method was characterized the salt species down the concentration gradient (away from the capillary
and optimized by defining duration time of CVD associated with resolution and into the sample) induces diffusiophoretic migration of DNA in the oppo-
of hydrophobic patterns. From a series of experiments with different sized site direction and towards the high ionic strength region. Surprisingly, this
patterns, cPMD technique enables formation of pattern as low as 500 µm migration occurs in the opposite direction of flow and results in DNA mole-
width on both chromatographic and nitrocellulose papers. With this opti- cules spontaneously accumulating in a small picoliter volume immediately
mized CVD conditions, we demonstrated glucose assay on the patterned adjacent to the capillary outlet. We have investigated the effects of flow
chromatographic paper. All paper-based glucose assay results were com- rate on the concentration factor and have demonstrated that the use of an
pared to assay results from a standard 96-well plate. With glucose assay optimized counter-flow rate can achieve higher concentration factors than
on this paper-based microfluidic device, various concentrations of glucose applying no flow. The concentration mechanism is also highly dependent
were prepared and then tested on the paper. With increasing of the glucose upon the buffer pair and geometry: concentration only occurs when the high
concentration, the color of each channel on the paper gets darker. Using ionic strength buffer flows into the low ionic strength sample.
iColorMeter app on a smart phone, the images of the assayed papers were
obtained and analyzed RGB intensity of each well to determine their glucose The concentrated bolus can be easily captured by quickly reversing the
concentration. All the results from paper-based glucose assay was then flow direction and injecting the small concentrated volume into the capillary.
compared to 96 well-plate results. From glucose assay, the detection limit Thus, CFC can be coupled to SML-FSHS sizing analysis by individually con-
of the device was found to be below 10 µg/mL. Compared to other glucose trolling the pressure at both sides of the capillary with separate valves. This
40 integrated platform is capable of fully automated DNA concentration, sep-
sensing devices, the paper-based microfluidic device can provide a proper
limit of detection. As compared to a commercially available glucose sensor, aration, and detection. We used this platform to quantify the concentration
factor of DNA molecules over a range of sizes (2 to 27 kbp) from a single