MICROTHERMAL TECHNIQUES FOR MIXING,CONCENTRATION, AND HARVESTING OF DNA
AND OTHER MICRODROPLET SUSPENSIONS
Amar S. Basu*, and Yogesh B. Gianchandani
EECS Department, University of Michigan, Ann Arbor, USAABSTRACT
The use of microprobes to manipulate, concentrate, and sample aqueous droplets within anoil phase on a blank substrate is explored. In particular, this paper introduces a number ofthermal-based techniques which make use of a point heat source to perform high speed
mixing of droplets at speeds up to 300 rpm, collection and merging of droplets,concentration of suspended particles through controlled evaporation of the droplet, andprecipitation of low concentration suspensions such as DNA onto a microprobe tip.
Quantities of DNA as low as 10 ng have been sampled on a 15 µm diameter tip.
Keywords: microdroplet, mixer, DNA precipitation, preconcentration
1. INTRODUCTION
Microscale investigations in cellular and biochemical analysis often call for flexibility
beyond what can be achieved with micromachined substrates. In such situations,microprobe-based tools can be used for localized sensing and actuation instead of fluidic
chips. Among the best known examples are microdroplet-based chemical analysis systems,
where micron-scale droplets of aqueous suspensions submerged within a continuous oilphase serve as low-volume, high-speed chemical reactors for quantifying single cell
enzyme kinetics [1], concentrating nanoparticles and dissolved solutes [2], detecting low
concentrations of molecules [3], and amplifying single molecules of DNA [4]. In contrast
with the traditional approach of processing liquids in a microfluidic chip, samples andreagents are manipulated in droplet form using a variety of localized techniques, including
micropipettes, optical pressure [5], and electrostatic tips [6]. These systems attain many ofthe benefits of conventional microfluidics including fast reaction times and low reagent
consumption; however, they also offer additional benefits such as reduced adhesion of cells
and hydrophobic molecules to channel walls, as well as simplicity and flexibility since noprefabricated chips are required.
This paper introduces a number of micro-thermal techniques for common procedures in
microdroplet systems, all of which make use of a heated microprobe tip; namely, wedemonstrate high speed mixing of droplets, collection and merging of droplets,
concentration of suspended particles, and the aggregation of DNA onto a microprobe tip.
2. CONVECTIVE MIXING OF DROPLETS
A heated metal tip (φ=5-620 µm, T=35-45°C) placed in contact with the surface of athin layer of mineral oil (200-1500 µm) establishes a micro-scale temperature gradientextending radially from the region directly beneath the heat source. The resulting
convection currents flow radially outward on the top surface of the liquid pool, and inward
below the surface, forming self-circulating toroidal streamlines which have been used totrap small solid particles and droplets [7]. In the previous work, the particles are small
compared to the cross-sectional height of the convective flow region, and, as a result, they
follow the toroidal streamlines. In the present work, we show that when the height of theflow region is approximately the same as the droplet diameter (Fig. 1a), the convective
currents can rotate and mix the droplet in various patterns depending on the size of theheated tip. In the case where the tip diameter is approximately the same size as the droplet,the droplet rotates about a single axis at speeds up to 300 rpm (Fig. 1b). As the droplet
diameter is progressively increased, the rotational speeds fall, and the flow pattern begins tochange (Fig. 1c). Eventually, when the tip diameter is small compared to the droplet, aflow pattern composed of two vortices and turbulent eddies is observed instead of rotation(Fig. 1d). Both patterns can be useful for micro mixing within a single droplet.

Fig 1: (a) A microprobe tip in contact with a thin oil layer generates a micro-thermal gradient which
drives convective flows around and within the droplet. (b) Single axis rotation occurs when a 650µm tip is placed near a droplet of approximately the same dimensions. (c) The effective rotational
speeds using a 50 µm tip fall as droplet diameter is increased due to the change in flow geometry.
(d) Vortices and turbulent eddies can be seen instead of rotation when a small (15 µm) tip is used inconjunction with a larger (φ=1000 µm) droplet. In both experiments, the tip temperature was ~3545°C. Flow patterns are visualized using immersed fluorescein particles and a 0.5 second CCD
exposure with 490 nm/500 nm excitation/emission filters.

3. DROPLET COLLECTION AND MERGING
The ability to merge discrete droplets is important capability in microdroplet systems,
as it allows reagents to be mixed at time scales fast enough to study chemical kinetics [1,6].
To collect and merge droplets, the heated tip is suspended just above the oil layer, and heattransferred to the oil surface drives convection rolls in the same manner as described above.
Rotating droplets trapped in the flow collide and merge together without the aid of asurfactant (Fig. 2). The rotation and reduced surface tension due to heating may both assist
in droplet merging. By scanning the heat source laterally, several droplets over a large area
can be collected and merged.

Fig 2: A 650 µm diameter, 80°C wire tip
suspended < 100 µm above mineral oil is used tocollect and merge four droplets (φ=200-400 µm).
(Top) The droplets are trapped in the selfcirculating convective flow. (Right) The droplets
merge together, one by one, eventually forming asingle, φ=600 µm droplet in 9 seconds.

4. CONCENTRATION OF SUSPENDED PARTICLESUnlike droplet evaporation on a solid surface, where suspended particles eventuallydeposit themselves in a circle (the commonly observed ‘coffee ring’ [8]), particles in an oilimmersed microdroplet aggregate towards the center instead, eventually forming a
concentrated solid precipitate after the liquid has completely evaporated. Microdroplet
evaporation is, therefore, an effective means to concentrate particles dissolved solutes, but
evaporation times for even small (φ=10 µm) droplets can be greater than 1 hour [2]. A
heated tip placed next to a suspended microdroplet enhances the evaporation rate, allowing
controlled evaporation of an 1800 µm droplet in less than 3 minutes (Fig. 3). The linear
reduction of the droplet surface area over time is consistent with previous reports [2]. Afterthe droplet has evaporated, the concentrated solids remain in the oil, trapped in theconvective flow described above.

Fig 3: (a) A 60°C, 50 µm tip is placed in contact with the oil near an 1800 µm droplet containing
suspended 3 µm polystyrene beads. As the liquid evaporates, the dissolved particles concentrate in
the center of the droplet, forming a solid micro-particle suspended in the oil. (b) The surface area ofthe droplet decreases linearly with time until all the liquid is evaporated at 150 s.

5. AGGREGATION DNA ON A MICROPROBE TIP
If a droplet is evaporated with the heated tip immersed within it, any suspended or
dissolved compounds aggregate on the tip as the droplet evaporates. This provides a means
to concentrate and ‘nanosample’ small amounts of solutes onto a probe tip for subsequent
analysis using methods such as micro-IR spectroscopy [9]. Fig. 4 shows the aggregate

obtained from 1 µL samples of high concentration calf thymus DNA (10 µg/µL, Invitrogen)and low concentration human male DNA (10 ng/µL, Applied Biosystems) on a φ=15 µm
metal tip. When the tips are placed in a DNA staining solution, much of the aggregate iswashed away; however, fluorescent images show that adsorbed DNA residues remain.

Fig 4: (a) A 15 µm, 40°C tip is lowered into a floating droplet containing DNA suspension. As theliquid evaporates, DNA aggregates onto the tip, and the tip is then removed from the oil. (b) DNA
precipitate collected from 1 uL samples of 10 µg/uL Calf Thymus DNA (top) and 10 ng/µL HumanMale DNA (bottom), shown in a bright field image before staining (left) and in 490 nm/500 nm
fluorescence after immersing in SYBR Green I DNA stain (right).

6. CONCLUSIONSThe experiments described in this effort demonstrate that a heated microprobe tip is aversatile tool that accomplishes several functions in chipless microdroplet analysis systems,
including droplet mixing, merging, concentration, and precipitation.
ACKNOWLEDGEMENTS
Funding provided by The Whitaker Foundation (AB) and the University of Michigan.
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* 1301 Beal Avenue, Ann Arbor MI 48109, USA; Tel: (734) 709-6688; Email: basua@umich.edu