Figure 2 shows the merging of two 10 μL droplets of deionized water (from Millipore Simplicity, Molsheim, France) on a superhydrophobic surface, clearly demonstrating two basic droplet manipulations: moving and merging. Movements were repeatable over the same line more than 10 times, regardless of the content of droplets. Movements were also successful for 5 and 20 μL droplets. Droplets were removed simply by tilting the surface and no further cleaning/rinsing was performed. Similar experiments were performed using polystyrene surfaces (plastic Petri dishes from Fisher Scientific; Pittsburg, PA, USA), but movements were not successful.

The water contact angles (θ) were 155 ± 2° on superhydrophobic surfaces, and those for 0.02% w/v antibody-conjugated particles and target solutions were not significantly different from those of water (154° to 156° with standard deviations of 2°), as measured using FTÅ200 (from First Ten Ångstroms, Portsmouth, VA, USA). The liquid surface tensions (γ_L) were also similar regardless of solution, and were measured at 73 ± 1 mN/m also using FTÅ200. The contact area of 10 μL droplet was measured as 2.0 ± 0.2 mm² (by FTÅ 200). Therefore, the work of adhesion (W_a) of 10 μL droplets to the superhydrophobic surfaces is:

(73 mN/m) (1 + cos 155°) (2.0 mm²) = 14 nJ.

Similar analysis can be made for the metal wires. (Note that a resistor was used merely as a metal wire; no voltage was applied.) Since the diameter of the metal wire was 0.5 mm and the insertion depth was 2 mm, the contact area between the metal wire and the droplet was 0.39 mm². Since most clean metal surfaces have a water contact angle of 10° 11, W_a can be estimated as:

(73 mN/m) (1 + cos 10°) (0.39 mm²) = 57 nJ,

which is larger than that of a droplet to a superhydrophobic surface, consequently enabling droplet movement.

The water contact angles on the polystyrene surfaces were 91 ± 5° and the contact area of 10 μL droplets were 10.5 ± 0.4 mm² (again using FTÅ200). The W_a of 10 μL droplet to a polystyrene surfaces is:

(73 mN/m) (1 + cos 91°) (10.5 mm²) = 750 nJ,

indicating a droplet cannot be moved with a metal wire on a polystyrene surface.

Figures 3, 4 and 5 show the maximum light intensities taken from the merged droplets. All results are the averages of three different experiments (i.e. each taken from different merged droplets). Error bars indicate standard deviations. Paired, two-tailed t-tests were performed by comparing each dilution with a blank (10 mM PBS). Dilutions with significant differences from the blank are indicated by the grey color. The detection limit for mouse immunoglobulin G (mIgG) was 50 pg mL^-1, equivalent to 0.5 pg of mIgG in a 10 μL target droplet.

The detection limit for bovine viral diarrhea virus (BVDV) was 2.5 TCID₅₀ mL^-1 (TCID₅₀ = 50% tissue culture infectious dose), equivalent to 0.025 TCID₅₀ for a 10 μL target drop. Although this detection limit is much lower than many other assays, it is still a possible number since there are at least hundreds or up to a few tens of thousands of virus particles in 1 TCID₅₀. The detection limit for E. coli was 85 CFU mL^-1 (CFU = colony forming unit), equivalent to 0.85 CFU for a 10 μL target drop and subsequently less than one bacterium (assuming 1 CFU = 1 viable cells). We have to recall that BVDV solutions were washed by centrifuging, eliminating a bulk of cell fragments and free antigens that may be recognized by anti-BVDV (specific or non-specific). We may attribute the smaller standard deviations for BVDV compared to mIgG and E. coli to this washing treatment. In contrast, E. coli solutions were not washed by centrifuging, and they possibly contained many cell fragments and free antigens 12. This indicates that the number of E. coli antigens may be much larger than one when the viable cell count = 1.

All three figures follow the so-called Heidelberger-Kendall curve 10, characterized by initial increase at lower target (antigen) concentrations, followed by a decrease at higher concentrations. The left-hand side (initial increase) can serve as a calibration curve, as well as providing a linear range of immunoassay. The right-hand side (decrease at higher concentrations) represents the antigen-excess region (i.e. too much target antigens for a fixed amount of antibodies bound to the particles), which inhibits antibody-antigen binding. The apparent advantage of this droplet manipulation is that the liquids are in minimal contact with solid surfaces. The bottom superhydrophobic surface consists largely of air pockets and the contact area of a top wire is extremely small. This minimized contact reduces biomolecular adsorption (biofouling), enabling repeated use of a platform. In fact, a single superhydrophobic surface could be repeatedly used for > 100 times for all droplets used in this experiments (blank and target solutions as well as antibody-conjugated particle suspensions). In this sense, we believe this setup can replace the standard microwell plate assays, where liquids are in direct contact with surfaces (thus resulting in more biofouling). Liquid volume is smaller than that of microwell plate assays, which may be further lowered to nanoliter scale (presumably in oil immersion to prevent rapid evaporation).

To demonstrate the automation possibility of these wire-guide droplet manipulations, we constructed a three-axis droplet manipulator using stepping motors and subsequent microcontrollers. Figure 6 shows the snapshots of pre-programmed movements of a droplet, starting from (1) taking a 10 μL droplet of 0.02% (w/v) antibody-conjugated particle suspension by inserting the wire into the droplet, (2) its linear movements, (3) merging with a 10 μL target droplet, and (4) linear movements of this merged 20 μL droplet towards the detection site. Complete movie is also available as Additional file 1, which also shows the movements controlled by a game pad.

We also demonstrated the rapid mixing of a merged droplet, by mechanically vibrating the wire with a vibration motor. Figure 7 shows the snapshots of this movie. Complete movie is also available as Additional file 1. The total cost of materials and supplies were less than $230 (excluding the experimenter's labor), which demonstrates the feasibility of this new droplet manipulation. Additionally, smaller stepper motors and integrated circuits (all commercially available) could greatly reduce the overall size of this setup.

These automated and programmed droplet manipulations eventually lead to "reprogrammable" digital microfluidics, where the reaction protocols can be altered by the user's program input or simply by the user's control of a game pad. Potential applications include, but are not limited to: potentiometric/conductometric titration, immunoassay, serial dilution, polymerase chain reaction (PCR) and single cell analysis.

Immunoglobulin G from murine serum, or mouse immunoglobulin G (mIgG), was chosen as a model protein (from Sigma-Aldrich; St. Louis, MO, USA). It was dissolved in 10 mM, pH 7.4 phosphate buffered saline (PBS) and various dilutions were made from a single stock solution. Bovine viral diarrhea virus (BVDV) was selected as a model virus pathogen. As its name indicates, BVDV causes diarrhea in cattle, leading to productivity loss and death. BVDV was cultured in Madin-Darby bovine kidney cells (MDBK) with appropriate tissue culture media (containing 5–10% fetal calf serum), followed by cell denaturation and centrifugal washing (from NVRQS; National Veterinary Research and Quarantine Service; Anyang, South Korea). Various dilutions were made from this stock solution; the tissue culture infectious dose 50 (TCID₅₀) value was provided by the manufacturer (NVRQS). Finally, E. coli was selected as a model bacterial pathogen. Lyophilized E. coli K-12 powder was purchased from Sigma-Aldrich and cultured in brain heart infusion broth (from Remel; Lenexa, KS, USA) at 37°C for 20 h. The colony forming units (CFU) was evaluated by plating some of the above dilutions on eosin methylene blue agar (from DIFCO; Lawrence, KS, USA), incubating at 37°C for 20 h and counting the number of colonies with a light microscope (from Nikon; Tokyo, Japan). Serial dilutions from the stock solution were made using 10 mM phosphate buffered saline (PBS).

Latex particles were purchased from Bangs Laboratories (Fishers, IN, USA). These particles were highly carboxylated, with 10.3 Ų parking area per carboxyl surface group, and a mean diameter of 920 nm according to the manufacturer's specifications. These particles should be mixed faster with target solution through their higher diffusivity, without using any surfactants, as we have recently demonstrated in microfluidic platforms 20. Maximum signals were obtained at 2 min, and all data points were taken at 2 min after merging two droplets. No significant evaporation was observed for this time frame. The particles were conjugated with three different polyclonal antibodies: anti-mIgG from Sigma-Aldrich, anti-BVDV from Jeno Biotech (Chuncheon, South Korea) and anti-E. coli from Abcam (Cambridge, MA, USA). These antibodies were conjugated to the particles by physical adsorption following the same protocol published previously 12. Antibody-conjugated particles were centrifuged twice, until no antibodies could be found in supernatants with the absorbance measurements at 280 nm. The surface coverage of antibodies was set to approximately 33% of its maximum possible value 21. The solid content of antibody-conjugated particles was 0.02% w/v.

Superhydrophobic surfaces made from nanocoatings of fluoropolymer on standard glass microscope slides were purchased from Surface Innovations (Durham, England). These nanocoatings create air pockets and put a microdrop in metastable Fakir state 22.

Figure 8 shows the experimental setup for the droplet manipulations and subsequent backscattering detection. Briefly, 10 μL each particle and target solution droplets were placed on a single superhydrophobic surface that was sitting on the positioning stages (from Edmund Optics; Blackwood, NJ, USA) capable of X-, Y- and Z-movements. A metal wire was inserted into the rightmost particle droplet by adjusting the positioning stages. The positioning stage was moved to the right so that the target droplet moved to the right with the superhydrophobic surface, while the particle droplet was fixed with a metal wire. Once the two droplets were merged, the positioning stage was moved downward to remove the metal wire. The merged droplet was then moved to the right so that it was positioned underneath the backscattering probe. The backscattering probe with optical fibers was purchased from Ocean Optics (Dunedin, FL, USA); it consists of a core-shell bundle of optical fibers and is shown on the right in Figure 8. A single fiber at the core delivers 375-nm light from a light emitting diode (LED; LS-450 from Ocean Optics) and six fibers at the shell side collect 180° backscattered light and transfer the signal to a USB4000 miniature spectrometer (from Ocean Optics). The positioning stages were moved along the X-, Y- and Z-directions to collect the maximum light intensity. SpectraSuite software (from Ocean Optics) was used to collect light intensities at 375 nm, without averaging over adjacent wavelengths (i.e. no smoothing). The integration time for data collection was 100 ms.

Figure 9 shows the experimental setup for motorized and programmed manipulations of droplets. The computer controlled three-axis droplet manipulator utilizes a microcontroller with a USB interface, 1.8° stepper motors, and an aluminium structure with THK SSR25 linear bearings. The aluminium components were machined from 6061 alloy blocks and extruded t-channels via three-axis Lagun vertical mill to within ten-thousandths of an inch accuracy. The t-channels serve as a bracket to create a rigid body with the stepper motors and linear bearings. Spacers attached between the linear bearings allow the output shaft of the stepper motors to rotate a threaded bolt that passes through it, transforming rotational motion to linear displacement along the direction of the output shaft. The microcontroller is an Arduino, an open-source electronics prototyping board manufactured by SmartProjects (Italy). The stepper motors are two-phase with a 1.8° step angle by SparkFun Electronics (Boulder, CO, USA). This means the stepper motors can complete 200 steps in a full 360° rotation. When combined with the EasyDriver V3 Stepper Motor Driver, also by SparkFun Electronics, the A3967 integrated circuit allows for microstepping, which allows the stepper motors to complete 8 microsteps per step. Thus, the stepper motors are capable of undergoing 1600 individual steps per rotation. The thread pitch is 0.79375 mm, therefore with 1600 steps per rotation, the theoretical displacement per step is 0.496 μm. With 200 steps per rotation, the displacement per step is 3.96 μm.

The Arduino board outputs simultaneously to three stepper motor driver boards, one per axis, through digital outputs integrated on the board. An original Nintendo controller wired into the digital inputs on the Arduino board allows the user to control the wire's position on-the-fly. The directional pad controls the X and Y axis movements, and the A and B buttons control the Z axis movement (i.e. insertion/retraction of a wire into a droplet). In conjunction with the controller, the system can be easily programmed to complete certain movements precisely and with consistency each time. The stage can be adjusted to ensure level surface conditions when working with superhydrophobic surfaces.