Mixing fluids for biochemical assays is problematic when volumes are very small (on the order of the 10 µL typical of single drops), which has inspired the development of many micromixing devices. In this paper, we show that micromixing is possible in the simple open wells of standard laboratory consumables using appropriate acoustic frequencies that can be applied using cheap, conventional audio components. Earlier work has shown that the phenomenon of acoustic microstreaming can mix fluids, provided that bubbles are introduced into a specially designed microchamber or that high-frequency surface acoustic wave devices are constructed. We demonstrate a key simplification: acoustic micromixing at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature. The meniscus of a drop in a small well provided an appropriately small radius, and so an introduced bubble was not necessary. Microstreaming showed improvement over diffusion-based mixing by 1–2 orders of magnitude. Furthermore, significant improvements are attainable through the utilization of chaotic mixing principles, whereby alternating fluid flow patterns are created by applying, in sequence, two different acoustic frequencies to a drop of liquid in an open well.

Introduction

An increasing range of assays are performed on very small sample volumes (1). Often a single drop on the order of 10 µL must be mixed with a reagent that has a similar volume, to make the resulting concentration uniform. Rapid mixing requires that the interfacial area between the two fluids be greatly enlarged, so that the average distance between the two fluids’ molecules becomes small enough for diffusion to work on a short time scale (2,3). For fluid volumes greater than a few tens of microliters, this is achieved through turbulence, which is readily induced by actions as simple as shaking the container. However, turbulence is absent for drop-wise volumes (4), requiring artificial means of setting the fluids into motion. The challenge is to mix drops over a time scale of tens of seconds rather than the hours typical of molecular diffusion. There are many aspects to micro-mixing (2,3) for which many special devices have been engineered (5,6,7,8,9). In some cases, however, the use of existing well-plate methods would minimize disruption to existing protocols.

Acoustic microstreaming is where sound waves propagating around a small obstacle create a noticeable flow in the vicinity of the obstacle. It is usually noticeable when the obstacle is a bubble, because a bubble has particular abilities to locally amplify and transform an acoustic field (10,11,12,13,14,15). Liu et al. (5,6) used the excitation of air bubbles trapped in specially designed pockets inside a 50-µL chamber to show that cavitation microstreaming (a form of acoustic microstreaming) could be used for micromixing. More elaborate microdevices containing trapped bubbles for acoustic micromixing have recently been engineered (16,17). It has also been shown that surface acoustic waves at frequencies of ≥ 100 MHz, which must be generated by specially manufactured piezoceramic devices, could generate acoustic streaming in open wells (18,19). Apart from engineered micro-mixers, microstreaming in general has many applications: for example, enhancing mass transfer in sonochemistry (20). Recent biological applications include sonothrombolysis (21) and sonoporation (22).

Fundamentally, the generation of acoustic microstreaming is due to a nonlinear rectification of the oscillatory fluid motion due to the sound waves. This creates a mean flow (23) that is noticeable only if there is a large gradient in the acoustic field. If the interface between a liquid and a gas has a small radius of curvature, there will be a locally large gradient in the sound field where it is forced to distort around the small radius. A small bubble is one way to create a large gradient in the sound field in the surrounding liquid. Another way is to use very high frequencies (18,19,24) that rapidly decay spatially, thus providing a large gradient.

Once the underlying physics is appreciated, it becomes clear that the trapped-bubble (5,6,12,16,17) or high-frequency surface-acoustic wave (18,19,24) systems hitherto investigated are different cases of the acoustic microstreaming phenomenon, and that each demands a specially designed microdevice. Of course, in the context of novel lab-on-a-chip devices where many other functions need to be performed, a specially designed microdevice is the objective. However, there are assays where micromixing is the bottleneck in a process that otherwise works well. In the present work, we tried to generate microstreaming with fluid containers more typical of standard laboratory consumables, and by using audio frequencies. Furthermore, principles of chaotic mixing were applied to see if further improvements were possible. Here, chaos is created in the topology of the interface between the two fluids, creating fine striations that greatly enlarge the interfacial area.

Materials and methods

The experimental chamber (well) was a 4-mm diameter hole drilled through an acrylic plate, 76 × 195 mm in size and approximately 1.63 ± 0.02 mm thick. The chamber top was open to the air and the bottom was sealed with office sticky tape (Cat. no. 87250; Marbig, Sydney, NSW, Australia). In prior experiments (12), acoustic streaming was generated by piezoelectric transducers at frequencies ranging from hundreds of Hertz to >1 MHz; however, more powerful conventional audio speakers can generate streaming velocities significantly higher than noted (see Reference 12). Therefore, in the present work, a pair of audio speakers (75 mm diameter, 4 Ohm, 20 kHz) were mounted firmly on the base of a microscope (Model no. PZMTIII; World Precision Instruments, Sarasota, FL, USA) and the acrylic plate was taped to them with the well in between the speakers.