Siavash
Ahrar
,
Michelle
Hwang
,
Philip N.
Duncan
and
Elliot E.
Hui
*
Department of Biomedical Engineering, University of California, Irvine, 3120 Natural Sciences II, Irvine, CA 92697-2715, USA. E-mail: eehui@uci.edu; Tel: +1 (949) 824-8723
First published on 6th November 2013
Serial dilution is a fundamental procedure that is common to a large number of laboratory protocols. Automation of serial dilution is thus a valuable component for lab-on-a-chip systems. While a handful of different microfluidic strategies for serial dilution have been reported, approaches based on continuous flow mixing inherently consume larger amounts of sample volume and chip real estate. We employ valve-driven circulatory mixing to address these issues and also introduce a novel device structure to store each stage of the dilution process. The dilution strategy is based on sequentially mixing the rungs of a ladder structure. We demonstrate a 7-stage series of 1:
1 dilutions with R2 equal to 0.995 in an active device area of 1 cm2.
A number of microfluidic devices for serial dilution have been previously reported. Continuous flow approaches utilize a tiered network of resistors to mix two fluids at ratios specified by the circuit design.1,2 These devices output an entire dilution series in parallel and are composed of simple channel networks with no moving parts. However, the need to sustain continuous flow requires a relatively large initial sample volume (5–100 μL),1 and long channel lengths are required to enable good mixing, resulting in fairly large devices (∼12 cm2).1 Additionally, the total fluidic resistance of this approach increases nonlinearly as the number of dilution steps increases, hence the scaling potential is unclear.
A second approach utilizes integrated valves to meter and mix defined volumes of sample and buffer. Paegel and colleagues implemented such an approach based on peristaltic pumping around a single ring mixer.3 A small sample (400 nL) was diluted by a fixed ratio during each dilution step, in which a volume of buffer was metered into the ring and circulated until fully mixed with the sample. The recirculating design eliminated the need for banks of long mixing channels, thus resulting in a compact device (∼1 cm2). Additionally, the possible number of sequential dilution steps was essentially unlimited. However, only the final dilution was stored, and the intermediate steps in the dilution series were discarded.
Here, we present an evolution of the Paegel dilution ring that is capable of storing every stage of dilution. As illustrated in Fig. 1, a series of chambers are arranged like rungs on a ladder and primed with a buffer solution. A small sample volume of 240 nL is loaded into the top rung. To begin the dilution process, the top two rungs are connected to form a loop, and the liquid contents are mixed by peristaltic pumping to perform a 1:
1 dilution. The second and third rungs are then connected and mixed to perform a subsequent dilution, and the process is repeated all the way down the ladder. When completed, the full dilution series is stored on the rungs of the ladder. The design is readily scalable, as dilution stages can be added with additional rungs. We demonstrate a 7-stage dilution in an active device area of only 1 cm2.
Food dye was first diluted into water to facilitate visualization of device operation (Fig. 1b, ESI, Movie 1†). Next, in order to quantify dilution efficiency, fluorescein was diluted into TE buffer and imaged by fluorescence microscopy. The log2 plot of fluorescence intensity for each dilution stage showed excellent linearity (R2 = 0.995 to 0.997 over 3 trials), demonstrating that the device can perform a 7-stage 1:
1 serial dilution with very high accuracy (Fig. 3).
Extending the design to perform more dilution stages should be straightforward, requiring only the addition of more rungs to the ladder. The 7-stage device is fairly compact, with the ladder structure measuring 8 mm wide by 13 mm long. In comparison, a previously reported continuous flow design required a 6 cm long device to achieve 8 dilution stages.1
Our device builds off of the circulatory mixing of the Paegel device,3 but adds the ability to store each stage of dilution. This can also be accomplished by a different strategy, which utilizes valves to direct packets of liquid around a 8 × 8 grid structure.7 This versatile device is capable of a variety of complex liquid manipulations, and by mixing and splitting different volumes of liquid, the device can perform multistage serial dilutions with storage of each dilution stage. Rather than pursuing this level of flexibility and modularity, we designed a simple device focused solely on serial dilution. The strategy is straightforward and should be readily incorporated into larger microfluidic systems.
Alternative strategies for serial dilution have also been demonstrated by using droplet microfluidics.8,9 As with our method, these approaches are capable of processing small samples and can store each stage of dilution in a separate droplet. While droplet devices have the advantage of simplicity, with no moving parts, valve-based systems offer more flexibility. If serial dilution is not the end goal but rather the beginning of a multi-step procedure, the required liquid-handling manipulations may be complex enough that valves become required even in a droplet device. Furthermore, droplet systems require off-chip pumps that add to the complexity of the total system. In our design, the pumps are integrated into the device itself. While in the current implementation these on-chip pumps require off-chip actuation, we have shown elsewhere that pump control can be accomplished by on-chip microfluidic circuits.10,11 The emergence of microfluidic digital logic technologies12 offers the possibility that complex control algorithms can be programmed directly into microfluidic circuits, allowing devices to operate autonomously without the need for external controllers. The serial dilution method presented here may be well suited for integration into such systems, thus allowing complex sample processing to be combined with very simple user operation.
Footnote |
† Electronic supplementary information (ESI) available: Operation of serial dilution ladder. For illustrative purposes, video playback is 4× real time, and only the first 4 seconds of mixing (16 s in real time) are shown for each stage. See DOI: 10.1039/c3an01710a |
This journal is © The Royal Society of Chemistry 2014 |