©20 05 TONY FERNANDEZ
The Quest for HighSpeed and LowVolume Bioanalysis
Thomas Laurell Johan Nilsson György Marko-Varga
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ithin a cell, clusters of proteins and protein complexes are tightly packed in a confined space. Each of these proteins has a role or a function, either as a free operator or as an agent within a complex. In either case, this microenvironment allows delicate biological communication—for example, receptor activation, pathway signaling, and transcription factor initiation—in which splicing mechanisms will determine the final protein product. Many of these cellular events occur at the nanoscale, and the desire to understand the detailed mechanisms that control the dynamics of these systems is a core issue in current life science research. In particular, on the clinical side, considerable interest exists in identifying proteins that Nanovials, microarrays, and microfluidics can serve as biomarkers for human disease (1). It seems are brought together for the exploration likely that many biomarker proteins of complex biological problems. are present in vivo at very low levels. Hence, their identification is a formidable analytical task, and the resolving power needed for these clinical proteomics studies is not fully understood yet (2). However, our group has been exploring the combination of dense ar© 2005 AMERICAN CHEMICAL SOCIETY
Lund University (Sweden) rays of nanovials in microfluidic platforms as a way to perform single-component analysis in samples of biological fluids. In this article, we discuss the trade-offs in overcoming the challenges inherent in this approach to reach the expected benefits.
Is smaller better? The trend toward smaller, faster, and denser arrays and assays with the goal of massively multiplexed mapping of components in biological samples is well established. One of the most obvious and widespread examples has been the breakthrough of DNA microarray technology—a format that has been heavily explored and is constantly being improved. On the industrial scale, the need for higher throughput has driven the shift from microwell plates with 96 wells to those with 384 or 1536 wells. This 4- to 16-fold reduction in size means that less reagent is needed per assay, and the resulting cost savings has evidently been beneficial. But there is plenty of room for further miniaturization. The 1536-well standard only approaches the boundary of the microscale domain; it does not come anywhere near the nanoscale (Figures 1a and 1b). Another motivation for
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moving to a smaller scale is the potential for higher sample yield—due, in part, to reduced adsorption of analytes in smaller vessels. In addition, merging micro- or nanotechnology with chemistry offers the possibility of fabricating high-density devices that have integrated higher-order functionalities, such as sensing structures. Finally, working at small scales offers analysts the choice of traditional modes of analysis or new readout modes, which are made possible by physicochemical scaling effects that become prominent in the micro- and nanoscale domain. However, the move from the macroworld to the nanoscale format poses inherent challenges. One critical issue is that if the assay dimensions are reduced but the sample volume remains large, the probability of actually finding a molecule becomes a severe statistical problem. Therefore, new strategies to enrich and confine the original sample during the preprocessing steps are important. Fluidic interfacing between the sample volume and the assay volume is also a challenge. Furthermore, massively parallel assay formats produce enormous amounts of information. For example, a clinical proteomics experiment can easily generate a terabyte of data, and handling and storing the gigantic raw files are not trivial tasks (3). Intelligent algorithm developments and up-to-date database tools are mandatory to correlate and cluster the data with biological functions and states. Unless new informatics and information technology principles are developed, high-density nanoscale formats would need orders of magnitude more space for data storage and processing.
trochemistry and various aspects of analyte diffusion in a confined space (11, 12). The integration of electrochemistry was demonstrated by fabricating 200-µm-diam polyimide nanovials with a photolithography-defined three-electrode system at the bottom (13). These nanovials were also used to study purine release from individual myocytes (14). A further advance was the manufacture of screen-printed electrodes in a layered nanovial structure. In this process, the final fabrication step was laser ablation of all screen-printed layers, which left a nanovial in which the side walls encompassed the electrodes (15). The development of new microfabrication technologies has been instrumental in a wider range of approaches to nanovial construction. For example, a laser-ablation direct-write strategy followed by molding steps was used to produce PDMS-based microfluidic structures and nanovial arrays, which could work as microreactors/containers for controlled crystallization processes (4). A disposable array with 1200 400-µm nanovials, arranged in groups of 100, was adapted for MALDI TOFMS applications. The array was fabricated in polycarbonate by cold embossing (16 ). Similar arrays were recently reported for performing cellfree protein expression and functional assays of large sets of protein libraries (17 ). In vitro transcription and translation analysis before expression, as well as enzyme inhibition assays, were demonstrated. (a)
(b)
Early nanovials Typically, true nanoscale well plates have been realized in microarray formats with individual vials of 20–500 µm (4 –8). One example of a well plate, with nanovials of 43 and 66 nm diam, has been realized in 89-nm-thick gold films for academic use (9). This cutting-edge approach made it possible to monitor singlemolecule interactions in a massively parallel format by capitalizing on the confined optical properties that result when work is done at such small dimensions. In particular, the fluorescence background interference was suppressed by the zero-mode waveguide domain; this was accomplished by working at the nanoscale. In another example, ~30-nm-diam nanopores were used to separate DNA fragments for individual biomolecule monitoring. This approach involves pressurization steps in an electrophoretic microfluidic chip (10) and enables DNA fragments of up to 15 kilobase pairs to be analyzed in a cycle time of 100 s. Nanovials are also a natural development in the ongoing efforts to miniaturize electrochemical detection. In the late 1990s, microfabricated nanovials were made in hot-embossed polyurethane with microstructured silicon as the embossing master. The transparent polymer offered simultaneous optical supervision of single-cell experiments and electrochemical readout. Fundamental studies on scaling effects were performed to assess the elec266 A
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FIGURE 1. Piezoelectric microdispensing. (a, b) Conventional microwell plate and pipette tip compared with a nanovial array. (c) Actual stroboscopic image of a high-speed (5-kHz) piezoelectric dropletgeneration sequence. To see a video of high-speed microdispensing, go to pubs.acs.org/subscribe/journals/ancham-a /77/free/ 705feature.avi.
(Figure 1c). This step increases the surface-area-to-volume ratio of the sample, which, in turn, increases the evaporation rate. Thus, samples can be rapidly enriched in a fixed spot area and position, for example, in a nanovial, just by the transition to the micrometer domain. Nevertheless, evaporation can be problematic at very small scales. In an open environment, it can be difficult to maintain the chemistry in a wet state. The combination of Working in humidity chambers solves the problem to some extent. Other posnanovials, microarrays, sible solutions include liquid compensation via capillaries to match the evaporation rate (6) and the use of a volatile and microfluidics in liquid cover to prevent evaporation (20). Although it seems to be a delicate “megadense” formats task to use controlled flow through a capillary to compensate exactly for evapoffers new ways to oration, the system is self-controlled. If the liquid compensation rate is slightly too high, a positive liquid meniscus will address complicated develop in the nanovial, providing a larger free liquid surface for evaporabiological problems. tion. As the surface grows, the evaporation rate eventually matches the liquid supply rate. This self-controlled evapoSample enrichment and ration also has been used to control liquid volumes in acoustievaporation The paradox of reduced dimensions is that they inherently di- cally levitated droplets for wall-less nanochemistry by continuous minish the possibility of detecting low-abundance proteins be- solvent supply via a piezoelectric microdispenser (21). cause, statistically, the molecule of interest may not be present in the selected volume. That is, if one monitors a species present in Sample handling a micromolar to nanomolar concentration in a volume of 100 In terms of instrument performance, small dimensions also offer 100 100 nm3, on average,
