Clockwork PCR including sample preparation.

With a few exceptions, the micro total analysis systems (mTASs) currently available have failed to live up to the ideal of the miniaturization of multiple laboratory operations onto a single chip. These systems perform sample preparation off chip and only pursue a single function. Hence, the definitive challenge is to interface the processing of real-world biological samples with downstream applications. To this end, the manipulation of individual droplets on a planar surface offers an attractive option for a mTAS. Herein, we transform a free droplet containing surface-functionalized superparamagnetic particles into a virtual mTAS with a (sub)microliter (mL) volume. Aside from being force mediators for actuating the droplet in a magnetic field, the superparamagnetic particles serve as a solid support for the sequential performance of laboratory or (bio)chemical processes. Depending on its particular task, the droplet temporarily becomes a pump, valve, mixer, extractor, or thermocycler. In an automated experiment, 30 green-fluorescent protein (GFP) transfected THP-1 cells are isolated from 25 mL of blood, 100-fold preconcentrated, purified, lysed, and subjected to a realtime PCR (RT-PCR) targeting the transfection vector, all within 17 min. Fast thermocycles of 8 s take place on a disposable substrate under time–space conversion by rotating the droplet clockwise over different temperature zones. Other PCR-based (bio)assay formats are easily adaptable, which makes this mTAS an attractive candidate for decentralized diagnostics. Most bench-scale thermocyclers rely on a thermoelectrically heated metal block holding plastic tubes containing up to 50 mL of PCR mixture. This setup results in a high thermal mass and the PCR run time—typically hours—is limited by the low heating and cooling rates. Downscaling and/or the utilization of highly heat-conductive materials can overcome these limitations and a chip-based (sub)microscale PCR can perform the job within minutes. There are two ways of conducting an on-chip PCR. In the time domain, a stationary PCR mixture is thermocycled between three different temperatures. Under time–space conversion, a PCR mixture is driven through a microchannel that is constantly held at three different temperatures. This is why the PCR mixture reaches its thermal equilibrium quickly and a PCR in the space domain allows for fast thermocycling. With a few exceptions, 10,11] the on-chip PCR is based on template DNA that has already been preprocessed off chip by using established bench-scale procedures. Complex samples like blood, saliva, or cell-culture medium contain (bio)chemicals, air bubbles, particulates, food residues, cell debris, etc. that have to be removed because they hamper the microfluidic operations and/or the subsequent PCR. This front-end sample workup is highly specific and cannot always be done on the (sub)microscale. Clearly, the ability to integrate a basic set of laboratory operations into a single device and to directly handle real-world biological samples will be key in defining the commercial success of any mTAS. Almost all bench-scale (bio)chemical protocols depend on handling fluid boluses by using a pipette. A droplet-based (bio)chemical protocol is functionally equivalent to its benchscale version: its reconfiguration or scale-down simply requires the rearrangement or volume variation of the droplets. This flexibility cannot be matched by conventional microfluidic architectures that rely on a continuous flow of liquids in rigid microchannels permanently micromachined into silicon, glass, or polymer substrates. Electrowetting-on-dielectric, dielectrophoresis, surface-acoustic waves, and (electro)magnetic forces are popular techniques that are used to actuate droplets either sandwiched between two plates or positioned on an open surface. Among these, (electro)magnetic actuation is unique in that it is unaffected by surface charges, pH values, or ionic strength. Thus, it is compatible with a wide range of substrate materials and (bio)chemical processes. Furthermore, external permanent or electromagnets that remotely control the superparamagnetic particles make the running of a dedicated test on a low-cost disposable possible. Notably, the most important tasks within a bioassay—sample isolation/preconcentration, labeling, and detection—can be assigned to superparamagnetic particles. In our approach, a free droplet spontaneously selforganizes on a Teflon-coated glass substrate by emulsifying an aqueous suspension of superparamagnetic particles in an immiscible liquid (Figure 1a). Sealing of the droplet in mineral oil prevents the aqueous phase from evaporating and renders a complicated chip design for the perpetuation of a humidifying atmosphere unnecessary. An external permanent magnet is used for droplet actuation. The magnetic-field gradient exerts a translational force on the superparamagnetic particles suspended in the aqueous phase, a force that is transferred onto the inner aqueous phase/mineral oil interface. To maximize the mag[*] Dr. J. Pipper, Y. Zhang, Dr. P. Neuzil, T.-M. Hsieh Institute of Bioengineering and Nanotechnology 31 Biopolis Way, The Nanos, #04-01 Singapore 138669 (Singapore) Fax: (+ 65)6478-9080 E-mail: jpipper@ibn.a-star.edu.sg