Juergen Pipper

Catching bird flu in a droplet

It is assumed that a timely mass administration of antiviral drugs, backed by quarantines and social distancing, could contain a nascent influenza epidemic at its source, provided that the first clusters of cases were localized within a short time. However, effective routine surveillance may be impossible in countries lacking basic public health resources. For a global containment strategy to be successful, low-cost, easy-to-use handheld units that permit decentralized testing would be vital. Here we present a microfluidic platform that can detect the highly pathogenic avian influenza virus H5N1 in a throat swab sample by using magnetic forces to manipulate a free droplet containing superparamagnetic particles. In a sequential process, the viral RNA is isolated, purified, preconcentrated by 50,000% and subjected to ultrafast real-time RT-PCR. Compared to commercially available tests, the bioassay is equally sensitive and is 440% faster and 2,000–5,000% cheaper.

An integrated fluorescence detection system for lab-on-a-chip applications

We present a low-cost miniaturized fluorescence detection system for lab-on-a-chip applications with a sensitivity in the low nanomolar range; a built-in lock-in amplifier enables measurements under ambient light.

Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes

We have designed, fabricated and tested a real-time PCR chip capable of conducting one thermal cycle in 8.5 s. This corresponds to 40 cycles of PCR in 5 min and 40 s. The PCR system was made of silicon micromachined into the shape of a cantilever terminated with a disc. The thin film heater and a temperature sensor were placed on the disc perimeter. Due to the systems thermal constant of 0.27 s, we have achieved a heating rate of 175°C s−1 and a cooling rate of −125°C s−1. A PCR sample encapsulated with mineral oil was dispensed onto a glass cover slip placed on the silicon disc. The PCR cycle time was then determined by heat transfer through the glass, which took only 0.5 s. A real-time PCR sample with a volume of 100 nl was tested using a FAM probe. As the single PCR device occupied an area of only a few square millimeters, devices could be combined into a parallel system to increase throughput.

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

Disposable real-time microPCR device: lab-on-a-chip at a low cost

We have designed, fabricated and tested a real-time micro polymerase chain reaction (microPCR) system. It consists of a microscope glass cover slip placed on top of a micromachined silicon chip integrated with a heater and a temperature sensor. A single microL of a sample containing DNA was placed on the glass and encapsulated with mineral oil to prevent the evaporation of water, thus forming a virtual reaction chamber (VRC). The PCR chip required half a second to heat up from 72 to 94 degrees C and two seconds to cool from 94 to 55 degrees C, corresponding to a cooling rate of -20 K s(-1). The real-time PCR yield was determined by a fluorescence method. The melting curve analysis method as well as capillary electrophoresis was performed to determine the purity of the PCR product. As the glass slip is disposable, cross-contamination from sample to sample is eliminated. The total cost of running the PCR is given by the value of the cover slip and its treatment.

Clinical Evaluation of a Low Cost, In-House Developed Real-Time RT-PCR Human Immunodeficiency Virus Type 1 (HIV-1) Quantitation Assay for HIV-1 Infected Patients

Objectives HIV-1 viral quantitation is essential for treatment monitoring. An in-house assay would decrease financial barriers to access. Materials and Methods A real-time competitive RT-PCR in house assay (Sing-IH) was developed in Singapore. Using HXB2 as reference, the assays primers and probes were designed to generate a 183-bp product that overlaps a portion of the LTR region and gag region. A competitive internal control (IC) was included in each assay to monitor false negative results due to inhibition or human error. Clinical evaluation was performed on 249 HIV-1 positive patient samples in comparison with the commercially available Generic HIV Viral Load assay. Correlation and agreement of results were assessed for plasma HIV-1 quantification with both assays. Results The assay has a lower limit of detection equivalent to 126 copies/mL of HIV-1 RNA and a linear range of detection from 100–1000000 copies/mL. Comparative analysis with reference to the Generic assay demonstrated good agreement between both assays with a mean difference of 0.22 log10 copies/mL and 98.8% of values within 1 log10 copies/mL range. Furthermore, the Sing-IH assay can quantify HIV-1 group M subtypes A–H and group N isolates adequately, making it highly suitable for our region, where subtype B and CRF01_AE predominate. Conclusions With a significantly lower running cost compared to commercially available assays, the broadly sensitive Sing-IH assay could help to overcome the cost barriers and serve as a useful addition to the currently limited HIV viral load assay options for resource-limited settings.