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Finite-element analysis has allowed researchers in Singapore to make good time with their experiments on clockwork PCR (polymerase chain reaction) for carrying out biomedical research. Their lab-in-a-drop device using components adapted from a standard computer CD-ROM drive, potentially making it commercially viable even for cash-strapped laboratories in the developing world. Juergen Pipper and his team at the Institute of Bioengineering and Nanotechnology in Singapore explain in the current issue of Angewandte Chemie how analysis and chip-based diagnostics will come of age once sample preparation techniques that overcome practical limitations are available. They point out that until now samples usually have to be prepared separately and on a relatively large scale prior to feeding into such chip-based analytical devices. "With a few exceptions, the micro total analysis systems (micro-TAS) currently available have failed to live up to the ideal of the miniaturization of multiple laboratory operations on to a single chip," say the researchers, "These systems perform sample preparation off chip and only pursue a single function." Pipper and colleagues want to change this mindset and have now developed a rapid genetics test that combines sample preparation with the polymerase chain reaction (PCR) on a single chip. Their "laboratory device" can carry out all necessary steps in the system with the addition of a drop of mineral oil containing emulsified magnetic nanoparticles. These act as carriers for target cells in the sample. PCR has been the mainstay of biomedical research into genes and genetics for many years. It allows gene sequences to be duplicated repeatedly to produce adequate samples for a wide range of tests and diagnostics. The key to success is to cycle the sample and reagents through a specific sequence of temperatures rises and falls, but this means laboratory PCR can take several hours to complete. Pipper's new chip PCR takes mere minutes, which astoundingly includes the sample preparation time. Aside from significant time savings, the new approach means a sample, such as a drop of blood, can be placed directly on to the device. It mixes with the droplet containing the magnetic particles at this stage, and surface antibodies on these particles bind specifically to cells of interest in the sample. In their tests, cells of a rare acute form of monocytic leukaemia were analysed this way. The researchers found that by moving a magnetic field beneath the chip they could relocate only those cells that have bonded to the magnetic particles to the next "station" on the chip, the washing station, and then into another droplet containing enzymes and reagents necessary for breaking open the cells, lysis. Finally, the mixture reaches the PCR station, and after combination with a reagent droplet, the magnetic droplet is then moved around the station like clockwork, passing repeatedly through four different zones set to the appropriate temperatures for PCR. The team modelled the temperature of the Teflon-coated glass substrate surface using finite-element analysis and infrared imaging. Each turn of the clock lasts 8 seconds, with fluorescence monitoring being used to indicate whether the target gene sequence is present and in what amount. The researchers have demonstrated proof of principle by isolating 30 cells spiked with the genetic information for green-fluorescent protein in a 25 microlitre blood sample. They were able to prepare the sample, concentrate it 100-fold, wash, carry out cell lysis, and detect the gene using PCR in just over a quarter of an hour. "Handheld units for point-of-care diagnostic tests in low-resource settings [such as the developing world] call for low-cost instruments and/or disposables," the researchers add. Aside from the thermal management equipment, all components used in their prototype clockwork PCR were scavenged from a standard computer CD-ROM drive, including the power supply, the stepper motor for the circular microfluidic actuation, and the optics for fluorescence detection. "At present, we are working on optical multiplexing by interrogating the rotating droplet at different locations among temperature zones 2-4 by using a miniaturized detector array working at different wavelengths," the researchers add. Related links:
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