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

Abstract

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 system's 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.

Micro real-time PCR device using a circulation pump

“…However, standard PCR techniques using the end-point detection method has drawbacks such as poor precision, difficult quantitation, and low sensitivity, among others (Richard et al 2003). As such, the standard PCR device is rapidly being replaced by miniaturized realtime PCR devices in order to meet the requirements of fast analysis, portability, and low cost (Xiang et al 2005;Neuzil et al 2006;Lien et al 2009;Hettiarachchi et al 2012).…”

Lab-on-a-chip fluorescence detection with image sensor and software-based image conditioning

“…From point of view of LOC material, two main requirements have to be fulfilled: (1) optical transparency necessary for introduction of the fluorescence excitation light and collection of the fluorescence light from an area of interest within the chip, and (2) low autofluorescence of LOC's material to minimize optical background signal. The chips can be whole made as multilayer construction of light-transparent materials -like glass, PDMS, SU-8, COC or other polymers, or only a top cover of the chip is made of these materials whereas body of the chip is made of silicon or ceramic [4][5][6][7][8][9][10][11]. It is also possible to make LOC from non-transparent materials like low-temperature co-fired ceramics (LTCC) but an integration of optical waveguides enabling introduction of the fluorescence inducing light into a detection chamber and covered with glass or PDMS is necessary [12].…”

“…In the detection channel, fluorescence light emitted by the fluorochrome is collected by the microscope objective with proper magnification, and guided to a photodetector by passing through filter(s) and dichroic mirror(s) to exclude the excitation light. Common detectors are cooled photomultiplier tubes (PMT) [5,6,11], semiconductor photodiodes [7,9,10], rather cooled charge coupled devices (CCD) as sensing matrix in video cameras [4] or lines in spectrophotometers [8].…”

CdTe quantum dots accelerate the speed of Pfu-based polymerase chain reaction

“…[2] Furthermore, microfluidic systems were introduced for fast thermocycling. [3,4] For instance, Woolley et al designed a fast PCR system in which every PCR chamber was equipped with a miniaturised heater. [5] Moreover, highspeed PCR was performed using a silicon-based microchamber array.…”