Real Time PCR on Disposable PDMS Chip with a Miniaturized Thermal Cycler

Abstract

This paper presents the design and implementation of a low-cost miniature PCR device consisting of a disposable reactor chip and a miniature thermal cycler. The simple fabrication of the PCR chip by PDMS (Polydimethylsiloxane) does not need micro-machining or photolithography processes. The thermal cycler was built with a thin film heater for heating and a fan for rapid cooling. This device can perform PCR tests in a single well chip or a multiple-well chip. It can run PCR reactions of different volumes to meet specific application requirements. The smallest reaction volume tested in this work is 0.9 microL. In addition, this device fits any standard fluorescence microscope for real time detection, which makes real time PCR affordable for most research labs and clinics with a fluorescence microscope. Real-time PCR of E. coli stx1 has been demonstrated with the device described.

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].…”

Low-Cost Temperature Logger for a Polymerase Chain Reaction Thermal Cycler

“…To measure the temperature of the well within a thermal cycler, a temperature probe and data logger are required [6,[8][9][10][11]. The probe with a temperature sensor should be designed to provide tight thermal-coupling with the well of the aluminum block, and the logger should read and store the temperature of the sensor.…”

“…The probe with a temperature sensor should be designed to provide tight thermal-coupling with the well of the aluminum block, and the logger should read and store the temperature of the sensor. In a common research laboratory, the temperature is read through a high-cost data acquisition system and a probe composed of a reagent container with a thermocouple is inserted [6,[8][9][10]. A PCR tube made of polypropylene is adapted as the reagent container which is susceptible to wear out over the course of time and therefore is not permanently reusable, and should be built anew for every test.…”