Microfluidics application note
Published on 27 August 2019
Fluorescence reader for microfluidic qPCR: faster, more sensitive and less expensive than most optical microscopes, it is a smart alternative for real-time fluorescence measurements of your on-chip qPCR signal. It offers a compact and cost-effective instrument compared to microscopes with a high numerical aperture, low magnification objective epifluorescence.
The FluoReader can be used to quantify on-chip qPCR signals in tiny chambers with an SNR that is more than 10 times higher than measurements done with most fluorescent microscopes equipped with a CCD camera. Thanks to its exceptional sensitivity, the FluoReader eliminates bleaching problems, reduces acquisition time and enhances the precision of acquisitions, allowing a more exact fitting of data and Cp calculation.
In this application note, we showcase the use of the FluoReader to perform qPCR.
The experiments shown in this application note have been realized only with Elveflow instruments and accessories. For any advice on your research project and experimental needs, do not hesitate to consult our team of specialists.
The complete qPCR system (figure on the left) consists of a heater controller (Cherry Biotech) commanding two heat exchangers. These two heat exchangers adjust the temperatures of heat-transfer liquids injected in the microfluidic chip in order to obtain the two desired qPCR temperatures for molecular samples. The switch between the two heat-transfer liquids within the microfluidic chip is achieved by a pressure controller (OB1 Mk3, Elveflow). The fluorescence detection is carried out by a FluoReader (figure on the right).
For qPCR assays, extracted and purified Bacillus atrophaeus (BG) bacteria DNA has been amplified. For each assays, 125 ng of initial DNA were used. 2 different qPCR master mix kits were used at a 1x concentration. An initial denaturation was conducted at 96°C and a temperature of 64°C was used for the annealing/elongation. The sample is contained in 1 mm x 1 mm x 100 µm transparent chamber.
RESULTS
The figure on the right describes the acquire signal during the qPCR. The lower panel shows the excitation light. Here, time-lapse detection is used to limit the light exposure of the fluorophore and the studied molecules.
400 µs light pulses are turned on every 1s. The light pulse consists of a binary signal of 0.5mW and 0 mW with of duty cycle 1/2. This osculating signal allows background removal by subtracting the signal detected when the light is ON to when the light is OFF. The signal is averaged with 0.4 s window and plotted in the upper panel.
The plot in the upper panel exhibits cycles of fluorescence signal which follows the thermalisation cycles. In fact the fluorescence of SYBR green changes inversely with the temperature. By following the upper part of the signal (when SYBR is attached to the DNA i.e. at low temperature), we see clearly an exponential increase of the fluorescence signal which shows the DNA amplification.
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The FluoReader can be used to quantify on-chip qPCR signals in tiny chambers with an SNR that is more than 10 times higher than measurements done with most fluorescent microscopes equipped with a CCD camera. Thanks to its exceptional sensitivity, the FluoReader eliminates bleaching problems, reduces acquisition time and enhances the precision of acquisitions, allowing a more exact fitting of data and Cp calculation.
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