Application note - Microfluidic qPCR using FluoReader

Fluorescence reader for microfluidic qPCRFluorescence 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 with high numerical aperture, low magnification objective epifluorescence microscopes.

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 show case the the use of the FluoReader to perform qPCR. 

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Instruments & Components

fluorescence detection for microfluidic device 8The experiments shown in this application note have been realized only with Elveflow’s instruments and accessories. For any advice on your research project and experimental needs, do not hesitate to consult our team of specialists.

 

 

 

FluoReader

Optical detection for microfluidics... See More

FluoReader

Chemical reaction tracking, flow throw ultra-sensitive fluorescence detection, on-chip qPCR, cell culture fluorescence measurement ...

FLOW CONTROLLER

Fast, stable and pulse free flow control... See More

FLOW CONTROLLER

The only microfluidic flow control system with patented piezoelectric regulators to enable flow control with 20-fold precision and 10-fold speed

Heater controller

Heater controller for live cell imaging.. See More

Heater controller

This heater cooler enables you to control the temperature of your sample in seconds during live cell imaging ...

Sample reservoirs

Our complete line of standard and specifically designed microfluidic reservoirs provides a number of solutions that will meet any of your requirements.

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Experiments

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 MkIII, Elveflow). The fluorescence detection is carried out by a FluoReader (figure on the right).

Fastgene Publication_Figure 1

Experimental set-up for the qPCR system

 

The qPRC chamber is placed on the FluoReader for the fluorescence monitoring of the DNA amplification

The qPRC chamber is placed on the FluoReader for the fluorescence monitoring of the DNA amplification

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.

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qPCR curve of Bacillus atrophaeus with Sybr Green ©

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 is turned on every 1s. The light pulse consists of binary signal of 0.5mW and 0 mW with of duty cycle 1/2. This osculating signal  enables 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 and exponential increase of the fluorescence signal which evidences the DNA amplification.

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Conclusion

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.