An integrated droplet digital PCR gene chip for absolute quantification of nucleic acid

An integrated droplet digital polymerase chain reaction (ddPCR) gene chip that can quantify nucleic acid absolutely was constructed to avoid problems such as droplet fusion, fragmentation and sample contamination during pipetting. The integrated ddPCR gene chip was fabricated by photolithography and cyclic olefin copolymer (COC) injection mold. The 20 mu L sample was distributed to about 60,000 water-in-oil droplets with a diameter of about 87 mu m within 3 min by flow focus microstructure, and the volume of each droplet was about 0.27 nL. The generation of bubbles was reduced by reducing the width of the collection chamber and increasing the deceleration step. In addition, the efficiency of PCR was improved using COC materials. The sample of human epidermal growth factor receptor (EGFR) exon18 gene was quantitatively detected by this chip. Experimental results show that the detection signal has a good linear relationship with the DNA concentration from 10(1) to 10(5) copies/mu L (R-2 = 0.999) so that the integrated ddPCR gene chip can realize absolute quantification of nucleic acid. It is verified that the integration of droplet generation, PCR amplification and fluorescence detection are realized on one chip, reduce bubble generation and improve PCR efficiency. Compared with the current independent split chip, the integrated ddPCR gene chip ensures a high degree of anti-pollution performance, avoids the operation of droplet movement, reduces human interference, and standardizes the results. It has wide application prospects in nucleic acid quantification and trace detection.