A scalable self-priming fractal branching microchannel net chip for digital PCR

As an absolute quantification method at the single-molecule level, digital PCR has been widely used in many bioresearch fields, such as next generation sequencing, single cell analysis, gene editing detection and so on. However, existing digital PCR methods still have some disadvantages, including high cost, sample loss, and complicated operation. In this work, we develop an exquisite scalable self-priming fractal branching microchannel net digital PCR chip. This chip with a special design inspired by natural fractal-tree systems has an even distribution and 100% compartmentalization of the sample without any sample loss, which is not available in existing chip-based digital PCR methods. A special 10 nm nano-waterproof layer was created to prevent the solution from evaporating. A vacuum pre-packaging method called self-priming reagent introduction is used to passively drive the reagent flow into the microchannel nets, so that this chip can realize sequential reagent loading and isolation within a couple of minutes, which is very suitable for point-of-care detection. When the number of positive microwells stays in the range of 100 to 4000, the relative uncertainty is below 5%, which means that one panel can detect an average of 101 to 15 374 molecules by the Poisson distribution. This chip is proved to have an excellent ability for single molecule detection and quantification of low expression of hHF-MSC stem cell markers. Due to its potential for high throughput, high density, low cost, lack of sample and reagent loss, self-priming even compartmentalization and simple operation, we envision that this device will significantly expand and extend the application range of digital PCR involving rare samples, liquid biopsy detection and point-of-care detection with higher sensitivity and accuracy.