The
“sample-to-answer” integration and automation
of circulating tumor DNA (ctDNA)-based liquid biopsy using digital
PCR (dPCR) has been hampered by the complicated operations of liquids
with volumes ranging from milliliter samples to nanoliter droplets.
On the basis of a “3D extensible” design paradigm proposed
previously, an integrated droplet digital PCR (IddPCR) microdevice
was successfully developed to automate the entire process of liquid
biopsy, from the extraction of ctDNA in 2 mL of plasma using magnetic
beads to the generation, amplification, and screening of over 30 000
droplets for detection. A series of reagent mixing structures, including
macro-, meso-, and micromixers, was designed to enable efficient
reagent handling and mixing at different volume scales. The volume
thresholds of the microscale and macroscale in the IddPCR device were
calculated to be 40 and 100 μL, respectively, based on the fluid
dynamics and sizes of the device structures, so that different mixers
can be selected according to the reagent volumes. The DNA extraction
efficiency obtained on the device was determined to be ∼60%,
and the on-chip ddPCR demonstrated a high correlation with an R
2 of 0.9986 between the readouts and the estimations
by a Poisson distribution. Finally, the IddPCR microdevice was able
to detect rare tumor mutations (T790M) with an occurring frequency
as low as ∼1% from 2 mL of human plasma in a “sample-to-answer”
manner. This work offers a feasible solution for the automation of
liquid biopsy and paves the way for its broad applications in clinics.
Microfluidics is facing critical challenges in the quest of miniaturizing, integrating, and automating in vitro diagnostics, including the increasing complexity of assays, the gap between the macroscale world and the microscale devices, and the diverse throughput demands in various clinical settings. Here, a "3D extensible" microfluidic design paradigm that consists of a set of basic structures and unit operations was developed for constructing any application-specific assay. Four basic structures-check valve (in), check valve (out), double-check valve (in and out), and on-off valve-were designed to mimic basic acts in biochemical assays. By combining these structures linearly, a series of unit operations can be readily formed. We then proposed a "3D extensible" architecture to fulfill the needs of the function integration, the adaptive "world-to-chip" interface, and the adjustable throughput in the X, Y, and Z directions, respectively. To verify this design paradigm, we developed a fully integrated loop-mediated isothermal amplification microsystem that can directly accept swab samples and detect Chlamydia trachomatis automatically with a sensitivity one order higher than that of the conventional kit. This demonstration validated the feasibility of using this paradigm to develop integrated and automated microsystems in a less risky and more consistent manner.
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