High Dynamic Range Digital Assay Enabled by Dual-Volume Centrifugal Step Emulsification

Abstract

We implement dual-volume centrifugal step emulsification on a single chip to extend the dynamic range of digital assays. Compared to published single-volume approaches, the range between the lower detection limit (LDL) and the upper limit of quantification (ULQ) increases by two orders of magnitude. In comparison to existing multivolume approaches, the dual-volume centrifugal step emulsification requires neither complex manufacturing nor specialized equipment. Sample metering into two subvolumes, droplet generation, and alignment of the droplets in two separate monolayers are performed automatically by microfluidic design. Digital quantification is demonstrated by exemplary droplet digital loop-mediated isothermal amplification (ddLAMP). Within 5 min, the reaction mix is split into subvolumes of 10.5 and 2.5 μL, and 2,5k and 176k droplets are generated with diameters of 31.6 ± 1.4 and 213.9 ± 7.5 μm, respectively. After 30 min of incubation, quantification over 5 log steps is demonstrated with a linearity of R 2 ≥ 0.992.

Auto Flow-Focusing Droplet Reinjection Chip-Based Integrated Portable Droplet System (iPODs)

“…Currently, the dLAMP mainly contains two categories: microwell-based dLAMP and droplet-based dLAMP . The microwell-based dLAMP has narrow detection ranges due to limited compartment numbers, and the complicated chip fabrication will increase the cost and labor intensity. , An alternative method is based on droplets; the compartment numbers can be adjusted on demand by much simpler and smaller chips, and the method is gradually establishing itself as a robust tool with higher throughput and improved automatization and detection sensitivity. , However, traditional droplet-based dLAMP relies on precise fluid control and high-precision pumps for droplet generation and reinjection. − Recently, several novel pump-free droplet generators have been developed for highly parallel emulsion generation by centrifugation, − vibration, − acoustic pulses, surface-wetting, capillary, etc. Nevertheless, to form a spaced droplet flow during the reinjection step for the accurate measurement of the microreaction number and signal, high-precision pumps are required to provide high stability and velocity of the fluids (separation oil phase and droplet phase) for flow focusing.…”

Automated Centrifugal Microfluidic Chip Integrating Pretreatment and Molecular Diagnosis for Hepatitis B Virus Genotyping from Whole Blood

“…Unlike other microfluidic devices, the centrifugal microfluidic chip can be driven only by rotation rather than extra bulky syringe pumps and cumbersome tubing systems. Using physical forces including centrifugal force, the Coriolis force, and the Euler force on centrifugal platforms, fluids can be controlled precisely to realize various functions such as transporting, valving, metering, aliquoting, mixing, and so on, − which provides a great potential for integrating a variety of complicated functions into a sample-in-answer-out platform for POC molecular diagnosis. Furthermore, the centrifugal microfluidic chip can also incorporate adjustable valves easily for new exploration, such as thermopneumatic valves, laser-actuated valves, wax valves, and so on.…”

Self-Assembled Inkjet Printer for Droplet Digital Loop-Mediated Isothermal Amplification

“…Finally, the target DNA concentration in the original sample is calculated using Poisson distribution [13,14]. A variety of microfluidic-based LAMP methods have been developed for molecular detection, which allows for the design of different precise micropatterns for compartmentalizing a sample into small-volume partitions, generating large numbers of droplets for digital analysis [15][16][17][18][19][20]. However, many of them still require complex processing devices and precise microfeatures, leading to its implementation in a cumbersome manner.…”