Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode

Abstract

Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA analysis in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification. This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions with high throughput processing up to 120μL of DNA dilution at flow rates ranging from 1 to 5μL/min without compromising performance. The fluidized bed was 20-50% more efficient than a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based microarray and tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostics.

Microfluidic Magnetic Mixing at Low Reynolds Numbers and in Stagnant Fluids

“…This makes them beneficial to diagnostic systems that require capillary filling (reducing the cost/complexity of additional microfluidic pumps and valves) or involve stagnant fluids within microchamber reactors. In addition, for applications that require the capturing of analytes, the magnetic beads offer the possibility of applying a functional coating based on antibodies [140]. These coatings can also prevent aggregation (e.g., carboxylic group coating).…”

Sensors Based on Bio and Biomimetic Receptors in Medical Diagnostic, Environment, and Food Analysis

“…The systems differ in the way they organize the flow. Continuous Flow Based Microfluidics provide a continuous flow of reagents through microchannels [ 244 , 245 ]. They are suitable for solving analytical problems that do not require complex liquid manipulations (flow chemical monocomponent analysis).…”

Microfluidic Technologies for cfDNA Isolation and Analysis

“…Such DNA extraction method in a segmented flow microfluidic system successfully removed 90% or even 95% of the original sample volume. More recently, magnetic bead-based approach has been applied to cfDNA isolation (Figure 2), with a constant position of external magnetic field and without the need for an incubation step, which had comparable efficiencies with standard column-based method [38,39]. The emergence of new technology, like 3D-printing, makes magnetic bead-based microdevices more diversified [40].…”