A magnetic nanoparticle-based microfluidic device fabricated using a 3D-printed mould for separation of Escherichia coli from blood

Abstract

Herein, A microfluidic device is described, produced with a 3D-printed master mould that rapidly separates and concentrates Escherichia coli directly from whole blood samples, enabling a reduction in the turnaround time of bloodstream infections (BSIs) diagnosis. Moreover, it promotes the cleansing of the blood samples whose complexity frequently hampers bacterial detection. The device comprises a serpentine mixing channel with two inlets, one for blood samples (spiked with bacteria) and the other for magnetic nanoparticles (MNPs) functionalized with a (bacterio)phage receptor-binding protein (RBP) with high specificity for E. coli. After the magnetic labelling of bacteria throughout the serpentine, the microchannel ends with a trapping reservoir where bacteria-MNPs conjugates are concentrated using a permanent magnet. The optimized sample preparation device successfully recovered E. coli (on average, 66%) from tenfold diluted blood spiked within a wide range of bacterial load (10² CFU to 10⁷ CFU mL^-1). The non-specific trapping, tested with Staphylococcus aureus, was at a negligible level of 12%. The assay was performed in 30 min directly from diluted blood thus presenting an advantage over the conventional enrichment in blood cultures (BCs). The device is simple and cheap to fabricate and can be tailored for multiple bacterial separation from complex clinical samples by using RBPs targeting different species. Moreover, the possibility to integrate a biosensing element to detect bacteria on-site can provide a reliable, fast, and cost-effective point-of-care device.

Keywords: 3D-printed; Bacteriophage receptor binding protein (RBP); Blood; Escherichia coli; Magnetic nanoparticles; Microfluidic.

Fig. 1

A COMSOL simulation results for the flow velocity profile mixing. B Tests with food colorants in the microfluidic structures with 600 µm channel width, 200 µm channel height, and 300 µm curve width

Fig. 2

A Microfluidic device fixed in the dedicated support holding a permanent magnet. B Micromixer during the assay with blood spiked with bacteria introduced at the 450-µm-wide inlet and MNP-RBP introduced at the 225-µm inlet. The MNP-bacteria conjugates were trapped by a permanent magnet placed under the trapping reservoir

Fig. 3

Bacterial trapping efficiency (TE) in buffer (A) and in tenfold diluted blood (B) for MNPs functionalized with a polyclonal antibody and MNPs functionalized with the RBP. For both the target (E. coli) and negative control (S. aureus), a cell concentration of 10⁶ CFU mL⁻¹ was used. All the assays were performed in bulk

Fig. 4

Comparison of bacteria trapping efficiency (TE) in bulk and in the microfluidic structure for assays performed with spiked buffer samples (A) and spiked tenfold diluted blood samples (B) for target bacteria, E. coli, at different concentrations and the negative control, S. aureus, at 10⁷ CFU mL⁻¹. Pairwise comparisons between the TE of the E. coli samples (from 10² to 10⁷ CFU mL⁻¹) and the negative control (S. aureus) show statistically significant differences (p < 0.001)