This study is a continuation of the previous study of the kinetics of capture of S. aureus bacteria spiked into clear solutions such as PBS (phosphate buffered saline), and apple juice.[1] In the present study, S. aureus bacteria were spiked into whole blood, blood plasma at various concentrations, suspensions of washed red blood cells (RBCs) at various concentrations, and protein solutions, all with the goal of understanding capture kinetics in order to produce a diagnostic that can capture bacteria from infected blood.
Magnetic (Fe3O4) nanoparticles (MNPs) were prepared by a high pressure autoclave procedure, which produced spherical clusters of particles about 120 nm in diameter. These were coated with about 5 nm of polydopamine (pDA) and were found to be very efficient in capturing S. aureus bacteria from PBS solutions.[1] In the present experiments, fresh human blood was collected (under IRB# X2021-135 of Brigham Young University) and used as collected, or separated by centrifugation into plasma and RBCs. The RBCs were washed in PBS twice and resuspended in PBS at various hematocrits. The plasma was used at normal concentration, or in 10-fold dilutions down to 10-3. In some experiments, human albumin, fibrinogen or gamma-globulin solutions were made to various concentrations. To these solutions or suspensions, bacteria were added and mixed. Then at time zero, pDA-coated MNPs were added. At selected time points, the suspension was subjected to a strong magnetic field to pull the MNPs to the side while the supernatant was collected and plated on nutrient agar plates to count bacteria concentration remaining in the supernatant. From these data, the kinetics of bacterial capture were computed, assuming first-order capture in both bacteria and MNP concentrations.
Results showed that the presence of plasma reduced the rate of capture significantly until the plasma concentration was below 1%. Likewise the presence of individual plasma proteins (albumin, fibrinogen, etc.) reduced the capture kinetics. We presume that plasma proteins are bound by the pDA surface on the MNPs, and this blocks or reduces attraction between the pDA and the bacteria. An alternative hypothesis is that the proteins coat the bacteria and block the interactions from the bacterial side.
As for RBCs, we saw no adhesive interaction between the RBCs and the pDA-coated MNPs, nor between the bacteria and RBCs. There were no plasma proteins nor plasma present in these experiments. Yet the presence of RBCs progressively attenuated the capture rate constant at the hematocrit increased. This data fit a model of increased pathlength between bacteria and pDA-coated MNPs due to the presence of much larger RBCs. The best fit was to a common model of increased “tortuosity” created by the presence of large particles.
This report shows our progress in our effort to capture bacteria directly from blood. The blood would need to be diluted to about 1% in PBS, and then the MNPs can capture the bacteria without interference from plasma proteins and RBCs. Our data suggests that protein, not RBCs are responsible for slower kinetics, but the presence of RBCs at high concentrations slows the capture kinetics by physically blocking the fast attachment of particles to bacteria.
Reference
[1] Bowen J. Houser, Alyson N. Camacho, Camille A. Bryner, Masa Ziegler, Justin B. Wood, Ashley J. Spencer, Rajendra P. Gautam, Tochukwu P. Okonkwo, Victoria Wagner, Stacey J. Smith, Karine Chesnel, Roger G. Harrison, William G. Pitt, “Bacterial Binding to Polydopamine-Coated Magnetic Nanoparticles”, ACS Applied Materials and Interfaces, 24(16), 58226-58240 (2024). https://doi.org/10.1021/acsami.4c11169