Research ArticleMEDICAL ROBOTS

Catalytic antimicrobial robots for biofilm eradication

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Science Robotics  24 Apr 2019:
Vol. 4, Issue 29, eaaw2388
DOI: 10.1126/scirobotics.aaw2388
  • Fig. 1 Catalytic and magnetic iron oxide NPs as building blocks for small-scale robots designed for biofilm killing and removal.

    (A) Diagram illustrating the challenges of biofilm removal due to the EPS matrix that provides protection against antimicrobials and mechanical stability (3, 4). (B) Diagram depicting the magnetic-catalytic NPs and their bacterial killing and EPS degradation mechanisms via reactive free radicals generated from hydrogen peroxide (H2O2) via peroxidase-like activity. The EPS degrading activity was enhanced by addition of mutanase/dextranase to digest extracellular glucans. (C) Catalytic-magnetic NPs in suspension served as multifunctional building blocks to form CARs. In the first CAR platform, biohybrid CARs with bristle-like structures were assembled from NPs suspended in H2O2 and mutanase/dextranase solution by a permanent magnet attached to a micromanipulator and used to remove biofilms from accessible surfaces. In a second platform, catalytic-magnetic NPs were embedded into gels to form 3D molded CARs having specialized vane and helicoid structures.

  • Fig. 2 Characterization of NPs in a model solution.

    (A) Cylindrical magnet placement using a micromanipulator and magnetic flux density. (B) NP cluster in suspension before and after magnetic actuation and (C) time-lapse quantification of NP cluster using MATLAB. (D) Surface area of the cluster versus suspension density of NP determined from still images 300 s after magnetic flux application using an optical thresholding method. (E) Motion of NP cluster under magnetic control. Initially, an NP cluster with a circular footprint was formed, centered above the magnet. Suspended NPs moved toward the NP cluster, which was moved in a preprogrammed trajectory, clearing the solution.

  • Fig. 3 Characterization and formation of biohybrid CARs.

    (A) NP actuation in biofilm in the presence of magnetic field. S. mutans, a biofilm-forming model organism and a bacterial oral pathogen, was used to form biofilms. NPs, absent catalysis, did not aggregate owing to adhesive interactions with the biofilm. Conversely, NPs with catalysis degraded the biofilm and moved toward the magnet to assemble into a cluster. (B) Quantitative imaging analysis shows the size of cluster in NP with and without catalysis [as shown in (A)]. (C) Data from bacterial viability assays using fluorescence imaging and culturing methods. Green fluorescence indicates live bacteria and red indicates dead bacteria, and the number of viable cells as determined by CFU counting. The data show that NPs alone were devoid of antibacterial activity, whereas NPs with catalysis potently killed bacteria. Error bars indicate standard deviation; ND, not detected. (D) Biohybrid CAR superstructure is revealed by high-resolution confocal microscopy. Absent catalytic activity, only small NPs assemblages formed (top panel). With catalytic activity, biohybrid CARs formed; they consisted of a complex bio-inorganic hybrid assemblage comprising spatially oriented, rod-like, micrometer-size superstructures of NP enmeshed with bacterial mass and debris (bottom and right panels).

  • Fig. 4 Platform 1: Biofilm removal using biohybrid CARs.

    (A) Orthogonal view of biofilms treated with biohybrid CARs reveals the rod-like superstructure forming bristles (highlighted by white dashed lines) through the compromised biofilms. After incubation, biohybrid CARs were manipulated precisely using a magnetic field gradient to completely remove biofilms and biofilm debris, including dead bacteria and degraded EPS, from surfaces. Scale bar indicates 10 µm. (B) Diagram depicting cleaning of large areas of a biofilm-coated surface by magnetically controlled sweeping of biohybrid CARs (after bacterial killing/EPS degradation). (C) The biohybrid CAR morphed into C-shaped aggregate (see arrows) as it moved over the surface to plow through and remove biofilm (labeled with SYTO 9 green fluorescence). (D) Fluorescent images indicating complete cleaning of an S. mutans biofilm (labeled in green) grown on a glass surface by using biohybrid CARs sweeping effect. (E) Biofilm-removed surfaces were incubated for additional 24 hours with the biofilm growth medium (supplemented with 1% sucrose) to assess bacterial regrowth using confocal microscopy and culturing methods. There is no biofilm regrowth on biofilm-removed surfaces by CARs even after 24 hours incubation. Control and NP-treated biofilms were also incubated using the same conditions, both showing abundant bacterial cells (in green) and EPS (in red); quantitative analyses show high amounts of biomass and high number of viable cells. Quantitative data also confirm that there was no detectable biomass or viable cells with treatment by CARs. Error bars indicate standard deviation. (F) The controlled movement of focused biohybrid CARs resulted in precise removal of biofilms from surfaces with defined geometrical shapes. (G) Fluorescent microscopy confirms the complete removal of bacteria from the surface.

  • Fig. 5 Platform 2: Small-scale CARs with functional shapes for specific biofilm disruption tasks.

    (A) Model representations of vane-like and helicoid-shaped CARs fabricated from 3D micromolding agar gel embedded with NPs. Both vanes and helicoids measure 5 mm in diameter and 10 mm in length. The final robot composition is 3% (w/v) agar and 10% (w/v) NPs. (B) Helmholtz coil system used to drive 3D molded CARs through cylindrical tubes. Sinusoidal time-varying currents (Ix and Iz) were applied to each coil pair to generate uniform, rotating magnetic fields. Robots were driven using fields measuring 3.4 mT and rotating at 4 Hz. (C) Robots were driven through the cylindrical tube with linear velocity, v, which was generated by applying a torque, T, to the robot body. The torque was generated when the magnetic dipole moment, M, sought to align with the rotating field, B. (D) Schematic diagrams of biofilms on the wall of a cylindrical tube and wall cleaning by vane CAR. Vane CARs were used to clean the curved surfaces of glass tubes, rotated with an applied magnetic torque, T, and driven forward in the tube at a velocity, v, by applying a force using a magnetic field gradient. (E) Vane CARs advanced forward in the tube, sweeping the walls and generating a pile of debris behind them. The integrated fluorescence intensity from the biofilm debris increased with time behind the advancing robot. (Left) Accumulated biomass behind the vane CAR as it moved forward through the tube. (F) Schematic diagrams of biofilm clogs in cylindrical tubes and drilling by helicoid CARs. Helicoid CARs were used to clean biofilm clogs at various locations within the glass tube. The robots were propelled along linear paths within glass tubes through an applied magnetic torque, T. Their forward propulsion was enabled by their chirality. (G) A helicoid CAR advanced forward in the tube to drill through the biomass clog. (Left) Accumulated biomass behind the helicoid CAR as it moved forward through the tube increased with time. The robot drilled by overcoming the biofilm mechanical resistance associated with the conical shape of the biofilm (for t < 30 s for outer biomass removal and t > 60 s with additional force to penetrate the conical portion for the inner biomass removal) accumulated at the dead end of the confined space. (H) Helicoid CARs could also drill and restore biofilm-occluded paths. Fluorescent images showing the action and biofilm removal efficacy of vane-like (E) and helicoid (G and H) robots: green color indicates S. mutans biofilms or clogs.

  • Fig. 6 Potential applications for CARs platforms.

    (A) CARs can be used to treat biofilms on biotic (e.g., teeth) and abiotic (catheter or implant) surfaces. (B) Demonstration of using CARs to access the interior of human teeth. Cross sections of the tooth canal show the isthmus, which is a narrow corridor (200 to 600 μm in width) between the root canals. A longitudinal section (across the tooth length) shows the tooth canal. Biohybrid CARs could access the isthmus, one of the most challenging anatomical areas of teeth, where bacterial biofilms are commonly found. Aggregated NPs could readily transverse the isthmus as directed by the external magnetic field. Lower middle panels show disruption of biofilms in the isthmus by CARS using fluorescently labeled biofilms. (C) For 3D molded CARs, miniaturized 3D molded helicoidal robots could be magnetically actuated through the canal of the tooth, another common location of dental biofilm formation.

Supplementary Materials

  • robotics.sciencemag.org/cgi/content/full/4/29/eaaw2388/DC1

    Materials and Methods

    Discussion

    Fig. S1. Optimization of concentrations of NPs and enzymes and application/biological functions of NPs with catalysis.

    Fig. S2. Characterization of NPs in model solution.

    Fig. S3. Biohybrid CAR formed from bio-inorganic superstructure.

    Fig. S4. Biohybrid CAR assembly and biofilm removal.

    Fig. S5. Viability of bacterial cells in biofilms after CAR treatment.

    Fig. S6. Biofilm removal using biohybrid Feraheme (ferumoxytol) CARs.

    Fig. S7. Catalytic activity of 3D molded CARs.

    Fig. S8. Vane-like CARs for cleaning the curved surfaces of glass tubes.

    Fig. S9. Characterization of response of helicoid robot to various conditions.

    Fig. S10. Assessment of biofilm removal using 3D molded CARs with and without catalytic activity.

    Movie S1. Application of controlled movement of focused CARs for precision removal of biofilms from surface (Fig. 4F).

    Movie S2. Application of helicoid CARs for removal of biofilms from the curved surfaces of glass tubes (Fig. 5G).

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Discussion
    • Fig. S1. Optimization of concentrations of NPs and enzymes and application/biological functions of NPs with catalysis.
    • Fig. S2. Characterization of NPs in model solution.
    • Fig. S3. Biohybrid CAR formed from bio-inorganic superstructure.
    • Fig. S4. Biohybrid CAR assembly and biofilm removal.
    • Fig. S5. Viability of bacterial cells in biofilms after CAR treatment.
    • Fig. S6. Biofilm removal using biohybrid Feraheme (ferumoxytol) CARs.
    • Fig. S7. Catalytic activity of 3D molded CARs.
    • Fig. S8. Vane-like CARs for cleaning the curved surfaces of glass tubes.
    • Fig. S9. Characterization of response of helicoid robot to various conditions.
    • Fig. S10. Assessment of biofilm removal using 3D molded CARs with and without catalytic activity.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Application of controlled movement of focused CARs for precision removal of biofilms from surface (Fig. 4F).
    • Movie S2 (.mp4 format). Application of helicoid CARs for removal of biofilms from the curved surfaces of glass tubes (Fig. 5G).

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