Research ArticleAPPLIED SCIENCES AND ENGINEERING

Real-time tracking of fluorescent magnetic spore–based microrobots for remote detection of C. diff toxins

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Science Advances  11 Jan 2019:
Vol. 5, no. 1, eaau9650
DOI: 10.1126/sciadv.aau9650
  • Fig. 1 Schematic illustration of the preparation and potential application of FMSMs.

    The obtained FMSMs can perform controlled locomotion in predefined tracks to detect the toxins in the supernatant from cultured bacteria and even in a redilution of patients’ stool by observing the fluorescence change when being driven continuously in a rotating magnetic field.

  • Fig. 2 Structures and compositions of the prepared samples.

    (A) Scanning electron microscopy (SEM) images of the original spores at different magnifications. (B) Low- and high-magnification SEM images of spore@Fe3O4 hybrids. (C) SEM images of hybrid spore@Fe3O4@CDs microrobots. Scale bars, 2 μm. XRD patterns (D), FTIR spectra (E), and zeta potential (F) of the original spores and their hybrids. a.u., arbitrary units.

  • Fig. 3 Swimming and fluorescence performance of FMSMs.

    (A) Schematic illustration of the magnetic actuation for an FMSM, wherein B represents the strength of the rotating magnetic field, f indicates the input frequency of the magnetic field, v represents the translational velocity, ω denotes the rotation velocity, γ denotes the tilt angle between the rotation axis, and α denotes the direction angle. (B) Superimposed snapshots of the controlled locomotion of an FMSM within 7 s with different motion modes in a rotating magnetic field (10 mT and 4 Hz, taken from movie S1). (C) Speed-frequency relationship at 10 mT with a pitch angle of 40°. (D) Speed-pitch angle relationship at 10 mT with a frequency of 4 Hz. (E) Fluorescence motion trajectories of the FMSM in DIW, PBS, DMEM, FBS, mucus, and intestinal mucus (Imucus) in a rotating magnetic field within 6 s (10 mT and 4 Hz). Scale bar, 30 μm (taken from movie S3). (F) Visual fluorescence trajectories of the autonomous navigation of the FMSM in DIW according to the preset O-, ∞-, and CU-like paths (taken from movie S4). Scale bar, 100 μm.

  • Fig. 4 Fluorescence response of the FMSMs to C. diff toxins.

    (A) Changing plot of fluorescence intensity showing the fluorescence quenching in different samples at different times. Here, C = 37.60 ng/ml, as determined by enzyme-linked immunosorbent assay (ELISA). F. nucleatum, Fuso. (B) Fluorescence intensity during the FMSMs navigated for 5 min in toxin-contaminated solutions in response to the toxin concentration and the resultant fitting equation. Inset shows the linear relationship between the fluorescence intensity and the natural logarithm of the toxin concentration.

  • Fig. 5 Fluorescence detection in stool supernatant using the FMSMs.

    The changing trend of the fluorescence intensity of static and mobile FMSMs (A) navigated for 10 min in different stool samples and (B) traveling for different times in 0.1C (C = 8.66 ng/ml) stool samples. (C) Fluorescence time-lapse images of static and mobile FMSMs with a time interval of 1 s at different times in different clinical stool supernatants (taken from movie S5). (D) Tracking for 8 s showing the propulsion of FMSMs in the infectious stool supernatant at the beginning and after 30 min. (E) Speed comparison of the FMSMs navigated for 0 and 30 min in the clinical infectious stool supernatant. (F) Tracking fluorescence trajectories of the FMSMs locomoting for approximately 18 min in different samples. Scale bars, 30 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaau9650/DC1

    Fig. S1. Cross-section SEM images of the prepared samples.

    Fig. S2. Magnetization curves of the prepared samples.

    Fig. S3. Optical properties of the prepared samples.

    Fig. S4. Magnetization direction of multiple FMSMs and single FMSM.

    Fig. S5. Motion trajectories of an FMSM in various media over 5-s time frames.

    Fig. S6. Optical images of the FMSMs under different illuminations.

    Fig. S7. Fluorescence time-lapse images of static and moving FMSMs in bacterial supernatants.

    Fig. S8. MSD of the microtracers caused by the motion of the FMSMs.

    Fig. S9. Fluorescence responses of the FMSMs toward different substances.

    Fig. S10. The survey results of the alignment of CROP segment of C. diff toxin B (amino acids 1830 to 2366) using Smart BLAST provided by the National Center for Biotechnology Information online.

    Fig. S11. Schematic for the structures of C. diff toxins and mechanism of quenching by the CROP segment of C. diff toxins to CDs on the FMSMs.

    Fig. S12. Fluorescence changes in different motion modes.

    Fig. S13. Detection calibration curves of the FMSMs.

    Fig. S14. Future automated control detection strategy.

    Table S1. Specific surface areas and pore parameters of the samples.

    Movie S1. Magnetic actuation of the FMSMs using a rotating magnetic field.

    Movie S2. Swimming of the FMSMs in different media.

    Movie S3. Fluorescence tracking of the FMSMs in different media.

    Movie S4. Automated fluorescence signal–based servoing of the FMSMs in predefined trajectories.

    Movie S5. Fluorescence detection using the FMSMs in clinical stool supernatant.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Cross-section SEM images of the prepared samples.
    • Fig. S2. Magnetization curves of the prepared samples.
    • Fig. S3. Optical properties of the prepared samples.
    • Fig. S4. Magnetization direction of multiple FMSMs and single FMSM.
    • Fig. S5. Motion trajectories of an FMSM in various media over 5-s time frames.
    • Fig. S6. Optical images of the FMSMs under different illuminations.
    • Fig. S7. Fluorescence time-lapse images of static and moving FMSMs in bacterial supernatants.
    • Fig. S8. MSD of the microtracers caused by the motion of the FMSMs.
    • Fig. S9. Fluorescence responses of the FMSMs toward different substances.
    • Fig. S10. The survey results of the alignment of CROP segment of C. diff toxin B (amino acids 1830 to 2366) using Smart BLAST provided by the National Center for Biotechnology Information online.
    • Fig. S11. Schematic for the structures of C. diff toxins and mechanism of quenching by the CROP segment of C. diff toxins to CDs on the FMSMs.
    • Fig. S12. Fluorescence changes in different motion modes.
    • Fig. S13. Detection calibration curves of the FMSMs.
    • Fig. S14. Future automated control detection strategy.
    • Table S1. Specific surface areas and pore parameters of the samples.
    • Legends for movies S1 to S5

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Magnetic actuation of the FMSMs using a rotating magnetic field.
    • Movie S2 (.mp4 format). Swimming of the FMSMs in different media.
    • Movie S3 (.mp4 format). Fluorescence tracking of the FMSMs in different media.
    • Movie S4 (.mp4 format). Automated fluorescence signal–based servoing of the FMSMs in predefined trajectories.
    • Movie S5 (.mp4 format). Fluorescence detection using the FMSMs in clinical stool supernatant.

    Files in this Data Supplement:

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