Bacterial isolation by lectin-modified microengines

Abstract

New template-based self-propelled gold/nickel/polyaniline/platinum (Au/Ni/PANI/Pt) microtubular engines, functionalized with the Concanavalin A (ConA) lectin bioreceptor, are shown to be extremely useful for the rapid, real-time isolation of Escherichia coli (E. coli) bacteria from fuel-enhanced environmental, food, and clinical samples. These multifunctional microtube engines combine the selective capture of E. coli with the uptake of polymeric drug-carrier particles to provide an attractive motion-based theranostics strategy. Triggered release of the captured bacteria is demonstrated by movement through a low-pH glycine-based dissociation solution. The smaller size of the new polymer-metal microengines offers convenient, direct, and label-free optical visualization of the captured bacteria and discrimination against nontarget cells.

Figure 1

Lectin-modified microengines for bacteria isolation. Schemes depicting A) the selective pick-up, transport and release of the target bacteria by a ConA-modified microengine, and B) surface chemistry involved on the microengines functionalization with the lectin receptor. Upon encountering the cells, the ConA-functionalized microengines recognize the E. coli cell walls by O-antigen structure binding-allowing for selective pick-up and transport. Inset (in Scheme A, top left side), a SEM image of a portion of a ConA-modified microengine loaded with an E. coli cell. Scheme A, right side: Release of the capture bacteria by navigation in a 10 mM glycine solution, pH 2.5. Scheme B, Steps involved in the microengines gold surface functionalization: 1) self-assembling of MUA/MCH binary monolayer; 2) activation of the carboxylic terminal groups of the MUA to amine-reactive esters by the EDC and NHS coupling agents; 3) reaction of NHS ester groups with the primary amines of the ConA to yield stable amide bonds.

Figure 2

Selective interaction between the ConA-functionalized microengines and the E. coli target bacteria in a fuel-enhanced and E. coli inoculated human urine sample. Time-lapse images - taken from Supporting Video S1A- before, during, and after interaction of the ConA-modified microengines with S. cerevisiae negative control (a–c, respectively) and E. coli target (d-f, respectively) cells. Urine samples are inoculated with E. coli (2.25×10⁷ colony forming units (cfu/ml) or 4.5×10⁴ cfu on the glass slide) and a 5-fold excess of S. cerevisiae and finally diluted 4 times in the glass slide to include the functionalized microengines and the fuel solutions (See Methods Section for additional details). Final fuel conditions: 7.5% (w/v) H₂O₂, 1.25% (w/v) Triton X-100. The E. coli and S. cerevisiae cells are accented by dashed green and red circles, respectively.

Figure 3

Isolation of the E. coli target bacteria from different real samples using ConA-functionalized microengines. Images -taken from Supporting Video S3- demonstrating the E. coli pick-up and transport in peroxide-fuel containing samples: a) drinking water, b) apple juice and c) seawater samples inoculated with E. coli 1.8×10⁷ cfu/mL (3.6×10⁴ cfu on the glass slide). Other conditions, as in Fig. 2. E. coli cells are accented by dashed green circles.

Figure 4

Release of the captured bacteria from the ConA-modified microengines. Images taken from Supporting Video S6A showing an E. coli-ConA-modified microengine before a) and after b) incubation (20 min) in a 10 mM glycine solution (pH 2.5). Other experimental conditions, as in Fig. 2. E. coli cells are accented by dashed green circles.

Figure 5

Dual capture and transport of E. coli and polymeric (PLGA) drug-carrier particles. Images taken from Supporting Video S7 with a ConA-modified microengine picking-up first the target bacteria (b), followed by the PLGA magnetic loading (c) and transport of both cargoes (d). Cartoon depicting the dual capture capability of a ConA-modified microengine (e). Other experimental conditions, as in Fig. 2. Attached E. coli cell and PLGA microparticle are accented by dashed green and blue circles, respectively.