Cite this: RSC Advances, 2013, 3,2390

Multifunctional nanoparticles for rapid bacterialcapture, detection, and decontamination3

Received 25th September 2012,Accepted 7th December 2012

DOI: 10.1039/c2ra22286h

www.rsc.org/advances

Longyan Chen,a Fereidoon S. Razavi,b Abdul Mumin,a Xiaoxuan Guo,c Tsun-Kong Shamc and Jin Zhang*a

Fluorescent magnetic nanoparticles (FMNPs) with a core–shell structure are synthesized through a one-pot

chemical method followed by the bioconjugation of gentamycin (Gm). The average diameter of the

FMNPs is estimated to be 65 ¡ 8 nm. The results of transmission electron microscopy (TEM), X-ray

absorption near edge structure spectroscopy (XANES), and fluorospectrometry indicate that the FMNPs

consist of a Fe3O4 core and a fluorescent silica (SiO2) shell. The FMNPs show typical superparamagnetic

properties with a blocking temperature (TB) of 120 K. We demonstrate that gentamicin (Gm)-

bioconjugated FMNPs can capture gram-negative bacteria, i.e. E. coli, (1 6 107 CFU mL21 from 10 mL

of solution) within 20 min. TEM micrographs clearly show that the Gm-FMNPs disrupt the cell wall of E. coli

prior to the lysis of E. coli as the interaction time (t) increases; whereas FMNPs without Gm are inert

towards E. coli. In addition, the Gm-FMNPs are able to detect diluted E. coli cells at a concentration as low

as 1 6 103 CFU mL21, which is revealed by a slight red-shift in fluorescent emissions from 517 nm to 528

nm along with a dramatic decrease in intensity. The Gm-conjugated FMNPs can be a multifunctional

platform for simultaneous rapid capture, sensitive detection, and decontamination of bacteria.

1. Introduction

Most bacteria, single-celled prokaryotic microorganisms, candouble their population in less than 20 min, and a singlebacterium can produce 10 000 cells within 4 h due to theirexponential growth in the early stages of growth.1 Escherichiacoli (E. coli) is a very common gram-negative bacterium. Caseswhere drinking water and food have become contaminated byE. coli have been found world wide.2 Some strains of E. coli caneven cause serious bacterial infections. Antibiotics have beendeveloped to treat bacterial infections, but the effective dosageis not well-controlled. Enzyme-linked immunosorbent assay(ELISA) and polymerase chain reaction (PCR) are normallyused to detect bacteria, and this can take several days.3,4

Consequently, the rapid capture, detection, and decontamina-tion of bacteria are needed to avoid or minimize contamina-tion of the environment, food, and infections.

Engineered nanoparticles (NPs) have shown promise inapplications in drug delivery, sensors, and bio-imaging.5,6

Unlike the passive internalization of NPs within eukaryotic

cells for cell tracking and drug/gene delivery, very few NPs (d ,

5 nm) can penetrate the bacterial cell wall.7–9 Therefore, well-tailored surface modification is the key to actively enhance theinteraction between NPs and bacteria.

Quite recently, magnetic NPs with bioconjugations thathave the ability to capture bacteria have been studied. Xu andhis co-workers conjugated Fe-based magnetic NPs withvancomycin, and through peptide binding captured E. coli ata low concentration of 3 6 104 cells mL21.10 El-Boubbou et al.conjugated lectin to magnetic NPs and used mannose to bind,capture and kill E. coli.11 Optical nanoparticles have also beenapplied in detecting bacteria. For instance, Zhao et al. appliedfluorescent nanoparticles to quantitatively detect singlebacteria.12 Further efforts are needed to explore new biocon-jugations and advanced NPs for simultaneous rapid bacterialcapture, sensitive detection, and efficient decontamination.

Fluorescent magnetic nanoparticles (FMNPs) are emergingmultifunctional NPs with a core–shell structure, i.e. a magneticcore coated with a fluorescent shell. The fluorescent shell canbe composed of polymers and inorganic materials loaded withorganic dye,13–15 quantum dots,16 or other complexes.17,18

Silica is one of the well-studied materials that can act as a shelldue to its functional surface, and its unique porous nature.19–21

Previous studies demonstrated that fluorophore-loaded meso-porous SiO2 NPs are 20 times brighter than semiconductorquantum dots.22,23 They can be taken up by dendritic cellsfor cell tracking and cancer treatment.24,25 The magnetic coresof most FMNPs are intended to be Fe2O3-rich materials.26

aDepartment of Chemical & Biochemical Engineering, University of Western Ontario,

London, Ontario, N6A 5B9, Canada. E-mail: jzhang@eng.uwo.cabDepartment of Physics, Brock University, St. Catharines, Ontario, L2S 3A1, CanadacThe Chemistry Department at the University of Western Ontario, London, Ontario,

N6A 5B9, Canada

3 Electronic supplementary information (ESI) available. See DOI: 10.1039/c2ra22286h

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2390 | RSC Adv., 2013, 3, 2390–2397 This journal is � The Royal Society of Chemistry 2013

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Compared to hematite (Fe2O3) and other magnetic materials,magnetite (Fe3O4) shows higher magnetization, about 65 emug21,27 and has various applications in biosensor, contrastagents.28,29 Here, a one-pot method is applied to produceFMNPs with a core of magnetite (Fe3O4), and a shell offluorescent SiO2.30

In this paper, a new bioconjugation is developed to modifyFMNPs. As shown in Fig. 1, gentamicin (Gm) is a FDAapproved thermal-resistant antibiotic for the treatment ofinfection caused by gram-negative bacteria. The amino acidsof Gm show positive charges through protonation in physio-logic solutions, which may contribute to the interaction of Gmwith lipopolysaccharides (LPS) on the surface of gram-negative

bacteria before entering the cytoplasm.31 Once bound to theribosomes of the bacteria, Gm suppresses protein synthesis.32–34

The challenge lies in accumulating a high concentration of Gmon the surface of bacteria. Thus, to efficiently deliver Gm to E.coli, Gm-conjugated FMNPs (Gm-FMNPs) are developed here.We thoroughly investigate the interaction between E. coli andFMNPs with/without Gm-bioconjugation. Meanwhile, the sensi-tivity of Gm-FMNPs to detect E. coli, is studied.

2. Experimental procedures

2.1 Materials

The following analytical-grade chemicals were purchased fromSigma-Aldrich and used without further purification: cetyl-trimethylammonium bromide (CTAB, 98%), toluene, ammo-nium hydroxide solution (NH4OH, 28%), iron(II) chloride(FeCl2, 98%), iron(III) chloride (FeCl3, 97%), fluoresceinisothiocyanate isomer (FITC, .90%), aminopropyltriethoxysi-lane (APTS, 98%), glutaraldehyde (Glu) (Grade I, 25%), bovineserum albumin (BSA) powder (96%), ethanolamine (EA, 98%)and gentamicin sulfate powder.

2.2 Synthesis and bioconjugation of FMNPs

The FMNPs were produced by base-catalyzed oxidization ofiron chlorides (FeCl2/FeCl3) followed by the condensation oftetraethylorthosilicate (TEOS), and the in situ encapsulation offluorescein isothiocyanate (FITC) in the shell as shown inFig. 1. Briefly, 7.3 g of cetyl-trimethylammonium bromide(CTAB) was added to 100 mL of toluene. The mixture wasstirred at 600 rpm for 4 h, followed by the slow addition ofaqueous FeCl2/FeCl3 solution (0.22 g/0.56 g, 7.3 mL) under anN2 atmosphere. The mixture (solution A) was stirred vigorouslyand purged with N2 for 8 h. Then, 1 mL NH4OH solution (28%in water) was added drop-wise to the solution under an N2

atmosphere. Solution A was stirred for another 4 h.Meanwhile, 5.5 mg fluorescein isothiocyanate (FITC) and 25mL aminopropyltriethoxysilane (APTS) were mixed under an N2

atmosphere to form N-1-(3-triethoxysilylpropyl)-N9-fluoresceylthiourea (FITC-APTS).35 After 2 h, 2.5 mL of tetraethylortho-silicate was added to the FITC-APTS solution. This mixture(solution B) was stirred continuously for an additional 3 hunder an N2 atmosphere using aluminum foil to protect thedye conjugate from light. Solution B was then added drop-wiseto the main reaction of the solution A pot under an N2

atmosphere and continuously stirred for 5 days. Aluminumfoil covered the reactor to protect the products from photo-degradation. The FMNPs were then freeze-dried and stored inthe dark.

The dried FMNPs were transferred to a glass vial anddispersed in 5 mL distilled water to obtain a final concentra-tion of 2.5 mg mL21. Following dispersion, the FMNPs weremixed and reacted with 200 mL of 25% commercial gradeglutaraldehyde (Glu) solution overnight in the dark to formGlu-FMNPs. The mixture was then purified by centrifugation(5800 rpm for 5 min) to remove the supernatant, and furtherre-suspended in 10 mL water. The purification process wasrepeated twice, and the FMNPs were then freeze-dried. To

Fig. 1 Schematic illustration of the synthesis of FMNPs used for E. coli captureand magnetic separation in solution.

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immobilize the Gm onto the surface of the FMNPs, Gm sulfate(0.5 mg) powder was dispersed in 0.5 mL distilled water andmixed for 1 h to obtain a Gm solution. Next, the solution wasadded to 4 mL of the Glu-FMNPs solution (2.5 mg mL21), wassealed, and stirred overnight. The unbound Gm was removedusing a series of centrifugation and washing steps, asdescribed above. The solution was then mixed with 1% bovineserum albumin (BSA) solution for 1 h to secure the unreactedglutaraldehyde (Glu) onto the surface of the FMNPs. Finally,the purified Gm-FMNPs were freeze-dried and stored in thedark at 4 uC.

In addition to the negative control samples of FMNPs andGlu-FMNPs, another negative control sample used to evaluatethe Gm-bioconjugation was ethanolamine (EA). EA and Gmhave the same functional group which conjugates to FMNPs.EA is inert towards E. coli, so 10 mL ethanolamine (EA) wasadded to 4 mL Glu-FMNPs (2.5 mg mL21) according to theconjugation process described above.

2.3 Materials characterization

Fourier transform infrared (FTIR) transmittance spectrometrywas carried out to identify the Gm bioconjugation. A BrukerVector 22 was used in the range of 600–4500 cm21 with aresolution of 4 cm-1 and 64 scans.

TEM and TEM-energy dispersive X-ray spectrometry (TEM-EDX) were performed to measure the core–shell structure andto verify the interaction between the bacteria and the Gm-FMNPs. The TEM images were obtained using a Philips CM-10microscope operating at 80 kV.

2.4 X-ray absorption near edge structure spectroscopy (XANES)study

The Fe K-edge X-ray absorption near edge structure spectro-scopy (XANES) study was conducted using the Soft X-rayMicrocharacterization Beamline (SXRMB) at the CanadianLight Source (CLS). The specimens were placed on the samplemanipulator in a vacuum chamber (y1028 Torr). XANES werecollected in Total Electron Yield (TEY) by measuring thespecimen current in the presence of a voltage bias.36 Inaddition, the XANES spectra have been normalized to I0, theintensity of the incoming photon beam.

2.5 Fluorescent properties of FMNPs

Fluorescence microscopy (Zeiss Axio Imager Z1) analysis wasconducted to study the interaction between the E. coli cells andthe FMNPs with and without Gm bioconjugation by placingglass slides of the samples under an excitation wavelength (lex)of 492 nm. In addition, the fluorescent emission signals of theNPs were detected by fluorophotometry (QuantaMsterTM 30,PTI) with an excitation wavelength (lex) of 492 nm.

2.6 Magnetic properties of FMNPs

An additional 1.52 mg of the core–shell FMNPs in a plasticcapsule was studied by using a superconducting quantuminterference device (SQUID, Quantum Design). The hysteresisloop was measured at room temperature under 60 kOe (6 T).Zero-field-cooled (ZFC) and field-cooled (FC) magnetizationcurves of the FMNPs were also recorded at 50 Oe over a

temperature range of 5 to 300 K. The sample capsule wasdiamagnetic and did not contribute to the measured result.

2.7 Capture of E. coli by Gm-FMNPs

Non-pathogenic E. coli (strain W3110) were grown at 37 uC for24 h in broth media. The optical density (O.D.) of this culturewas adjusted to having a concentration of approximately 107

CFU mL21. The cells were harvested by centrifugation (8000rpm, 5 min) and further re-suspended in 10 mL phosphate-buffered saline (PBS, 0.01 M, pH 7.4) buffer containing Gm-FMNPs (1 mg). The mixtures were further incubated for 20 and60 min, respectively. Two samples with different interactiontimes (t) were separated from the solution using magneticconfinement. Both samples were washed three times, and thenre-suspended in 1 mL PBS for TEM analysis.

The response of E. coli to the Gm-FMNPs was furtherinvestigated by comparing it to the response of E. coli to thenegative control samples, i.e. FMNPs, Glu-FMNPs, and EA-FMNPs. In this experiment, the solution of E. coli cells (y1.06 103 CFU mL21, 900 mL in PBS) was incubated with 100 mLdifferent NPs (0.1 mg mL21), i.e. FMNPs, Glu-FMNPs, EA-FMNPs, and Gm-FMNPs, respectively, for 1 h at roomtemperature. After incubation, magnetic confinement wasperformed under an external magnetic field (0.2 T) from thesupernatants of the mixture of E. coli cells and FMNPs withand without Gm. The separated contents were then washedtwice and dispersed in 1 mL PBS. Next, 200 mL of bothsupernatants and re-dispersed particle solutions were spreadonto agar plates (petri dishes). All the agar plates were thenincubated overnight at 37 uC. The number of CFU of thebacteria was calculated following the incubation.

2.8 Thin sections for TEM

Thin sections for TEM and TEM-EDX were prepared by fixingthe above samples (E. coli incubated with and without Gm-FMNPs for 20 min and 60 min, respectively) with 2.5% (vol%)glutaraldehyde in sodium cacodylate buffer (0.1 M, pH 7.4).After 2.5 h fixation, the cells were washed three times incacodylate buffer with centrifugation (y4500 g for 6 min), andfurther fixed with 1% osmium tetroxide in a cacodylate buffersolution for 1 h. The samples were washed again, and the finalpellets were placed in drops of 5% Noble agar. The sampleswere further fixed in 2% uranyl acetate for 2 h, followed bydehydration in an ethanol solution with ascending gradientsof strength (50%, 70%, 85%, 95% with two changes inabsolute ethanol, 15 min each). The specimens were thenwashed in propylene oxide twice and infiltrated with an EPONresin : propylene oxide mixture (ratio 1 : 1 and 3 : 1, placed ineach once) and twice in pure EPON resin. Sample resin blockswere prepared by embedding the samples in resin andpolymerizing them at 60 uC for 2 days. The resin blocks werethen trimmed and sectioned (80–100 nm) on a Reichert Om-U3ultramicrotome with a diamond knife. Ultrathin sections wereplaced on 200-mesh formvar/carbon-coated nickel (Ni) grids.

The antimicrobial susceptibilities of E. coli to Gm-FMNPsand pure Gm were investigated by standard agar disc-diffusion. One isolated E. coli colony was incubated overnightin fresh broth at room temperature to reach a bacterial cultureof y107 CFU mL21. A 200 mL aliquot of this culture (y105 CFU

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mL21) was spread onto agar plates. Sterile filter paper discs (7mm) immersed in 1 mL PBS containing different standardconcentrations of Gm and FMNP solution samples (bothconjugated with or without Gm) were then applied to theplates and incubated for 18 h at 37 uC. The antibacterialefficiency of the Gm-FMNPs was then determined as afunction of E. coli colony inhibition diameters based on thecalibration exponential curve obtained in experiments withknown concentrations of the standard solutions.

3. Results and Discussion

3.1 Synthesis and characterization of FMNPs with/without Gm-bioconjugation

FMNPs were characterized by TEM as shown in Fig. 2a. Anobvious boundary between the core and the shell is observedin most of the particles. The average diameter of the iron oxidecore is estimated to be 50 ¡ 8 nm, while the thickness of thesilica shell is estimated to be 12 ¡ 5 nm. The mean particlesize is approximately 65 ¡ 8 nm. The FMNPs were investigatedusing XRD (ESI3). Two major phases were identified: the broadpeak of semi-crystalline SiO2 at 26u, and magnetite, Fe3O4,with an FCC structure. Thus, the semi-crystalline SiO2 shellhas a lighter color in the TEM micrograph, whereas the Fe3O4

core has higher electron density, and therefore, has a darkercolor. The oxidation status of the magnetic core is furtherinvestigated by the results of X-ray absorption near edgestructure spectroscopy (XANES). Fig. 2b shows the Fe K-edge inthe XANES spectra of the standard Fe3O4 sample and FMNPs.The Fe K-edge excitation threshold (E0) is the maximum of thefirst derivative of the XANES spectra. Clearly, the FMNPs havethe same E0 as that of Fe3O4.

In comparison with other Fe-based magnetic materials,Fe3O4 has high thermal stability with a high Curie temperature(Tc) of 858 K. Fig. 2c shows the superparamagnetic behavior ofthe FMNPs during field cooling (FC) and zero-field cooling(ZFC). The individual magnetic spin is randomly oriented at 5K. Upon applying the external magnetic field cooling (FC), the

original random distributed spins align along the direction ofthe applied field. The magnetization increased with tempera-ture, and reached the blocking temperature at 120 K.37 Ourcalculation shows the saturation magnetization (Ms) of thecore approaches the Ms of magnetite (Fe3O4), 65 emu g21, atroom temperature (ESI3).

To bioconjugate Gm with FMNPs, glutaraldehyde (Glu) wasemployed as a linker. In this bio-conjugation, two carbon–nitrogen double bonds (CLN), i.e. Schiff bases, are formed asshown in Fig. 1. The successful conjugation of Gm and FMNPsis confirmed by fourier transform infrared spectroscopy(FTIR). Fig. 3 shows the FTIR spectra of FMNPs, Glu-FMNPs,and Gm-FMNPs, respectively. The Si–O–Si stretching (1040cm21) can be recognized in the spectra. Furthermore, thetypical –CH2 stretch at 2930 cm21, the –CLN stretch of theimine group, and –CLN–R at 1640 cm21 appears in thespectrum of Gm-FMNPs. No –CLO stretch at 1760 cm21 isfound after Glu linking the FMNPs and Gm.

Fig. 2 (a) TEM micrograph of the core–shell structures of FMNPs. (b) Fe K-edge XANES of FMNPs (black line) and model compound, Fe3O4 (red line). (c) Magnetizationof FMNPs as a function of temperature in the applied field of 50 Oe at a temperature range of 5 to 300 K using field cooling (FC) and zero-field cooling (ZFC)procedures.

Fig. 3 FTIR absorption spectra of FMNPs, glutaraldehyde (Glu) modified FMNPs(Glu-FMNPs), and Gm-FMNPs.

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3.2 Using Gm-FMNPs to capture E. coli

Our experimental results demonstrate that Gm-conjugatedFMNPs (Gm-FMNPs, 1 mg) are able to capture and remove E.coli cells (1 6 107 CFU mL21) from 10 mL solution under anexternal magnetic field of 2.0 kOe as shown in Fig. 4.Approximately 90% of the Gm-FMNPs can be separated fromthe solution within 6 min, and no living E. coli is found in thesuspension after 20 min of magnetic confinement.

3.3. Interaction between E. coli and FMNPs with/without Gm

The responses of cultured E. coli to FMNPs with and withoutGm were further studied by TEM. FMNPs without Gm wereused as the negative controls, including FMNPs, glutaralde-hyde (Glu)-FMNPs, and ethanolamine (EA)-FMNPs. EA is inerttowards E. coli. Here, EA was conjugated with FMNPs usingGlu as a linker, using the same chemical method as describedfor the conjugation of Gm to FMNPs. The interactions betweenE. coli cells and the negative control samples are hardlyobserved (Fig. 5a). Thus, both FMNPs and possible excessglutaraldehyde remaining on the FMNPs are inert towards E.coli.

In addition, our previous study showed that mesoporoussilica NPs with a similar size and size distribution (d = 65 ¡ 8nm) have good internalization (over 50%) into dendriticcells.24 Therefore, these findings suggest that the E. coli cellwall plays a key role in blocking the entry of FMNPs, with d =65 ¡ 8 nm, into E. coli cells.

To investigate how the Gm-FMNPs interact with E. coli, weperformed TEM measurements of samples of E. coli mixedwith Gm-FMNPs which were cultured for 20 and 60 min asshown in Fig. 5b and 5c, respectively. Fig. 5b shows that Gm-

Fig. 4 10 mL E. coli suspension (1 6 107 CFU mL21) captured by 1 mg FMNPsunder an external magnetic field, 0.2 T, within 6 min (middle), and 20 min(right), respectively.

Fig. 5 Interactions between FMNPs with/without Gm and E. coli, (a) TEM micrographs of E. coli mixing with FMNPs without Gm. (b) E. coli mixing with Gm-FMNPswith less than 20 min of interaction time (t). (c) E. coli interacting with Gm-FMNPs when t = 60 min. (d) Thin section TEM analysis of E. coli mixing with Gm-FMNPswhen t = 20 min. (e) The TEM-EDX spectrum of the Gm-FMNPs. (f) Thin section TEM analysis of E. coli mixing with Gm-FMNPs when t = 60 min.

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FMNPs aggregate on the cell wall. Compared with the centralcylindrical region of the cell, the cell poles (dark hemispheres)are more aggressively attacked by Gm-FMNPs. The areas of thecell wall to which the Gm-FMNPs attach are clearly damagedand exhibit texture changes (see the small insert in Fig. 5b).Fig. 5c shows that the bacterial cell membranes are brokenand the cytoplasmic matrix appears to be leaking out. Suchphenomena are not observed in samples of bacteria mixedwith negative control samples, i.e. FMNPs (Fig. 5a), Glu-FMNPs, and EA-FMNPs, respectively. In addition, the corre-sponding thin-sectioned samples measured by TEM (Fig. 5dand 5f) were used to verify the interactions between the Gm-FMNPs and E. coli over time. In Fig. 5d, the internalization ofGm-FMNPs within E. coli cells, maintaining the integral‘‘envelope’’ structure, is observed (indicated by a blue arrow)when the interaction time (t) is 20 min. Significant disruptionof the membrane and cell lysis occur with an increase in theinteraction time from 20 to 60 min. TEM-EDX results (Fig. 5e)clearly show that the bioconjugated particles interacting withE. coli contain iron (Fe) from the core of the FMNPs and silicon(Si) from the mesoporous silica shell. The peaks of nickel (Ni)stem from the TEM sample grid. Furthermore, no bacterialcells with an integral structure are observed using TEM whenthe sample was incubated for 3 h. It is likely that the Gm-FMNPs damage the cell wall first, and are then adsorbedwithin the cell. The polycationic Gm-FMNPs mixing with E. colicells rapidly induce strong cell wall-adsorption at an earlystage.33 Previous studies indicated that cationic Gm may re-arrange the lipopolysaccharide (LPS) packing order throughionic bonding, consequently disrupting bacteria’s outermembrane and promoting membrane permeability.38,39

3.4 Detection of low concentrations of E. coli

The maximum fluorescent emission (lem) of FMNPs is centredat 517 nm, while free FITC with lem = 510 is shown in Fig. 6a.Meanwhile, Gm-FMNPs (0.1 mg mL21) were measured using afluorospectrometer with an excitation wavelength of 492 nmbefore and after interacting with E. coli. The results show thatthe maximum emission wavelength (lem) of Gm-FMNPs iscentered at 517 nm (Fig. 6a). A broad emission peak with asubstantial decrease in intensity is observed when the Gm-FMNPs directly interacted with the diluted E. coli cells, 1 6 103

CFU mL21 for 20 min. It is also noted that the peak has a slightred-shift to lem = 528 nm. No significant change is observedover time.

Fig. 6b1 shows there is no aggregation when the E. coli wereincubated with FMNPs alone, whereas E. coli cells attached toGm-FMNPs are aggregated in solution (Fig. 6b2). Thephenomenon may explain the significant decrease in fluor-escent intensity after mixing FMNPs with E. coli. It clearlyshows that Gm-conjugated with FMNPs are able to recognizevery low concentrations of E. coli.

3.5 Antibacterial activity of Gm-FMNPs

The response of E. coli to the Gm-FMNPs was furtherquantitatively studied by calculating the colony-forming units(CFU) of both supernatants and precipitates. A cultured E. colisample was used as a positive control. Negative controls areFMNPs without Gm, i.e. FMNPs, Glu-FMNPs, EA-FMNPs.

Diluted E. coli cells were incubated with Gm-FMNPs andnegative controls for 30 min, respectively. No significantprecipitation is observed in the E. coli suspensions mixed withnegative controls: FMNPs, Glu-FMNPs, and EA-FMNPs, respec-tively. However, obvious precipitation can be found in the E.coli suspension mixed with Gm-FMNPs. To evaluate theantibacterial activity of Gm-FMNPs, all samples were washedand freeze-dried several times to remove free Gm from thesample

Fig. 7a shows photographs of the resulting colonies in agarplates for all samples. The cultured E. coli mixed with FMNPs(Fig. 7a-A2), Glu-FMNPs (Fig. 7a-A3), and EA-FMNPs (Fig. 7a-A4), respectively, show a similar result with the positive control(Fig. 7a-A1); whereas Gm-FMNPs (0.1 mg) significantly inhibitthe growth of E. coli. No living cells can be found throughoutthe suspension of E. coli mixed with Gm-FMNPs as shown in(Fig. 7a-A5 and -A6). Further analysis indicates that thebacterial concentration of those FMNPs without Gm is 0.8 6103 CFU mL21, which is similar to the positive control (1.0 6103 CFU mL21). However, the bacterial concentration

Fig. 6 (a) Fluorospectra of free FITC, FMNPs, and the Gm-FMNPs before/afterdetecting E. coli cells (1 6 103 CFU mL21); (b1) FMNPs mixing with E. coli, 1 6103 CFU mL21, (with dark field (DF, left) and bright field (BF, right); (b2) E. coliinteracting with Gm-FMNPs with dark field (DF, left) and bright field (BF, right)when t = 20 min.

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decreases dramatically in the sample of Gm-FMNPs as shownin Fig. 7b. The results confirm that Gm-conjugation is playingthe key role to allow the FMNPs to interact with E. coli. Theantimicrobial susceptibility of E. coli to Gm-FMNPs wasfurther determined by the agar disk diffusion method withthe comparison of the calibration curve obtained by testingpure Gm (Fig. 7c). Here, 0.1 mg Gm-FMNPs show a similarantimicrobial efficiency to 12–15 mg of pure Gm. Ourexperimental results indicate that y8 mg of Gm could beconjugated on 0.1 mg FMNPs (see ESI3). Furthermore, Gm-FMNPs are able to maintain their bioactivity, even when storedat 6 uC for 3 months. The antimicrobial efficiency of Gm-FMNPs will be further investigated using different types ofbacteria.

4. Conclusion

In conclusion, FMNPs (diameter of 65 ¡ 8 nm), comprised ofa magnetic core (Fe3O4) and fluorescent shell (SiO2), aresuccessfully conjugated with Gm, an aminoglycoside antibio-tic. In this study, the interactions between E. coli and theengineered multifunctional NPs with and without Gm bio-conjugation are investigated. E. coli cells (y1 6 107 CFUmL21) can be magnetically captured within 20 min by 1 mgGm-FMNPs from 10 mL of solution under an externalmagnetic field of 20 kOe. TEM results clearly show thedynamic interactions between E. coli and the Gm-FMNPs withincreasing interaction time, whereas there are no interactionsbetween E. coli and FMNPs without Gm-conjugation. Our TEMresults first demonstrate that the Gm-FMNPs attempt to attachto the surface of the poles of E. coli cells. In addition, the Gm-FMNPs are able to detect diluted E. coli cells at a concentrationas low as 1 6 103 CFU mL21, which is revealed by a slight red-shift in fluorescent emissions from 517 nm to 528 nm alongwith a dramatic decrease in intensity. This phenomenon couldbe caused by the aggregation of FMNPs driven by theinteraction between Gm and E. coli. Clearly, Gm-FMNPs notonly inhibit the growth of E. coli, but also kill E. coli cells in ashort period. The results also indicate that Gm-conjugatedFMNPs are able to act simultaneously as a method for rapidbacterial capture, sensitive detection, and decontamination.

Acknowledgements

We thank Mr Brian Dennis from the Biochemical EngineeringLaboratory of Western, for generously providing the E. coli(strain W3110). Authors appreciate Dr David Litchfield, DrSusan Koval, and Judy Sholdice for discussing the TEM results.Special thanks to the Canadian Light Source (CLS). We alsothank the Biotron image center at Western for allowing us toaccess the facilities. This work was supported by the CanadaFoundation for Innovation - Leaders Opportunity Fund (CFI-LOF), Natural Sciences and Engineering Research Council ofCanada (NSERC) Engage, and NSERC Discovery Grant.

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2396 | RSC Adv., 2013, 3, 2390–2397 This journal is � The Royal Society of Chemistry 2013

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