Environ. Sci. Technol. 2010, 44, 7908–7913
Amine-Functionalized Magnetic Nanoparticles for Rapid Capture and Removal of Bacterial Pathogens Y A N - F E N G H U A N G , †,§ Y A - F A N W A N G , ‡ A N D X I U - P I N G Y A N * ,† Research Center for Analytical Sciences, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China, and School of Food Engineering and Biological Technology, Tianjin University of Science and Technology, 29 Avenue 13, TEDA, Tianjin 300457, China
Received July 8, 2010. Revised manuscript received September 7, 2010. Accepted September 12, 2010.
Interest in magnetic nanoparticles for capturing bacteria arises from a variety of attributes, including the similar size of nanoparticles, magnetic behavior, and attached biomolecules such as proteins and nucleotide probes. Here we report the application of amine-functionalized magnetic nanoparticles (AFMNPs) for rapid and efficient capture and removal of bacterial pathogens. The AF-MNPs are used without the need for any further modifications with affinity biomolecules. The positive charges on the surface of AF-MNPs can promote strong electrostatic interaction with negatively charged sites on the surface of bacterial pathogens to exhibit efficient adsorptive ability. The hydrophobic interaction between the pendant propyl group of the amine functionality and the bacteria also plays a supplementary role. The amine groups on the surface of the magnetic nanoparticle are robust and inexpensive ligands to ensure a high binding affinity to at least eight different species of Gram-positive and Gram-negative bacteria. The amount of AFMNPs, pH of phosphate buffer solution, and ionic strength are crucial in mediating fast and effective interactions between AF-MNPs and bacteria. The AF-MNPs allow rapid removal of bacteria from water samples, food matrixes, and a urine sample with efficiency from 88.5% to 99.1%. Though amino group offers less specificity/selectivity than biomolecules such as antibodies, AF-MNPs are attractive for capturing a wide range of bacteria.
Introduction In recent years, microbial contamination of water and food is more of a security issue due to the increasing risk of bioterrorism attacks (1-6). The low infectious dose of bacterial pathogens in food and contaminated water not only can pose a serious threat to human health, but also can cause widespread damage (5-7). Bacteria at low concentrations are hard to detect and usually require long induction times before further analysis (8). Magnetic nanoparticles have been utilized for magnetic capture of pathogens in the sample preparation giving aid to detection and identification, and * Corresponding author fax: (86)22-23506075; e-mail: xpyan@ nankai.edu.cn. † Nankai University. ‡ Tianjin University of Science and Technology. § On leave from National Engineering Technology Research Center for Preservation of Agricultural Products, Tianjin 300384, China. 7908
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 20, 2010
are promising candidates for microbial decontamination (8-25). For a specific interaction to occur, magnetic nanoparticles are usually functionalized with biorecognition molecules, such as antibody (15-17), aptamers (3), bioprotein (18), and carbohydrates (19-22), which allow for bacteria recognition and targeting. Several groups have confirmed superior performances of magnetic nanoparticles (MNPs) and demonstrated exciting and promising applications for capturing bacterial pathogens (8-25). For example, Gu et al. (23, 24) showed that E. coli at ultralow concentrations was effectively captured by vancomycin-conjugated FePt nanoparticles, whereas no E. coli was captured by the FePt nanoparticles capped with cystamine (FePt-NH2). Kell et al. (25) also developed a series of vancomycin-modified nanoparticles and employed them in magnetic confinement assays to isolate a variety of bacteria from aqueous solution. Here we report the utilization of amine-functionalized magnetic nanoparticles (AF-MNPs) for rapid, inexpensive, and effective capture of bacterial pathogens. We determine that the amount of AF-MNPs, pH of phosphate buffer solution (PBS), and ionic strength are crucial in mediating fast and effective interactions between the nanoparticles and the bacteria. Potential factors for efficient capture of several important bacterial pathogens, such as the amount of AFMNPs, the pH, and concentration of PBS are investigated and optimized. The mechanism for AF-MNPs to capture bacteria is also discussed. The developed method does not need functionalization of affinity biomolecules on the surface of MNPs for bacteria recognition and targeting, and is demonstrated for effective removal of bacterial pathogens from water, food, and urine samples.
Experimental Section Reagents and Materials. Ferric chloride (FeCl3 · 6H2O), ferrous sulfate (FeSO4 · 7H2O), disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium sulfate, N,N-dimethyl formamide (DMF), toluene, and aqueous ammonium hydroxide were obtained from Guangfu Fine Chemical Research Institute (Tianjin, China). Tetraethylorthosilicate (TEOS) and γ-aminopropyltriethoxysilane (APTES) were used as received from Wuhan University Silicone New Material Co. Ltd. (Wuhan, China). Luria-Bertani (LB) broth and granulated agar were obtained from Beijing Aoboxing Biotech Company Ltd. (Beijing, China). Apparatus. Fourier transform infrared (FT-IR) spectra (4000-400 cm-1) of bare Fe3O4 nanoparticles, silica coated MNPs and AF-MNPs were recorded on a Magna-560 spectrometer (Nicolet, USA). The magnetic properties of the nanoparticles were studied using a vibrating sample magnetometer (LDJ 9600-1, USA) at room temperature by cycling the field from -6 to 6 kOe. AF-MNPs and the nanoparticle pathogen conjugates were characterized by transmission electron microscopy (TEM, FEI Tecnai G2 F20 and JEOL 100 CXII). The optical density (OD) measurements were performed on a Shimadzu UV-3600 UV-vis-NIR spectrophotometer at 600 nm. Zeta potentials of MNPs and AF-MNPs (0.01% w/w) in a phosphate buffer solution (10 mM, pH 7.0) were measured on a zeta potential analyzer (Brookhaven Instruments Co., Holtsville, NY). Preparation and Characterization of AF-MNPs. The schematic diagram for the synthetic procedure is shown in Scheme 1. Fe3O4 nanoparticles were prepared by a coprecipitation method (19, 26). Briefly, 1 M FeCl3 and 1 M FeSO4 were prepared by dissolving the corresponding iron salt in 2 M HCl. Then, 10 mL of the FeCl3 solution was mixed with 10.1021/es102285n
2010 American Chemical Society
Published on Web 09/24/2010
SCHEME 1. Schematic Diagram for the Synthesis of AF-MNPs
5 mL of the FeSO4 solution in a degassed flask. Concentrated ammonia aqueous solution (about 30 mL, 25%-28% w/w) was added under nitrogen until a pH of 11 was reached. After vigorous stirring at room temperature for 40 min, the resulting black MNPs were isolated by applying a permanent external magnet, washed 6 times with ultrapure water and 3 times with ethanol, and redispersed in a mixture of ethanol (120 mL), ultrapure water (30 mL), and concentrated ammonia aqueous solution (2 mL) under ultrasonication for 5 min. Then, TEOS (1 mL) was added to the above dispersion, and it was sonicated for another 5 min. The reaction was allowed to proceed under mechanical stirring for 8 h. The harvested particles were collected, washed with the distilled ethanol and DMF, and redispersed in a mixture of DMF (90 mL) and toluene (60 mL) under ultrasonication for 5 min. APTES (10 mL) was then added to the above dispersion, and it was sonicated for 5 min. The reaction was allowed to proceed under mechanical stirring for 24 h. The AF-MNPs were collected and washed with ethanol several times with the help of an external magnet, then dried under vacuum at 60 °C for 12 h. To avoid possible oxygen, all the main synthesis steps were carried out by passing N2 gas through the solution. Preparation of Bacterial Samples. Stock cultures of Sarcina lutea (S. lutea), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Bacillus cereus (B. cereus), Proteus vulgaris (P. vulgaris), Salmonella, and Bacillus subtilis (B. subtilis) were kind gifts from Prof. Chang-Lu Wang and Mei-Fang Lu (Tianjin University of Science and Technology, Tianjin, China). Cultures were grown at 37 °C on a rotary shaker at 150 rpm for 16 h. The bacterial cells were centrifuged at 5000 rpm for 5 min, and washed with PBS (10 mM, pH 7.0). The bacterial concentration was adjusted to a desired level by measuring the optical density at 600 nm (OD600). For safety considerations, all of the bacterial samples were placed in an autoclave at 121 °C for 20 min to kill bacteria before disposal and all glassware in contact with bacteria was sterilized before and after use. Bacterial Capture Efficiency of AF-MNPs. The bacterial concentration was adjusted to a desired level, a certain amount of AF-MNPs suspended in PBS (10 mg mL-1) was then added into the bacterial solution, and the solution volume was fixed to 5 mL. The solution was incubated by a rotary shaker at 250 rpm for a specific period (when capture kinetics of E.coli in static state was evaluated, the suspension was allowed to settle for a specific period), then an external magnet was employed for magnetic separation. The supernatant was then carefully pipetted into a cell to measure its OD600 using absorption spectroscopy. The relative efficiencies of the magnetic capture of bacteria by AF-MNPs were calculated from the decrease of turbidity relative to a reference before magnetic capture (9, 11). Removal of Bacteria in Real Samples. The developed AF-MNPs based method was applied for the removal of bacterial pathogens from water, beverage, and urine samples. Two river water samples, two lake water samples, a groundwater sample, and a tap water sample were collected locally. Grape juice and green tea beverage samples were collected from local supermarkets. A urine sample was collected from a healthy volunteer. Plating microbial plate count method
(25) was adopted to verify the robustness of the method, and anionic silica coated MNPs was used as control. The nanoparticles were added to the two river water samples, two lake water samples, and a urine sample and the final concentration was 0.5 mg mL-1. The nanoparticles were generally incubated for 10 min using a rotary shaker at 250 rpm in order to bind with the bacteria effectively. The resulting nanoparticle-bacteria conjugates were magnetically confined for 10 min and the supernatant was carefully removed with a pipet. The nanoparticle-bacteria conjugates were resuspended with autoclaved PBS to the original volume (5 mL), and the magnetic separation was repeated. Resuspensions of the nanoparticle-bacteria conjugates (500 µL) were spread plated on a Petri dish containing LB agar medium at a certain dilution. The plates were then incubated at 37 °C for 24 h. The number of colonies grown on the plates was enumerated for both AFMNPs and the control nanoparticles. To ensure that all of the bacteria were accounted for, the supernatants from the two consecutive separations were combined, and then plated onto LB agar plates. The total number of resulting cfu’s from both the magnetically confined bacteria and the bacteria remaining in the supernatant was consistent with the total number of bacteria that was employed in the experiments. The capture efficiency was calculated by dividing the number of bacteria in AF-MNPs aggregate over the total number of cells in both the supernatant and the aggregate (25). E. coli and S. lutea were spiked into a groundwater sample and a tap water sample, E. coli and P. vulgaris were spiked into an autoclaved grape juice (10% v/v) sample and an autoclaved green tea beverage sample, and the microbial concentration was adjusted to 103-104 colony forming units (cfu) in 1.0 mL to examine the efficiency of AF-MNPs for removal of pathogens. Magnetic capture of bacterial pathogens was performed as above.
Results and Discussion Preparation and Characterization of AF-MNPs. The MNPs prepared by the coprecipitation method are easily coated with amorphous silica using conventional sol-gel method. The sol-gel process is based on the hydrolysis of TEOS, and subsequent condensation of hydrolyzed TEOS on surface iron hydroxyls. With controlled hydrolysis of TEOS, a Fe-O-Si chemical linkage is established between surface Fe atoms and TEOS, followed by lateral polymerization, and finally formation of a three-dimensional network via siloxane bond formation (Si-O-Si) with increasing TEOS concentration and degree of hydrolysis (27). The silica shell is favorable for the dispersion of the Fe3O4 particles in aqueous media, and also protects them from leaching in an acidic or basic environment (28, 29). Subsequently, postsynthetic modification of the magnetic silica nanoparticles is accessed by employing the common organosilane reagent APTES. Silanation of MNPs using a silane-coupling agent is attractive for improving stability and control of surface properties. APTES not only provides a common intermediate that is amenable to a wide range of subsequent modifications, particularly with respect to biomolecule immobilization, but also causes a drastic change in the electrokinetics of the particles in an aqueous solution through obtaining cationic amine groups on nanosized magnetic particles (30, 31). Furthermore, amino groups impart the AF-MNPs good solubility and dispersibility in aqueous media. TEM images show the size and shape of AF-MNPs at different magnifications (Figure S1 in the Supporting Information). FT-IR analysis provides direct proof for the amine functionalization (Figure S2A). The main IR bands at 617 and 453 cm-1 originate from Fe-O vibrations. The magnetic silica nanoparticles and the amine functionalized particles possess absorption bands in the region 980-1220 cm-1 which VOL. 44, NO. 20, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7909
FIGURE 1. Efficiency of AF-MNPs to capture E. coli from PBS buffer (10 mM, pH 7.0). Photo of (A) E. coli solution before magnetic capture, (B) solution after magnetic capture by silica coated MNPs (control), and (C) solution after magnetic capture by AF-MNPs. can be assigned to vibration modes of Si-O-Si and Si-OH bonds (19). N-H bending at 1650 cm-1 in Figure S2Ac can be attributed to characteristic of the presence of N-H groups (32). The presence of aminopropyl groups is identified by the appearance of C-H stretching band at 2931 cm-1 (33). APTES is thus believed to be coated on the surface of the silica coated nanoparticles. The content of nitrogen in AF-MNPs determined by elemental analysis is 3.22%, proving that APTES is bonded to the surface of the silica coated MNPs which possess sufficient charge densities. The zeta potentials of MNPs and AF-MNPs (0.1% w/w) in a phosphate buffer solution (10 mM, pH 7.0) were -46.05 mV and +7.66 mV, respectively. After the deposition of APTES on the MNPs, the zeta potential of AF-MNPs became positive due to the cationic amine group on the surface. Figure S2B shows hysteresis loops of the magnetic particles at room temperature. The magnetic saturation (Ms) values of the bare Fe3O4 nanoparticles, silica coated MNPs and AF-MNPs are 17.24, 13.86, and 11.22 emu g-1, respectively. The Ms values of these MNPs decrease gradually because of the deposited silane layers (TEOS and APTEs). AF-MNPs for Capture of Pathogens and Possible Mechanism. The ability of AF-MNPs to capture bacterial pathogens was examined using E. coli as a model microorganism and the cell concentration was adjusted to an OD600 of 1.0. The AF-MNPs suspension (10 mg mL-1) was added to E. coli solution of neutral pH (PBS, 10 mM, pH 7.0) and incubated for 1 min using a rotary shaker at 250 rpm. Rapid aggregation and microbial arrest occurred in E. coli solution. Aggregates of cells and AF-MNPs precipitated in several minutes in the presence of an external magnet and the supernatant solution became clear (Figure 1C). The capture efficiency of E. coli by AF-MNPs calculated from the decrease of turbidity relative to a reference before magnetic capture was greater than 97%. As the control, anionic silica coated MNPs showed inability to capture the pathogens because the optical density of its supernatant (Figure 1B) was much the same as that of E. coli solution without MNPs (Figure 1A). The AF-MNPs-bacteria conjugates are much more easily separated by a magnet than silica coated MNPs because more AF-MNPs are attached to one E. coli simultaneously. In addition, flocculation of bacteria and AF-MNPs is formed by charge neutralization (34), which differs significantly from buffer density and results in phase separation by rapid sedimentation of flocs, facilitating magnetic separation. The silica coated MNPs as the same negatively charged nanoparticles (pI ∼3) (35), however, keep free upon the mutual repulsion of negatively charged bacteria, and only respond weakly to magnet. Bacterial capture is influenced by bacteria surface charge, hydrophobicity, and surface properties of bacteria and matrix (36). AF-MNPs capture of bacteria is mainly driven by attractive electrostatic interaction between the negatively 7910
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 20, 2010
FIGURE 2. Bar graphs representing the magnetic capture efficiency for a variety of Gram-positive (+) and Gram-negative (-) bacteria by AF-MNPs and silica coated MNPs. charged bacteria and the positively charged AF-MNPs surface. Bacterial cell surfaces possess net negative electrostatic charge by virtue of ionized phosphoryl and carboxylate substituent on outer cell envelope macromolecules which are exposed to the extracellular environment (37). The isoelectric point (pI) of most bacterial cells lies within the pH range of 1.5-4.5. The majority of cells are negatively charged within the pH range 5.0-9.0 (38). In contrast, AFMNPs have a pI above 9.0 (39) and possess positive zeta potential under pH 9.0. The predominantly negative surface charge of the bacterial cellular membranes provides a clear target for separation via electrostatic interactions. Efficiency for Magnetic Capture of Different Species Bacteria by AF-MNPs. It is interesting to note that amine groups on the surface of AF-MNPs are potentially nonselective ligands to bacteria since the outer membranes of bacterial cells are generally negatively charged. With such a merit, decontamination from the medium could be carried out through magnetic separation. The capture efficiency of several bacteria was investigated at a cell concentration of OD600 ) 1.0. The results shown in Figure 2 suggest that AF-MNPs have the ability to efficiently confine at least eight different species of Gram-positive and Gram-negative bacteria. Control experiments carried out with silica coated MNPs suggest weak interaction between silica coated MNPs and the bacteria. The silica coated MNPs also showed high affinity to B. Cereus likely due to hydrophobic interaction (36, 40). To further confirm the effective capture of bacteria by AF-MNPs, we used TEM to study the aggregates of the AFMNPs and bacteria. The AF-MNPs were added to bacteria suspension, after mixing and magnetic separation, and the nanoparticle-pathogen conjugates were resuspended in 500 µL of PBS. A portion of the suspension (0.5 µL) was deposited onto 200 mesh carbon-coated copper grids, and after drying, the sample was subjected to TEM analysis. The TEM images in Figure S3 show that the bacteria indeed aggregated with the AF-MNPs, and AF-MNPs covered the most regions of the cells surface and distributed nonuniformly. Kinetics for Capturing Bacterial Pathogen by AFMNPs. The kinetics for AF-MNPs to capture bacterial pathogens was investigated using E. coli as a model microorganism (Figure S4). The capture behavior of E. coli was evaluated both under regular shaking and static conditions. In the case of regular shaking, AF-MNPs were added to E. coli suspension (PBS, 10 mM, pH 7.0, OD600 about 1.4) to make a final concentration of 1.0 mg mL-1 AF-MNPs. The resulting suspension was shaken on a rotary shaker at 250 rpm for different time periods. Bacterial clustering happened
TABLE 1. Comparison of the Present Method with Other Methods for Capturing Bacteria by MNPs MNPs
amount of MNPs
incubation time (min)
capture efficiency (%)
detection method
ref
plate-counting method UV-vis spectrophotometer mid-IR spectrometers
this work
0.5 mg mL-1
10
93.8 (E. coli, S. lutea)
1.0 mg mL-1
1
97 (E. coli)
1.8 mg mL-1 for spinach 0.04 mg mL-1 for 2% milk 0.57 mg mL-1
15-30
-
1.5
95 (E. coli)
vancomycin-immobilized MNPs magnetic glyco-nanoparticle FePt-Van nanoparticles
1.41 µg mL-1 (3.47 × 1011 nanoparticles) 2 mg mL-1
60
-
45 5 10
88 (E. coli) 65 (E. coli) -
vancomycin-modified MNPs
0.02-0.2 µg mL-1 (1011-1012 nanoparticles)
60
83 (E. coli)
amine-functionlized MNPs modified by anti-E.coli O157:H7MAbs (mouse) carboxyl-modified MNPs
11 µg mL
-1
5
15
immediately and the suspended bacteria flocculated within seconds of being mixed with AF-MNPs. After magnetic separation, the supernatant was then carefully pipetted into a cell to measure its OD600 using a UV-vis-NIR spectrophotometer. Figure S4A shows that the concentration of E. coli in the supernatant solution quickly decreases with increase of incubation time; even in 5 s of incubation, bacterial OD600 drops by more than 90%. The adsorption reaches equilibrium in only 30 s of incubation in contrast to dozens of minutes of incubation needed for other methods (19, 25). The capture kinetics of AF-MNPs for other bacteria is also fast under the same condition. In the case of P. aeruginosa or S. lutea, adsorption equilibrium is to attain only within 5 s. In the case of static incubation, the resulting suspension (AF-MNPs and E. coli) was allowed to settle for a specific period, and after magnetic separation the OD600 nm of its supernatant was measured. As shown in Figure S4B, equilibrium was reached within 20 min in such static state, and the capture efficiency was 96%. The magnetic capture of the bacteria by AF-MNPs in static state was also rapid and efficient, so AF-MNPs-based system is feasible for removing the bacteria from contaminated water, foods, and culture media. The AF-MNPs have high capture efficiency and fast kinetics mainly because the sizes of AF-MNPs are typically about 2 orders of magnitude smaller than a bacterium, which allows the attachment of multiple AF-MNPs onto a bacterial cell, rendering easy magnet-mediated separation. Moreover, their high surface-to-volume ratio, fast movement, and good dispersibility result in a faster binding rate between the AFMNPs and bacterial cells (23-25, 39). The more important reason is that flocculation of bacteria and AF-MNPs is formed by charge neutralization, facilitating magnetic separation immensely (33). Factors Affecting Magnetic Capture of Bacteria by AFMNPs. Factors such as the amount of AF-MNPs, and the concentration and pH of PBS for the adsorption of the bacteria were investigated using S. lutea, S. aureus, and E. coli, P. aeruginosa as Gram-positive and Gram-negative bacteria representative microorganisms. To determine the effect of the amount of AF-MNPs on capture efficiency, magnetic capture was performed in PBS (10 mM, pH 7.0) by adding various amounts of AF-MNPs to cell suspensions with an OD600 of 0.5. After incubation of AF-MNPs with bacterial solutions for 1 min, a magnetic field was applied to separate AF-MNPs/bacteria aggregates. The capture efficiency rapidly increased as the concentration of AF-MNPs increased up to 0.1 mg mL-1 for S. lutea, E. coli,
UV-vis spectrophotometer MALDI-MS fluorescent microscope microscopic analysis plate-counting method
9 11 19 23 25
and P. aeruginosa, and to 0.3 mg mL-1 for S. aureus, then leveled off with further increase in the concentration of AFMNPs (Figure S5). The binding capacity of the AF-MNPs toward E. coli estimated from a count of the bacterial growth on the LB agar plate is about 4.0 × 109 cfu mg-1. Since the capture of the bacteria by AF-MNPs is mainly driven by electrostatic interaction, the ionic strength and pH have important effects on the capture of pathogens. The effect of ionic strength on bacteria capture was studied in a broad range of PBS concentrations (Figure S6A). Cell suspensions were incubated with AF-MNPs (0.5 mg mL-1) in PBS in a concentration range of 5-200 mM. Although the concentration of PBS had little effect on the capture efficiency of S. lutea, it does significantly influence those of S. aureus, E. coli, and P. aeruginosa. The concentration of PBS for effective bacteria capture was in the range of 5-10 mM for S. aureus, 5-30 mM for E. coli, 5-50 mM for P. aeruginosa, and 5-150 mM for S. lutea. Excessive PBS led to a significant decrease in capture efficiency for S. aureus, E. coli, and P. aeruginosa likely due to the charge neutralization on the surface of the AF-MNPs or bacteria by excessive PBS. To further determine the effect of ionic strength on capture efficiency, Na2SO4 was added to adjust ionic strength and the concentration of Na2SO4 in 10 mM phosphate buffer (pH 7) was varied (Figure S6B). An increase of electrolyte concentration resulted in an obvious descrease in capture efficiency for E. coli, S. aureus and P. aeruginosa and S.lutea as a result of decreased electrostatic interaction in high ionic strength, which was proved by zeta potential change from +7.66 mV in 10 mM PBS alone to -0.192 mV in 10 mM PBS containing 300 mM Na2SO4. However, there was no significant change in the capture efficiency of S. lutea in the Na2SO4 concentration range of 0-300 mM, and for B. Cereus decrease in the Na2SO4 concentration range of 100-500 mM was observed. In these cases, hydrophobic interaction likely mediates adhesion as some bacterial cells surfaces show hydrophobic properties (36, 40, 41). The effect of pH on the capture efficiency of S. lutea, S. aureus, E. coli, and P. aeruginosa was studied in PBS from pH 2.0 to 11.0 (Figure S7). No pH effect on the capture of S. lutea and E. coli was observed in the studied pH range. The optimal pH ranged from 4.0 to 11.0 for P. aeruginosa, and from 5.0 to 8.0 for S. aureus. The results were generally in agreement with the speculation that the capture of the bacteria by AF-MNPs is driven by electrostatic force. The isoelectric point (pI) of AF-MNPs is above 9.0, and when the solution pH value is below the pI of AF-MNPs, the AFMNPs have a net positive surface charge, which favors VOL. 44, NO. 20, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7911
interaction with the bacteria that have a negative charge on its surface at pH > 4.5. The AF-MNPs are applicable as a coagulant and capture agent for the bacteria in a wide pH (5.0-8.0) range. Comparison of the Present AF-MNPs Based Method with Other Methods for Bacteria Capture. A comparison of the present method with other methods for capturing bacteria by functional MNPs in terms of amount of MNPs, capture efficiency, and incubation time is made in Table 1, revealing the advantages of the present method, such as no need for further modification with biomolecules, shorter incubation time, and high capture efficiency. Removal of Bacteria in Real Samples. We employed a microbial plate count method (25) to further investigate the trapping capability of the AF-MNPs toward Gram-positive and Gram-negative bacteria in water, beverage, and urine samples. Anionic silica coated MNPs were used as control. AF-MNPs were allowed to interact with their target bacteria for 10 min. The resulting conjugates were then separated and diluted, and cultured on a LB plate for 24 h at 37 °C. The results of the magnetic capture assays for AF-MNPs and silica coated MNPs are shown in Figure S8. AF-MNPs show efficient capture capability in water, beverage, and urine samples. Bacterial cells are removed by AF-MNPs with an efficiency of 88.5-99.1% in the studied water, beverage and urine samples. As the control, silica coated MNPs show much less efficiency than AF-MNPs for bacteria removal. In conclusion, we have reported a simple method for rapid and efficient capture of bacterial pathogens based on AF-MNPs. Although amino-group offers less specificity/ selectivity than antibodies, AF-MNPs are attractive for capturing a wide range of bacteria.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grants 20935001, 21077057), and the Tianjin Natural Science Foundation (Grants 10JCZDJC16300, 09JCYBJC14200).
Supporting Information Available Additional figures as described in the text (Figures S1-S8). This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) Yang, H.; Li, H.-P.; Jiang, X.-P. Detection of foodborne pathogens using bioconjugated nanomaterials. Microfluid. Nanofluid. 2008, 5 (5), 571–583. (2) Nayak, M.; Kotian, A.; Marathe, S.; Chakravortty, D. Detection of microorganisms using biosensors-A smarter way towards detection techniques. Biosens. Bioelectron. 2009, 25 (4), 661– 667. (3) Torres-Chavolla, E.; Alocilja, E. C. Aptasensors for detection of microbial and viral pathogens. Biosens. Bioelectron. 2009, 24 (11), 3175–3182. (4) Kaittanis, C.; Naser, S. A.; Perez, J. M. One-step, nanoparticlemediated bacterial detection with magnetic relaxation. Nano Lett. 2007, 7 (2), 380–383. (5) Ravindranath, S. P.; Mauer, L. J.; Deb-Roy, C.; Irudayaraj, J. Biofunctionalized magnetic nanoparticle integrated midinfrared pathogen sensor for food matrixes. Anal. Chem. 2009, 81 (8), 2840–2846. (6) Zhao, Y.; Ye, M.-Q.; Chao, Q.-G.; Jia, N.-Q.; Ge, Y.; Shen, H.-B. Simultaneous detection of multifood-borne pathogenic bacteria based on functionalized quantum dots coupled with immunomagnetic separation in food samples. J. Agric. Food. Chem. 2009, 57 (2), 517–524. (7) Wang, L.; Zhao, W.-J.; O’Donoghue, M. B.; Tan, W.-H. Fluorescent nanoparticles for multiplexed bacteria monitoring. Bioconjugate Chem. 2007, 18 (2), 297–301. (8) Gao, J.-H.; Gu, H.-W.; Xu, B. Multifunctional magnetic nanoparticles: Design, synthesis, and biomedical applications. Acc. Chem. Res. 2009, 42 (8), 1097–1107. 7912
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 20, 2010
(9) Shan, Z.; Wu, Q.; Wang, X.-X.; Zhou, Z.-W.; Oakes, K. D.; Zhang, X.; Huang, Q.-M.; Yang, W.-S. Bacteria capture, lysate clearance, and plasmid DNA extraction using pH-sensitive multifunctional magnetic nanoparticles. Anal. Biochem. 2010, 398 (1), 120–122. (10) Yitzhaki, S.; Zahavy, E.; Oron, C.; Fisher, M.; Keysary, A. Concentration of Bacillus spores by using silica magnetic particles. Anal. Chem. 2006, 78 (18), 6670–6673. (11) Lin, Y.-S.; Tsai, P.-J.; Weng, M.-F.; Chen, Y.-C. Affinity capture using vancomycin-bound magnetic nanoparticles for the MALDI-MS analysis of bacteria. Anal. Chem. 2005, 77 (6), 1753– 1760. (12) Chen, W.-J.; Tsai, P.-J.; Chen, Y.-C. Functional nanoparticlebased proteomic strategies for characterization of pathogenic bacteria. Anal. Chem. 2008, 80 (24), 9612–9621. (13) Kell, A. J.; Somaskandan, K.; Stewart, G.; Bergeron, M. G.; Simard, B. Superparamagnetic nanoparticle-polystyrene bead conjugates as pathogen capture mimics: A parametric study of factors affecting capture efficiency and specificity. Langmuir 2008, 24 (7), 3493–3502. (14) Bromberg, L.; Raduyk, S.; Hatton, T. A. Functional magnetic nanoparticles for biodefense and biological threat monitoring and surveillance. Anal. Chem. 2009, 81 (14), 5637–5645. (15) Zhao, X.-J.; Hilliard, L. R.; Mechery, S. J.; Wang, Y.-P.; Bagwe, R. P.; Jin, S.-G.; Tan, W.-H. A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles. Proc. Natl. Acad. Sci. U S A. 2004, 101 (42), 15027–15032. (16) Ryan, S.; Kell, A. J.; van Faassen, H.; Tay, L.-L.; Simard, B.; MacKenzie, R.; Gilbert, M.; Tanha, J. Single-domain antibodynanoparticles: Promising architectures for increased Staphylococcus aureus detection specificity and sensitivity. Bioconjugate Chem. 2009, 20 (10), 1966–1974. (17) Ho, K.-C.; Tsai, P.-J.; Lin, Y.-S.; Chen, Y.-C. Using biofunctionalized nanoparticles to probe pathogenic bacteria. Anal. Chem. 2004, 76 (24), 7162–7168. (18) Liu, J.-C.; Tsai, P.-J.; Lee, Y. C.; Chen, Y.-C. Affinity capture of uropathogenic Escherichia coli using pigeon ovalbumin-bound Fe3O4@Al2O3 magnetic nanoparticles. Anal. Chem. 2008, 80 (14), 5425–5432. (19) El-Boubbou, K.; Gruden, C.; Huang, X.-F. Magnetic glyconanoparticles: A unique tool for rapid pathogen detection, decontamination, and strain differentiation. J. Am. Chem. Soc. 2007, 129 (44), 13392–13393. (20) Lin, C.-C.; Yeh, Y.-C.; Yang, C.-Y.; Chen, C.-L.; Chen, G.-F.; Chen, C.-C.; Wu, Y.-C. Selective binding of mannose-encapsulated gold nanoparticles to type 1 pili in Escherichia coli. J. Am. Chem. Soc. 2002, 124 (14), 3508–3509. (21) Liu, L.-H.; Dietsch, H.; Schurtenberger, P.; Yan, M.-D. Photoinitiated coupling of unmodified monosaccharides to iron oxide nanoparticles for sensing proteins and bacteria. Bioconjugate Chem. 2009, 20 (7), 1349–1355. (22) Honda, H.; Kawabe, A.; Shinkai, M.; Kobayashi, T. Development of chitosan-conjugated magnetite for magnetic cell separation. J. Ferment. Bioeng. 1998, 86 (2), 191–196. (23) Gu, H.-W.; Ho, P.-L.; Tsang, K. W. T.; Wang, L.; Xu, B. Using biofunctional magnetic nanoparticles to capture vancomycinresistant enterococci and other gram-positive bacteria at ultralow concentration. J. Am. Chem. Soc. 2003, 125 (51), 15702– 15703. (24) Gu, H.-W.; Ho, P.-L.; Tsang, K. W. T.; Yu, C.-W.; Xu, B. Using biofunctional magnetic nanoparticles to capture Gram-negative bacteria at an ultra-low concentration. Chem. Commun. 2003, (15), 1966–1967. (25) Kell, A. J.; Stewart, G.; Ryan, S.; Peytavi, R.; Boissinot, M.; Huletsky, A.; Bergeron, M. G.; Simard, B. Vancomycin-modified nanoparticles for efficient targeting and preconcentration of Grampositive and Gram-negative bacteria. ACS Nano 2008, 2 (9), 1777–1788. (26) He, Y. P.; Wang, S. Q.; Li, C. R.; Miao, Y. M.; Wu, Z. Y.; Zou, B. S. Synthesis and characterization of functionalized silica-coated Fe3O4 superparamagnetic nanocrystals for biological applications. J. Phys. D: Appl. Phys. 2005, 38 (9), 1342–1350. (27) Liu, Q.-X.; Xu, Z.-H.; Finch, J. A.; Egerton, R. A Novel two-step silica-coating process for engineering magnetic nanocomposites. Chem. Mater. 1998, 10 (12), 3936–3940. (28) Lu, Y.; Yin, Y.-D.; Mayers, B. T.; Xia, Y.-N. Modifying the surface properties of superparamagnetic iron oxide nanoparticles through a sol-gel approach. Nano Lett. 2002, 2 (3), 183–186. (29) Zhang, Z.-C.; Zhang, L.-M.; Chen, L.; Chen, L.-G.; Wan, Q.-H. Synthesis of novel porous magnetic silica microspheres as adsorbents for isolation of genomic DNA. Biotechnol. Prog. 2006, 22 (2), 514–518.
(30) Takafuji, M.; Ide, S.; Ihara, H.; Xu, Z.-H. Preparation of poly (1-vinylimidazole)-grafted magnetic nanoparticles and their application for removal of metal Ions. Chem. Mater. 2004, 16 (10), 1977–1983. (31) Koh, I.; Wang, X.; Varughese, B.; Isaacs, L.; Ehrman, S. H.; English, D. S. Magnetic iron oxide nanoparticles for biorecognition: Evaluation of surface coverage and activity. J. Phys. Chem. B 2006, 110 (4), 1553–1558. (32) Chong, A. S. M.; Zhao, X. S. Functionalization of SBA-15 with APTES and characterization of functionalized materials. J. Phys. Chem. B 2003, 107 (46), 12650–12657. (33) Luan, Z.-H.; Fournier, J. A.; Wooten, J. B.; Miser, D. E. Preparation and characterization of (3-aminopropyl) triethoxysilane-modified mesoporous SBA-15 silica molecular sieves. Microporous Mesoporous Mater. 2005, 83 (1-3), 150–158. (34) Larsen, M. U.; Seward, M.; Tripathi, A.; Shapley, N. C. Biocompatible nanoparticles trigger rapid bacteria clustering. Biotechnol. Prog. 2009, 25 (4), 1094–1102. (35) Corr, S. A.; Rakovich, Y. P.; Gun’ko, Y. K. Multifunctional magnetic-fluorescent nanocomposites for biomedical applications. Nanoscale Res. Lett. 2008, 3 (3), 87–104.
(36) Dickson, J. S.; Koohmaraie, M. Cell surface charge characteristics and their relationship to bacterial attachment to meat surfaces. Appl. Environ. Microbiol. 1989, 55 (4), 832–836. (37) Wilson, W. W.; Wade, M. M.; Holman, S. C.; Champlin, F. R. Status of methods for assessing bacterial cell surface charge properties based on zeta potential measurements. J. Microbiol. Methods 2001, 43 (3), 153–164. (38) Horka´, M.; Ru ˚ zˇicˇka, F.; Hola´, V.; Sˇlais, K. Capillary isoelectric focusing of microorganisms in the pH range 2-5 in a dynamically modified FS capillary with UV detection. Anal. Bioanal. Chem. 2006, 385 (5), 840–846. (39) Gu, H.-W.; Xu, K.-M.; Xu, C.-J.; Xu, B. Biofunctional magnetic nanoparticles for protein separation and pathogen detection. Chem. Commun. 2006, (9), 941–949. (40) van Loosdrecht, M. C. M.; Lyklema, J.; Norde, W.; Schraa, G.; Zehnder, A. J. B. The role of bacterial cell wall hydrophobicity in adhesion. Appl. Environ. Microbiol. 1987, 53 (8), 1893–1897. (41) Vadillo-Rodríguez, V.; Busscher, H. J.; van der Mei, H. C.; de Vries, J.; Norde, W. Role of lactobacillus cell surface hydrophobicity as probed by AFM in adhesion to surfaces at low and high ionic strength. Colloids Surf. B 2005, 41 (1), 33–41.
ES102285N
VOL. 44, NO. 20, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7913
