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Abstract

Pathogenic contamination and resistant bacterial infections remain critical concerns in both developed and developing countries. Rapid and sensitive detection of pathogens is still a key requirement for both environmental and clinical settings. This article introduces a simple, colorimetric, cost-effective, and high-throughput system based on a positively charged iron oxide/enzyme complex for the detection of both gram-positive and gram-negative bacteria in water between 10³ and 10⁸ cfu/mL. This study provides an effective strategy for the identification and purification of pathogen contamination in drinking water.

Keywords

magnetic nanoparticles bacterial detection colorimetric assay

Introduction

Exposure to bacteria in drinking water is a serious public health concern. The World Health Organization estimates 502,000 diarrheal deaths induced by drinking contaminated water each year.¹ Any technology for rapid and ultrasensitive detection of pathogenic bacteria is highly desired for food and water safety. Advances in nanotechnology have allowed researchers to develop various strategies for rapid and sensitive bacteria detection in water. They have a similar sensitivity to the current technologies, like PCR,² plate counting, and enzyme-linked immunosorbent assay (ELISA).³ For example, Rotello’s group reported a colorimetric method based on gold nanoparticles (Au NPs) and β-galactosidase.^4–6 The activity of β-galactosidase was initially inhibited after precomplexation with Au NPs and recovered after being substituted by bacteria. The reactivated β-galactosidase allowed the quantitative detection of bacteria in both solution and strip paper with the detection limit of 100 cfu/mL. Based on this similar mechanism, other studies replaced Au NPs with ethylenediamine (EDA)-modified graphene oxide (GO), which could achieve the detection of Escherichia coli (E. coli) at concentrations as low as 100 cfu/mL and Staphylococcus aureus (S. aureus) at 200 cfu/mL.⁷

Due to their unique magnetic properties, magnetic nanoparticles (MNPs) are also widely explored for bacteria separation and detection.^8,9 For example, Nugen et al. prepared bacteriophage-tagged MNPs for the specific recognition and separation of E. coli.¹⁰ Lee et al. developed magneto-DNA probes capable of rapid and specific profiling of bacterial 16S rRNAs using a miniaturized nuclear magnetic resonance (NMR) device.¹¹ In general, MNPs act as either concentrating affinity probes to enrich bacteria or magnetic resonance contrast agents. However, existing methods required either an additional step with another agent for the bacteria quantification or fancy NMR equipment for the magnetic resonance detection.

This article aims to develop a strategy that combines the features of β-galactosidase and MNP. This strategy can achieve the simultaneous separation and quantification of bacteria without the need for additional agent or equipment. Briefly, a β-galactosidase/MNP complex is developed to enrich and remove the bacteria ( Fig. 1 ). The binding of bacteria on the complex proportionally detaches the β-galactosidase from MNPs, and the freed β-galactosidase can be quantified with colorimetric assay. Due to the high sensitivity of β-galactosidase, we demonstrate the detection of gram-negative E. coli and gram-positive S. aureus with the limit of 1000 cfu/mL in 2 h. In addition, the bacteria were removed from the water samples by applying magnetic field because of the complexation with MNPs.

Figure 1.

Schematic illustration of the synthesis (top) and working mechanism (bottom) of the β-galactosidase/MNP complex (β-Gal@Fe₃O₄-PEI NPs).

Materials and Methods

All materials except those mentioned specifically were obtained from Sigma-Aldrich (Singapore). Luria-Bertani (LB) broth and LB agar were bought from Becton, Dickinson and Company (Franklin Lakes, NJ). Chloroform (CHCl₃) was purchased from TEDIA (Fairfield, OH). E. coli ATCC 25922 and S. aureus ATCC 6538 were from ATCC (Manassas, VA).

Instruments for the Particle Characterization

The hydrodynamic diameter and zeta potential were examined with the ZS Particle Sizer (Malvern Instruments, Malvern, UK). FTIR spectra were acquired with a Fourier-transform infrared spectroscopy–attenuated total reflection (FTIR-ATR) spectrometer. Particle morphology was examined using a transmission electron microscope (TEM, JEOL 2010) operated at 200 kV.

Bacterial Culturing

E. coli and S. aureus were cultured overnight by transferring a colony from the agar plates to culture tubes containing sterilized LB broth medium. Then small aliquots were taken from the tubes and transferred to 10 mL of fresh LB medium to further culture for 4 h. Then obtained bacteria were washed and diluted with 5 mM phosphate buffer (pH 7.4)⁵ for experiments.

Plate Counting

Bacterial solution (~100 µL) was uniformly sprayed on the surface of LB agar and cultured in a 37 °C incubator. After 24 h of culture, colonies were counted to derive the initial concentration. All the samples were measured in triplicate.

Synthesis and Modification of Fe₃O₄ MNPs

Ten-nanometer Fe₃O₄ MNPs were synthesized by the reduction of Fe(acac)₃ in oleyl amine solvent at 300 °C for 2 h.¹² Modification of Fe₃O₄ MNPs with branched polyethylenimine (PEI, 25 kDa) was achieved by adding 1 mL of Fe₃O₄ MNP solution (1 mg/mL in chloroform) into 1 mL of CHCl₃ containing 3 mg of PEI under stirring. Solution was then dried under a nitrogen flow to obtain the Fe₃O₄/PEI film, which was dispersed in 0.7 mL of sterile deionized water to get a final concentration of 1 mg/mL. The Fe₃O₄/PEI complex was separated from free PEI through high-speed centrifugation and redispersed in 5 mM phosphate buffer (pH 7.4). The Fe concentration of the Fe₃O₄/PEI complex was quantified through inductively coupled plasma mass spectrometry (ICP-MS, Elan DRC-e, PerkinElmer, Waltham, MA).

Synthesis of Fe₃O₄/PEI/Enzyme Complex

Fe₃O₄/PEI MNPs were coupled to β-galactosidase via the electrostatic interaction. In brief, 1500 µL of Fe₃O₄/PEI solution with a concentration of 1 mg/mL was mixed with 50 µL of enzyme (1 mg/mL) in 5 mM phosphate buffer and incubated for 15 min at room temperature. The free enzyme in supernatant was removed by magnetic separation and subsequently quantified using a chlorophenolred-β-d-galactopyranoside (CPRG) assay.^4,13 The quantification of free enzymes allowed the determination of the mass ratio between bound enzymes and Fe₃O₄ MNPs.

Bacterial Enrichment and Quantification with Fe₃O₄/PEI/Enzyme Complex in Known Solutions

The Fe₃O₄/PEI/enzyme complex was incubated with known solutions containing various concentrations of bacteria ranging from 10² to 10⁷ cfu/mL for 1 h. During the incubation, gentle shaking (80 rpm) was applied to accelerate the replacement process. Supernatant containing free enzyme molecules was later separated from the MNP complex under the magnet, and free enzyme molecules were quantified by CPRG assay, recording the absorbance at 574 nm after reaction with 500 µM CPRG.

Bacterial Enrichment and Quantification with Fe₃O₄/PEI/Enzyme Complex in Unknown Solutions

The study was performed in a single-blinded manner, with the preparation and analysis kept separate. Solutions containing various amounts of bacteria were performed by investigator 1, who was kept separate from the data acquisition workflow. Investigator 2 collected the raw data and performed analysis. Thereafter, the identity of the various groups was revealed by investigator 1 and matched with the experimental data. The sample process was the same as that with known solutions.

Results

Synthesis and Characterization of Fe₃O₄/PEI NPs

Fe₃O₄ NPs with a radius of 10 nm were synthesized using the traditional high-temperature deposition method ( Fig. 2a ). The as-synthesized NPs were hydrophobic and made hydrophilic through complexation with cationic branched PEI. The free PEI postsurface modification was removed through high-speed centrifugation. We optimized the mass ratio between PEI and MNPs during the synthesis of the Fe₃O₄@PEI complex. As shown in Supplemental Figure S1 , the highest zeta potential of Fe₃O₄@PEI NPs was achieved when the ratio of PEI/MNP was 2. However, considering the productivity, 3 mg/ml PEI was utilized which compromised both good dispersity and higher productivity. At this point, there was a layer of PEI that closely bound on the surface of MNPs. Thus, the density of charge was high. When there was more PEI, more layers of PEI were deposited on the particle surface, and the density of charge on the slipping plane of the NP/PEI complex and the zeta potential decreased.

Figure 2.

Characterization of Fe₃O₄/PEI NPs. TEM images of (a) as-synthesized Fe₃O₄/PEI NPs and (b) Fe₃O₄/PEI NPs. (c) Aqueous solution of Fe₃O₄/PEI NPs. (d) Fe₃O₄/PEI NPs separation in aqueous solution under magnet. (e) Hydrodynamic diameters of Fe₃O₄ NPs, Fe₃O₄/PEI NPs, and Fe₃O₄/PEI/β-galactosidase NPs. (f) FTIR spectra of Fe₃O₄ NPs and Fe₃O₄/PEI NPs.

The modified particles dispersed well in aqueous solution and could respond to the magnet attraction ( Fig. 2c , d ). There was no significant change of particle size after the PEI modification under TEM ( Fig. 2b ); however, the hydrodynamic diameter changed from 21 to 59 nm ( Fig. 2e ). The FTIR result reveals that there are strong absorptions from N-H stretch vibration (3300–3500 cm^–1) after the modification, which confirms the successful modification of NPs with PEI ( Fig. 2f ).

Modification of Fe₃O₄/PEI NPs with β-Galactosidase

The coating of cationic PEI provided a positive surface charge (i.e., +22 mV) for Fe₃O₄/PEI NPs in phosphate buffer, which allowed the complexation with β-galactosidase via electrostatic interactions. Fe₃O₄/PEI/β-galactosidase was magnetically separated with free β-galactosidase. After the complexation, the zeta potential dropped from +20 to +10 mV. The prepared Fe₃O₄ NPs and Fe₃O₄ NP/PEI and Fe₃O₄ NP/PEI/β-galactosidase complexes had a hydrodynamic diameter of about 21, 59, and 78 nm, respectively ( Fig. 2e ). The PEI coating increased the hydrodynamic diameter of Fe₃O₄ NPs from 21 to 59 nm. The further increase from 59 to 78 nm was due to the binding of the enzymes.

To calculate the loading capacity of enzymes on the complexes, we first generated a standard curve between CPRG concentrations and maximum reaction velocities (V_max) ( Suppl. Fig. S2a ). We found that V_max reached a plateau when the CPRG concentration was higher than 500 µM, so 500 µM CPRG was used to acquire the standard curve between enzyme concentrations and V_max ( Suppl. Fig. S2b ),^14–16 which was later used to quantify the enzyme concentration in the complexes through measuring and fitting V_max of the solutions. We derived the concentration of β-galactosidase in Fe₃O₄ NP/PEI/β-galactosidase complexes to be 29 µg of enzymes per milligram of Fe₃O₄ NPs, or 21 enzyme molecules per particle.

Sensitivity of Fe₃O₄/PEI/β-Galactosidase Complexes in Bacterial Enrichment and Quantification

Fe₃O₄/PEI/β-galactosidase complexes were first used to confirm the already-known concentrations of gram-negative E. coli in a series of solutions. Briefly, solutions containing a range of bacterial concentrations were incubated with Fe₃O₄/PEI/β-galactosidase complexes. Enzymes on the complexes were proportionally replaced by E. coli due to electronic interaction between the bacterial membrane and Fe₃O₄/PEI/β-galactosidase NPs. The higher the concentration of bacteria was, the more enzymes that were freed. The enzymes detached from the complexes were quantified by the CPRG assay. Specifically, the supernatant containing detached β-galactosidase was mixed with CPRG and the dynamic change of absorbance at 574 nm was monitored ( Fig. 3a and Suppl. Fig. S3 ), from which V_max (i.e., initial reaction velocity) was derived and plotted against the bacterial concentrations ( Fig. 3b ). Here, V_max is the initial reaction velocity (the first 5 min), which was positively correlated with the initial absorbance changes at 574 nm. Results in Figure 3a , b were normalized against the highest absorbance and maximum reaction rate, respectively. The detection limit of this strategy is 10³ cells/mL. Solutions containing less than 10³ cells/mL did not produce sufficient signals. The bacteria left in the supernatant were quantified through traditional plate counting ( Fig. 3c ), which reveals the high enrichment efficiency of this methodology.

Figure 3.

Fe₃O₄/PEI/β-galactosidase complexes for the enrichment and analysis of E. coli in samples with known concentrations. (a) CPRG assay with the supernatants of E. coli solutions after incubation with Fe₃O₄/PEI/β-galactosidase complexes. (b) Correlation curve between the maximum reaction rates derived in panel a and the E. coli concentrations in the solutions. (c) Enrichment efficiency of Fe₃O₄/PEI/β-galactosidase complexes.

We further looked into the quantification of gram-positive S. aureus using a similar protocol to understand the sensitivity of this system ( Fig. 4a and Suppl. Fig. S4 ). V_max and the corresponding standard curve were derived as well ( Fig. 4b ). All the results in Figure 4a were normalized against the highest and lowest absorbances in Figure 3a , while Figure 4b was normalized against the maximum and minimum reaction rates in Figure 3b . The enrichment efficiency was similar to that in the E. coli assay ( Fig. 4c ).

Figure 4.

Fe₃O₄/PEI/β-galactosidase complexes for the enrichment and analysis of S. aureus in samples with known concentrations. (a) CPRG assay with the supernatants of S. aureus solutions after incubation with Fe₃O₄/PEI/β-galactosidase complexes. (b) Correlation curve between the maximum reaction rates derived in panel a and S. aureus concentrations in the solutions. (c) Enrichment efficiency of Fe₃O₄/PEI/β-galactosidase complexes.

To better understand the different performances of Fe₃O₄/PEI/β-galactosidase NPs between gram-positive and gram-negative bacteria, we studied the surface charge of two kinds of bacteria ( Fig. 5 ). EC-exp and SA-exp represent the surface charges of E. coli and S. aureus, respectively, at the exponential phase, while EC-sta and SA-sta represent the charges of E. coli and S. aureus, respectively, at the stationary phase. The surface charge of E. coli was around 10 mV lower than that of S. aureus for both exponential and stationary phases.

Figure 5.

Zeta potentials of E. coli and S. aureus (exponential phase and stationary phase).

Fe₃O₄/PEI/β-Galactosidase NPs for the Enrichment and Quantification of E. coli and S. aureus in Unknown Solutions

The standard curves derived above allowed us to predict the bacterial concentration in an unknown sample ( Fig. 6 ). To evaluate the efficacy of this system, a blind assay was conducted. Briefly, E. coli and S. aureus were cultured for 7–9 h randomly with different numbers of inoculation colonies. This step was carried out by one person, samples were labeled only by numbers, and the corresponding sample names were recorded at the same time. Then the measurements were blindly conducted by another person ( Table 1 ).

Figure 6.

Fe₃O₄/PEI/β-galactosidase complexes for the enrichment and analysis of bacteria in samples with unknown concentrations. (a) Kinetic absorbance intensity at the wavelength of 574 nm for various samples. (b) Maximum reaction rates of various samples derived from panel a.

Table 1.

Bacteria Samples Used in the Blind Assay and the Quantification from Both Plate Counting and the Fe₃O₄/PEI/β-Galactosidase Complex Assay.

Number	Sample	Concentration Derived from Plate Counting (cells/mL)	Concentration Derived from Fe₃O₄/PEI/β-Galactosidase Complex Assay
6	E. coli	(3.27 ± 0.84) × 10⁴	(4.21 ± 0.08) × 10⁴
2	E. coli	(3.27 ± 0.84) × 10⁸	(2.71 ± 0.52) × 10⁸
7	S. aureus	(7.93 ± 0.6) × 10³	(8.89 ± 0.11) × 10³
5	S. aureus	(7.93 ± 0.6) × 10⁴	(7.75 ± 0.18) × 10⁴
4	S. aureus	(7.93 ± 0.6) × 10⁶	(8.36 ± 0.16) × 10⁶
3	S. aureus	(7.93 ± 0.6) × 10⁸	(8.51 ± 0.23) × 10⁸
1	Phosphate buffer	0	78 ± 15

As shown in Figure 6a , the control group had the lowest absorbance at 574 nm, while samples 2–3 showed the highest absorbance. The derived V_max confirmed this observation ( Fig. 6b ). Samples 6–7 ended up with similar absorbance and V_max to the control. V_max values were brought back to the standard curves in Figures 3b and 4b for the calculation of bacterial concentrations. Simultaneously, the concentrations were examined with the standard plate counting. As shown in Table 1 , results from two kinds of assays matched well for all samples except sample 2 (E. coli at the highest concentration, 10⁷ cfu/mL).

Discussion

Fe₃O₄/PEI/β-galactosidase complexes were constructed with oleic acid-coated Fe₃O₄ NPs, 25 kDa branched PEI, and β-galactosidase. Fe₃O₄ NPs synthesized through thermal decomposition with oleic acid coating are well known for their uniform size and superior magnetic properties.¹⁷ PEI is also widely known for its capability to bring the hydrophobic Fe₃O₄ NPs to aqueous solution.^18,19 We compared the effectiveness of branched PEI with molecular weights of 0.8, 25, and 50–100 kDa ( Suppl. Fig. S5 ). NPs modified with 0.8 kDa PEI aggregated, which suggests that the 0.8 kDa PEI coating did not provide sufficient steric and/or electrostatic stabilization. The 50–100 kDa PEI did work, but the efficiency was very low.

The anionic enzyme (i.e., β-galactosidase) was then easily complexed with the Fe₃O₄/PEI NPs in solution. Similar to other systems reported,⁵ positively charged Fe₃O₄/PEI NPs bound with β-galactosidase at the negatively charged residues around its active site through electrostatic interactions, resulting in the inhibition of enzymatic activity ( Suppl. Fig. S6 ). The higher the ratio between NPs and enzyme, the more the inhibition. When in the presence of analyte bacteria, competitive binding of the anionic surface of the bacteria to the positively charged Fe₃O₄/PEI NPs released the enzymes, with concomitant restoration of their activity. The active enzymes converted the yellow substrate into the purple or red product, providing an amplified colorimetric readout ( Suppl. Fig. S7 ).

We examined the detection of two kinds of bacteria: gram-negative E. coli and gram-positive S. aureus. First, we generated a standard curve with the known concentrations of both bacteria ( Figs. 3b and 4b ). However, we realize that the sensitivity of this methodology is lower than the reported results from the Au NP/β-galactosidase and EDA-GO/β-galactosidase complexes, both of which allowed the detection of E. coli at concentrations as low as 100 cfu/mL. In comparison, Fe₃O₄/PEI/β-galactosidase complexes can provide a trustable readout when the bacterial concentration is above 1000 cfu/mL. This should be due to the strong absorption of Fe₃O₄ NPs at 574 nm, which presents a strong background and requires a higher concentration of CPRG metabolites. In addition, we noticed that there was aggregation of NPs when the bacterial concentration of E. coli was above 10⁸ cfu/mL. This suggests the disruption of the cationic PEI coating on the Fe₃O₄ NPs. In other words, the binding between cationic Fe₃O₄/PEI and anionic bacterial membrane destabilizes the PEI coating. With a higher bacteria versus Fe₃O₄/PEI ratio, the destruction becomes more severe. This is confirmed by the less severe aggregation of NPs in the S. aureus solution with the same 10⁸ cfu/mL concentration. S. aureus is less negatively charged than E. coli. ( Fig. 5 ), which means that more S. aureus is needed to achieve the same destabilization of the PEI coating.

Finally, we explored use of the derived standard curve to quantify the bacteria in unknown solutions ( Fig. 6 and Table 1 ). While the result obtained from the Fe₃O₄/PEI/β-galactosidase complexes is comparable to that from plate counting, we note that this method did not allow us to identify the type of bacteria. In other words, it only allows us to enrich and quantify a bacterium with the known species.

In summary, this article introduces an Fe₃O₄/PEI/β-galactosidase complex for the enrichment and quantification of both gram-negative and gram-positive bacteria. We can recurrently realize the quantification for bacteria between 10³ and 10⁸ cfu/mL. Despite the sensitivity needing further improvement, the method is universal for all kinds of bacteria, and the bacteria enriched can be used for further analysis. In the future, an antibody or aptamer could be modified onto the surface of NPs to further identify bacterial types in unknown samples.

Supplemental Material

Supplemental material for Detection of Bacteria in Water with β-Galactosidase-Coated Magnetic Nanoparticles

Supplemental material for Detection of Bacteria in Water with β-Galactosidase-Coated Magnetic Nanoparticles by Mingyue Cui, Hao Chang, Yang Zhong, Min Wang, Tianze Wu, Xiao Hu, Zhichuan J. Xu, and Chenjie Xu in SLAS Technology

Footnotes

Acknowledgements

We appreciate the support from Nanyang Environment & Water Research Institute, Nanyang Technological University.

Supplemental material is available online with this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

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Supplementary Material

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Detection of Bacteria in Water with β-Galactosidase-Coated Magnetic Nanoparticles

Abstract

Keywords

Introduction

Materials and Methods

Instruments for the Particle Characterization

Bacterial Culturing

Plate Counting

Synthesis and Modification of Fe3O4 MNPs

Synthesis of Fe3O4/PEI/Enzyme Complex

Bacterial Enrichment and Quantification with Fe3O4/PEI/Enzyme Complex in Known Solutions

Bacterial Enrichment and Quantification with Fe3O4/PEI/Enzyme Complex in Unknown Solutions

Results

Synthesis and Characterization of Fe3O4/PEI NPs

Modification of Fe3O4/PEI NPs with β-Galactosidase

Sensitivity of Fe3O4/PEI/β-Galactosidase Complexes in Bacterial Enrichment and Quantification

Fe3O4/PEI/β-Galactosidase NPs for the Enrichment and Quantification of E. coli and S. aureus in Unknown Solutions

Discussion

Supplemental Material

Supplemental material for Detection of Bacteria in Water with β-Galactosidase-Coated Magnetic Nanoparticles

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

References

Supplementary Material

Synthesis and Modification of Fe₃O₄ MNPs

Synthesis of Fe₃O₄/PEI/Enzyme Complex

Bacterial Enrichment and Quantification with Fe₃O₄/PEI/Enzyme Complex in Known Solutions

Bacterial Enrichment and Quantification with Fe₃O₄/PEI/Enzyme Complex in Unknown Solutions

Synthesis and Characterization of Fe₃O₄/PEI NPs

Modification of Fe₃O₄/PEI NPs with β-Galactosidase

Sensitivity of Fe₃O₄/PEI/β-Galactosidase Complexes in Bacterial Enrichment and Quantification

Fe₃O₄/PEI/β-Galactosidase NPs for the Enrichment and Quantification of E. coli and S. aureus in Unknown Solutions