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Efficient Enrichment and Analyses of Bacteria at Ultralow Concentration with Quick-Response Magnetic Nanospheres Cong-Ying Wen, Yong-Zhong Jiang, Xi-You Li, Man Tang, Ling-Ling Wu, Jiao Hu, Dai-Wen Pang, and Jing-Bin Zeng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16831 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on March 1, 2017

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ACS Applied Materials & Interfaces

Efficient Enrichment and Analyses of Bacteria at Ultralow Concentration with Quick-Response Magnetic Nanospheres Cong-Ying Wena,b,1, Yong-Zhong Jiangb,c,1, Xi-You Lia, Man Tangb, Ling-Ling Wub, Jiao Hub, Dai-Wen Pangb, Jing-Bin Zenga,∗ a

College of Science, China University of Petroleum (East China), Qingdao, 266580, P. R. China. b

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education),

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China. c

Hubei Provincial Center for Disease Control and Prevention, Wuhan 430072, P. R. China. 1

∗

These authors contributed equally to this work.

Corresponding author. Email: [email protected]. Fax: 0086-532-86983363.

ACS Paragon Plus Environment

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ABSTRACT: Enrichment and purification of bacteria from complex matrices are crucial for their detection and investigation, in which magnetic separation techniques have recently show great application advantages. However, currently used magnetic particles all have their own limitations: Magnetic microparticles exhibit poor binding capacity with targets, while magnetic nanoparticles suffer slow magnetic response and high loss rate during treatment process. Herein, we used a highly controllable layer-by-layer assembly method to fabricate quick-response magnetic nanospheres (MNs), and with Salmonella typhimurium as a model, we successfully achieve their rapid and efficient enrichment. The MNs combined the advantages of magnetic microparticles and nanoparticles. On the one hand, the MNs had a fast magnetic response, and almost 100% of the MNs could be recovered by 1 min attraction with a simple magnetic scaffold. Hence, using antibody conjugated MNs (immunomagnetic nanospheres, IMNs) to capture bacteria hardly generated loss and didn’t need complex separation tools or techniques. On the other hand, the IMNs showed much excellent capture capacity. With 20 min interaction, almost all of the target bacteria could be captured, and even only one bacterium existing in the samples wasn’t missed, comparing with the immunomagnetic microparticles which could only capture less than 50% of the bacteria. Besides, the IMNs could achieve the same efficient enrichment in complex matrices, such as milk, fetal bovine serum, and urine, demonstrating their good stability, strong anti-interference ability, and low nonspecific adsorption. What’s more, the isolated bacteria could be directly used for culture, polymerase chain reaction (PCR) analyses, and fluorescence immunoassay without a release process, which suggested our IMNs-based enrichment strategy could be conveniently coupled with the downstream identification and analysis techniques. Thus, the MNs provided by this work showed great superiority in bacteria enrichment, which would be a promising tool for bacteria detection and investigation.


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KEYWORDS: magnetic nanospheres · quick-response · bacteria · efficient capture · downstream analyses INTRODUCTION Foodborne illness has always been an important public health problem. The World Health Organization (WHO) reported that each year roughly 600 million people (almost 1 in 10 people in the world) got sick and 420 000 died of foodborne diseases, among which, children under 5 years of age carried 40% of the burden.1 The main causes lie in the infection of various pathogenic bacteria, especially Salmonella, Listeria monocytogenes, Campylobacter spp., and so on.1,

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Therefore, bacteria detection and related research, such as genotyping, pathogenesis

mechanism, and antimicrobial resistance, etc., have great significance for reducing foodborne illness. However, pathogens usually exist within complex food or biological matrices, and many of them show low infectious dose and high health risk (e.g. the doses for E. coli O157:H7 and Salmonella are as low as 10 cells.).3-5 Thus, efficient enrichment and purification of pathogens are highly demanded by many detection and analysis methods.6-8 For example, polymerase chain reaction (PCR) analyses often require intensive and careful pre-purification to eliminate the interference from the sample matrix, and efficient pre-concentration of the targets would definitely improve the sensitivity of enzyme-linked immunosorbent assays (ELISA).9, 10 In recent years, magnetic separation techniques have become a very promising tool for separation and enrichment, and the isolated objects can be from ions, small molecules or biomacromolecules, to viruses, bacteria, or cells.11-14 The basic principle is that magnetic particles coated with affinity tags are mixed with the mixture containing the targets, followed with a suitable incubation to allow the magnetic particles to bind to the targets, and then the


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obtained complexes are isolated by magnetic attraction to remove the contaminants, thereby achieving purification and enrichment.15 Magnetic separation gains wide acceptance mainly due to the fact that magnetic particles have controlled surfaces, versatile functionalization, good biocompatibility, and especially that they can be conveniently manipulated by a magnet which makes all the separation process accomplished in a tube without expensive liquid chromatography systems.16, 17 Besides, magnetic interaction is a non-contact force, which isn’t influenced by the variables such as pH, temperature, and concentration, et al.18 So far, both micro and nano magnetic particles have been used for separation and enrichment, which have their own advantages and limitations. In general, magnetic microparticles are more widely used than magnetic nanoparticles, mainly due to the fact that the formers have faster magnetic response and lower loss rate during the treatment process.19-22 Dynabeads are one of the most typical representatives of magnetic microparticles, which have come into market and been used in lots of scientific applications.23-25 While magnetic nanoparticles show a slow magnetic response and high loss during separation, despite of which, they still possess unique advantages greatly attracting researchers: their higher surface to volume ratio causes higher binding capacity; their faster binding kinetics enables rapid recognition; their negligible influence on the targets facilitates further analyses without a release process, etc.26-28 Thus, trials to use magnetic nanoparticles have always been continued.29-34 Usually, in practical applications, using magnetic nanoparticles requires stronger magnetic separation tools or more complex techniques.19 For instance, with the help of a high-gradient magnetic separation-column, the magnetic activated cell sorting technology (MACS-technology) developed by Miltenyi Biotech Corp., successfully utilized 50 nm magnetic nanoparticles for cell isolation.28, 35 And Cellsearch system was able to efficiently capture cells, attributing to the self-assemblies of the magnetic nanoparticles (120-200


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nm) which increased the number of the nanoparticles bound with the cells to achieve fast magnetic response.36,

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However, strong magnetic separation tools aren’t readily available in

most laboratories, and the methods based on magnetic nanoparticle aggregation introduce more complicated steps. Hence, it would be very helpful to develop a kind of nanosized particles with fast magnetic response, and their application to bacteria capture would combine the advantages of both magnetic microparticles and nanoparticles, improving the enrichment effect or simplifying the manipulation process. In our previous work, we developed a layer-by-layer (LBL) assembly method for constructing magnetic spheres.38 This method had much high controllability, and by tuning the coating layers of nano-γ-Fe2O3 particles, both the magnetic response and the size of the spheres could be tuned. Finally, magnetic nanosized spheres (MNs) with quick response, a compromise between size and response, were obtained, and had been used to capture tumor cells.37, 39, 40 On the previous basis, this work successfully utilized quick-response MNs to achieve rapid and efficient enrichment of Salmonella typhimurium (S. typhimurium, taken as a model bacteria) at ultralow concentrations. The main unique advantages of our MNs-based enrichment strategy could be listed as follows: (1) The MNs had a fast magnetic response, and almost 100% of the MNs could be recovered by 1 min attraction with a simple magnetic scaffold. Hence, using antibody conjugated MNs (immunomagnetic nanospheres, IMNs) to capture bacteria hardly generated loss and didn’t need complex tools or techniques. (2) In comparison with magnetic microparticles, the IMNs showed much more excellent capture capacity. The efficiency to capture S. typhimurium with the IMNs could reach 97%, while only 46% with 1.0-µm immunomicroparticles and 36% with 2.8-µm immunomicroparticles. Moreover, efficient recognition and separation could also be achieved even when the target bacteria were very rare (Only one bacterium existing in the samples could


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be definitely captured.). Besides, the binding between the bacteria and the IMNs was very fast, and 20 min incubation was enough for capturing almost all of the bacteria. (3) The IMNs had high stability, strong anti-interference ability and low nonspecific adsorption. Their direct application to the complex matrices, such as milk, fetal bovine serum (FBS), and urine, exhibited similar capture capacity with those in the ideal samples. (4) The IMNs’ convenient manipulation and negligible influence on the isolated bacteria facilitated the downstream coupling with various identification and analysis techniques: Without release process, the captured bacteria could be directly used for culture, PCR analyses, and fluorescence immunoassay. Therefore, we believed that our developed quick-response MNs possessed great superiority over the magnetic particles currently used in the bacteria enrichment, which would show high application value in bacteria detection and relative research. EXPERIMENTAL SECTION Reagents and Instruments Branched poly(ethylene imine) (PEI, MW 25 kDa and MW 750 kDa), polyvinylpyrrolidone (PVP-k30), tetraethyl orthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), bovine serum albumin (BSA), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and Cy3-labeled sheep anti-mouse IgG were purchased from Sigma-Aldrich. Mouse anti-S. typhimurium monoclonal antibody was obtained from Abcam. NH2-PEG-CM (MW 3400, and CM here refers to carboxymethyl) was purchased from Laysan Bio. Hoechst 33342, the primers for PCR, and carboxyl terminal magnetic spheres with 1.0-µm and 2.8-µm diameter were bought from Invitrogen Corp. Streptavidin conjugated CdSe/ZnS quantum dots (SA-QDs, emission wavelength: 605 nm) were bought from Wuhan Jiayuan Quantum Dots Co., Ltd. 2×Tag PCR master mix was got from Tiangen Biotech (Beijing) Co.,


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Ltd. S. typhimurium (ATCC: 14028) and Escherichia coli (E. coli, ATCC: 8099) were from Chinese Center for Disease Control and Prevention. Agar, tryptone, and yeast extract for preparing LB plate were obtained from Oxoid Ltd. Ultrapure water (18.2 MΩ·cm) was made by a Millipore Milli-Q system. Transmission electron microscopy (TEM) images were acquired by a FEI Tecnai G2 20 TWIN electron microscope. Magnetic hysteresis loops were obtained with a vibrating sample magnetometer (Lake Shore 7410 VSM). Fluorescence images were recorded with a CCD camera (Nikon digital sight DS-U3) mounted on an inverted fluorescence microscope (Ti-U, Nikon, Japan). Fluorescence emission spectra were measured with a fluorescence spectrometer (HORIBA JOBIN YVON). Magnetic separation was performed with the help of a magnetic scaffold (Invitrogen, 12320D, the field strength on the surface of the magnetic scaffold was 325 ± 25 mT). Fabrication and Biomodification of the MNs MNs were fabricated according to our previous work.38,

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Briefly, carboxyl-terminated

poly(styrene/acrylamide) copolymer nanospheres (prepared by emulsifier-free polymerization method) were firstly coated with a foundation layer of PEI (low MW 25 kDa) using carbodiimide chemistry. Then, hydrophobic nano-γ-Fe2O3 particles (synthesized by high-temperature pyrolysis) were assembled on the nanosphere surface in hexanol. The second layer was formed similarly: The above nanospheres were coated with another kind of PEI (high MW 750 kDa) in PBS, and then further attached with an additional layer of nano-γ-Fe2O3 in hexanol. After repeating this step four times, the obtained 5-layers-MNs were silanized with TOES in a seeded growth process, and by modification with APTES and succinic anhydride, MNs with carboxyl groups on their surfaces were finally obtained.


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For biomodification, typically, 5 mg of the MNs were washed with PBS (pH 6.8, 0.01 M) two times, and dispersed in 1 mL of PBS (pH 6.8, 0.01 M) containing 50 mM EDC and 50 mM NHS. The mixture was shaken gently at room temperature for activating the carboxyl groups on the nanosphere surface. After 0.5 h incubation, the nanospheres were washed with PBS (0.01 M pH 7.2) three times by magnetic separation, which were then dispersed in 1.0 mL of PBS (0.01 M pH 7.2) to react with 50 µg of anti-S. typhimurium monoclonal antibody with continuous shaking for about 4 h. Then, the resultant IMNs were washed with PBS to remove surplus antibody, and blocked with 1% BSA-PBS at 37 °C with gentle agitation for 30 min. Finally, the IMNs were stored in PBS (0.01 M pH 7.2) at 4 °C for use. Bacteria Culture The pure culture of S. typhimurium and E. coli was grown on the LB agar at 37 °C for 24 h. Colonies were picked and dispersed in sterile physiological saline (0.9% NaCl) to obtain bacteria suspension. The exact cell number of the bacteria suspension was determined with plate count: 50 µL of proper dilution of the sample were plated onto the LB agar, and after incubation at 37 °C for 24 h, the colonies grown on the plates were counted to calculated the bacteria concentration (the number of the colony forming units per milliliter of the bacteria suspension, CFU/mL). Considering biological safety, all experiments utilizing viable pathogens were done by trained personnel in a Biosafety level 2 laboratory. The bacteria were heat-inactive before they were used in ordinary laboratory. Capture of Target Bacteria Basically, a certain amount of IMNs were added to 1 mL of the sample (containing 1% BSA and 0.05% Tween 20), which were then shaken gently at 120 rpm at 37 °C. After incubation for


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some time, the bacteria combined with IMNs were isolated by a magnetic scaffold, and washed with physiological saline (containing 1% BSA and 0.05% Tween 20) three times for further treatment. For optimizing the dosage of IMNs, different concentrations of IMNs (0.06, 0.15, 0.30, 0.45, 0.60 mg/mL) were used to capture S. typhimurium (~105 CFU/mL). The numbers of the bacteria added (N0) and in the supernatant (Ns) were both determined, and the corresponding capture efficiencies were calculated by equation (1): capture efficiency = (N0 - Ns)/ N0×100%

(1)

where N0 and Ns are calculated with plate count (50 µL of proper dilution of the bacteria suspension added and the supernatant after magnetic separation were plated onto the LB agar for culture and counting colonies), and N0 - Ns is consistent to the number of the bacteria captured by IMNs. For optimizing incubation time, 0.30 mg/mL IMNs were used to capture S. typhimurium (~105 CFU/mL) with different incubation times (5, 10, 15, 20, 25, 30 min). Corresponding capture efficiencies were also calculated by equation (1). To investigate the specificity, IMNs were used to treat E. coli, and unmodified MNs were used to treat S. typhimurium. Further, samples with ultralow concentration of S. typhimurium (0~50 CFU/mL) were prepared: Typically, the colony of S. typhimurium was picked and dispersed in sterile physiological saline to obtain bacteria suspension with a concentration of about 108 CFU/mL according to the McFarland Standards, which was then serially diluted to obtain the rare bacteria samples. The IMNs were added to the above samples for capturing the rare target bacteria. After magnetic separation, the captured bacteria were resuspended in sterile physiological saline for plate count to determine their number (Nc). And all the supernatants were collected, which were plated to calculate the


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number of the uncaptured bacteria (Ns). Corresponding capture efficiencies were calculated by equation (2): capture efficiency = Nc/(Nc + Ns)×100%

(2)

where Nc + Ns is the total number of the bacteria employed in the experiments. As controls, MNs without antibody modified were used to treat rare S. typhimurium under the same condition. For capturing bacteria from synthetic samples, milk, FBS, and urine were mixed with S. typhimurium suspension (9:1 by volume), which were then directly added with IMNs for capture and isolation. It should be noted that, in the experiments involving bacteria culture, contamination of other bacteria must be avoided, for which, the solution, tubes, and other stuffs were all sterilized before use, and the plating was done in the biosafety cabinet. Fabrication of Immunomagnetic Microspheres for Bacteria Capture Magnetic microspheres (MMs, 1.0 µm and 2.8 µm) were employed to capture S. typhimurium. Two kinds of strategies were used for conjugating magnetic spheres (Ms, including MNs and MMs) with antibody. Strategy I: The carboxyl groups on Ms were directly conjugated with the amines of antibodies by carbodiimide chemistry, and the experimental procedures were basically the same with that of biomodifying MNs described above. Strategy II: NH2-PEG-CM (MW 3400) was introduced as a spacer between Ms and antibody. The conjugation of NH2-PEG-CM was similar to the conjugation of antibody. Briefly, 5 mg of Ms were activated by 50 mM EDC and 50 mM NHS in PBS (0.01 M pH 6.8). Then, the Ms were washed and dispersed in PBS (0.01 M pH 7.2) to react with 2 mg of NH2-PEG-CM. After 4 h incubation, the obtained MsPEG-COOH were washed to remove surplus PEG, which were then followed by the modification of antibody. The schematic diagrams of the two antibody-modification strategies were shown in


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Scheme 1. These immunomagnetic spheres were all used to capture S. typhimurium, and corresponding capture efficiencies were calculated for comparison.

Scheme 1. Schematically illustrating the two strategies for modifying Ms with antibody. PCR Assay PCR assays of the bacteria captured with IMNs were done by a Peltier thermal cycler (BioRAD). Referring to the previous work,10 a 605-bp fragment of the iroB gene of S. typhimurium was amplified with the specific primers of 5’- TACGTTCCCACCATTCTTCCC-3’ and 5’TGCGTATTCTGTTTGTCGGTCC-3’. The PCR was performed in a total volume of 25 µL containing 10.5 µL of sterile water, 12.5 µL of 2×Tag PCR master mix, 0.5 µL of each primer solution (10 µM), and 1 µL of the templates. The thermal profile was 95 °C (10 min), 30 cycles of 95 °C (30 s), 50 °C (30 s), and 72 °C (1 min), followed by a final extension at 72 °C (10 min). 1% (wt/vol) agarose gel was used to separate the PCR products, which were then stained with ethidium bromide for observation. Fluorescence Assay S. typhimurium and E. coli were stained by Hoechst 33342 (30 µg/mL) at 37 °C for 30 min, and then washed with physiological saline by centrifugation. The obtained bacteria were resuspended in 1% BSA-0.05% Tween 20-0.9% NaCl, which were treated with IMNs. After


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magnetic separation, the parts captured by IMNs were observed with a fluorescence microscope. Another control experiment was also performed: Unmodified MNs were used to treat stained S. typhimurium for observation. Fluorescent QDs were used to label and identify the captured bacteria.41 As illustrated in Scheme 2, after capture and isolation of bacteria, the IMNs-bacteria composites were resuspended in 200 µL of physiological saline (containing 1% BSA and 0.05% Tween 20) to react with 1 µg of biotinylated anti-S. typhimurium antibody (biotin-Ab) which were prepared with an EZ-link sulfo-NHS-LC-biotinylation kit purchased from Pierce Biotechnology (Rockford, IL). After 30 min incubation at 37 °C with gentle shaking, the immune complexes were washed by magnetic separation, which were then resuspended in 1% BSA-0.05% Tween 20-0.9% NaCl, and incubated with 1 µL of 10-7 M SA-QDs for 15 min at 37 °C. Finally, the immune complexes were rinsed for fluorescence spectrum measurement and microscopy observation.

Scheme 2. Schematic diagram for magnetic capture and fluorescence labeling of S. typhimurium.


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RESULTS AND DISCUSSION Characterization of the MNs and IMNs According to our previous work,38, 39 MNs were fabricated by LBL assembling nano-γ-Fe2O3 on copolymer nanospheres utilizing the coordination between the primary amines of PEI and the metallic atoms from nano-γ-Fe2O3. Further, an outer layer of silica was coated on the nanospheres and a modification with succinic anhydride was applied to increase the stability and biocompatibility. Figures 1A-1C visually illustrated this process. Copolymer nanospheres had a diameter of around 250 nm with smooth surfaces, just as shown in Figure 1A. After assembly of five layers of nano-γ-Fe2O3 (Figure S1 showed the size distribution and magnetic properties of the nano-γ-Fe2O3 particles.), the size of the nanospheres increased to about 320 nm, and numerous particles were clearly visible on their surfaces (Figure 1B). Finally, silanization and modification of the nanospheres made their surfaces relatively smooth again, and their sizes further increased (Figure 1C). A larger vision of the obtained MNs by TEM was shown in Figure S2A, from which it could be seen that the MNs were well dispersed with uniform size (370 ± 18 nm). The hydrodynamic diameter and the polydispersity index (PDI) of the MNs (Figure S2B) were 398.2 nm and 0.015, further indicating their high uniformity and good dispersibility in water, which helped providing for a very uniform reproducibility of magnetic separation. Moreover, the MNs exhibited excellent superparamagnetic properties: They had a high magnetic saturation value (34.1 emu/g) and a nearly zero coercivity at room temperature (Figure 1D), which enabled them to have a quick magnetic response. Just as shown in Figure 1E, with a commercial magnetic scaffold, almost 100% of the MNs could be captured by 1 min attraction, which made the MNs conveniently manipulated by a magnet (The capture efficiency of MNs at different attraction times was evaluated as our previous work.42). Besides, referring to our monitoring the properties of the MNs, the MNs showed high storage stability and strong anti-


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interference ability: They could retain superparamagnetic properties and good monodispersibility for at least 12 months, and could be directly used in complex matrix, even in whole blood.12 For specific recognition and capture of target bacteria, the MNs were modified with anti-S. typhimurium antibody by cross-linking the carboxyl groups on the MN surface and the amines of the antibody. To confirm the conjugation, the obtained IMNs were reacted with Cy3-labeled sheep anti-mouse IgG. As shown in Figure S3, nearly every IMN showed red fluorescence, while no fluorescence was observed in the control group (Unmodified MNs were treated with Cy3labeled sheep anti-mouse IgG.), indicating that mouse anti-S. typhimurium antibody was successfully conjugated to MNs. What’s more, our previous work had used secondary antibody as a model to modify the copolymer nanosphere and calculated that there were about 100 active affinity sites on each nanosphere, and further proved that the biomodified nanospheres could efficiently preserve their bioactivity for at least one year.43 Therefore, with unique magnetic properties, strong anti-interference ability, good bioactivity and high stability, the IMNs were ready for their application in separation and enrichment.


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Figure 1. (A) TEM image of the polymer nanospheres. (B) TEM image of the polymer nanospheres coated with nano-γ-Fe2O3. (C) TEM image of the finally obtained MNs. (D) Magnetic hysteresis loop of the MNs measured at room temperature. (E) Capture efficiencies of the MNs with a commercial magnetic scaffold at different attraction times. Error bars = ± SD (n = 3). Capability of the IMNs to Capture Bacteria In the capture of target bacteria with IMNs, the manipulation was very convenient: IMNs were directly added to the sample, followed by an incubation with gentle shaking. Afterward, the target bacteria combined with IMNs were separated and washed with a magnet, which were then collected for further study. Here, S. typhimurium was used as a model for the following investigation. For calculating the efficiencies to capture bacteria, two methods were used, which were described in the Experimental Section. At high bacteria concentration, equation (1) was used by determining the added bacteria number and the supernatant bacteria number with plate


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counting, which could avoid the errors from the bacteria aggregation caused by the multivalency of both IMNs and bacteria.44,

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At rare bacteria concentration, equation (2) was used by

determining the captured bacteria number and the supernatant bacteria number with plate counting. It was mainly due to the fact that when the bacteria suspension was diluted to very low concentration, the concentration of the obtained samples couldn’t be accurately calculated according to the dilution.10 Hence, the true number of the added bacteria was calculated by the summation of the captured bacteria number and supernatant bacteria number. Optimization of the Capture Conditions The IMN concentration and the incubation time were optimized. As shown in Figure 2A, with the IMN concentration increasing, the efficiencies to capture S. typhimurium increased until the IMN concentration reached 0.30 mg/mL, where 98% of the bacteria were captured. Further increasing IMN concentration did not obviously improve the capture efficiency, and thus 0.30 mg/mL IMNs were used to capture S. typhimurium. On the other hand, prolonging incubation time would facilitate the combination of the bacteria and the IMNs. From Figure 2B, it could be seen that the capture efficiency increased with incubation time, and 20 min incubation was enough for capturing almost all of the bacteria. Therefore, 20 min was chosen for the following experiments.


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Figure 2. (A) Efficiencies to capture S. typhimurium with different concentrations of IMNs. (B) Efficiencies to capture S. typhimurium with IMNs at different incubation times. Error bars = ± SD (n = 3). Reliability and Specificity Nonspecific adsorption of IMNs had two main sources: One was the nonspecific binding between the modified antibody with non-target bacteria, and the other was from the nonspecific adsorption of MNs. Hence, to investigate the specificity of IMNs, two control experiments were performed. Anti-S. typhimurium IMNs were used to treat E. coli (a common gram negative bacillus), and meanwhile, unmodified MNs were used to treat S. typhimurium. With the same procedure as IMNs to capture S. typhimurium, corresponding capture efficiencies were calculated, which were shown in Figure 3A. It could be seen that the IMNs were able to capture nearly 100% of S. typhimurium, while only 5% of E. coli were nonspecifically trapped, and unmodified MNs could hardly capture S. typhimurium either (capture efficiency: 3%), which suggested that the binding between IMNs and S. typhimurium was effective and specific. For further verification, a fluorescence microscopy analysis was done. S. typhimurium and E. coli were first stained by Hoechst 33342. Then the stained S. typhimurium were respectively treated with IMNs and MNs, and stained E. coli were treated with IMNs. The captured parts were observed with a fluorescence microscope. As shown in Figure 3B, many blue fluorescence spots, which indicated the stained bacteria, could be observed in the sample of IMNs to S. typhimurium. Moreover, the blue fluorescence spots matched well with the black spots (indicating the IMNs) in the bright field. While no obvious fluorescence was found in the samples of IMNs to E. coli or MNs to S. typhimurium. These results visually confirmed the good selectivity and specificity of IMNs. TEM imaging (Figures 3C) was performed to demonstrate the association of IMNs with S. typhimurium, which showed several IMNs (black spots) bound to one S. typhimurium,


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facilitating the effective capture by a simple magnetic scaffold. Besides, intra-assay and interassay variabilities of capturing S. typhimurium with IMNs were evaluated to be 0.36% and 1.98% respectively (Table S1), validating a good reproducibility and high stability.

Figure 3. (A) Capture efficiencies of S. typhimurium with IMNs, E. coli with IMNs, and S. typhimurium with MNs. Error bars = ± SD (n = 3). (B) Microscopy images of Hoechst 33342 stained S. typhimurium and E. coli treated with IMNs, and Hoechst 33342 stained S. typhimurium treated with MNs. (C) TEM image of S. typhimurium captured by IMNs. (The red arrow indicates the captured bacterium.) Capture of Bacteria at Ultralow Concentration For ultrasensitive detection and analysis of bacterial pathogens, highly efficient isolation and enrichment are necessary and crucial steps. Here, the capability of IMNs to capture rare bacteria was investigated. As controls, the MNs without antibody modified were used to treat ultralow concentration of S. typhimurium to evaluate the nonspecific interaction. The results were summarized in Tables 1 and 2. It could be seen that the efficiency to capture rare S. typhimurium


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could reach more than 96%, and even only one target bacteria existing in the samples wasn’t missed, suggesting that the IMNs had excellent capability to capture rare target bacteria. While the MNs hardly trapped any S. typhimurium, indicating the nonspecific adsorption of the MNs to S. typhimurium was slight and could be ignored. In Figure 4A, regression analysis of the captured bacterium number versus the spiked bacterium number was obtained: y=0.980x (R2=0.998), which further confirmed the powerful capture capability of the IMNs to their targets. Table 1. Efficiencies to Capture Ultralow Concentration of S. typhimurium with IMNs. Experiment No. 1

2

3

4

5

6

7

8

9

10

Ns

0

0

0

0

1

1

1

0

0

0

Nc

21

17

15

12

10

8

7

4

3

1

100

100

100

100

91

89

88

100

100

100

Capture efficiency (%) Average capture efficiency (%)

96.8

Table 2. Nonspecific Interaction of MNs to S. typhimurium at Ultralow Concentration Experiment No. 1

2

3

4

5

6

7

8

9

10

Ns

24

22

18

12

12

10

7

4

2

2

Nc

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

9

0

0

0

0

Capture efficiency (%) Average capture efficiency (%)

0.9


19


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Figure 4. (A) Capability of IMNs and MNs to capture ultralow concentration of S. typhimurium. (B) Efficiencies to capture S. typhimurium with IMNs in different synthetic samples. (C) Efficiencies to capture S. typhimurium with different diameters of immunomagnetic spheres. Error bars = ± SD (n = 3). Capture of Bacteria from Complex Samples To investigate whether IMNs can be used in complex matrices, milk, FBS, and urine were spiked with S. typhimurium to mimic real samples, which were directly captured and isolated with IMNs. As shown in Figure 4B, the efficiencies to capture S. typhimurium in the synthetic complex samples all could reach more than 95%, which were comparable to that in the ideal environment (bacteria in NaCl solution). The efficiencies in the four kinds of samples did not have significant differences at the 0.05 level (0.95 confidence level) by t-test. This suggested that the IMNs possessed a good anti-interference ability, and they had great potential applications in real samples. Comparison with Magnetic Microspheres The capture capability of the IMNs fabricated by our method was compared with those of the immunomagnetic microspheres (IMMs, 1.0 µm and 2.8 µm). During the capture process, the IMMs were applied at the same weight concentration with that of the IMNs. As shown in Figure 4C, with modification strategy I that the carboxyl groups on Ms were directly conjugated with the amines of antibodies, the 370-nm IMNs generated the highest capture efficiency, which was 97%


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compared with 46% with 1.0-µm IMMs and 36% with 2.8-µm IMMs. This might be due to the fact that with the sphere size decreasing, their reaction kinetics became faster and their surfaceto-volume ratio became higher, enabling rapider recognition and higher binding capacity, which resulted in a higher capture efficiency.26, 27 After introducing PEG as a spacer between the sphere and the antibody (modification strategy II), the capture capability of the IMMs improved significantly: For 1.0-µm IMMs, the capture efficiency increased from 46% to 89%, and for 2.8µm IMMs, the capture efficiency increased from 36% to 46%. This might be explained by that using PEG as a spacer allowed the antibody to extend out from the sphere surface, which could partly reduce the binding steric hindrance to improve the binding efficiency.10, 45, 46 While for 370-nm IMNs, the addition of a PEG spacer did not obviously benefit bacteria capture, and both of the two conjugation strategies achieved high capture efficiencies. This indicated that the small size of the IMNs enabled them to process high reaction flexibility and low steric hindrance when they were bound with their targets. Therefore, compared with IMMs, the IMNs fabricated by our method would be a more excellent separation tool for capturing bacteria. Identification of the Captured Bacteria The above results confirmed the excellent capture capacity of the IMNs: They were able to effectively, specifically, and rapidly isolate the target bacteria, and even only one bacterium existing in the samples could be definitely captured; They had strong anti-interference ability, and could be directly used in complex matrices. All these helped to lead to effective purification and enrichment of the target bacteria. What’s more, the IMNs-based separation could be conveniently coupled with various identification and analysis techniques, which were investigated as follows. Culture


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Figures 5A-5D were the typical photographs of the colonies formed by the captured S. typhimurium in the experiments of capturing rare bacteria with IMNs, which respectively corresponded to the captured 15, 10, 3, and 1 S. typhimurium from the samples. Figures 5E-5H were the typical photographs of the colonies formed by the uncaptured S. typhimurium in the experiments of treating rare bacteria with MNs, which respectively corresponded to the 22, 12, 12, 4 S. typhimurium in the supernatants. It could be seen that there was no obvious difference between the morphologies of colonies formed by the captured and uncaptured bacteria. In fact, our previous work had confirmed that the IMNs had low cytotoxicity and their combination with cells hardly affected the viability of the cells.39 Hence, the captured bacteria could be directly used for cultivation without disassociating IMNs, which would lead to a substantial enrichment of the targets, thereby greatly facilitating the following analysis or detection.

Figure 5. (A-D) Photographs of the colonies of the captured S. typhimurium on the LB agar. (EH) Photographs of the colonies of the uncaptured S. typhimurium on the LB agar. PCR


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The S. typhimurium captured with IMNs was directly used as template for PCR analysis, in which the 605 bp fragment of the iroB gene of S. typhimurium was amplified (Figure 6). Using the mixture of S. typhimurium and IMNs as template was able to get corresponding gene fragment (lane 3), which suggested that IMNs had a negligible influence on PCR, thereby making PCR performed without disassociating IMNs feasible. As expected, experimental groups with captured S. typhimurium (107, 106, 105, 104, 103 CFU/mL, lanes 4~8) as templates all got the 605 bp fragment of the iroB gene. While no fragment was found in the case without target bacteria (lane 9) or in the case using MNs to treat S. typhimurium (lane 10), which further confirmed the specific binding between IMNs and bacteria. Overall, all these results indicated that the IMNs captured bacteria were suitable for molecular biological analyses, enabling further research (e.g., genotyping, investigation of pathogenesis mechanism and antimicrobial resistance, etc.) more convenient.

Figure 6. Agarose gel electrophoresis of the PCR products: lane 1, DNA ladder (5 kbp); lane 2, positive control using pure S. typhimurium as template; lane 3, positive control using the mixture of S. typhimurium and IMNs as template; lanes 4~9, experimental groups using templates of S. typhimurium captured respectively at the concentrations of 107, 106, 105, 104, 103, 0 CFU/mL;


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lane 10, negative control using MNs trapped S. typhimurium as template; lane 11, negative control using sterile water as template. Fluorescence Observation Fluorescence detection is an important identification method due to its rapid and simple manipulation and its high sensitivity. Hence, in this work, IMNs captured bacteria were labeled with QDs for identification to verify its coupling with fluorescence observation. QDs are versatile fluorescent nanoparticles with unique optical properties, which have been newly developed in recent decades and quickly applied to the labeling and detection.47 As shown in Scheme 2, after S. typhimurium was captured by IMNs, biotin-Ab was added to form a sandwich immune complex, followed with QD labeling via biotin-streptavidin conjugation. The immune complexes were measured with a fluorescence spectrometer, whose fluorescence intensities were shown in Figure 7A and typical fluorescence spectra were provided in the inset. It could be seen that significant fluorescence signal was produced in the positive samples. When using E. coli or physiological saline to replace S. typhimurium, or using unmodified MNs to treat S. typhimurium, no detectable QD fluorescence was obtained. These suggested that the nonspecific binding was minimal and the QD labeling had good selectivity and specificity. Fluorescence microscope was also used to observe the IMNs captured and QDs labeled bacteria (Figure 7B), and the results showed good agreement with those of the fluorescence spectrum measurements. In the positive sample, many fluorescence spots (~ 2 µm), which indicated the bacteria,10, 48 were found. Besides, some aggregation was produced due to the multisite binding between the IMNs and the bacteria. While no obvious fluorescence spot was found in the negative controls. To further confirm the binding of the target bacteria and QDs, co-localization analysis was performed. As shown in Figure 7C, there was a high co-localization efficiency (Figure 7C (d)) between the Hoechst 33342 stained S. typhimurium (Figure 7C (b)) and the QDs (Figure 7C (c)), indicating that S.


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typhimurium were efficiently recognized and identified by the QDs. All these results showed that IMNs-based isolation was able to be successfully coupled with fluorescence identification, which presented little non-specific adsorption and convenient manipulation.

Figure 7. (A) Histogram of fluorescence intensity after identified with QDs. (“S. typhimurium” corresponds to IMNs captured S. typhimurium followed with QD identification. “E. coli” corresponds to IMNs treated E. coli followed with QD identification. “MNs” corresponds to MNs treated S. typhimurium followed with QD identification. “Blank” was the treatment of physiological saline. Corresponding fluorescence spectra are shown in the inset.) (B) Microscopy images after identified with QDs. (C) Co-localization analysis of the captured S. typhimurium after identified with QDs: (a), Bright field; (b), Hoechst 33342 stained S. typhimurium (excitation


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filter: 330-380 nm, emission filter: bandpass 455±25 nm); (c), QDs labeled S. typhimurium (excitation filter: 510~560 nm, emission filter: longpass 590 nm); (d), Merge of (b) and (c).

CONCLUSIONS In summary, we had successfully fabricated quick-response MNs for rapid and efficient enrichment of bacteria, which showed great superiority over currently used magnetic micro/nanoparticles. Compared with magnetic nanoparticles, the MNs had faster magnetic response and could be manipulated by a simple magnetic scaffold with little loss. Using IMNs to capture bacteria didn’t need complex separation tools or techniques, facilitating their application to common laboratories, and even to on-site analyses. While compared with magnetic microparticles, the IMNs showed much more excellent capacity to capture bacteria, including their high capture efficiencies and rapid binding rates. With 20 min interaction, almost all of the target bacteria could be captured, and even only one bacterium in the samples wasn’t missed. Besides, the IMNs had good stability, strong anti-interference ability, and low nonspecific adsorption, which could be directly used in complex matrices. Moreover, the IMNs-based enrichment strategy could be conveniently coupled with the downstream identification and analysis techniques, validating their great application advantages in bacteria detection and investigation.

AUTHOR INFORMATION Corresponding Author * Email: [email protected]


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ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (No. 21505157), the Fund for Young Scientists of Shandong Province (No. BS2015SF001), the Talent Project of China University of Petroleum (East China) (No. 2014010578), the Applied Basic Research Projects of Qingdao (15-9-1-94-JCH), and the National Key Research and Development Program (2016YFC1201404).

SUPPORTING INFORMATION AVAILABLE The Supporting Information is available free of charge on the ACS Publications website. Figure S1, TEM image and magnetic hysteresis loop of the nano-γ-Fe2O3 particles. Figure S2, TEM image and hydrodynamic diameter distribution of the MNs. Figure S3, microscopy images of the IMNs and MNs after they were reacted with Cy3-labeled sheep anti-mouse IgG. Table S1, reproducibility analysis of the capture of bacteria with IMNs.

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