Sensitive Detection of Staphylococcus aureus with Vancomycin

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Sensitive Detection of Staphylococcus aureus with Vancomycin-Conjugated Magnetic Beads as Enrichment Carriers Combined with Flow Cytometry Xiangyu Meng, Guotai Yang, Fulai Li, Taobo Liang, Weihua Lai, and Hengyi Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 07 Jun 2017 Downloaded from http://pubs.acs.org on June 9, 2017

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ACS Applied Materials & Interfaces

Sensitive

Detection

of

Staphylococcus

aureus

with

Vancomycin-Conjugated Magnetic Beads as Enrichment Carriers Combined with Flow Cytometry

Xiangyu Meng, Guotai Yang, Fulai Li, Taobo Liang, Weihua Lai, Hengyi Xu*

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P. R. China

*Correspondence to: Dr. Hengyi Xu State Key Laboratory of Food Science and Technology, Nanchang University. Address: 235 Nanjing East Road, Nanchang 330047, P.R. China. Phone: +0086-791-8830-4447-ext-9520. Fax: +0086-791-8830-4400. E-mail: [email protected] or [email protected].

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ABSTRACT: A novel sandwich strategy was designed to detect Staphylococcus aureus. The strategy is based an antibacterial agent that captures bacterial cells and a fluorescein-labeled antibody that acts as the signal-output probe. Vancomycin (Van), which exerts a strong antibacterial effect on Gram-positive bacteria, was utilized as a molecular recognition agent to detect pathogenic bacteria. To effectively concentrate S. aureus, bovine serum albumin (BSA) was used as the amplification carrier to modify magnetic beads (MBs), which were then functionalized with Van. To improve the specificity of the method for S. aureus detection, fluorescein isothiocyanate (FITC)-tagged pig immunoglobulin G (FITC-pig IgG) was adopted as the signal probe and the second recognition agent that bound between the Fc fragment of pig IgG and protein A in the surface of S. aureus. To quantify S. aureus, the fluorescence signal was measured by flow cytometry (FCM). The use of multivalent magnetic nanoprobes (Van–BSA–MBs) showed a high concentration efficiency (>98%) at bacterial concentrations of only 33 colony-forming units (CFU)/mL. Furthermore, the sandwich mode (FITC-pig IgG/SA/Van-BSA-MBs) also showed ideal specificity because Van and IgG bound with S. aureus at two distinct sites. The detection limit for S. aureus was 3.3 × 101 CFU/mL and the total detection process could be completed within 120 min. Other Gram-positive bacteria and Gram-negative bacteria, including

Listeria

monocytogenes,

Bacillus

cereus,

Cronobacter

sakazakii,

Escherichia coli O157:H7, and Salmonella Enteritidis, negligibly interfered with S. aureus detection. The proposed detection strategy for S. aureus possesses attractive characteristics, such as high sensitivity, simple operation, short testing time, and low 2 / 34


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cost. KEYWORDS: Staphylococcus aureus, vancomycin, magnetic beads, fluorescence, flow cytometry

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INTRODUCTION Staphylococcus aureus (SA) is a widely distributed Gram-positive pathogen that causes many dangerous infectious diseases, such as septicemia, osteomyelitis, pneumonia, toxic shock syndrome, and endocarditis.1,2 Thus, the identification and quantitation of S. aureus have become a crucial point in medical diagnosis, food safety, environmental hygiene, and drug discovery.3,4 In the past decades, numerous efforts have been exerted to develop rapid, sensitive, and specific assay methods for S. aureus detection.5 The conventional culture and colony counting-based method is the gold standard for bacteria detection given its high sensitivity, ideal specificity, and good reliability.6 This method, however, requires labor-intensive operation, time-consuming culture, and substantial laboratory apparatuses. Immunological assays, such as enzyme-linked immunoabsorbent assay (ELISA), can rapidly detect S. aureus but suffer from relatively low sensitivity.7 Nucleic acid-based assays, such as polymerase chain reaction (PCR), have high sensitivity and high specificity for S. aureus. Nevertheless, the PCR method requires complicated procedures and skillful technicians.8,9 Other methods such as quartz crystal microbalance (QCM),10 surface plasmon resonance (SPR),11

fluorescence

resonance

energy

transfer

(FRET),3,12

electrochemiluminescence,13,14 and flow cytometry (FCM),15 have been developed to detect pathogenic bacteria. Among these methods, FCM is the best option given its rapid and automatic detection and quantification of various bacterial species at the single-cell level. Moreover, FCM does not require labor-intensive nucleic acid 4 / 34


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extraction, time-consuming bacterial detection, and complicated manipulation.16 FCM combined with the molecular recognition mode has been recently used to detect pathogenic bacteria.15 The molecular recognition mode has attracted increasing interest because of its suitability for direct assaying, molecular recognition is conducted with a molecule recognition agent that specifically binds to bacteria without conducting cell disruption.4,17 Therefore, the molecular recognition mode combined with FCM is an ideal bacterial detection assay for rapid screening in a public food safety emergency. This recognition mode utilizes molecular recognition agents, including antibodies,18 aptamers,19 bacteriophage,20 and specific proteins.21 These biomaterial-based recognition agents, however, have poor stability, high cost, and difficult preparation methods; moreover, some of them are commercially unavailable.18 To solve the problems and to enlarge the application latent capacity of this mode, readily prepared molecular recognition agents with lower cost and higher stability should be identified. Antibiotics are a batch of small molecules used for treating and preventing bacterial infection.22,23 As a broad-spectrum glycopeptide antibiotic, vancomycin (Van) can bind with the D-Ala-D-Ala moieties in most Gram-positive bacteria cell wall via five-point hydrogen-bond binding.24-26 As a commercially available conventional drug, Van has many outstanding properties, such as high stability, low cost, and good quality controllability. Thus, as a molecular recognition agent that acts on the bacterial cell wall, Van is an ideal candidate for pathogen identification.1,4 Magnetic separation technology offers the advantage of maintaining bacterial 5 / 34



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viability while eliminating sample matrix interference.27-29 Magnetic beads (MBs) that have been functionalized with Van can act as highly sensitive, easily manipulated concentration carriers. Van-functionalized MBs have been widely studied for the concentration, detection, and identification of bacteria.27,30,31 Bovine serum albumin (BSA) is a depot and transport protein in the blood and is widely used as a blocking agent to reduce the non-specific absorption or incorporation of the target molecule.32-34 The surface of the BSA protein molecule presents numerous amine groups, which may allow multiple Van molecules to couple to the surface of one BSA protein.35,36 Given that Van is a wide-spectrum antibiotic that can identify Gram-positive bacteria, Van–BSA–MBs are nonspecific for S. aureus.17,36 Fortunately, based on the report that the surface of S. aureus comprises ample protein A, which can specific combine with the Fc region of mammal IgG. In the present study, fluorescein isothiocyanate (FITC)-pig IgG is utilized as the second recognition agent to tremendously enhance the specificity of the antibiotic-affinity strategy for S. aureus detection.23,35,37, In this paper, we report two different recognition mechanisms to effectively enrich and specifically detect S. aureus as described above. In brief, Van-functionalized BSA–MBs (Van–BSA–MBs) probes are used to recognize and enrich S. aureus. This BSA-mediated multivalent conjugated system has the following advantages: (1) the surface of MBs can load more Van, thus enabling the Van–BSA–MBs to rapidly and intensely connect to the S. aureus cell wall and improve capture efficiency (CE); (2) Van–BSA–MBs decrease the nonspecific absorption of nontarget bacteria; and (3) the 6 / 34


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BSA linker provides a flexible molecular backbone, reduces steric hindrance, and maintains the biological activity of Van molecules. Meanwhile, FITC-tagged pig IgG is adopted as the second recognition agent for S. aureus detection. Then, the fluorescence intensity (FI) of FITC-pig IgG/SA/Van–BSA–MBs is measured by FCM. Finally, S. aureus is quantified via the changes in FI (△FI).

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MATERIALS AND METHODS Materials and Reagents. Carboxylated MBs (180 nm, 10 mg/mL) were purchased from

Allrun

Nano Science &

1-(3-(dimethylamino)

Technology

Co.

Ltd.

propyl)-3-ethylcarbodiimide

(Shanghai, China).

hydrochloride

(EDC),

Nhydroxysuccinimide sodium salt (NHSS), and vancomycin hydrochloride was purchased from Aladdin Industrial Corporation (Shanghai, China). BSA were obtained from Sigma-Aldrich Chemical Co. (St. Louis, U.S.A.). FITC-conjugated pig IgG was purchased from Shanghai Biotechnology Co., Ltd (Shanghai, China). Luria-Bertani (LB) broth and Baird-Parker Agar Base was bought from Land Bridge Technology Co. Ltd. (Beijing, China). 0.01 M phosphate-buffered saline (PBS, pH = 7.4, 0.01 M, 8.0 g NaCl, 0.2 g KCl, 0.24 g KH2PO4, 1.44 g Na2HPO4, adjusted pH using NaOH). Distilled deionized water was used for the preparation of all aqueous solutions. Apparatus. The Van-BSA-MBs complex was characterized by Fourier transform infrared spectroscopy (FT-IR) (Nicolet 5700 FTIR spectrometer, Thermo Fisher Scientific, Inc., Waltham, MA), zeta potential and hydrodynamic diameter (Zeta Sizer Nano ZS90, Malvern Instruments Ltd., Britain) and scanning electronic microscopy (SEM) (JSM-6701F, JEOL Ltd., Tokyo, Japan) was used to confirm the modified BSA and Van on the surface of MBs. BD FACSCalibur flow cytometry (FCM) (FACSCalibur, Becton Dickinson, New Jersey, USA) was used to detect FI of FITC-pig IgG/SA/Van-BSA-MBs. Bacterial Strains and Culture Conditions. The bacterial strains used in this study 8 / 34


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including Staphyloccocus aureus CMCC26001, Listeria monocytogenes ATCC13932, Bacillus cereus JX-CDC JDZ0121LY (Jiang Xi Province Center for Disease Control and Prevention, China.), Cronobacter sakazakii CMCC45401, Escherichia coli O157:H7 ATCC43888, Salmonella Enteritidis ATCC13076. All bacterial strains were grown overnight in LB broth medium at 37 °C. The collected bacteria pellets were resuspended and serial 10-fold dilution with sterile PBS (0.01 M, pH = 7.4). The number of viable cell was determined using conventional plate counting method in LB plates and incubated at 37 °C for 18 h. The captured bacteria was incubated in Baird-Parker Agar Base overnight. All the plate counting results were counted for colony-forming units (CFU). Considering the safety, the bacteria samples were also sterilized to kill the bacteria after use. Preparation and Characterization of Van-BSA-MBs. Van-BSA-MBs were prepared using EDC/NHSS reaction and outlined in Scheme 1A. It was synthesized as our previously reported.38 Firstly, 2.0 mL of carboxy groups-coated MBs suspension was washed thrice with sterile PBS (0.01 M, pH = 7.4), then resuspended in 10 mL of the same buffer containing 5.8 mg of EDC and 6.5 mg NHSS. Following 1h activation, the MBs were washed thrice sterile PBS and dispersed in 10 mL of the same buffer containing 24 mg of BSA. Following 2 h reaction, BSA was coupled to the surface of MBs (BSA-MBs), the BSA-MBs were washed thrice and dispersed in sterile PBS. 240 mg vancomycin hydrochloride were mixed with 150 mg of EDC and 50 mg NHSS (the mole ratio at 1:4:1). After 10 min activation at room temperature added into BSA-MBs. After 6 h reaction under constant shaking, the resulted 9 / 34



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Van-BSA-MBs were washed thrice with sterile PBS with final concentration at 2 mg/mL and stored at 4 °C. To understand and ensure the properties of the synthesized Van-BSA-MBs, material characterizations were performed with fourier transform infrared spectroscopy (FT-IR), bactericidal halo test, scanning electronic microscopy (SEM), zeta potential and hydrodynamic diameter. Procedures for Detection Assay of S. aureus. In 10 mL volume, added S. aureus with the concentration at 104 CFU/mL and 700 µg Van-BSA-MBs, then incubated at 37 °C for 45 min, after magnetic separation 3 min. Resuspended the SA/Van-BSA-MBs complex in 500 µL sterile PBS, and 15 µL of FITC-labeled pig IgG at 1 mg/mL was mixed together and incubated under constant shaking for 1 h at 37 °C. Following a thorough washing and resuspended FITC-labeled pig IgG/SA/Van-BSA-MBs complex in 300 µL, FI signal was measured with an excitation wavelength of 488 nm and an emission wavelength of 525 nm by FCM. Van-BSA-MBs was used to enrich S. aureus and the CE was determined by the equation (1). The FI signal value was calculated based on the equation (2). CE = [S / (S + C) ] × 100%

(1)

Where S is the number of bacteria in the supernatant (CFU/mL), and C is the number of bacteria captured by Van-BSA-MBs (CFU/mL). △FI = FI - FI0

(2)

where FI = the FI from the sandwich structure with target bacteria, FI0 = the FI from the sandwich structure with no bacteria. 10 / 34


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Detection of S. aureus in Mock Sample. The drinking water, milk, and fruit juice sample purchased from a local supermarket. Then S. aureus was added to the treated drinking water, milk, and fruit juice sample with three known concentrations, then measured the CE and △FI as described above.

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RESULTS AND DISCUSSION Principle of the Antibiotic-Affinity-Based Detection Method for S. aureus. The principle of sandwich assay for S. aureus detection is described in Scheme 1B. First, BSA is modified on the surface of MBs. Then, Van is conjugated with BSA–MBs to form the multivalent magnetic nanoprobes Van–BSA–MBs that is used to enrich S. aureus. In these nanoprobes, the BSA carrier could couple with multiple Vans given its multiple binding sites, and decreasing the nonspecific absorption of the other non-target bacteria. Although Van is linked to BSA–MBs via the carbodiimide reaction, it still retains its antibiotic viability S. aureus. Van, however, suffers from inadequate specificity given its nature as a broad-spectrum antibiotic that recognizes multiple bacterial species. Some research have already reported that using PE-tagged Van and FITC-pig IgG (Fc) can simultaneously stain S. aureus.23,35,39 This phenomenon demonstrates that pig IgG and Van simultaneously bind with S. aureus to form a sandwich complex. Finally, FITC-pig IgG was added to bind protein A and form FITC-pig IgG/SA/Van-BSA-MBs sandwich complex that can be allowed to detect the whole S. aureus cells via FCM. Characterization of Van-BSA-MBs. The FT-IR spectra in Fig. 1A indicate that the absorption peaks at 519 and 1019 cm–1 may be related to the –S–S and –NH2 of BSA.40 The absorption peaks at 1019 cm–1 indicated the presence of aromatic ether, whereas the spectrum absorption peak at 1415 cm–1 indicated the phenolic hydroxy of the Van molecule.4,41 These results confirmed the presence of BSA and Van on the surface of MBs. 12 / 34


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As shown in Fig. 1B and C, the hydrodynamic diameter and zeta potential of MBs, BSA–MBs, and Van–BSA–MBs were measured. The actual particle size of the bare MBs was 185 nm; after modification with BSA and Van, the particle size of the bare MBs increased to 226.7 and 254.9 nm, respectively. The surface potential of the MBs was −33.65mV. After functionalization with BSA and Van, the surface potential of the bare MBs decreased to −52.65 and −44.2 mV, respectively. The surface potential of BSA and Van was −22.65 and −5.13 mV, respectively. The potential of MBs significantly decreased after surface modification with BSA. After modification with Van, the surface potential of the Van–BSA–MBs increased compared with those of BSA–MBs but decreased compared with those of MBs. The change in surface potential was caused by the reaction of amino and carboxyl groups. The changes in hydrodynamic diameter and zeta potential signified that Van and BSA were successfully conjugated on the surface of MBs. A bactericidal halo test was conducted on LB agar to investigate the antibacterial activity of Van, which was chemically modified on the surface of BSA–MBs. As shown in Fig. 1D, MBs and BSA–MBs exerted no inhibitory effect on S. aureus, as evidenced by the almost invisible inhibition zones. Intact Van and Van–BSA–MBs showed visual inhibition zones with diameters of 2.1 and 1.3 cm, respectively, which illustrated that the conjugated Van retained its antibacterial activity. The images of MBs, BSA-MBs, S. aureus, and the attachment of Van–BSA–MBs to S. aureus was confirmed via SEM. The obtained SEM image, which is presented in

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Fig. 1E, shows that Van–BSA–MBs are bound to the cell wall of S. aureus. This phenomenon indicated that Van–BSA–MBs effectively captured S. aureus. Optimization of Separation Experimental Conditions. To obtain the optimal performance of the enrichment assay, some important factors, including the amount of Van–BSA–MBs, incubation time, and magnetic separation time were optimized using S. aureus at 3.3 × 104 CFU/mL. Figure 2A shows that the CE increased with the amount of Van–BSA–MBs and reached saturation when 700 µg of Van–BSA–MBs was used. Hence, 700 µg of Van–BSA–MBs was used for the following procedure. Compared with that used in our previous studies, the amount of functionalized MBs decreased by 300 µg.38 The effect of incubation time on CE was also investigated. Figure 2B suggests that the CE attained saturation when Van–BSA–MBs and S. aureus were incubated for 45 min. Thus, 45 min was selected as the incubation time in the next experiment. As illustrated in Fig. 2C, the CE reached a peak value at 3 min of magnetic separation time. Extending the magnetic separation time beyond this period did not significantly change the CE. In the following experiments, the incubation time was set as 3 min. The CE of the Van–BSA–MBs was further investigated under optimal assay conditions with S. aureus concentrations of 3.3 × 101 CFU/mL to 3.3 × 106 CFU/mL. As shown in Fig. 2D, the CE of the Van–BSA–MBs was 82% even at high concentration (106 CFU/ml) of S. aureus. Specificity of Sandwich Assay for Detecting S. aureus. As a broad-spectrum glycopeptide antibiotic, Van can anchor a wide range of Gram-positive bacteria. Given that Gram-negative bacteria have complex lipopolysaccharide structures in the 14 / 34


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outer cell wall, Van cannot easily contact and bind to terminal D-Ala–D-Ala moieties.4 As shown in Fig. 3A, the CE of Gram-negative bacteria, including Cronobacter sakazakii, Escherichia coli O157:H7, and Salmonella Enteritidis are less than 15%. S. aureus, mixture 1 (including C. sakazakii, E. coli O157:H7 and S. Enteritidis), and mixture 2 (including S. aureus, C. sakazakii, E. coli O157:H7 and S. Enteritidis), exhibited CEs of approximately 92.10%, 14.32%, and 88.72%, respectively. This result showed that Van–BSA–MBs can efficiently capture S. aureus instead of Gram-negative bacteria, and that Gram-negative bacteria have little influence on the CE of the mixture. To verify that only protein A on the surface of S. aureus can be identified by FITC-pig IgG, two Gram-positive bacteria, including Listeria monocytogenes and Bacillus cereus, were investigated.4,37 As presented in Fig. 3B, L. monocytogenes, B. cereus, and mixture 1 (L. monocytogenes and B. cereus) had weak △FI signals, whereas S. aureus and mixture 2 (S. aureus, L. monocytogenes and B. cereus) showed an intense △FI signal. These results indicated that L. monocytogenes, B. cereus, and mixture 1 could be captured by Van–BSA–MBs but could not bind with FITC-pig IgG to form the sandwich complex given the absence of protein A in their cell walls. Evaluation of 10 mL Van–BSA–MBs Combined with FCM for the Detection of S. aureus. Given the high capture efficiency of Van–BSA–MBs and intense, sensitive FI-based detection by FITC-pig IgG, the experimental results indicated the excellent sensitivity of our proposed system for S. aureus detection. In Fig. 4A, the △FI signal continuously and steadily increased as S. aureus concentrations increased from 3.3 × 15 / 34



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101 CFU/mL to 3.3 × 105 CFU/mL, and then reached saturation at 3.3 × 106 CFU/mL. Figure 4B illustrates that the detection of S. aureus is linear in the range of 3.3 × 101 to 3.3 × 105 CFU/mL. The regression equation is Y = 27.01x + 34.962, with a correlation coefficient of 0.9904. The limit of detection (LOD) was estimated as 3.3 × 101 CFU/mL, which is considerably lower than those of bacterial detection methods that are based on magnetic separation (Table 1). Therefore, our method can sensitively detect S. aureus. Mock Sample Detection. To verify the detection reliability of our proposed method, drinking water, milk, and fruit juice were selected as mock samples for the quantitative detection of S. aureus. These samples were sterilized prior to the addition of S. aureus at three known concentrations. The results are listed in Table 2, CE was acceptable and ranged from 80.48% to 97.29%. This result indicated that the multivalent nanocarriers have a favorable enrichment effect on spiked samples. As Fig. 5 illustrates, visible △FI signals are obtained from the experiments. Both concentration and fluorescence detection results showed that our detection method has excellent practical application in the detection of S. aureus in samples.

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CONCLUSION In this study, we developed a sensitive and specific antibiotic-affinity strategy for the direct detection of whole S. aureus cells without cell disruption. In our strategy, Van–BSA–MBs were used to enrich a 10 mL-volume solution of S. aureus, and FITC-pig IgG was used as the second recognition molecule and signal tracer for S. aureus. More than 82% S. aureus can be concentrated by Van–BSA–MBs even at high concentration (106 CFU/ml) of S. aureus, making these multivalent magnetic nanoprobes a powerful screening method for S. aureus. Under optimized conditions, the linear range of the Van–BSA–MB combined with FCM assay was 3.3 × 101 to 3.3 × 105 CFU/mL with an LOD of 3.3 × 101 CFU/mL. The whole detection time was approximately 120 min. The constructed strategy was slightly influenced by Gram-negative bacteria and other Gram-positive bacteria. In conclusion, this approach demonstrated ideal sensitivity and specificity because the Van–BSA–MBs complex showed excellent capture capability and FITC-pig IgG possessed favorable specificity. Thus, our constructed strategy is a powerful tool for the rapid enrichment and identification of S. aureus in food and environmental samples. In the next work, we will attempt to use this methodology, replace Van and IgG with lectin and aptamers as recognition molecules for detecting Gram negative bacteria.

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ACKNOWLEDGEMENTS This work was supported by the Research Foundation for Young Scientists of State Key Laboratory of Food Science and Technology, Nanchang University, China (Grant no. SKLF-QN-201504) and Training Plan for the Young Scientist (Jinggang Star) of Jiangxi Province (Grant no. 20142BCB23004).

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1130-1136. (8) Johnson, W.; Tyler, S.; Ewan, E.; Ashton, F.; Pollard, D.; Rozee, K. Detection of Genes for Enterotoxins, Exfoliative Toxins, and Toxic Shock Syndrome Toxin 1 in Staphylococcus aureus by the Polymerase Chain Reaction. J. Clin. Microbiol. 1991, 29 (3), 426-430. (9) Cheng, J. C.; Huang, C. L.; Lin, C. C.; Chen, C. C.; Chang, Y. C.; Chang, S. S.; Tseng, C. P. Rapid Detection and Identification of Clinically Important Bacteria by High-Resolution Melting Analysis After Broad-Range Ribosomal RNA Real-Time PCR. Clin. Biochen. 2006, 19 / 34



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Tables

Table 1. Comparison of the current approach and relevant methods for analysis pathogenic bacteria by functionalized magnetic nanoparticles. Different functional group modified MNPs antibody -coated magnetic beads monoclonal antibody -coated magnetic beads monoclonal antibody -coated MBs vancomycin-functionalized mNPs vancomycin-bound magnetic nanoparticles vancomycin modified PEGylated-MBs vancomycin modified BSA coated-MNPs

Target Volume bacteria (mL) Listeria 2 monocytogenes Listeria 1 monocytogenes Listeria 10 monocytogenes sulphate reducing 1 bacteria Staphylococcus 3 aureus Staphylococcus 10 aureus Staphylococcus 10 aureus

Detection method flow cytometry impedance biosensor mPCR quartz crystal microbalance MALDI-MS analysis PCR flow cytometry

Whole assay LOD time (min) (cfu/mL) 120 103

42

120

300

43

420

10

28

70

1.8 × 104

44

120

7.8 × 104

45

240

30

38

120

33

This study

24 / 34


Reference

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Table 2. The capture tests of S. aureus in spiked samples using the proposed Van-BSA-MBs complex (n = 3). Food sample Drinking water Milk a Fruit juice

102 97.29 ± 4.46 86.25 ± 5.65 95.19 ± 3.08

CE (%) SA concentration (CFU/mL) 103 94.68 ± 3.02 82.12 ± 4.08 93.70 ± 2.72

104 91.02 ± 3.45 80.48 ± 4.91 93.64 ± 3.29

a The sample was 20-time diluted.

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Scheme Scheme 1. Detection of S. aureus by Van-BSA-MBs separation combined with FCM in 10 mL volume. (A) procedures of constructing Van-BSA-MBs; and (B) the assay of S. aureus enrichment and detection.

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Figure captions

Fig. 1 Characterization. (A) FT-IR spectrum of (a) MBs, (b) BSA-MBs, and (c) Van-BSA-MBs, (B) hydrodynamic diameter of MB and Van-BSA-MBs, (C) zeta potential of MB, BSA-MBs, and Van-BSA-MBs, (D) bactericidal halo test of (a) intact vancomycin, (b) BSA-MBs, (c) MBs, and (d) Van-BSA-MBs, (E) SEM image of MBs, BSA-MBs, S. aureus, and S. aureus-Van-BSA-MBs complex. Vertical bars represent standard deviation (n = 3).

Fig. 2 Optimization of Van-BSA-MBs magnetic separation parameters. (A) the amounts of Van-BSA-MBs (incubation time at 45 min, magnetic separation time 3 min), (B) incubation time of Van-BSA-MBs with bacteria (magnetic separation time at 3 min), (C) magnetic separation time, (D) the capture efficiency of Van-BSA-MBs using S. aureus concentrations from 3.3 × 101 CFU/mL to 3.3 × 106 CFU/mL. Vertical bars represent standard deviation, *p

Sensitive Detection of Staphylococcus aureus with Vancomycin

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