Efficient Capture and T2 Magnetic Resonance Assay of Candida

Jun 3, 2019 - Efficient Capture and T2 Magnetic Resonance Assay of Candida albicans with Inorganic Nanoparticles: Role of Nanoparticle Surface Charge ...
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Article Cite This: ACS Biomater. Sci. Eng. 2019, 5, 3270−3278

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Efficient Capture and T2 Magnetic Resonance Assay of Candida albicans with Inorganic Nanoparticles: Role of Nanoparticle Surface Charge and Fungal Cell Wall Wei Tian,†,‡ Fan Li,§ Shengming Wu,‡ Gen Li,∥ Lieying Fan,∥ Xue Qu,*,†,⊥ Xinming Jia,*,§ and Yilong Wang*,‡

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†

Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China ‡ The Institute for Translational Nanomedicine, Shanghai East Hospital, The Institute for Biomedical Engineering & Nano Science, Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, P. R. China § Clinical Translational Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, P. R. China ∥ Department of Clinical Lab, Shanghai East Hospital, Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, P. R. China ⊥ State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, P. R. China S Supporting Information *

ABSTRACT: The early detection of fungi through a facile and straightforward method is desirable. The isolation of fungi from a body fluid sample plays a major role in effective detection. In the past, concanavalinA (conA), one of the lectins, interacted with Candida albicans through the surface component of the yeast cell wall. The development of a facile method with a robust binding affinity and an efficient capture of the yeast in addition to conA could pose a potential for the sandwich-like assay of C. albicans. In this study, the feasibility of an electrostatic interaction-mediated yeast capture was investigated as compared to conA-mediated binding. Also, the optimal parameters for the efficient isolation of C. albicans by surface-charged nanoparticles were studied, and the mechanism of the binding site through the electrostatic interaction on the surface of the yeast cell wall was explored by a blocking experiment. Furthermore, the yeast strains were found to interact uniformly only via the positively charged nanoparticles, and the captured yeast could be analyzed by FITC-conA fluorescence staining and T2 magnetic resonance assay. Thus, this strategy established a rapid and highly efficient method for the isolation of fungi and analysis. KEYWORDS: Candida albicans, yeast strains, surface charge, lectin, mannan, fluorescence labeling, T2 magnetic resonance

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INTRODUCTION Candida albicans is the most common fungal pathogen causing nosocomial bloodstream infections worldwide. About 50% of candidiasis is caused by C. albicans, with a total mortality rate of 43%.1,2 This symbiotic organism primarily existed in the human oral, digestive, and reproductive cavities, causing systemic infection under certain conditions, especially in immunocompromised patients.3 The prognosis of the fungal infection depends on early treatment as it can improve the survival rate. In the case of a total of 77 suspected patients with deep mycosis, 62% of the early treatment patients showed improved clinical symptoms and serological test results as compared to 21% counterparts with nonearly treatment.4 The effective and simple method for the enrichment of C. albicans is an urgent requisite for the profound study of yeast strains, © 2019 American Chemical Society

which might provide critical information about the infection degree and the guide for optimal treatment. Although the isolation of C. albicans yeast by functionalized magnetic nanoparticles (MNs) has been recently combined with some diagnosis platforms including PCR and magnetic resonance assays,5,6 few yeast strains interacting with lectin−mannose glycans hinder the further development of an effective and straightforward diagnosis method utilizing MNs.7,8 In addition, the discrete and heterogeneous distribution of the capsular polysaccharides on the surface of fungi influences the interaction of lectin with glycan motifs.9 Received: January 16, 2019 Accepted: June 3, 2019 Published: June 3, 2019 3270

DOI: 10.1021/acsbiomaterials.9b00069 ACS Biomater. Sci. Eng. 2019, 5, 3270−3278

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Figure 1. Schematic illustration of the distribution of glycan on the surface of C. albicans and differential functionalized nanoparticles−fungal yeast interaction in various conditions. (A) Differential interaction between the surface glycan of C. albicans and three kinds of magnetic nanoparticles including positively charged magnetic nanoparticles (PCMNs), negatively charged magnetic nanoparticles (NCMNs), and conA-conjugated magnetic nanoparticles (ConA-MNs). PCMN refers to polyethylenimine (PEI)-functionalized magnetic nanoparticles. NCMN refers to the poly(acrylic acid) (PAA)-functionalized magnetic nanoparticles. ConA-MN refers to lectin-conjugated nanoparticles. (B) The strong electrostatic attraction between positively charged MNs and C. albicans can be blocked by mannose-functionalized on the surface of PCMNs. This experiment is helpful to study the interacting mechanism and binding site of C. albicans and positively charged nanoparticles.

Therefore, in this study, C. albicans yeast was used as the model to investigate the following four aspects: (1) design and preparation of magnetic beads with concanavalinA (conA) conjugation and positive or negative surface charges and comparison of C. albicans yeast capture efficiency with these magnetic beads; (2) differences in the enrichment efficiency of fungal strains via a surface-charge-mediated way at different concentrations of magnetic beads in different media and with different initial fungal strain concentrations; (3) study of the mechanism of strong interaction between fungi yeast and the positively charged MNs through a strategy similar to free antibody competition in the cell targeting experiments; (4) stains of the enriched fungal strains with fluorescein-labeled conA to determine the availability of conA active sites on the fungal surface bound with positively charged beads, in order to explore the possibility of a sandwich-like immunoassay for fungi. Finally, the dramatic change in the T2 magnetic resonance signal from dispersed nanoparticles to aggregates adhered to the fungal surface was measured. The experimental results suggest the potential for straightforward and efficient capture of C. albicans and immunoassay through either fluorescence or a T2MR signal.

In a previous study, we demonstrated the effective binding of cancer cells, derived from unique glycolysis, with electricitymediated surface-charged MNs.10,11Several studies reported that the surface property of bacteria affected the biobehaviors and its interaction with nanoparticles of tuned shape, size, and surface charge.12−16For fungal cells, C. albicans exists in several growth states, including yeast, pseudohyphae, and hyphae.16In each state, the cell wall of C. albicans displays different surface proteins and molecular composition,17,18 which alter the surface chemistry, including cell surface hydrophobicity and surface charge. The future improved design of antifungal diagnostics and therapeutics is also affected; the frontiers of research on fungal cell walls are progressing from a descriptive phase defining the components of the fungal walls to a dynamic analysis of the assembly, cross-linking, and modification of various components in response to environmental signals.19,20 Among them, the effect of location of the glucomannan and polyglucosan on the nanoparticle−fungal yeast strains interaction and the comparison of binding efficacy and sites through either lectin−mannose affinity or electronicmediated affinity is yet to be elucidated. Lyden studied the electrostatic interaction between surface-functionalized nanoparticles and C. albicans and mutant strains deficient in various C. albicans surface proteins; however, only a robust interaction was observed between hyphae and carboxylate-functionalized nanoparticles.21 To date, only little is known about the surface charge and glucan composition-dependent interaction between fungal yeast strains and functionalized nanoparticles.

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RESULTS AND DISCUSSION Figure 1 gives a schematic illustration of composition and distribution of glycan on the surface of C. albicans yeast, and several ways for C. albicans to interact with different functionalized magnetic nanoparticles. It is proposed that the positively charged magnetic nanoparticles (PCMNs) can bind 3271

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Figure 2. TEM images of (A) NCMNs and (B) PCMNs and SEM images of (C) NCMNs and (D) PCMNs. The scale bars are all 200 nm.

Figure 3. Physiochemical properties of the surface-charged magnetic nanoparticles (MNs): dynamic size and distribution of (A) negatively charged MNs (NCMNs) and (B) positively charged MNs (PCMNs) measured by DLS instrument. (C) Zeta potential of NCMNs and PCMNs dispersed in ultrapure water. (D) Hysteresis loops of surface-charged MNs.

to a C. albicans yeast cell wall through an electrostatic attraction. (See the left part of Figure 1A.) On the contrary,

negatively charged magnetic nanoparticles (NCMNs) hardly bind to yeasts due to the electrostatic repulsion. (See the 3272

DOI: 10.1021/acsbiomaterials.9b00069 ACS Biomater. Sci. Eng. 2019, 5, 3270−3278

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Figure 4. (A) Capture efficiency of C. albicans (107 cfu/mL) in aqueous solution by different surface-charged magnetic particles and conAconjugated magnetic particles (ConA-MNs). (B) The capture efficiency of C. albicans (107 cfu/mL) in aqueous solution using different working concentrations of surface-charged MNs.

Preparation and Characterization of the ConAConjugated MNs. For specific recognition and capture of C. albicans yeast strains, the MNs were modified with conA lectin by cross-linking the N-hydroxysuccinimide (NHS) groups on the commercialized NHS-MNs surface with the amine groups of the conA. To confirm successful conjugation, FITC-conA was allowed to react with NHS-MNs under identical conditions, and the fluorescent microscopy images of the conA-modified nanoparticles were illustrated. In Figure S2A, almost all the MNs show a bright green fluorescence signal corresponding to the MNs observed in the bright field image in Figure S2B, indicating the successful conjugation of the lectin to MNs. In addition, the BCA assay characterized the amount of coupled lectin. As shown in Figure S2C, the standard curve of the BCA protein has a good linear fit relationship with the R2 value of 0.996. The amount of lectin conjugated to MNs was calculated by the OD value of the remaining lectin in the supernatant. After optimization of the reaction parameters, the amount of coupling of conA was 0.037 mg/mg of MNs. Comparison of Capture Efficiency of C. albicans Isolation by the Surface-Charged MNs and ConA Coupled MNs. Previous studies demonstrated that the fungi cell wall with dynamic structures was composed of a multilayer of polyglycans.18 In C. albicans yeast strains, the outer layer of the cell wall is mainly composed of glucomannan. The zeta potential of the C. albicans was measured to be −20 mV in deionized (DI) water by a Malvern DLS analyzer. Furthermore, to investigate whether the isolation of C. albicans can be effectuated by electrostatic interaction and the efficiency of yeast compared between the surface-charged nanoparticles and conA-conjugated nanoparticles, a simple binding and magnet-assisted isolation of the yeast was performed in DI water. An aqueous dispersion of the yeast at the original concentration was prepared. ConA-MNs, NCMNs, and PCMNs were added into the spiked fungal sample, followed by incubation under gentle agitation at room temperature for 30 min. Then, the yeast strains bound with MNs were separated and washed three times using a magnet. The resulting supernatant was collected for further characterization. The capture efficiency of C. albicans was estimated by hemocytometer. As shown in Figure 4A, at the same concentration of C. albicans (107 cfu/mL), PCMNs exhibit nearly 100% capture efficiency as compared to 18% by NCMNs, while conA-MNs present an intermediate level of 30% capture efficiency. Thus, the capture efficiency of PCMNs was 5-fold more than that of NCMNs and 3-fold more than

middle part of Figure 1A.) ConA-conjugated magnetic nanoparticles (conA-MNs) interacted with C. albicans yeast through a well-known lectin−mannan specific binding. (See the right part of Figure 1A.) For C. albicans yeast binding and isolation, the parameters that may influence the nanomaterials−fungi interaction will be investigated here. To understand the interaction mechanism and binding site between fungi and PCMNs, the isolation of C. albicans at the same incubation condition by mannose- or glucose-modified electric nanoparticles was performed. Figure 1B gives the schematic illustration of the electrostatic interaction between fungi, and PCMNs could be blocked by functionalized mannose on the surface of PCMNs. Synthesis and Characterization of the SurfaceCharged Nanoparticles. According to our previous study,11 NCMNs were synthesized through a solvothermal process. Subsequently, the NCMNs were functionalized with poly(acrylic acid) (PAA). As a result, the products exhibited a strong negative surface charge due to a large number of carboxyl groups on the surface. Furthermore, the synthesis of the positively charged composite nanoparticles and polyethylenimine (PEI, MW = 10 000) polymers was modified on the surface of NCMNs via a facile process. The surface-charged nanoparticles showed a satisfactory dispersity and spherical shape under TEM and SEM (Figure 2). The average size of both negative and positive nanoparticles is 111 nm, as observed by TEM, due to the invisible layer of the polymer molecules on the nanoparticles. The DLS data (Figure 3A,B) demonstrated that the hydrodynamic size of PCMNs is about 20 nm larger than that of NCMNs in the deionized water. Moreover, the average diameter of NCMNs is approximately 100 nm (Figure 3A), while that of PCMNs is approximately 120 nm (Figure 3B). In order to prove the successful PEI modification on the surface of NCMNs, the zeta potentials of nanoparticles were characterized (Figure 3C) as −20 and +40 mV for NCMNs and PCMNs, respectively. The reversal of the zeta potential of the negatively charged nanoparticles demonstrated the successful PEI polymer modification on the surface of NCMNs. The hydrophilicity of surface-charged nanoparticles could be seen in Figure S1. The hysteresis loops of the surface-charged MNs are shown in Figure 3D. Both NCMNs and PCMNs were characteristically superparamagnetic without the coercive force and exhibit a high magnetization of 51.5 and 48.9 emu/g, respectively. This optimal superparamagnetism of the surface-charged nanoparticles ensured a rapid response to the magnet during the separation. 3273

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nanoparticles (Figure 5B,C). For each micrometer-scaled yeast strain, the majority of the fungal cell wall is occupied by a monolayer of positively charged MNs, facilitating the isolation using a magnet. To preliminarily investigate the difference in isolation of C. albicans yeast by surface-charged MNs in the clinical sample, the mimic samples from a bronchoalveoar lavage fluid (BALF) sample of a patient with a negative diagnostic result were used. The cultured C. albicans yeasts were first spiked into the negative BALF sample and transferred to 200 μL of different reaction media with a certain ratio of BALF/water (200:0, 100:100, 0:200) in the test tubes after centrifugation at 6000 rpm for 2 min. The incubation and separation were performed with a typical procedure. Figure S4 gives the comparison of capture efficiencies of the C. albicans by two kinds of surface-charged MNs in different media. It can be seen that, in the presence of BALF, the capture efficiency of C. albicans by PCMNs rises gradually with increased water partition, while that by NCMNs decreases gradually. The biggest gap between the capture efficiency of C. albicans by two kinds of MNs occurred when the C. albicans spiked in the BALF was transferred into ultrapure water. This result indicated that the saline solution in BALF obviously influenced the isolation of C. albicans by electric nanoparticles. However, we can isolate C. albicans from BALF by positively charged MNs after a simple centrifugation and resuspension. Mechanism of Electrostatic Interaction of C. albicans with PCMNs. Previous results have shown that the positively charged MNs exhibited an overwhelming ability for the isolation of C. albicans yeast as compared to the negatively charged or conA-conjugated MNs. To identify the primary binding site of the positively charged nanoparticles to fungi yeast, two kinds of (monosaccharides) mannose- and glucosemodified PCMNs were applied to capture the C. albicans yeast. To ensure the successful modification of PCMNs by the monosaccharide, various ratios of MNs and monosaccharide input and the zeta potentials to final products were characterized. Table 1 shows the zeta potentials of the

that of conA-MNs. This phenomenon might be attributed to the highly efficient charge-mediated interaction in the isolation of C. albicans as compared to the conventional lectin−sugar recognition. Parameters for Efficient Isolation of C. albicans Yeast Strains by the Surface-Charged MNs. Several parameters affected the electrostatic interaction between the C. albicans yeast strains and surface-charged nanoparticles. First, the working concentration of the nanoparticles was studied. As shown in Figure 4B, the increasing concentration of PCMNs led to an increased capture efficiency of C. albicans; it increased until the concentration of PCMNs reached 100 μg/mL which captured 100% of C. albicans. A further increase of the PCMN concentration did not improve the capture efficiency. Also, the gap between the capture efficiency of yeast by PCMNs and NCMNs is distinct at a working concentration of 100 μg/mL (Table S1). Thus, 100 μg/mL of PCMNs was used in the subsequent experiments. Second, the effect of the dispersing medium with respect to the interaction on the capture efficiency was studied. As shown in Figure 5A, the absolute

Table 1. Zeta Potentials of PCMNs Coated with Monosaccharide of Mannose or Glucose at Various Input Ratios Figure 5. (A) Comparison of capture efficiencies of C. albicans (107 cfu/mL) by PCMNs and NCMNs in different media: ultrapure water, NaCl aqueous solution (0.9% w/w), and PBS solution. (B, C) Scanning electron microscopy images of C. albicans yeast bound with positively charged nanoparticles at a different amplification. Scale bar: (B) 4 μm, (C) 800 nm.

PCMNs/monosaccharide 1 1 1 1 1

value of the capture efficiency of yeast by PCMNs and the gap between positively and negatively charged MNs were high in DI water as compared to in the NaCl aqueous solution (0.9% w/w) and PBS. This result might be attributed to the disruption of the colloidal stability of the positively charged nanoparticles in the presence of salt. Third, the capture efficiency of the fungi samples with different yeast concentrations by surface-charged nanoparticles was investigated. In addition to107 cfu/mL, fungi in an aqueous dispersion at concentrations from 102 to 105 cfu/mL were treated by 100 μg/mL MNs. Even at the low concentration of fungi, positively charged MNs can separate the fungi yeasts efficiently (Figure S3). Furthermore, the robust interaction between the PCMNs and yeast strains was demonstrated by scanning electron microscopy images of the yeast bound with dozens of

mg/0 mg mg/50 mg mg/500 mg mg/1000 mg mg/1500 mg

mannose

glucose

40 mV 25.1 mV 10.6 mV 40 mV 2.2 mV

40 mV 29.2 mV 28.1 mV 28.7 mV 25.7 mV

PCMNs coated with mannose and glucose at various input ratios. With the increase in the amount of mannose coated on the surface of PCMNs, the zeta potentials of the final product after several washes were decreased as compared to those of pure PCMNs, indicating that mannose was successfully coated on the surface of PCMNs. The original zeta potential of the PCMNs was +40 mV. In the wide range of the ratio of the monosaccharide to PCMNs (50−1500), the zeta potential of the mannose-modified PCMNs decreased continuously from 25.1 to 2.2 mV, while that of glucose-modified PCMNs is almost consistent from 29.2 to 25.7 mV. As shown in Figure 6, the capture efficiency of C. albicans yeast strains by the glucosemodified PCMNs does not alter from that of the original PCMNs. However, the capture efficiency of C. albicans by 3274

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fluorescence dye, which is critical for developing a practical detection method. Although the above results confirmed the ability of the PCMNs in the isolation, detection, or analysis of C. albicans, the feasibility of an immunostaining assay of the captured yeast strains by the fluorescence dye-labeled lectin is also essential. To this aim, FITC-conA was used to stain the yeast after separation by different kinds of magnetic nanoparticles. Figure S5 provides the bright field and fluorescent image of the C. albicans successfully labeled by FITC-conA, indicating the possibility of immunoassay of captured yeast by the FITC-conA. In addition, the cellular viability of C. albicans bound with positively charged nanoparticles was studied. It s found that the survival rate of C. albicans was 75.4% even when the material concentration was as high as 200 μg/mL. The viability of C. albicans is 85.4% for the typical working concentration of the PCMNs in this study (Figure S6). The data proves that the magnetic nanoparticles have excellent biocompatibility for fungi detection. Figure 7 shows the paired fluorescent microscopy and bright field images of the magnetically separated C. albicans yeast stained with FITCconA. Also, schematic illustrations of the binding status of MNs and the corresponding FITC-conA are provided below the images. Figure 7A,B demonstrates the fluorescence and bright field images of yeast cells treated with PCMNs and stained with FITC-conA. The uniform layer of MNs surrounding the yeast cell does not repel a distinct fluorescence

Figure 6. Comparison of the capture efficiency of C. albicans yeast (107 cfu/mL) by mannose- and glucose-modified PCMNs using various nanoparticle to monosaccharide ratios (w/w).

mannose-modified PCMNs declines dramatically; especially at the ratio of 1:1500, only 1/8th of the capture efficiency is remaining. These results implied that the positively charged nanoparticles interacted with C. albicans through the mannan component of the cell wall instead of the glucans, which was similar to that reported previously.18 Isolation and Immunostaining of C. albicans Yeast by Nanomaterials and FITC-ConA. After isolation, the yeast is detected for the remaining site for binding of the tag, such as

Figure 7. Fluorescence microscopy and bright field images of captured C. albicans yeast by different MNs and subsequently stained by FITC-conA: (A, B) PCMNs, (C, D) NCMNs, (E, F) conA-MNs, and (G, H) fluorescence microscopy and bright field images of pure PCMNs treated by FITCconA in the same condition. The schematic illustrations of the simultaneous binding of yeast strains by the different functionalized magnetic nanoparticles and FITC-conA are indicated by arrows. The scale bars are all 20 μm. 3275

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Table 2. T2 Magnetic Resonance Signal of Pure PCMNs Dispersion, PCMNs Bound C. albicans Dispersion (Fungi/PCMNs), NCMNs Dispersion, and NCMNs Bound C. albicans Dispersion (Fungi/NCMNs)a T2 (ms) PCMNs fungi/PCMNs (107 cfu/mL) NCMNs fungi/NCMNs (107 cfu/mL)

10.81 65.22 7.31 12.03

12.01 54.52 7.27 8.45

11.81 70.20 7.54 8.11

mean T2 (ms)

CV (%)

11.54 63.31 7.37 9.53

5 10 2 19

ΔT2 (%) 448.48 29.25

a

The concentration of fungi is 107 cfu/mL.

to the mannosan moiety on the yeast cell wall. However, the branched structure of the mannose layer on C. albicans facilitated the efficient isolation as well as sequential fluorescence labeling of the fungi. Furthermore, the T2 magnetic resonance signals of positively charged magnetic nanoparticles were found to change dramatically due to their strong electrostatic interaction with C. albicans strains. Taken together, this strategy could serve as a potential approach for effective detection of fungi.

signal of FITC on the yeast via the conA moiety. Thus, it was proposed that, due to the branched structure of the glucomannan layer on the yeast cell wall, several mannan residues could be reached by the conA molecules. Conversely, the cell wall of the C. albicans yeast strains was isolated during the rapid magnetic separation of NCMNs as the NCMNs seldom adhere to the yeast (Figure 7D). Consequently, the strongest FITC fluorescence signal arises from the yeast strains due to quintessential adhesion of the nanoparticles (Figure 7C). The quantitative data of the fluorescence intensity upon area of the yeast strain was analyzed using ImageJ software. If the fluorescence intensity of the yeast strain in Figure 7C was set at the standard of 1.0, the relative fluorescence intensities of the PCMN-treated yeast and conA-MN-treated yeast are about 0.65 and 0.74, respectively (Figure 7A,E). Figure 7G,H shows the noise of the fluorescence signal present on the surface of pure PCMNs. Moreover, the nonspecific adsorption of FITCconA with positively charged MNs was negligible. Besides the semiquantitative results from the fluorescence signal, the T2 magnetic resonance signal of magnetic nanoparticles before and after binding to C. albicans yeast was measured using a nuclear magnetic resonance analyzer (minispec mq60, Bruker, USA). The results based on positively and negatively charged magnetic nanoparticles are shown in Table 2. The big variation of the T2 magnetic resonance signal is obtained only in the group of positively charged nanoparticles, because they bind to C. albicans strains. It has been reported that the variation of the T2 magnetic resonance signal showed good potential for accurate detection of fungi aided with DNA hybridization.22 So, after fungi capture, both fluorescence labeling and the T2 signal were proven to be practical ways to detect fungi. The study of sensitive detection of C. albicans with this strategy is still underway.

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MATERIALS AND METHODS

Materials. Polyacrylic acid (PAA, MW = 3000), mannose, and glucose were purchased from Aladdin. Branched polyethylenimine (PEI, MW = 10 000) was purchased from Alfa Aesar. Concanavalin A (ConA), FITC-labeled conA (FITC-conA), ferric trichloride hexahydrate (FeCl3·6H2O), urea, methanol, ethylene glycol, ethanol, potassium chloride (KCl), glycerol, and ethanolamine were purchased from Sinopharm Chemical Reagent Co., Ltd. 4-(2-Hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), yeast extract peptone dextrose (YPD), and ethylenediaminetetraacetic acid (EDTA) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. NHS-magnetic beads for conA conjugation were purchased from Tamagawa Seiki Co., Ltd. The BCA kit was purchased from Beyotime. The CCK-8 kit was purchased from Donjingdo Molecular Technologies, Inc. Synthesis of NCMNs, PCMNs, and ConA-Conjugated MNs. The superparamagnetic monodispersed Fe3O4 nanoparticles were synthesized by the solvothermal method according to our previous study.11 Briefly, NCMNs were synthesized by the following experimental procedure: 0.81 g of iron trichloride hexahydrate and 1.8 g of urea were added to 30 mL of ethylene glycol, followed by sonication for 10 min. Then, 0.29 g of PAA was added to the ethylene glycol. After magnetic stirring for 30 min, 1 mL of ultrapure water was added, and after magnetic stirring for 3 min, the mixture was transferred to a reaction kettle and placed in an oven at 200 °C for 12 h. Subsequently, the product was washed three times with ethanol and DI water, respectively, using a magnet, and the concentration of the product was quantified by vacuum drying of an aliquot of dispersion and weighing. Then, the product was sealed and stored at room temperature. PCMNs were synthesized by the following experimental procedure: 20 mg of NCMNs was added to 25 g of methanol under ultrasonic agitation. After stirring for 10 min, 20 mg of PEI was added under mechanical stirring for 2 h at 350 rpm. Subsequently, the product was washed three times with methanol and DI water, respectively, using a magnet, and the concentration of the product was quantified by vacuum drying of an aliquot of dispersion and weighing. Here, conA-MNs were synthesized by conjugation of conA protein to NHS-group-functionalized magnetic beads. Typically, 1 mg/mL of conA protein immobilization buffer was prepared by dissolving conA protein in the HEPES buffer (25 mM HEPES−NaOH, pH = 7.9). A 1 mg portion of magnetic beads was dispersed in 50 μL of protein immobilization buffer and 50 μL of HEPES buffer. The conjugation reaction lasted for 30 min at 4 °C in an end-over-end shaker. The excess protein was removed from the magnetic beads by centrifugation at 15 000 rpm for 5 min at 4 °C. Then, 250 μL of 1 M ethanolamine (pH = 8) was added to block the excess carboxyl

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CONCLUSIONS In summary, we successfully developed surface-charged superparamagnetic nanoparticles for the facile and efficient enrichment of C. albicans yeast strains in the pure medium. Also, the putative mechanism and potential application for rapid detection of the yeast with the help of a fluorescence probe were studied. Serial comparisons of the capture efficiency of C. albicans by the positively charged, negatively charged, and conA-conjugated MNs were performed in different conditions involving several parameters to detect the optimal binding condition. In the range 102−107 cfu/mL initial yeast concentrations, the positively charged magnetic nanoparticles exhibited better yeast isolation efficiency. The underlying mechanism for the high binding ability of the positively charged nanoparticles to C. albicans yeast and the binding site was explored by control experiments performed using monosaccharide-treated PCMNs based on the composition of the yeast cell wall. Notably, both the surface chargemediated interaction and lectin-dependent binding are related 3276

DOI: 10.1021/acsbiomaterials.9b00069 ACS Biomater. Sci. Eng. 2019, 5, 3270−3278

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ACS Biomaterials Science & Engineering groups and reacted in a refrigerator at 4 °C for 16 h. To calculate the conjugating efficacy of conA, the original and remaining amounts of conA in the process were measured by the BCA kit. The resulting product was washed with wash/storage buffer (10 mM HEPESNaOH, pH = 7.9, 50 mM KCl, 1 mM EDTA, 10% glycerol) three times with a magnet. To directly display the successful conjugation, FITC-labeled conA (FITC-conA) was used to replace the conA to react to the MNs under the same condition. Then, the product was sealed and stored in a refrigerator at 4 °C. C. albicans Culture. C. albicans (SC5314/ATCC MYA-2876) was cultured in 1 mL of YPD solution at 30 °C for 18 h in a shaking incubator. Colonies were picked and dispersed in sterile water to obtain a C. albicans suspension. The cell number of the C. albicans was determined using a hemocytometer in a 10 μL suspension under the microscope at 20× magnification. All experiments utilizing viable pathogens were carried out by trained personnel in a Biosafety Level II Laboratory. Isolation of C. albicans by Magnetic Nanoparticles in Ultrapure Water. A specific amount of MNs was added to 200 μL of C. albicans suspension. After incubation for 10 min at room temperature, the yeasts combined with MNs were isolated by a magnet and washed with sterile water three times before further treatment. The number of fungal yeasts captured (Nc) could be calculated by the number of cells in the supernatant (Ns). If the total number of cells added initially was expressed as N0, the capture rate could be expressed by the formula

nuclear magnetic tube, and T2 magnetic resonance data was tested by the Bruker mq60 NMR analyzer. The original surface-charged magnetic nanoparticles were used as a control.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.9b00069. Table of capture efficiency ratios and figures including photographs, fluorescence microscopy images, capture efficiency comparisons, and cellular viabilities (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yilong Wang: 0000-0001-7483-9296 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants from the National Natural Science Foundation of China (31571018, 51573047) and the Fundamental Research Funds for the Central Universities (22120170166). Partial fund of State Key Laboratory of Molecular Engineering of Polymers of Fudan University.

capture efficiency = (N0 − Ns)/N0 × 100% In order to reduce the error, three sets of parallel samples in each set of experiments were conducted. Isolation of C. albicans by Magnetic Nanoparticles in Mimic BALF Sample. The Candida albicans sample was initially shaken for 18 h in a YPD culture medium and then was spiked into the negative BALF after centrifugation at 6000 rpm for 2 min. All experiments were performed in accordance with the Guidelines of “Management Measures for Medical Science and Technology Research Involving Human Subjects”, and experiments were approved by the ethics committee at Tongji University. Informed consents were obtained from human participants of this study. Candida albicans spiked in the BALF, and surface-charged magnetic nanoparticles were added to 200 μL of different reaction media with a certain ratio of BALF/water (200:0, 100:100, 0:200) in the test tubes. After incubation for 10 min at room temperature, the yeasts bound with MNs were separated by a magnet and washed with sterile water three times. The capture efficiency of fungal yeasts was calculated similarly to the method described above. Fluorescence Assay. In the sandwich-immunoassay procedure, C. albicans yeast was first bound by three kinds of MNs under a condition similar to that described above and isolated with the help of a magnet. Then, the isolated yeasts through different interaction mechanisms shown in Figure 1A were stained by an FITC-conA aqueous solution (500 μg/mL) at room temperature for 60 min. Subsequently, the product was washed with sterile water three times with the help of a magnet. Finally, C. albicans was resuspended in sterile water, and the complexes were observed by a fluorescence microscope. CCK-8 Assay. First, 107 cfu/mL of C. albicans was added into a 96well plate, and a certain concentration of PCMNs was added into the plate. The volume of the solution was 200 μL. The control group is pure 107 cfu/mL of C. albicans without PCMNs. Then, the plate was placed in a fungal incubator at 30 °C for 6 h. After that, 10 μL of CCK-8 detection solution was added into each well, and then the plate was placed in an incubator at 30 °C for 6 h. The result was obtained by using a microplate reader under 450 nm irradiation. T2 Magnetic Resonance Assay. C. albicans strains were captured by positively charged and negatively charged magnetic nanoparticles using the same method, respectively. The C. albicans species bound with positively charged and negatively charged magnetic nanoparticles were resuspended with 200 μL of sterile water, separately. After that, the dispersion was transferred into a

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DOI: 10.1021/acsbiomaterials.9b00069 ACS Biomater. Sci. Eng. 2019, 5, 3270−3278