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Fluorescent Immunoassay for the Detection of Pathogenic Bacteria at the Single-Cell Level Using Carbon DotsEncapsulated Breakable Organosilica Nanocapsule as Labels Lu Yang, Wenfang Deng, Chang Cheng, Yueming Tan, Qingji Xie, and Shouzhuo Yao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18714 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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ACS Applied Materials & Interfaces

Fluorescent Immunoassay for the Detection of Pathogenic Bacteria at the Single-Cell Level Using Carbon Dots-Encapsulated Breakable Organosilica Nanocapsule as Labels Lu Yang, Wenfang Deng,* Chang Cheng, Yueming Tan,* Qingji Xie, and Shouzhuo Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China

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KEYWORDS: breakable organosilica nanocapsule, carbon dot, immunoassay, pathogenic bacteria, Staphylococcus aureus.

ABSTRACT: Herein, carbon dots (CDs)-encapsulated breakable organosilica nanocapsules (BONs) were facilely prepared and used as advanced fluorescent labels for ultrasensitive detection of Staphylococcus aureus (S. aureus). CDs were entrapped in organosilica shells by cohydrolyzation of tetraethylorthosilicate and bis[3-(triethoxysilyl)propyl]disulfide to form coreshell CDs@BONs, where hundreds of CDs were encapsulated in each nanocapsule. Immunofluorescent nanocapsules, i.e. anti-S. aureus antibody-conjugated CDs@BONs， were prepared to specifically recognize S. aureus. Before fluorescent detection, CDs were released from the BONs by simple NaBH4 reduction. The fluorescent signals were amplified by two orders of magnitude because of hundreds of CDs encapsulated in each nanocapsule, compared with a conventional immunoassay using CDs as fluorescent labels. A linear range was obtained at the S. aureus concentration from 1 to 200 CFU mL-1. CDs@BONs are also expected to expand to other systems and allow the detection of ultralow concentrations of targets.


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1. INTRODUCTION Rapid and sensitive detection of pathogenic bacteria is significant since pathogenic bacteria can cause a serious health risk.1-4 Culture-based methods are accurate but time-consuming and cannot provide on-site feedback for detection of pathogenic bacteria.5 Polymerase chain reaction amplification and enzyme-linked immunosorbent assay possess high specificity and relatively low detection limits, but the complicated procedures and high cost still retard their widespread applications in point-of-care detection.6-8 To address the issues of conventional methods, electrochemical, colorimetric, chemiluminescence, and fluorescence detection of pathogenic bacteria have been developed by utilizing biological recognition molecules (e.g. antibody) to specifically recognize bacterial cells.9-17 Fluorescent detection of pathogenic bacteria has advantages of good selectivity, high sensitivity, fast response, and simplicity.18,

19

Recently, fluorescence dyes, semi-conducted

quantum dots, upconversion nanoparticles, and carbon dots (CDs) have been widely used as fluorescence labels for rapid detection of pathogenic bacteria.11, 16, 18, 20-23 CDs have recently arisen as a new class of fluorescence nanomaterial in biosensing and bioimaging, owing to their intriguing fluorescence properties and good biocompatibility.24 For instance, CDs have been used as fluorescence labels to detect Staphylococcus aureus (S. aureus), showing a detection limit of 9.40 ×104 CFU mL-1.25 Despite the great progress in fluorescence detection of pathogenic bacteria, rapid fluorescent detection of pathogenic bacteria at the single-cell level is still challenging at present. Therefore, it is important to efficiently amplify the fluorescence signal for improving the sensitivity and reducing the detection limit in point-of-care detection. Various nanocapsules including liposomes, polymers and inorganic nanostructures have been widely used for drug/gene delivery, bioimaging, catalysis, and biosensing.26, 27 Especially, silica


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nanocapsules have received great attention, because they can be synthesized under mild reaction conditions and can offer strong barrier to the leakage of the encapsulated nanoparticles and molecules.28-30 For instance, organosilica nanocapsules were used for the delivery of native proteins into in cancer cells.29 However, to the best of our knowledge, fluorescent nanoparticles encapsulated in organosilica nanocapsules have not been prepared and used for rapid and ultrasensitive detection of pathogenic bacteria up to now. Immunomagnetic nanoparticles can specifically capture and recognize targets from complex samples, and avoid complicated pretreatment procedures, so immunomagnetic nanoparticles have been widely used for biosensing.9, 11, 31 Inspired by the above-mentioned points, we will introduce a novel fluorescent immunoassay for the detection of pathogenic bacteria by coupling the nanocapsule-amplification strategy with immunological recognition and magnetic separation. Herein, CDs-encapsulated breakable organosilica nanocapsules (BONs) were prepared and used as advanced fluorescent labels for rapid detection of S. aureus at the single-cell level. CDs were encapsulated in BONs to form core-shell CDs@BONs, where hundreds of CDs are encapsulated in each nanocapsule. To specifically recognize bacterial cells via antibody-antigen interaction, immunofluorescent nanocapsules were prepared by modification of anti-S. aureus antibodies on the surfaces of CDs@BONs. As illustrated in Scheme 1a, immunofluorescent nanocapsules and immunomagnetic nanoparticles specifically captured and recognized S. aureus simultaneously. After magnetic separation and NaBH4 reduction, CDs were released from the BONs and then fluorescent detection was carried out (Scheme 1a). Compared with the conventional fluorescence immunoassay, where only a few fluorescent nanoparticles were linked to each bacterial cell (Scheme 1b), and the immunofluorescent nanocapsules enable significant amplification of the fluorescence signals because of hundreds of CDs encapsulated in each


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nanocapsule. As a result, the sensitivity of this fluorescence immunoassay was boosted by two orders of magnitude, compared with the conventional method using CDs as fluorescence labels.


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2. EXPERIMENTAL SECTION Preparation of CDs. CDs were synthesized following the reported synthesis strategy.32 Briefly, 7.0 mL of ethylenediamine and 20 g of citric acid were dissolved in 200 mL of ultrapure water. The solution was hydrothermally treated at 200 oC for 5 h in a 500 mL Teflon-lined autoclave. After natural cooling to room temperature, dialysis was carried out to purify the CDs. Preparation and Surface amination of CDs@BONs. 17 mL of Triton X-100 and 18 mL of n-hexanol were dissolved in 75 mL of cyclohexane to form solution A. 3 mL of aqueous dispersion of CDs (0.45 µg mL-1) was mixed with 0.40 mL of tetraethyl orthosilicate and 0.9 mL bis[3-(triethoxysilyl)propyl]disulphide to form solution B. Then solution A was mixed with solution B, and 0.5 mL of 30% aqueous ammonia solution was added to this mixture. After stirred for 20 h at 26 oC, CDs@BONs were obtained. For surface amination of CDs@BONs, 0.5 mL of 3-aminopropyl triethoxysilane was added to the above mixture. After reacting for 12 h, 200 mL of acetone was subsequently added to precipitate the amino-functionalized CDs@BONs (CDs@NH2-BONs). Finally, the CDs@NH2BONs collected by centrifugation was washed with ethanol and water, and dried in a freeze dryer until dried. Preparation of immunofluorescent nanocapsules and immunomagnetic nanoparticles. Immunofluorescent

nanocapsules

were

prepared

as

follows.

480

mg

N-(3-

(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride and 72 mg N-hydroxysuccinimide were added in 20 mL of aqueous dispersion of CDs@NH2-BONs (1 mg mL-1) with vigorous shaking. Then, 500 µL of 0.1 mg mL-1 anti-S. aureus antibody was added into the above dispersion with vigorous shaking for 4 h at room temperature. The mixture was centrifuged and washed with 0.1 M phosphate buffer solution (PBS, pH 7.4) to remove the excess reagents, and


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then re-dispersed in 20 mL of 0.1 M PBS. The final product was blocked with 1 mL of PBS containing 1 wt.% bovine serum albumin at room temperature with gentle agitation for 30 min, then centrifuged, re-dispersed in 0.1 M PBS, and stored at 4 °C for future use. Immunomagnetic nanoparticles were prepared similarly to immunofluorescent nanocapsules except that aminofunctionalized Fe3O4 nanoparticles were used instead of CDs@NH2-BONs. Procedures for the detection of S. aureus. 400 µL of 0.1 M PBS containing 1 mg mL-1 immunofluorescent nanocapsules was first mixed with 400 µL of 0.1 M PBS containing 0.5 mg mL-1 immunomagnetic nanoparticles, and then the mixture was incubated with 200 µL of 0.1 M PBS containing different concentration of S. aureus cells (0, 5, 25, 150, 300, 450, 750, 1000, 1250 CFU mL-1) for 60 min at 37 oC. Thus the final concentrations of S. aureus cells in the PBS are 0, 1, 5, 30, 60, 90, 150, 200, 250 CFU mL-1, respectively. Then magnetic separation was carried out, and the product collected by a controlled magnetic field was re-dispersed in 1 mL 0.1 M PBS (pH 7.4). Subsequently, 10 µL of 100 mg mL-1 NaBH4 solution was added into the above solution to release CDs. After reacting for 30 min, the fluorescence spectrum of the obtained solution was recorded at the excitation wavelength of 353 nm. The detection of S. aureus in milk and orange juice (from the local market) was also carried out. The impurities in milk and orange juice were separated by centrifugation (3000 rpm, 5 min). Then the orange juice and milk after centrifugation were diluted 10 times. The cultured S. aureus cells were spiked into the diluted orange juice and milk with final concentrations of 20 and 50 CFU mL-1. Ultimately, the prepared samples were used for bacteria detection based on the general procedure without special treatment. The traditional plate-counting method for assaying the bacteria supplemented in orange juice and milk served as the controls.


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3. RESULTS AND DISCUSSION The size and morphology of CDs were characterized by transmission electron microscopy (TEM). As shown in Figure 1a-c, CDs are spherical and monodisperse with an average diameter of 3 nm. Figure S1 shows Fourier transform infrared (FT-IR) spectrum of CDs. Three vibrational absorption peaks at 1578, 1635, and 3452 cm-1 are observed, which are related to bending vibrations of N-H, bending vibrations of C=O, and stretching vibrations to O-H, respectively. Figure S2 shows ultraviolet-visible (UV-Vis) absorption spectrum of CDs. Two absorption peaks at 239 and 339 nm are observed, which are assigned to π-π* transition of C=C and n-π* transition of C=O, respectively.33 The fluorescent spectra in Figure 1d indicate that CDs have an optimal excitation wavelength at 353 nm and an optimal emission wavelength at 446 nm. As shown in Figure S3, at the excitation wavelength of 353 nm, the fluorescent intensity at 446 nm is proportional to the concentration of CDs in the range of 0.03 - 0.47 µg mL1

. Despite the high fluorescence response at higher pH (Figure S4), the following experiments

were carried out at pH 7.4 due to the convenience in detecting S. aureus at physiological pH. CDs were entrapped in organosilica shells to form CDs@BONs. Figure S5 illustrates the synthesis pathway of CDs@BONs and the structure of organosilica shells. Organosilica shells were

prepared

by

co-hydrolyzation

of

tetraethylorthosilicate

and

bis[3-

(triethoxysilyl)propyl]disulfide. The morphology of CDs@BONs was characterized by scanning electron microscopy (SEM) and TEM. The SEM and TEM images in Figure 2a-c indicate that the nanoparticles are monodisperse and spherical with an average diameter of 80 nm. The high resolution TEM (HRTEM) image in Figure 2d clearly reveals that hundreds of CDs were encapsulated in the organosilica shells.


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To enable surface modification of antibodies on the CDs@BONs, the nanocapsules were subjected to surface amination. Figure 3a shows FTIR spectra of CDs@BONs before and after surface amination. The CDs@BONs before surface amination show vibrational stretching peaks of Si-O at 1109, 798, and 478 cm-1 and the band of C-S at 698 cm-1.34, 35 After surface amination, two peaks at 1587 and 3434 cm-1 in the FTIR spectrum are observed, which are associated with bending vibrations and stretching vibrations of amino groups, respectively. Hence, amino groups are successfully modified on the surfaces of CDs@BONs. Figure S6 shows SEM image, FT-IR spectrum, and magnetic hysteresis loop of aminofunctionalized Fe3O4 nanoparticles. Spherical nanoparticles with a diameter of 100 - 200 nm are observed (Figure S6a). A sharp stretching vibration peak at 3426 cm-1 is observed (Figure S6b), confirming the presence of –NH2 on the surfaces of Fe3O4 nanoparticles. The magnetic hysteresis curve (Figure S6c) confirms that these nanoparticles have an excellent superparamagnetic property at room temperature. To specifically capture and recognize S. aureus, immunofluorescent nanocapsules and immunomagnetic nanoparticles were prepared by modification of anti-S. aureus antibodies on the surfaces of CDs@NH2-BONs and amino-functionalized Fe3O4 nanoparticles, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as a molecular weightdependent method was carried out to confirm the successful modification of anti-S. aureus antibodies on the surfaces of various samples.36-38 As shown in Figure 3b, a visible band appears at approximately 50-60 kDa for anti-S. aureus antibodies alone. Obvious retarded electrophoretic mobility is observed for nanocapsule-antibody, Fe3O4-antibody, and CD-antibody conjugates, confirming the successful modification of anti-S. aureus antibodies on the surfaces of various samples.


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The binding of immunofluorescent nanocapsules with S. aureus was studied by SEM. As show in Figure 4, a large number of immunofluorescent nanocapsules linked to S. aureus cell can be observed, confirming that immunofluorescent nanocapsules can efficiently recognize S. aureus. As reported previously, fluorescent nanoparticles encapsulated in the nanocapsules can quench their neighboring fluorescent nanoparticles, resulting in weakened fluorescent signal.39,

40

To

achieve the optimal sensing response, the immunofluorescent nanocapsules should be destructed by NaBH4 reduction and CDs are released from the nanocapsules before fluorescent detection. Because bispropyldisulfide groups are contained in the organosilica shells (Figure S5), the organosilica shells can be facilely destructed by NaBH4 reduction,29 and CDs can be released from the nanocapsules. As shown in Figure 5a, after NaBH4 reduction for 30 min, the nanocapsules are totally destructed, and CDs are released from the nanocapsules. As shown in Figure 5b, fluorescence intensity of immunofluorescent nanocapsules in 0.10 M PBS (pH 7.4) at 446 nm increases 2.1 times after NaBH4 reduction. To obtain the optimal release efficiency, NaBH4 concentration and reaction time were optimized. As shown in Figure S7, the optimal NaBH4 concentration and reaction time are 1 mg mL-1 and 30 min, respectively. By measuring the fluorescence intensity of the released CDs from immunofluorescent nanocapsules by NaBH4 reduction, we found that 1 mg immunofluorescent nanocapules contain ca. 0.45 µg CDs. Quantitative detection of S. aureus in PBS (pH 7.4) was carried out. As illustrated in Scheme 1a, S. aureus cells were incubated simultaneously with immunofluorescent nanocapsules and immunomagnetic nanoparticles in PBS. The weight ratio of immunofluorescent nanocapsules and immunomagnetic nanoparticles was optimized to be 4:1 (Figure S8), and the optimal incubation time is 60 min (Figure S9). After magnetic separation and NaBH4 reduction, CDs were released from the nanocapsules and then fluorescent detection was carried out. For


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comparison, a conventional immunoassay of S. aureus using antibody-conjugated CDs as labels was also carried out (Scheme 1b). Figure 6a shows fluorescence spectra for the determination of S. aureus using immunofluorescent nanocapsules as fluorescent labels. As shown in Figure S10, even at a S. aureus concentration as low as 1 and 5 CFU mL-1, obvious fluorescent signals can be observed. In our experiment, the number of immunofluorescent nanocapsules is far more than the number of S. aureus cells, so the number of immunofluorescent nanocapsules linked to each bacterial cell is comparable. As a result, the fluorescent intensity increases with the increase of S. aureus concentration (Figure 6a). Note here that the fluorescent emission of S. aureus at the excitation wavelength of 353 nm can be ignored (Figure S11), comparing with the high fluorescent response for the determination of S. aureus using immunofluorescent nanocapsules as fluorescent labels. Figure 6b shows fluorescent intensity at 446 nm as functions of S. aureus concentration. The linear range for S. aureus detection is from 1 to 200 CFU mL-1, with a linear regression equation of y=94.6+25.7x (R2=0.9980). The detection limit of our fluorescent sensor for S. aureus is much lower than most reported pathogen sensors (Table S1). A conventional immunoassay of S. aureus was also carried out using antibody-conjugated CDs as labels (see additional experimental section in the Supporting information for details). Briefly, the detection procedures of the conventional immunoassay were similar to those of the proposed immunoassay using immunofluorescent nanocapsules as fluorescent labels except that antibody-conjugated CDs were used instead of immunofluorescent nanocapsules. Note here that the concentration of antibody-conjugated CDs was optimized to be 0.45 µg mL-1 (Figure S12). As shown in Figure 6c and d, the linear range for S. aureus detection is from 1×102 to 1×104 CFU mL-1, with a detection limit of 30 CFU mL-1 (S/N=3) and a linear regression equation of y=104+0.239x


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(R2=0.9961). The sensitivity (slope of the regression curve) of the proposed immunoassay using immunofluorescent nanocapsules as labels is 108 times that of the conventional immunoassay using antibody-conjugated CDs as labels. Compared with the conventional fluorescence immunoassay using CDs as labels, more CDs can be linked to each bacterial cell by using the immunofluorescent nanocapsules as labels, resulting in the greatly improved sensitivity. The selectivity of the proposed immunoassay was studied. As shown in Figure 7, compared with the high fluorescent signal for the detection of S. aureus, the fluorescent signals for E. coli O157:H7, E. coli K12, Salmonella, and L. monocytogenes are negligible. The good selectivity can be attributed to the high immunoaffinity between the anti-S. aureus antibodies and surface antigens of S. aureus cells. The binding of immunofluorescent nanocapsules with S. aureus cells in milk (10 times diluted) was studied by fluorescence microscopy. Experimentally, immunofluorescent nanocapsules and immunomagnetic nanoparticles specifically captured and recognized S. aureus simultaneously, and then S. aureus cells collected by a controlled magnetic field were used for fluorescence microscopy image study. As shown in Figure 8, S. aureus cells linked with a large number of immunofluorescent nanocapsules can be observed, implying the application potential of the proposed immunoassay. The fluorescent immunoassay of S. aureus in milk and orange juice was carried out. As listed in Table S2, the recoveries are between 95% and 105%. Thus we can conclude that the detection of S. aureus in real samples using immunofluorescent nanocapsules is feasible.

4. CONCLUSIONS


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In summary, novel CDs@BONs were prepard by co-hydrolyzation of tetraethylorthosilicate and bis[3-(triethoxysilyl)propyl]disulfide in the presence of CDs. By using CDs@BONs as fluorescent labels, a novel signal-amplified strategy was developed for fluorescent detection of S. aureus. By coupling the nanocapsule-amplification strategy with immunological recognition and magnetic separation, a rapid, ultrasensitive, and selective method for fluorescent detection of S. aureus was developed. The sensitivity of the proposed immunoassay using immunofluorescent nanocapsules as labels was 108 times that of the conventional immunoassay using carbon dots as labels. This work provides a potential method for highly sensitive fluorescent detection of pathogen bacteria at point-of-care.

ASSOCIATED CONTENT Supporting Information The following files are available free of charge. Additional experimental description, FT-IR spectrum of CDs (Figure S1), UV-Vis absorption spectrum of CDs (Figure S2), fluorescent intensity as functions of the concentration of carbon dots (Figure S3), effect of pH on the fluorescence intensity of CDs (Figure S4), schematic illustration of the synthesis and structure of the CDs@BONs (Figure S5), Characterizations of amino-functionalized Fe3O4 nanoparticles (Figure S6), optimization of NaBH4 concentration and reaction time (Figure S7), optimization of the weight ratio of immunofluorescent nanocapsules and immunomagnetic nanoparticles (Figure S8), optimization of incubation time (Figure S9), Fluorescence spectra for the determination of low concentration of S. aureus using


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immunofluorescent nanocapsules as fluorescent labels (Figure S10), the fluorescent emission of S. aureus at the excitation wavelength of 353 nm (Figure S11), effect of the concentration of antibody-conjugated CDs on the fluorescent intensity for the detection S. aureus (Figure S12),

performance comparison (Table S1), detection results in real samples (Table S2).

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] * E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21705045, 21305041 and 21475041), the Hunan Provincial Innovation Foundation for Postgraduate (CX2016B167), the Scientific Research Fund of Hunan Provincial Education Department (17A125), Science and Technology Project of Changsha (KQ1707009), and Program for Excellent Talents in Hunan Normal University (ET1503).

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Scheme 1. Illustration of the detection of pathogen bacteria with the proposed method (a) and conventional method (b).


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Figure 1. TEM image (a), HRTEM image (b), and the corresponding size distribution (c) of CDs. Fluorescence spectra (d) of 0.3 µg mL-1 CDs in 0.10 M PBS (pH 7.4).


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Figure 2. SEM image (a), TEM image (b), size distribution (c), and HRTEM image (d) of CDs@BONs.


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Figure 3. (a) FTIR spectra of CDs@BONs before (black curve) and after (red curve) surface amination. (b) SDS-PAGE for various nanoparticles conjugated with antibodies. Lane 1: 120 kDa protein marker; Lane 2: anti-S. aureus antibody; Lane 3: immunofluorescent nanocapsules; Lane 4: immunomagnetic nanoparticles; Lane 5: antibody-conjugated CDs.


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Figure 4. SEM images of S. aureus cell before (a) and after (b) binding with immunofluorescent nanocapsules.


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Figure 5. (a) TEM image of CDs released from immunofluorescent nanocapsules. (b) Fluorescence spectra of immunofluorescent nanocapsules in 0.10 M PBS (pH 7.4) before and after release.


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Figure 6. Fluorescence spectra (a) and standard curve (b) for the determination of S. aureus using immunofluorescent nanocapsules as fluorescent labels. Fluorescence spectra (c) and standard curve (d) for the determination of S. aureus using antibody-conjugated CDs as fluorescent labels.


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Figure 7. Fluorescent intensities at 446 nm for the detection of S. aureus, E. coli O157:H7, E. coli K12, Salmonella, and L. monocytogenes (200 CFU mL-1 for each).


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Figure 8. Fluorescence microscopic images of S. aureus cells linked with immunofluorescent nanocapsules.


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Fluorescent Immunoassay for the Detection of Pathogenic Bacteria at

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