Fluorescent Immunoassay for the Detection of Pathogenic Bacteria at

Research Article www.acsami.org

Cite This: ACS Appl. Mater. Interfaces 2018, 10, 3441−3448

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 S Supporting Information *

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. The CDs were entrapped in organosilica shells by cohydrolyzation of tetraethyl orthosilicate and bis[3-(triethoxysilyl)propyl]disulfide to form core− shell 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 2 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. KEYWORDS: breakable organosilica nanocapsule, carbon dot, immunoassay, pathogenic bacteria, Staphylococcus aureus

1. INTRODUCTION Rapid and sensitive detection of pathogenic bacteria is significant because pathogenic bacteria can cause a serious health risk.1−4 Culture-based methods are accurate but time-consuming and cannot provide the on-site feedback for the 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 the 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, semiconducted quantum dots, upconversion nanoparticles, and carbon dots (CDs) have been widely used as fluorescence labels for the 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, 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 © 2018 American Chemical Society

level is still challenging. Therefore, it is important to efficiently amplify the fluorescence signal for improving the sensitivity and reducing the detection limit in the 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 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 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. Received: December 8, 2017 Accepted: January 4, 2018 Published: January 4, 2018 3441

DOI: 10.1021/acsami.7b18714 ACS Appl. Mater. Interfaces 2018, 10, 3441−3448

Research Article

ACS Applied Materials & Interfaces

Scheme 1. Illustration of the Detection of Pathogen Bacteria with the Proposed Method (a) and Conventional Method (b)

Figure 1. TEM image (a), high-resolution TEM (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).

particles specifically captured and recognized S. aureus simultaneously. After magnetic separation and NaBH4 reduction, the 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), the immunofluorescent nanocapsules enable significant amplification of the fluorescence signals because of hundreds of CDs encapsulated in each nanocapsule. As a result, the sensitivity of this fluorescence immunoassay was boosted by

Herein, CDs-encapsulated breakable organosilica nanocapsules (BONs) were prepared and used as advanced fluorescent labels for the rapid detection of S. aureus at the single-cell level. The 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 the modification of anti-S. aureus antibodies on the surfaces of CDs@BONs. As illustrated in Scheme 1a, immunofluorescent nanocapsules and immunomagnetic nano3442


Research Article


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

Figure 3. (a) FTIR spectra of CDs@BONs before (black curve) and after (red curve) surface amination. (b) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (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. solution A was mixed with solution B and 0.5 mL of 30% aqueous ammonia solution was added to this mixture. After stirring for 20 h at 26 °C, CDs@BONs were obtained. For the 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 aminofunctionalized CDs@BONs (CDs@NH2-BONs). Finally, the CDs@ NH2-BONs collected by centrifugation was washed with ethanol and water and dried in a freeze dryer. 2.3. Preparation of Immunofluorescent Nanocapsules and Immunomagnetic Nanoparticles. Immunofluorescent nanocapsules were prepared as follows. N-(3-(Dimethylamino)propyl)-N′ethylcarbodiimide hydrochloride (480 mg) and 72 mg N-hydroxysuccinimide were added in 20 mL of aqueous dispersion of CDs@NH2BONs (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

2 orders of magnitude, compared with the conventional method using CDs as fluorescence labels.

2. EXPERIMENTAL SECTION 2.1. 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 °C for 5 h in a 500 mL Teflon-lined autoclave. After natural cooling to room temperature, dialysis was carried out to purify the CDs. 2.2. Preparation and Surface Amination of CDs@BONs. Triton X-100 (17 mL) and 18 mL of n-hexanol were dissolved in 75 mL of cyclohexane to form solution A. An aqueous dispersion of CDs (3 mL, 0.45 μg mL−1) was mixed with 0.40 mL of tetraethyl orthosilicate and 0.9 mL bis[3-(triethoxysilyl)propyl]disulfide to form solution B. Then, 3443


Research Article


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

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.

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. 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, redispersed in 0.1 M PBS, and stored at 4 °C for

vigorous shaking for 4 h at room temperature. The mixture was centrifuged and washed with 0.1 M phosphate-buffered saline (PBS, pH 7.4) to remove excess reagents and then redispersed in 20 mL of 0.1 M 3444


Research Article

ACS Applied Materials & Interfaces future use. Immunomagnetic nanoparticles were prepared similarly to immunofluorescent nanocapsules except that amino-functionalized Fe3O4 nanoparticles were used instead of CDs@NH2-BONs. 2.4. Procedures for the Detection of S. aureus. PBS (400 μL, 0.1 M) 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. Then, the mixture was incubated with 200 μL of 0.1 M PBS containing different concentrations of S. aureus cells (0, 5, 25, 150, 300, 450, 750, 1000, and 1250 CFU mL−1) for 60 min at 37 °C. Thus, the final concentrations of S. aureus cells in the PBS were 0, 1, 5, 30, 60, 90, 150, 200, and 250 CFU mL−1, respectively. Then, magnetic separation was carried out, and the product collected by a controlled magnetic field was redispersed 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 the 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.

1a−c, CDs are spherical and monodispersed with an average diameter of 3 nm. Figure S1 shows the Fourier transform infrared (FTIR) spectrum of CDs. Three vibrational absorption peaks at 1578, 1635, and 3452 cm−1 are observed, which are related to the bending vibrations of N−H, the bending vibrations of CO, and the stretching vibrations of O−H, respectively. Figure S2 shows the ultraviolet−visible (UV−vis) absorption spectrum of CDs. Two absorption peaks at 239 and 339 nm are observed, which are assigned to the 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 mL−1. 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 are prepared by cohydrolyzation of tetraethyl orthosilicate and bis[3-(triethoxysilyl)propyl]disulfide. The morphology of CDs@BONs is characterized by scanning electron microscopy (SEM) and TEM. The SEM and TEM images in Figure 2a−c show that the nanoparticles are monodispersed and spherical with an average diameter of 80 nm. The high-resolution TEM (HRTEM) image in Figure 2d clearly reveals that hundreds of CDs are encapsulated in the organosilica shells. To enable the surface modification of antibodies on the CDs@ BONs, the nanocapsules are subjected to surface amination. Figure 3a shows the 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 the surface amination, two peaks at 1587 and 3434 cm−1, which are associated with the bending vibrations and the stretching vibrations of amino groups, respectively, in the FTIR spectrum are observed. Hence, the amino groups are successfully modified on the surfaces of CDs@BONs. Figure S6 shows the SEM image, the FTIR spectrum, and the magnetic hysteresis loop of amino-functionalized 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

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).

3. RESULTS AND DISCUSSION The size and morphology of CDs are characterized by transmission electron microscopy (TEM). As shown in Figure

Figure 8. Fluorescence microscopic images of S. aureus cells linked with immunofluorescent nanocapsules. 3445


Research Article


the fluorescent intensity increases with an increase in 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), compared with the high fluorescent response for the determination of S. aureus using immunofluorescent nanocapsules as fluorescent labels. Figure 6b shows the fluorescent intensity at 446 nm as a function 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 that of most reported pathogen sensors (Table S1). A conventional immunoassay of S. aureus is 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 are similar to those of the proposed immunoassay using immunofluorescent nanocapsules as fluorescent labels, except that antibody-conjugated CDs are used instead of immunofluorescent nanocapsules. Note here that the concentration of antibody-conjugated CDs is optimized to be 0.45 μg mL−1 (Figure S12). As shown in Figure 6c,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 (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 antibodyconjugated 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 is studied. As shown in Figure 7, compared with the high fluorescent signal for the detection of S. aureus, the fluorescent signals for Escherichia coli O157:H7, E. coli K12, Salmonella, and Listeria 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) is studied by fluorescence microscopy. Experimentally, immunofluorescent nanocapsules and immunomagnetic nanoparticles specifically capture and recognize S. aureus simultaneously, and then S. aureus cells collected by a controlled magnetic field are 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 is 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.

have an excellent superparamagnetic property at room temperature. To specifically capture and recognize S. aureus, immunofluorescent nanocapsules and immunomagnetic nanoparticles are 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 is 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. The binding of immunofluorescent nanocapsules with S. aureus was studied by SEM. As shown 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 a 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 reduction29 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, the 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 are optimized. As shown in Figure S7, the optimal NaBH 4 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 find that 1 mg immunofluorescent nanocapsules contain ca. 0.45 μg CDs. Quantitative detection of S. aureus in PBS (pH 7.4) is carried out. As illustrated in Scheme 1a, S. aureus cells are incubated simultaneously with immunofluorescent nanocapsules and immunomagnetic nanoparticles in PBS. The weight ratio of immunofluorescent nanocapsules and immunomagnetic nanoparticles is optimized to be 4:1 (Figure S8), and the optimal incubation time is 60 min (Figure S9). After magnetic separation and NaBH4 reduction, the CDs are released from the nanocapsules and then fluorescent detection is carried out. For comparison, a conventional immunoassay of S. aureus using antibody-conjugated CDs as labels is also carried out (Scheme 1b). Figure 6a shows the 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,

4. CONCLUSIONS In summary, novel CDs@BONs were prepared by cohydrolyzation of tetraethyl orthosilicate 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 3446


Research Article


(4) Shorten, P. R.; Pleasants, A. B.; Soboleva, T. K. Estimation of Microbial Growth Using Population Measurements Subject to a Detection Limit. Int. J. Food Microbiol. 2006, 108, 369−375. (5) Deisingh, A. K.; Thompson, M. Strategies for the Detection of Escherichia coli O157:H7 in Foods. J. Appl. Microbiol. 2004, 96, 419− 429. (6) He, Q.; Velumani, S.; Du, Q.; Lim, C. W.; Ng, F. K.; Donis, R.; Kwang, J. Detection of H5 Avian Influenza Viruses by Antigen-Capture Enzyme-Linked Immunosorbent Assay Using H5-Specific Monoclonal Antibody. Clin. Vaccine Immunol. 2007, 14, 617−623. (7) 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. Chem. 2006, 52, 1997−2004. (8) Lee, M.-S.; Chang, P.-C.; Shien, J.-H.; Cheng, M.-C.; Shieh, H. K. Identification and Subtyping Of Avian Influenza Viruses by Reverse Transcription-PCR. J. Virol. Methods 2001, 97, 13−22. (9) Wang, S.; Deng, W.; Yang, L.; Tan, Y.; Xie, Q.; Yao, S. CopperBased Metal−Organic Framework Nanoparticles with Peroxidase-Like Activity for Sensitive Colorimetric Detection of Staphylococcus aureus. ACS Appl. Mater. Interfaces 2017, 9, 24440−24445. (10) Tokel, O.; Inci, F.; Demirci, U. Advances in Plasmonic Technologies for Point of Care Applications. Chem. Rev. 2014, 114, 5728−5752. (11) Wu, S.; Duan, N.; Shi, Z.; Fang, C.; Wang, Z. Simultaneous Aptasensor for Multiplex Pathogenic Bacteria Detection Based on Multicolor Upconversion Nanoparticles Labels. Anal. Chem. 2014, 86, 3100−3107. (12) Shen, Z.; Huang, M.; Xiao, C.; Zhang, Y.; Zeng, X.; Wang, P. G. Nonlabeled Quartz Crystal Microbalance Biosensor for Bacterial Detection Using Carbohydrate and Lectin Recognitions. Anal. Chem. 2007, 79, 2312−2319. (13) Yang, S.; Ouyang, H.; Su, X.; Gao, H.; Kong, W.; Wang, M.; Shu, Q.; Fu, Z. Dual-Recognition Detection of Staphylococcus aureus Using Vancomycin-Functionalized Magnetic Beads as Concentration Carriers. Biosens. Bioelectron. 2015, 78, 174−180. (14) Liu, H.; Zhou, X.; Liu, W.; Yang, X.; Xing, D. Paper-Based Bipolar Electrode Electrochemiluminescence Switch for Label-Free and Sensitive Genetic Detection of Pathogenic Bacteria. Anal. Chem. 2016, 88, 10191−10197. (15) Zhang, H.; Zhang, Y.; Lin, Y.; Liang, T.; Chen, Z.; Li, J.; Yue, Z.; Lv, J.; Jiang, Q.; Yi, C. Ultrasensitive Detection and Rapid Identification of Multiple Foodborne Pathogens with the Naked Eyes. Biosens. Bioelectron. 2015, 71, 186−193. (16) Wang, R.; Xu, Y.; Zhang, T.; Jiang, Y. Rapid And Sensitive Detection of Salmonella typhimurum Using Aptamer-Conjugated Carbon Dots as Fluorescence Probe. Anal. Methods 2015, 7, 1701− 1706. (17) Ng, B. Y. C.; Xiao, W.; West, N. P.; Wee, E. J. H.; Wang, Y.; Trau, M. Rapid, Single-Cell Electrochemical Detection of Mycobacterium tuberculosis Using Colloidal Gold Nanoparticles. Anal. Chem. 2015, 87, 10613−10618. (18) Edgar, R.; McKinstry, M.; Hwang, J.; Oppenheim, A. B.; Fekete, R. A.; Giulian, G.; Merril, C.; Nagashima, K.; Adhya, S. High-Sensitivity Bacterial Detection Using Biotin-Tagged Phage and Quantum-Dot Nanocomplexes. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4841−4845. (19) Kempf, V. A.; Trebesius, K.; Autenrieth, I. B. Fluorescent In Situ Hybridization Allows Rapid Identification of Microorganisms in Blood Cultures. J. Clin. Microbiol. 2000, 38, 830−838. (20) Heyduk, E.; Heyduk, T. Fluorescent Homogenous Immunosensors for Detecting Pathogenic Bacteria. Anal. Biochem. 2010, 396, 298−303. (21) Pan, W.; Zhao, J.; Chen, Q. Fabricating Upconversion Fluorescent Probes for Rapidly Sensing Foodborne Pathogens. J. Agric. Food Chem. 2015, 63, 8068−8074. (22) Arcidiacono, S.; Pivarnik, P.; Mello, C. M.; Senecal, A. Cy5 Labeled Antimicrobial Peptides for Enhanced Detection of Escherichia coli O157:H7. Biosens. Bioelectron. 2008, 23, 1721−1727.

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

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b18714. Additional experimental description, FTIR 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 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), and detection results in real samples (Table S2) (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (W.D.). *E-mail: [email protected] (Y.T.). ORCID

Yueming Tan: 0000-0003-3356-9079 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS 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), Program for Excellent Talents in Hunan Normal University (ET1503), and Key Project of Research and Development Plan of Hunan Province (2016SK2020).

■

REFERENCES

(1) Brecher, M. E.; Hay, S. N. Bacterial Contamination of Blood Components. Clin. Microbiol. Rev. 2005, 18, 195−204. (2) Law, J. W.-F.; Ab Mutalib, N.-S.; Chan, K.-G.; Lee, L.-H. Rapid Methods for the Detection of Foodborne Bacterial Pathogens: Principles, Applications, Advantages and Limitations. Front. Microbiol. 2015, 5, 770−789. (3) Pandey, P. K.; Kass, P. H.; Soupir, M. L.; Biswas, S.; Singh, V. P. Contamination of Water Resources by Pathogenic Bacteria. AMB Express 2014, 4, 51−67. 3447


Research Article

ACS Applied Materials & Interfaces (23) Xiong, L.-H.; Cui, R.; Zhang, Z.-L.; Yu, X.; Xie, Z.; Shi, Y.-B.; Pang, D.-W. Uniform Fluorescent Nanobioprobes for Pathogen Detection. ACS Nano 2014, 8, 5116−5124. (24) Lim, S. Y.; Shen, W.; Gao, Z. Carbon Quantum Dots and Their applications. Chem. Soc. Rev. 2015, 44, 362−381. (25) Zhong, D.; Zhuo, Y.; Feng, Y.; Yang, X. Employing Carbon Dots Modified With Vancomycin for Assaying Gram-Positive Bacteria Like Staphylococcus aureus. Biosens. Bioelectron. 2015, 74, 546−553. (26) Gu, Z.; Biswas, A.; Zhao, M.; Tang, Y. Tailoring Nanocarriers for Intracellular Protein Delivery. Chem. Soc. Rev. 2011, 40, 3638−3655. (27) Rother, M.; Nussbaumer, M. G.; Renggli, K.; Bruns, N. Protein Cages and Synthetic Polymers: A Fruitful Symbiosis for Drug Delivery Applications, Bionanotechnology and Materials Science. Chem. Soc. Rev. 2016, 45, 6213−6249. (28) Niu, D.; Li, Y.; Shi, J. Silica/Organosilica Cross-Linked Block Copolymer Micelles: A Versatile Theranostic Platform. Chem. Soc. Rev. 2017, 46, 569−585. (29) Prasetyanto, E. A.; Bertucci, A.; Septiadi, D.; Corradini, R.; Castro-Hartmann, P.; De Cola, L. Breakable Hybrid Organosilica Nanocapsules for Protein Delivery. Angew. Chem., Int. Ed. 2016, 55, 3323−3327. (30) Chen, Z.-Z.; Cai, L.; Chen, M.-Y.; Lin, Y.; Pang, D.-W.; Tang, H.W. Indirect Immunofluorescence Detection of E. coli O157:H7 with Fluorescent Silica Nanoparticles. Biosens. Bioelectron. 2015, 66, 95−102. (31) Wang, J.-J.; Jiang, Y.-Z.; Lin, Y.; Wen, L.; Lv, C.; Zhang, Z.-L.; Chen, G.; Pang, D.-W. Simultaneous Point-of-Care Detection of Enterovirus 71 and Coxsackievirus B3. Anal. Chem. 2015, 87, 11105− 11112. (32) Zhu, S.; Meng, Q.; Wang, L.; Zhang, J.; Song, Y.; Jin, H.; Zhang, K.; Sun, H.; Wang, H.; Yang, B. Highly Photoluminescent Carbon Dots for Multicolor Patterning, Sensors, and Bioimaging. Angew. Chem., Int. Ed. 2013, 52, 3953−3957. (33) Yang, Z.; Xu, M.; Liu, Y.; He, F.; Gao, F.; Su, Y.; Wei, H.; Zhang, Y. Nitrogen-doped, carbon-rich, highly photoluminescent carbon dots from ammonium citrate. Nanoscale 2014, 6, 1890−1895. (34) Hao, N.; Han, L.; Yang, Y.; Wang, H.; Webley, P. A.; Zhao, D. A Metal-Ion-Assisted Assembly Approach to Synthesize Disulfide-Bridged Periodical Mesoporous Organosilicas with High Sulfide Contents and Efficient Adsorption. Appl. Surf. Sci. 2010, 256, 5334−5342. (35) Imamoglu, M.; Pérez-Quintanilla, D.; Sierra, I. Bifunctional Periodic Mesoporous Organosilicas with Sulfide Bridges as Effective Sorbents for Hg(II) Extraction from Environmental and Drinking Waters. Microporous Mesoporous Mater. 2016, 229, 90−97. (36) Haid, A.; Suissa, M. Immunochemical Identification of Membrane Proteins after Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. Methods Enzymol. 1983, 96, 192−205. (37) Xing, H.; Zhang, C. L.; Ruan, G.; Zhang, J.; Hwang, K.; Lu, Y. Multimodal Detection of a Small Molecule Target Using StimuliResponsive Liposome Triggered by Aptamer−Enzyme Conjugate. Anal. Chem. 2016, 88, 1506−1510. (38) Li, W.; Jiang, W.; Dai, S.; Wang, L. Multiplexed Detection of Cytokines Based on Dual Bar-Code Strategy and Single-Molecule Counting. Anal. Chem. 2016, 88, 1578−1584. (39) Zhou, F.; Li, B. Exonuclease III-Assisted Target Recycling Amplification Coupled with Liposome-Assisted Amplification: OneStep and Dual-Amplification Strategy for Highly Sensitive Fluorescence Detection of DNA. Anal. Chem. 2015, 87, 7156−7162. (40) Park, J.; Park, Y.; Kim, S. Signal Amplification via Biological SelfAssembly of Surface-Engineered Quantum Dots for Multiplexed Subattomolar Immunoassays and Apoptosis Imaging. ACS Nano 2013, 7, 9416−9427.

3448


Fluorescent Immunoassay for the Detection of Pathogenic Bacteria at

Recommend Documents