Dual Recognition Strategy for Specific and Sensitive Detection of

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A Dual Recognition Strategy for Specific and Sensitive Detection of Bacteria Using Aptamer-Coated Magnetic Beads and Antibiotic-Capped Gold Nanoclusters Dan Cheng, Mengqun Yu, Fei Fu, Weiye Han, Gan Li, Jianping Xie, Yang Song, Mark T. Swihart, and Erqun Song Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03320 • Publication Date (Web): 07 Dec 2015 Downloaded from http://pubs.acs.org on December 7, 2015

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Analytical Chemistry

A Dual Recognition Strategy for Specific and Sensitive Detection of Bacteria

Using

Aptamer-Coated

Magnetic

Beads

and

Antibiotic-Capped Gold Nanoclusters Dan Cheng,#† Mengqun Yu,#† Fei Fu,† Weiye Han,† Gan Li,† Jianping Xie,‡ Yang Song,† Mark T. Swihart,║Erqun Song†,* †

Key Laboratory of Luminescence and Real-Time Analytical Chemistry, Ministry of Education,

College of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, People’s Republic of China. Fax: +86-2368251225; Tel: +86-2368251225. E-mail: [email protected] ‡

College of Life Sciences, Southwest University, Chongqing, 400715, People’s Republic of China.

║

Department of Chemical and Biological Engineering, University at Buffalo, State University of

New York, Buffalo, NY 14260, USA.

Abstract Food poisoning and infectious diseases caused by pathogenic bacteria such as Staphylococcus aureus (SA) are serious public health concerns. Method of specific, sensitive and rapid detection of such bacteria is essential and important. This study presents a strategy that combines aptamer and antibiotic-based dual recognition units with magnetic enrichment and fluorescent detection to achieve specific and sensitive quantification of SA in authentic specimens and in the presence of much higher concentrations of other bacteria. Aptamer-coated magnetic beads (Apt-MB) were employed for specific capture of SA. Vancomycin-stabilized fluorescent gold nanoclusters (AuNCs@Van) were prepared by a simple one-step process and used for 1

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sensitive quantification of SA in the range of 32-108 cfu/mL with the detection limit of 16 cfu/mL via fluorescence intensity measurement. And using this strategy, about 70 cfu/mL of SA in complex samples (containing 3×108 cfu/mL of other different contaminated bacteria) could be successfully detected. In comparison to prior studies, the developed strategy here not only simplifies the preparation procedure of the fluorescent probes (AuNCs@Van) to a great extent, but also could sensitively quantify SA in the presence of much higher concentrations of other bacteria directly with good accuracy. Moreover, the aptamer and antibiotic used in this strategy are much less expensive and widely available compared to common-used antibodies, making it cost-effective. This general aptamer- and antibiotic-based dual recognition strategy, combined with magnetic enrichment and fluorescent detection of trace bacteria shows great potential application in monitoring bacterial food contamination and infectious diseases. Keywords: aptamer; antibiotic; gold nanocluster, bacteria; dual recognition

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INTRODUCTION Food contamination1 and disease infection2 caused by pathogenic bacteria,

especially as the gram-positive bacterium (G+) of staphylococcus aureus (SA), have become a growing threat to public health,3-6 which makes the specific, sensitive, simple, and inexpensive detection of SA great interest. Classical strategies for the detection of SA, including traditional microbiological methods7 and polymerase chain reaction based methods,8 show the limit of time-consuming and false-positive results respectively.7,9 Molecular recognition-based strategy of assaying SA is an effective alternative for the traditional strategies, which employs the molecules of antibody,10-12 aptamer,13-15 antibiotic,16-18 or others19 to detect SA. Among them, antibody-based immunoassays are simple, sensitive and selective but rely on animal-derived antibodies that are expensive with poor reproducibility.20, 21 Nucleic acid aptamers are single-stranded oligonucleotide (DNA or RNA) molecules that can bind to targets with high specificity and affinity.22 Compared with antibodies, they are relatively stable, inexpensive, easy to prepare and modify, and minimally immunogenic. Several aptamer-based strategies for the detection of SA have been reported based on light-scattering,13 fluorescence,14,15,23 raman spectroscopy,24 electrochemical,25 and colorimetric detection.26 These methods showed high specificity and relative sensitivity for SA. However, they involved complicated and time-consuming procedures for preparation of aptamer-based probes. Antibiotics, which feature small size, low price, and broad availability, are capable of inhibiting bacterial proliferation

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by preventing cell wall synthesis or by blocking transcription/translation processes. Their specific interactions with bacteria make them interesting recognition molecules for the detection of bacteria as well.27-28 For example, the antibiotic of vancomycin (Van) has been used to design strategy to detect SA.17, 29-32 Unfortunately, published procedures for preparation of Van-based nanoprobes are relatively complex and time-consuming. Moreover, Van-based methods are not specific for SA, as Van is a broad-spectrum antibiotic for all G+ bacteria. Thus, accurate, simple, rapid and cost-effective SA assays remain unavailable and are of great interest. As is known to all, magnetic particle-based separation and enrichment,15, 33-36 and gold nanoclusters-based fluorescence detection37-39 are powerful tools for assay due to their controllability and high sensitivity respectively. Meanwhile, as described above, aptamers and antibiotics can provide specific or broad-spectrum recognition of bacteria, respectively, and are much less expensive and more readily available than antibodies. Inspired by the above-mentioned points, here we propose and demonstrate a new method for sensitive, specific, simple, cost-effective detection of SA by combining the two recognition molecules (aptamer and antibiotic) with magnetic enrichment and fluorescent detection. Based on this strategy (Scheme1), the SA in a mixture containing other non-target bacteria were quantified by using aptamer-coated magnetic beads (Apt-MB) and Van-functionalized fluorescent gold nanoclusters (AuNCs@Van) together, with the detection recoveries from 96.94% to 101.24% for SA in authentic milk and human serum samples. Such an aptamer- and antibiotic-based dual recognition strategy combined with magnetic enrichment and 4


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fluorescence detection has significant potential for application in screening for food-borne pathogens and in clinical diagnosis of bacterial infection.

Scheme 1 Schematic illustrations of (A) one-step preparation of AuNCs@Van, and (B)determination of SA in mixtures using the Apt-MB and AuNCs@Van dual recognition strategy.

EXPERIMENTAL SECTION Materials and Apparatus. Vancomycin.HCl (Van) and streptavidin-modified

magnetic beads (with 1 µm particle size) were purchased from Sigma-Aldrich Co. Ltd.; chloroauric acid (HAuCl4) was obtained from Tianjin Guangfu Chemical Research Institute; SA(ATCC 29213), Methicillin-resistant Staphylococcus aureus (MRSA: ATCC 6538), Escherichia coli (E.coli: ATCC 11864), and mycobacterium smegmatis (M. smegmatis: ATCC 607) were obtained from China General Microbiological Culture Collection Center; and all buffers were prepared with ultrapure water which was purified through a Milli-Q system (Millipore, Synergy, USA) to a specific resistance of 18.2 MΩ.cm. Biotinylated SA aptamer23 (biotin-aptamer), Cy3 labeled SA aptamer (Cy3-aptamer), FITC labeled SA aptamer (FITC-aptamer), and random DNA sequence were obtained from Sangon Biotech 5



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(Shanghai) Co., Ltd with the sequences information presented in Table S1 in the supporting information. Fluorescence spectra were obtained using a fluorescence spectrophotometer (F-7000, Hitachi). Fluorescence micrographs were recorded using an Olympus IX71 fluorescence microscope and a confocal laser scanning microscopy (IX2-DSU, Olympus, Japan). UV-Vis absorption spectra were recorded on a Shimadzu UV-2450 spectrometer. Fourier transform infrared spectroscopy (FTIR) was carried out on a Shimadzu IRPrestige-21. Morphology and microscopic structure were characterized using a transmission electron microscope (TEM) (LIBRA 200PE, Carl Zeiss SMT). X-ray photoelectron spectroscopy (XPS) was conducted using a Thermo escalab 250Xi. Energy dispersive X-ray (EDX) analysis was conducted by an energy dispersive spectrometer (Oxford Instrument, X-Max). Zeta potential was measured using a Malvern Zetasizer (Nano ZS ZEN3600, Malvern, UK). Synthesis of AuNCs@Van. The AuNCs@Van was synthesized by minor modifications of our previously reported procedures.40-41 In a typical synthesis, 500 µL of 1% (W/V) aqueous HAuCl4 solution was diluted in 49.5 mL ultrapure water in a bottle with stirring. The mixture was heated to 100 ºC, and then 250 µL of 1% (W/V) Van aqueous solution (nVan:nHAuCl4=1:7.2) was added and the mixture was held at 100 ºC for 50 min. After cooling to room temperature, the supernatant product was collected by centrifugation at 12000 rpm/10 min to remove clusters with larger size. And then the product was subjected to lyophilization and stored at 4 ºC after excess Van removed by dialysis with ultrapure water. 6


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Synthesis of Apt-MB. A dispersion of streptavidin-modified magnetic beads aqueous solution (1 mg/mL, 500 µL) was incubated with 75µL of 10 µM biotin-aptamer for 1 h at 37 ºC. Then, the excess biotin-aptamer in supernatant was removed by magnetic separation, and the product Apt-MBs were re-dispersed in 100µL phosphate-buffered saline buffer (PBS, pH=7.4) after washing three times with PBS. Stability of AuNCs@Van. First, the stability of AuNCs@Van was investigated upon storage by measuring the fluorescence signal versus storage time at 4 ºC in the dark. Secondly, the photobleaching stability of AuNCs@Van was investigated by measuring the fluorescence signals versus time under exposure to 365 nm UV-light with power of 150W. Thirdly, the effect of different buffer solutions on the stability of AuNCs@Van was studied. AuNCs@Van was respectively treated with PBS, britton-robison buffer (BR), boric acid buffer solution (BB), Tris-HCl buffer, and 10% human serum (10%HS). The fluorescence signals were recorded after 30 minutes. Subsequently, the effect of pH on the stability of AuNCs@Van was studied. AuNCs@Van was dispersed in BR buffer at pH from 2 to 11, and the fluorescence signals were recorded after 30 minutes. Detection of SA with Apt-MBs and AuNCs@Van. Varying concentrations of SA (0, 1×101.5, 1×103, 1×105, 1×106, 1×108, 2×108, and 1×109 cfu/mL) were each incubated with the Apt-MBs (1.25 µM) for 30 min at 37 ºC in 400µL binding buffer (40 mM HEPES buffer pH 8.0, 5 mM KCl, 1 mM CaCl2, 2 mM MgCl2, and 150 mM NaCl).23 The complex of Apt-MBs@SA were dispersed in 100µL PBS after washing 7



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three times with PBS. Then AuNCs@Van (with final concentration of 70 µg/mL) was added to the dispersion and incubated for 3 h at 37 ºC. After excess AuNCs@Van removed by magnetic separation, the bound Apt-MBs on the surface of SA were eluted with NaOH13 and then removed by magnetic separation using a magnet. Finally, the fluorescence signal of SA@AuNCs@Van was recorded at λex/em=303/412 nm. Separation and detection of SA from mixed samples. Varying concentrations of SA were mixed with E.coli, M. smegmatis, and MRSA (each at about 1×108 cfu/mL) to construct an artificial complex specimen. The mixture was then subjected to the SA assay as described above. Detection of SA in authentic smaples. Before assay, the qualified milk samples obtained from a local supermarket and human serum samples from health volunteers were sterilized. Then pure SA with varying known concentrations was added to the treated milk and serum samples, followed measuring amount of SA in the samples as described above.

RESULTS AND DISCUSSION Synthesis and characterization of AuNCs@Van. AuNCs@Van were

prepared from chloroauric acid in one step by employing Van as both reducing and stabilizing agents (as shown in Scheme 1 A) at empirically optimized conditions of nVan:nHAuCl4 ratio=1:7.2 and incubation time of 50 min (see Figure S1). And then their morphology, size, composition, optical properties, and stability were studied. The formation of AuNCs@Van was confirmed by TEM imaging and XPS. TEM images

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from Figure 1A and 1B showed that the AuNCs@Van were uniform spherical and monodisperse with a diameter of 2.0±0.6 nm while XPS spectrum of Figure 1C showed a typical peak of gold element at the binding energy of 83.6eV (Au 4f7/2). FTIR spectrum (Figure 1D) showed that AuNCs@Van had the same infrared absorbance spectrum as Van. Together, these results demonstrate the successful synthesis of AuNCs@Van. As for their optical properties, Figure 2A shows that AuNCs@Van have monotonically increasing absorbance with decreasing wavelength below about 400 nm, and absorb strongly below about 300 nm. Figure 2B show that the wavelengths of maximum excitation and emission of AuNCs@Van were 303 nm and 412 nm, respectively, and the inset illustrates the blue fluorescence of the AuNCs@Van under UV light. Their fluorescence quantum yield was measured about 13% (compared to quinine sulphate). Chemical, photochemical, and colloidal stability are essential features for biomedical applications of nanoprobes. The stability of the fluorescence of AuNCs@Van nanoprobes with storage time, UV illumination, and buffer environment was investigated next. As shown in Figure S2A, the fluorescence intensity decreased by only 0.05% after storage for 100 days in aqueous solution at 4ºC in the dark. Figure S2B shows that the AuNCs@Van fluorescence intensity increased by 3% after 24 h of UV illumination (365nm, 150W). Figure S2C and S2D demonstrate that AuNCs@Van fluorescence was stable in different buffers and at varying pH, only falling off significantly at pH below 4.

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Figure 1 TEM image (A), size distribution (B), XPS (C) and infrared spectra (D) of AuNCs@Van. The inset in Figure 1A shows an HR-TEM image of AuNCs@Van.

Figure 2 Absorption (A) and fluorescence excitation and emission (B) spectra of AuNCs@Van. The inset in (B) shows a photograph of AuNCs@Van under ambient (a) and UV(b) light.

Binding of AuNCs@Van with SA. Next, the recognition ability of AuNCs@Van nanoprobes to SA was tested. According to the published research work,17,

29-32

Van can combine with SA by binding onto terminal residues

D-alanyl-D-alanine of N-acetylmuramic acid and N-acetylglucosamine peptide subunits on the cell wall of the G+ bacteria, which will make the AuNCs adhere to the surface of SA. The attachment of AuNCs@Van to SA was confirmed by fluorescence microscopy and TEM imaging (Figure 3). The fluorescence image of Figure 3B shows many blue fluorescent dots on the same sites of where the SA locate in the 10


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bright-field image of Figure 3A while there no obvious blue fluorescent dots observed in Figure 3D for E.coli (Figure 3C). Since the two kinds of bacteria only treated with AuNCs@Van before fluorescence imaging, the blue fluorescent dots should come from AuNCs@Van, suggesting the binding of AuNCs@Van with SA but not with E.coli indirectly. Specifically, TEM images show some small black dots (the blue arrows indicated features in Figure 3E) located on the surface of SA but nothing on E. coli (Figure 3F). And the following EDX analysis (Figure S3) for the black dots around SA confirmed that they contained gold element and should be AuNCs@Van, directly demonstrating the binding of AuNCs@Van with SA.

Figure 3 Fluorescence microscopy images of SA (A, B) and E.coil (C, D), and TEM images of SA (E) and E.coil (F) after incubated with AuNCs@Van.

Detection of SA based on the dual-recognition of Apt-MBs and AuNCs@Van. The Apt-MBs were prepared via the interaction between the streptavidin-magnetic beads and biotin-aptamer as described in the experimental section. Compared with streptavidin-magnetic beads only, the zeta potential of Apt-MBs shifted from -12.8±0.9mV to -19.5±1.1 mV, which suggested the successful binding of aptamer to the magnetic beads, as the aptamer has a net negative charge at neutral pH. 11



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Before SA assay with Apt-MBs and AuNCs@Van, the dual recognition of both aptamer and Van with SA were first fully confirmed by using confocal laser scanning microscopy after SA incubated with Cy3-aptamer and AuNCs@Van (data was shown in Figure S4). And then a control experiment with a non-aptamer sequence coated magnetic beads (random DNA-MBs) and AuNCs@Van for SA sensing was conducted. The negligible fluorescence signals from the control experiment compared with the signals from Apt-MBs based experiment (Figure 4A) demonstrated that the random DNA will not bring obvious background signals and the strong fluorescence signals is due to the specific binding of aptamer with target bacteria but not due to nonspecific adsorption. After the assay conditions including reaction temperature, concentration of nanoprobe, and reaction time were optimized (Figure S5), the linear range and detection limit of SA by Apt-MBs and AuNCs@Van were studied. Specifically, a series of SA solution with different concentrations were incubated with Apt-MBs and AuNCs@Van nanoprobes successively, followed by the determination of the fluorescence intensity of SA@AuNCs@Van complex of each specimen. As shown in Figure 4B, the enhanced fluorescence intensity ∆F (∆F=F-F0, F0 and F stands for the fluorescence intensity of sediment when AuNCs@Van nanoprobes before and after reacting with SA respectively) was correlated with the concentration of the SA in the range

of

32-108

cfu/mL,

with

a

detection

limit

of

16

cfu/mL

(△F=212.71LogNSA+12.41, R2=0.9936, here NSA stands for the quantity of SA in cfu/mL).

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Figure 4 (A) Fluorescence spectra of detected SA with Apt-MBs or random DNA-MBs and AuNCs@Van. (B) The linear regression curve of the enhanced fluorescence intensity (∆F, at 412 nm) vs. the concentration of SA (Log NSA/cfu.mL-1 ).

The accuracy of the Apt-MBs and AuNCs@Van based dual recognition strategy for SA detection was investigated by testing pure SA samples at three different concentrations (64, 5000 and 1×108 cfu/mL). The results in Table S2 showed that the recovery efficiency for pure SA detection was in the range of 99.75-103.33% in different specimens with RSDs from 0.3-3.8%, demonstrating the good accuracy of the Apt-MBs and AuNCs@Van based dual recognition strategy for SA detection. In addition, as SA is usually accompanied by many other bacteria, including both G+ bacteria and gram-negative(G-) bacteria, the selectivity of the Apt-MBs and AuNCs@Van based dual recognition strategy for SA detection should be confirmed. To investigate the selectivity of the developed strategy, SA, MRSA(G+), M. smegmatis(G+), and E.coli (G-) (each at a concentration of about 1×105 cfu/mL) were respectively treated with Apt-MBs and AuNCs@Van according the protocol as done to SA described in the experimental section. The fluorescence signal changes which were induced by the binding of three other pathogenic bacteria (E.coli, M. smegmatis and MRSA) were compared with the fluorescence signal changes from SA. As shown

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in Figure 5, compared with the obvious increase of fluorescence intensity for SA, the three other bacteria exhibited negligible increase of fluorescence intensity which is in agreement with the data shown in Figure S6, suggesting the high selectivity of the as-developed dual recognition assay.

Figure 5 The enhanced fluorescence intensity (∆F) of four different kinds of bacteria after analyzed with the as-developed method. The error bars show the standard deviation of three replicate determinations.

Sensitive detection of SA from complex samples. To further demonstrate the selectivity and sensitivity of the current approach for SA in complex samples, a series of bacterial mixtures (containing E.coli, M. smegmatis, MRSA and SA together) were prepared and used as model samples. The samples were prepared by mixing SA (0, 10, 50, 70, 80, 100, 150, 300, and 400 cfu/mL) with E.coli (1×108 cfu/mL), M. smegmatis(1×108 cfu/mL) and MRSA (1×108 cfu/mL) together respectively. Figure 6 shows that when just 70 cfu/mL of SA was mixed with 3×108 cfu/mL of other bacteria, the fluorescence signal for SA was still detectable, which means that one SA in a hundred thousand other bacteria could be detected using the approach demonstrated here, showing high selectivity and sensitivity.

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Figure 6 The enhanced fluorescence intensity (∆F) of detected SA with different concentration (50, 70, 80, 100, 150, 300 and 400 cfu/mL) mixed with three other bacteria: E.coli (1×108 cfu/mL), M. smegmatis(1×108 cfu/mL) and MRSA (1×108 cfu/mL).

Detection of SA in authentic samples. In order to demonstrate the practical applicability of the method for SA detection, qualified milk from a local supermarket and sterile human serum samples spiked with SA were analyzed. The results are shown in Table 1. The recoveries varied from 96.94% to 101.24% with variation coefficients of 0.5-5.87%, indicating that the proposed method could be applied for detection of SA in authentic samples. Table 1 Recovery efficiency of SA spiked in milk and serum samples based on the proposed strategy

Samples

Added (Log cfu/mL)

Found (Log cfu/mL)

Recovery (%)

RSD (%, n =3)

Milk 1

1.60

1.65

96.94

5.87

Milk 2

3.60

3.65

98.54

3.42

Milk 3

7.20

7.11

101.24

0.50

Serum 1

1.60

1.66

96.67

2.28

Serum 2

3.60

3.61

99.75

1.10

Serum 3

7.20

7.35

97.92

0.50

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CONCLUSION In summary, we have shown that aptamer-bound magnetic beads and

antibiotic-functionalized fluorescent gold nanoclusters can be used in a dual recognition strategy for specific and sensitive detection of bacteria from complex samples. Compared with similar prior studies, the strategy demonstrated this study has several advantages. The AuNCs@Van fluorescent nanoprobes were obtained by a very simple one-step procedure, and both the aptamer and Van antibiotic are much less expensive and more readily available than antibodies, making the strategy simple, facile and cost-effective. Moreover, this proposed strategy could quantify SA in the presence of much higher concentrations of other bacteria (achieve to the level of ppm), showing good feasibility. Such an Apt-MBs and AuNCs@Van based dual recognition assay could detect SA in milk and human serum samples with good recovery efficiencies, showing potential application for the SA-related food contamination assay and infectious diseases diagnosis.

ASSOCIATED CONTENT

Supporting information The information of DNA sequences and modifications, the optimized synthesis protocol and the data of stability characterization of AuNCs@Van, the EDX analysis results of AuNCs@Van binding with SA, the Cy3-aptamer and AuNCs@Van based dual recognition with SA, the optimization of binding condition of AuNCs@Van with SA, the recovery efficiency of pure SA detected with the as-proposed strategy, and the 16


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specific recognition of aptamer with SA. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected].

Fax: (+86)2368251225

Author Contributions #

Dan Cheng and Mengqun Yu contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (21575118, 21477098), Science and Technology Talent Cultivation Project of Chongqing (cstc2014kjrc-qnrc00001), Fundamental Research Funds for the Central Universities (XDJK2015A017, XDJK2016A004).

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Figure Legends: Scheme 1 Schematic illustrations of (A) one-step preparation of AuNCs@Van, and (B) determination of SA in mixtures using the Apt-MB and AuNCs@Van dual recognition strategy.

Figure 1 TEM image (A), size distribution (B), XPS (C) and infrared spectra (D) of AuNCs@Van. The inset in Figure 1A shows an HR-TEM image of AuNCs@Van.

Figure 2 Absorption (A) and fluorescence excitation and emission (B) spectra of AuNCs@Van. The inset in (B) shows a photograph of AuNCs@Van under ambient (a) and UV(b) light.

Figure 3 Fluorescence microscopy images of SA (A, B) and E.coil (C, D), and TEM images of SA (E) and E.coil (F) after incubated with AuNCs@Van.

Figure 4 (A) Fluorescence spectra of detected SA with Apt-MBs or random DNA-MBs and AuNCs@Van. (B) The linear regression curve of the enhanced fluorescence intensity (∆F, at 412 nm) vs. the concentration of SA (Log NSA/cfu.mL-1).

Figure 5 The enhanced fluorescence intensity (∆F) of four different kinds of bacteria after analyzed with the as-developed method. The error bars show the standard deviation of three replicate determinations.

Figure 6 The enhanced fluorescence intensity (∆F) of detected SA with different concentration (50, 70, 80, 100, 150, 300 and 400 cfu/mL) mixed with three other bacteria: E.coli (1×108 cfu/mL), M. smegmatis (1×108 cfu/mL) and MRSA (1×108 cfu/mL).

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For TOC only

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Dual Recognition Strategy for Specific and Sensitive Detection of

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