Sandwich Fluorimetric Method for Specific Detection of

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A sandwich fluorimetric method for specific detection of Staphylococcus aureus based on antibiotic-affinity strategy Weijun Kong, Jie Xiong, Huan Yue, and Zhifeng Fu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02301 • Publication Date (Web): 09 Sep 2015 Downloaded from http://pubs.acs.org on September 13, 2015

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Analytical Chemistry

A sandwich fluorimetric method for specific detection of antibiotic-affinity strategy Staphylococcus aureus based on antibioticWeijun Kong, Jie Xiong, Huan Yue, Zhifeng Fu* Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China

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*Corresponding author. Tel: +86 23 6825 0184; Fax: +86 23 6825 1048 E-mail address: [email protected] (Z.F. Fu)

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ABSTRACT: A novel antibiotic-affinity strategy was designed for fluorimetric detection of pathogenic bacteria based on the strong affinity of antibiotic agent to the cell wall of bacteria. In this proof-of-concept work, vancocin, a glycopeptide antibiotic for Gram-positive bacteria, was used as a molecular recognition agent to anchor Staphylococcus aureus (S. aureus) cell. To improve the specificity of this method for S. aureus detection, IgG was adopted as the second recognition agent utilizing the binding between Fc region of IgG and S. aureus protein A in the cell wall, to form a sandwich complex. By using fluorescein isothiocyanate as the signal probe, S. aureus whole cells could be directly assayed within a linear range of 1.0 × 103 - 1.0 × 109 CFU mL-1 with a detection limit of 2.9 × 102 CFU mL-1. The whole assay process could be completed within 130 min when a ready-for-use microplate was adopted. This proposed strategy for pathogenic bacteria detection possessed some attractive characteristics such as high sensitivity, wide linear range, simple manipulation, short assay time and low cost. Furthermore, this sandwich mode also showed ideal specificity because vancocin and IgG bound with S. aureus at two distinct sites. It opened up a new pathway for high-throughput screening of pathogenic bacteria in medical diagnosis, food safety, bioterrorism defense and drug discovery.

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Bacterial infection has been one of the major threats to public health for decades and remains the leading cause of disease and mortality in most developing and underdeveloped countries. The identification and quantitation of pathogenic bacteria has become a crucial point in medical diagnosis, food safety, bioterrorism defense and drug discovery. In the past decades, numerous efforts have been made to develop rapid, sensitive and specific assay method for pathogenic bacteria.1,2 Traditionally, culture and colony counting-based method has been used as the gold standard for bacteria identification and quantitation owing to its high sensitivity, ideal specificity and good reliability.3,4 Although this approach is powerful and error-proof, it is not fit for point-of-care test (POCT) and rapid screening because it typically demands labor-intensive manipulation, time-consuming culture and highly trained personnel. Polymerase chain reaction (PCR) is a culture-free technique allowing rapid and multiplexed assay of pathogenic bacteria.5,6 However, this approach usually requires cell disruption and nucleic acid extracting, thus limits its usage in POCT. In the recent years, molecular recognition mode has attracted increasing interest since it is suitable for direct assaying whole cells of pathogenic bacteria. This mode utilizes molecular recognition agents such as antibody,7-11 aptamer,12-15 polypeptide,16-18 bacteriophage19,20 and its tailspike protein21,22 to bind specifically with bacteria cells. It doesn’t require time-consuming bacteria culture and labor-intensive nucleic acid extracting, thus shows remarkable merits such as high assay speed, simple manipulation and ease of developing portable POCT device. Nevertheless, these biomaterial-based recognition agents always suffer from poor stability, high cost and varying activity depending on batch and source. Furthermore, some of them are commercially unavailable,

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needing home preparation and screening. To overcome these problems, some non-biological small molecular compounds, such as carbohydrate,23-25 metal complex,26,27 crystal violet28 have also been employed as recognition agents for pathogenic bacteria detection. The main drawback of these small molecular recognition agents is their poor specificity since most of them can bind with many different bacteria. Nowadays, numerous antibiotics have been widely used to kill pathogens and treat bacterial infections. Some antibiotics, including β-lactam antibiotics, glycopeptide antibiotics and lantibiotics, exert their antimicrobial effect by anchoring the cell wall or the outer membrane of bacteria, and then destroying the cell wall. For example, as a broad-spectrum glycopeptide antibiotic, vancocin can anchor firmly most Gram-positive bacteria via a five-point hydrogen bond binding between this agent and D-Ala-D-Ala moieties in the cell wall of bacteria.29 As commercial conventional drugs, antibiotics show many outstanding properties such as high stability, low cost, and good quality controllability. Therefore, antibiotics acting on the cell wall or the outer membrane of bacteria should be ideal candidates of molecular recognition agents for pathogens. However, like the previously reported non-biological small molecular recognition agents, most of them cannot distinguish different bacteria because they are broad-spectrum antibiotics. Staphylococcus aureus (S. aureus) is well known to be one of the major pathogens that causes many serious infectious diseases such as abscesses, nosocomial pneumonia, endocarditis, and even toxic shock syndrome. Due to its high health risk, developing rapid and low-cost assay method for S. aureus would have a significant impact on improving the treatment of these diseases. Here, an antibiotic-affinity strategy was

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designed for fluorimetric assay of S. aureus based on the affinity of vancocin to the cell wall of S. aureus. The cell wall of S. aureus was reported to highly express S. aureus protein A (SPA) that could bind specifically with the heavy chain in Fc region of IgG of many mammalian species.30 Thus IgG was utilized as the second recognition agent to improve the specificity of this antibiotic-affinity strategy. Since vancocin and IgG recognized S. aureus at two distinct sites to form a sandwich complex, a complementary effect in the specificity was achieved in this protocol.

EXPERIMENTAL SECTION Reagents and materials R-phycoerythrin

(R-PE),

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride (EDC) and vancocin hydrochloride were purchased from Sigma-Aldrich (USA). Fluorescein isothiocyanate (FITC)-labeled porcin IgG was provided by Beijing Biosynthesis Biotechnology Co., Ltd (China). Strains of Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), Salmonella typhimurium (S. typhimurium), Micrococcus luteus (M．luteus) and S. aureus were obtained from Chongqing Center for Disease Control and Prevention (China). N-hydroxysuccinimide (NHS) were purchased from Aladdin Chemistry Co., Ltd (China). Bovine serum albumin (BSA) was obtained from Dingguo Biotechnology Company (China). Apple juice was purchased from the local supermarket. All aqueous solutions were prepared using the ultrapure water (18.2 MΩ) treated by ELGA PURELAB Classic system (France). High-affinity 96-well polystyrene microplate for fluorimetry was provided by Greiner Bio-One Biochemical Co., Ltd. (Germany).

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Buffers Dilution buffer for FITC-labeled porcin IgG, vancocin-conjugated BSA and bacteria was 0.010 M phosphate buffer saline at pH 7.4 (PBS). Coating buffer was 0.10 M Tris-HCl buffer at pH 8.5. Blocking buffer was PBS containing 3.0% BSA and 0.05% Tween-20. Washing buffer was PBS containing 0.05% Tween-20. All buffers were sterilized at 121 °C for 20 min in an autoclave sterilizer. Apparatus Fluorescence signal was detected by an Infinite M200 PRO multifunctional microplate reader (TECAN Group Ltd., Switzerland). Optical density value of bacteria solution was detected by an UV-2700 spectrophotometer (Shimadzu Co., Ltd., Japan). Fluorescence micrographs were obtained with a LSM710 laser confocal fluorescence microscopy (Carl Zeiss Co., Ltd., Germany). Purification of vancocin-protein conjugates was conducted on an AKTAprimeTM protein purification system (GE Healthcare Co., Ltd., USA) equipped with a Sephadex G-25 column. Bacteria culture and counting Bacteria strains were grown overnight in Luria-Bertani broth medium at 37°C with constant shaking till optical density value at 600 nm (OD600) reached 1.0. Bacteria were harvested at the exponential growth phase by a centrifugation at 5000 rpm for 5 min. The collected pellets were resuspended and diluted serially in PBS. Cell number of bacteria were determined using a conventional plate counting method, and the results were converted to OD600 values. Preparation, purification and antibiotic activity investigation of vancocin-protein 6


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conjugate R-PE and BSA were conjugated with vancocin by a conventional EDC/NHS chemistry. In a typical protocol, 500 µL of vancocin hydrochloride solution at 20 mg mL-1 was mixed with 300 µL of EDC solution at 20 mg mL-1 and 300 µL of NHS solution at 1.0 mg mL-1 to active vancocin. Then 250 µL of protein solution at 1.0 mg mL-1 was added to conjugate with the actived vancocin, followed by an overnight reaction at 4 oC. The reaction was stopped by adding 40 µL of glycine solution at 2.0 M. Using the protein purification system, the product was purified with a flow rate of 0.5 mL/min and a detection wavelength of 280 nm. With such a protocol, approximately 4.5 mL of purified vancocin-protein conjugate was collected. The antibiotic activity of the obtained vancocin-protein conjugates was investigated by a standard bactericidal halo test using Luria-Bertani broth medium. Confocal microscopy imaging of fluorescence-stained S. aureus One hundred microliter of bacteria solution at 108 CFU mL-1 was mixed with 50 µL of R-PE-labeled vancocin (100 µg/mL) and the same amount of FITC-labeled porcin IgG, followed by an incubation of 1 h at 37 °C. The stained bacteria cells were centrifugally washed thrice and resuspended in PBS. Then 12 µL of stained bacteria solution was dropped onto a microscope slide, and covered by a cover slip. The prepared specimen was observed under the laser confocal fluorescence microscopy with a magnification of 1000. The excitation wavelengths for R-PE and FITC were 542 nm and 488 nm, respectively, while the emission wavelengths for R-PE and FITC were 575 nm and 525 nm, respectively.

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Procedure for fluorimetric assay of S. aureus A sandwich fluorimetric method was adopted to assay S. aureus with a 96-well polystyrene microplate. In a typical protocol, the 96-well microplate was coated with vancocin-conjugated BSA at 25 µg mL-1 (100 µL/well) overnight at 4 °C. The coated microplate was washed thrice with washing buffer (300 µL/well) after removal of the coating solution. Afterward, the microplate was blocked with the blocking buffer (150 µL/well) for 60 min at 37 °C, and washed thrice. Subsequently, 150 µL of S. aureus solution was pipetted into the well and incubated at 37 °C for 60 min. After washing, 100 µL of FITC-labeled porcin IgG at 50 µg mL-1 was pipetted into the well and incubated for another 60 min. Following a thorough washing, fluorescence signal was measured with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

RESULTS AND DISCUSSION Principle of sandwich assay of S. aureus based on antibiotic-affinity strategy The principle of sandwich assay of S. aureus is described in Scheme 1. Vancocin was conjugated with BSA to enable physical adsorption of this agent onto the polystyrene microplate. The purification chromatogram of vancocin-conjugated BSA showed that the desired product was completely separated from excessive vancocin (Figure 1A). Although vancocin was conjugated to BSA through EDC/NHS chemistry, it still remained its antibiotic activity, which was demonstrated by the bactericidal halo test (Figure 1B). As a Gram-positive pathogen, S. aureus was captured by the coated vancocin-conjugated BSA through the firm five-point hydrogen bond binding between vancocin and D-Ala-D-Ala moieties in the cell wall. Since SPA was one of the dominant

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proteins in the cell wall of S. aureus, FITC-labeled porcin IgG bound with the captured S. aureus through the strong and specific binding between SPA and Fc region of IgG. Thus a FITC-labeled sandwich complex was formed on the microplate, which allowed a direct fluorimetric assay of whole cells of S. aureus. Binding behavior of vancocin and IgG to S. aureus To investigate the binding behavior of vancocin and IgG to S. aureus, R-PE-labeled vancocin and FITC-labeled porcin IgG were simultaneously incubated with S. aureus cells, and the dual fluorescence-stained cells were observed under the laser confocal fluorescence microscopy. As shown in Figure 2, both red fluorescence from R-PE and green fluorescence from FITC were observed on the cells after the bacteria solution was incubated with the two fluorescence tracers. The fluorescence signals were found to be mainly concentrated on the outer layer of cells, implying that both vancocin and IgG bound with the cell wall. Since the two molecular recognition agents anchored the cell of S. aureus at two binding sites, a sandwich complex of vancocin/S. aureus/IgG could be formed, allowing detection of bacteria with a principle similar to classic sandwich immunoassay. Binding capability of IgGs from different animals SPA in the cell wall of S. aureus is known to be capable of binding with IgGs from many mammalian species, and the binding capability depends on the species they originate from.30 Therefore, IgGs from some mammals such as porcin, human, rabbit, mouse and goat were used to perform sandwich assay of S. aureus at 1.0 × 108 CFU mL-1 to compare their affinity to SPA. Porcin IgG was found to show the strongest affinity to SPA, allowing sensitive sandwich detection of S. aureus. The signals by using IgGs from 9



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human, rabbit, mouse and goat were 91%, 84%, 40% and 23%, respectively, of that by using porcin IgG (Figure 3). Therefore, porcin IgG was adopted as the second recognition agent in the whole investigation due to its strong affinity to SPA. Optimization of experimental conditions Some factors influencing the performance of the proposed method were investigated using S. aureus at 1.0 × 108 CFU mL-1. The whole assay process involved two incubations for forming sandwich complex. Thus the effect of the incubation time on the fluorescence signal was investigated at 37 °C in detail. It was found that the both incubation processes tended to be saturated after 60 min (Figure 4). Therefore the both incubations were conducted for 60 min. The influence of the concentrations of vancocin-conjugated BSA and FITC-labeled porcin IgG on fluorescence response was also studied, and the optimal concentrations for the two proteins were chosen to be 25 µg mL-1 and 50 µg mL-1, respectively (data not shown). Analytical performance Under the chosen optimal conditions, the fluorescence response increased with the concentration of S. aureus in a linear range of 1.0 × 103 to 1.0 × 109 CFU mL-1. For a seven-point calibration curve, the linear regression equation was lg I (a. u.) = 0.3414 lg C (CFU mL-1) + 1.56 (R2 =0.992), where I and C were the fluorescence response and the concentration of S. aureus, respectively. The detection limit was 2.9 × 102 CFU mL-1 at a signal to noise ratio of 3. The relative standard derivations (RSDs) for five replicated detections of S. aureus at low (1.0 × 103 CFU mL-1), medium (1.0 × 106 CFU mL-1) and high (1.0 × 109 CFU mL-1) concentrations were 7.4%, 8.9% and 4.9%, respectively,

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showing acceptable repeatability. By using a ready-for-use microplate which had already been coated and blocked, the whole assay process could be completed within 130 min. Specificity This antibiotic-affinity strategy utilized vancocin as the first molecular recognition agent to capture the whole cells of S. aureus. As a broad-spectrum antibiotic, vancocin showed noticeable affinity to many types of bacteria, thus its specificity was a key concern for ensuring its application reliability. Adoption of IgG as the second molecular recognition agent could greatly improve the specificity because SPA was a specially expressed protein in the cell wall of S. aureus. The two recognition agents bound with S. aureus at two distinct sites, thus a complementary effect in the specificity could be easily achieved. To demonstrate this claim, P. aeruginosa, E. coli, S. typhimurium and M．luteus all at 1.0 × 108 CFU mL-1 were assayed with the proposed method, and the obtained fluorescence signals were compared with those from blank sample (PBS) and S. aureus at the same concentration. As seen in Figure 5, the four interfering bacteria only showed very weak response close to that from blank sample. The interference factor (IF) values of these potential interferents were obtained according to the following formula: IS BS ×100% (1) SS BS Here, IS, BS and SS were signals from interferent sample, blank sample and S. aureus IF =

sample, respectively. The IF values of P. aeruginosa, E. coli, S. typhimurium and M．luteus were calculated to be 0.26%, 0.68%, 0.85% and -0.64%, respectively, implying negligible interference of these bacteria. Mixture 1 composed of the four interfering bacteria all at 1.0 × 108 CFU mL-1 was prepared and assayed with the same protocol. This mixture also showed a very low IF value of 1.38%. Then, Mixture 2 was prepared by

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mixing S. aureus and the four interfering bacteria at the same concentration. The fluorescence response of Mixture 2 only showed a small change of 6.01% in comparison with that of S. aureus sample. The above results revealed ideal specificity of the proposed antibiotic-affinity strategy for S. aureus detection.

Application in real sample assay Three sterile apple juice samples were spiked with S. aureus standard solutions at different known concentrations and assayed with the proposed method. The results listed in Table 1 showed acceptable recoveries ranging from 85% to 130%, demonstrating its reliability and application potential in real sample assay.

CONCLUSION In conclusion, an antibiotic-affinity strategy was proposed to directly detect whole cells of pathogenic bacteria based on the strong affinity between antibiotic and cell wall. This strategy was demonstrated by fluorimetric assay of S. aureus using vancocin as the molecular recognition agent. As a molecular recognition mode-based approach, it didn’t demand such pretreatments as bacteria culture and nucleic acid extracting, thus allowed rapid and simple assay of pathogens. Compared with the previously reported biomaterial-based recognition agents, antibiotics were much more unexpensive and stable. To improve its specificity for S. aureus detection, FITC-labeled IgG was adopted as the second recognition agent and signal tracer since its Fc region showed strong and specific binding capability to SPA that was highly expressed in the cell wall of S. aureus. This approach also showed ideal sensitivity and wide linear range compared with the previously reported methods for bacteria detection. It should be kept in mind that it is only a proof-of-concept work for pathogenic bacteria assay. In the near future we will try 12


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to extend this strategy to assay other pathogens using other antibiotic agents. A further developing of this approach to POCT device is also ongoing.

ACKNOWLEDGEMENTS We thank the National Natural Science Foundation of China (21475107, 21175111), the Program for Innovative Research Team in University of Chongqing (2013), the Natural Science Foundation of Chongqing (CSTC2013jjB0096) and the Fundamental Research Funds for the Central Universities (XDJK2013A025). We also thank Professor Guojian Liao and Professor Chong Li for their extremely valuable discussion and suggestion for this research.

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Figure Captions Scheme 1 The principle of sandwich fluorimetric detection of S. aureus based on antibiotic-affinity strategy.

Figure 1 (A) Purification chromatogram of vancocin-conjugated BSA and (B) photograph of bactericidal halo test for vancocin-conjugated BSA. 1: blank; 2, 3 and 4: vancocin-conjugated BSA.

Figure 2 Fluorescence micrographs of S. aureus stained with R-PE-labeled vancocin and FITC-labeled porcin IgG. (A) bright field image, (B) image of green fluorescence channel, (C) image of red fluorescence channel, (D) image merged from green and red.

Figure 3 Fluorescence responses for S. aureus at 1.0 × 108 CFU mL-1 using IgGs from different mammalian species as the second recognition agents. All other assay conditions were the chosen optimal conditions.

Figure 4 Influence of the incubation time of (A) S. aureus and (B) FITC-labeled porcin IgG on the fluorescence signal for detection of S. aureus at 1.0 × 108 CFU mL-1. All other assay conditions were the chosen optimal conditions.

Figure 5 Fluorescence responses for S. aureus and the interfering bacteria using the proposed antibiotic-affinity strategy. The concentrations of all bacteria were 1.0 × 108 CFU mL-1. All assay conditions were the chosen optimal conditions.

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Scheme 1

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Table 1 Recovery test of S. aureus spiked in three apple juice samples using the proposed method. Sample No. 1

Spiked (CFU mL-1) 1.0 × 104 1.0 × 105 1.0 × 106

Found (CFU mL-1) (9.5 ± 1.2) × 103 (1.2 ± 0.1) × 105 (1.1 ± 0.1) × 106

Recovery (%) 95 120 110

2

1.0 × 104 1.0 × 105 1.0 × 106

(1.2 ± 0.2) × 104 (1.3 ± 0.1) × 105 (8.7 ± 1.0) × 105

120 130 87

3

1.0 × 104 1.0 × 105 1.0 × 106

(9.1 ± 1.5) × 103 (8.5 ± 0.8) × 104 (1.3 ± 0.1) × 106

91 85 130

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Sandwich Fluorimetric Method for Specific Detection of

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