OnâOffâOn Gold Nanocluster-Based Fluorescent Probe for Rapid

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An On-Off-On Gold Nanocluster-Based Fluorescent Probe for Rapid Escherichia coli Differentiation, Detection and Bactericide Screening Rong Yan, Zhangxuan Shou, Jie Chen, Hao Wu, Yuan Zhao, Lin Qiu, Pengju Jiang, Xiao-Zhou Mou, Jian-Hao Wang, and Yong-Qiang Li ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00112 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 11, 2018

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ACS Sustainable Chemistry & Engineering

An On-Off-On Gold Nanocluster-Based Fluorescent Probe for Rapid Escherichia coli Differentiation, Detection and Bactericide Screening Rong Yan,†,∆ Zhangxuan Shou,║,∆ Jie Chen,§,∆ Hao Wu,‡ Yuan Zhao,‡ Lin Qiu,‡ Pengju Jiang,‡ Xiao-Zhou Mou,*,║ Jianhao Wang,*,‡ and Yong-Qiang Li*,† †

State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and

Protection, Collaborative Innovation Center of Radiological Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China. ‡

School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou

213164, China. §

║

Suzhou Hospital Affiliated to Nanjing Medical University, Suzhou 215002, China. Clinical Research Institute, Zhejiang Provincial People’s Hospital, People’s Hospital of

Hangzhou Medical College, Hangzhou 310014, China. *Corresponding Author: [email protected] (X.-Z. Mou), [email protected] (J. Wang), [email protected] (Y.-Q. Li) KEYWORDS: bacterial infections, gold nanocluster, on-off-on fluorescent probe, bacterial detection, bactericide screening, environmental monitoring

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ABSTRACT: The bacterial differentiation, detection and bactericide screening are critical for environmental monitoring and human health maintenance. In this work, we present a novel strategy for rapid Escherichia coli (E. coli) differentiation, detection and bactericide screening, by employing an on-off-on gold nanocluster (AuNC)-based fluorescent probe. We demonstrate that by hijacking their copper-binding and redox pathways to capture and reduce Cu2+, E. coli bacteria can selectively and efficiently recover the fluorescence of AuNC from Cu2+-caused quenching. Based on this on-off-on AuNC-based fluorescent probe, rapid differentiation and detection of E. coli bacteria in artificial contaminated water sample with trace concentration of bacteria (around 100 CFU/mL) is successfully realized within 0.5 h, showing great potential for rapid point-of-care detection of pathogenic E. coli bacteria in environmental monitoring and clinical bedside diagnosis. Furthermore, this on-off-on AuNC-based fluorescent probe can enable rapid and accurate bactericide screening test for multidrug-resistant E. coli in sepsis blood sample, which is greatly helpful to accelerate the pace of treatment and reduce the mortality of infectious diseases.

INTRODUCTION Bacterial infectious diseases are potentially life-threatening conditions, and constitute a big challenge facing public healthcare today due to the increasingly serious microbe contamination of food, drinking water and biomedical reagents.1-6 Escherichia coli (E. coli), one of the most common clinical pathogens, cause a series of life-threatening diseases (e.g., sepsis, haemolytic uremic syndrome) and enormous medical and financial burden.3,4 To combat E. coli infectious diseases and effectively decrease their damage, rapid E. coli differentiation, detection and


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bactericide screening are the foremost crucial steps.7,8 Currently, bacterial culture methods and polymerase chain reaction (PCR)-based assays are two typical strategies widely used for pathogenic bacteria detection.9-12 The time-consuming biochemical characterization processes in bacterial culture methods and the tedious procedures in PCR-based assay with the occurrence of false-positive results,9,11 restrict the applications of these two techniques in clinical settings. In addition, the agar diffusion test used in conventional bactericide screening is bacterial culturebased and generally requires more than one day to report the outcomes,13 which is far from satisfying the aim of rapid detection. To address these issues, novel strategy for rapid and accurate E. coli differentiation, detection as well as bactericide screening should be developed. In recent years, fluorescent probes have attracted extensive attention in the fields of sensing and diagnostics.14-16 As a fluorescent candidate, metal nanoclusters with ultrafine size is attractive due to their extraordinary physical and chemical properties.17-24 In particular, gold nanocluster (AuNC) possessing bright fluorescence, high stability and good biocompatibility, represents a fascinating option to produce label-free and fast responsive fluorescence signals for biosensing (e.g., bacterial detection) and bioimaging.21-27 Lately, by exploiting the phenomena of metal ions (e.g., Cu2+ and Hg2+)-induced fluorescence quenching and analyte-triggered recovery, several exquisite AuNC-based nanoprobes have been developed for rapid detection of various biomolecules.28-31 Inspired by these studies, rapid bacterial detection is highly expected to be achieved by investigating the bacteria-responsive fluorescence recovery of AuNC-metal ion ensemble. Meanwhile, recent studies on copper-homeostasis mechanisms in E. coli have found that bacteria can adapt to copper-rich environments through copper-binding system and redox enzyme cascades to efficiently bind toxic Cu2+ and quickly reduce it to Cu+.32-34 The cupric reductase NDH-2 in E. coli is responsive for Cu2+ reduction in this process, and the formed Cu+


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can then be transmitted to the CopA (a Cu+-translocating P-type ATPase) for cytoplasmic Cu+ detoxification.32 Therefore, we hypothesize that by hijacking the copper-binding and redox pathways of bacteria to recover the fluorescence of AuNC-Cu2+ ensemble, a novel strategy amenable for rapid differentiation and detection of pathogenic E. coli as well as bactericide screening, could be developed. Herein, we present a simple but effective strategy for rapid E. coli differentiation, detection and bactericide screening by employing an on-off-on fluorescent probe. This probe is constructed based on the bovine serum albumin (BSA)-stabilized fluorescent AuNC. Scheme 1 illustrates the working principle of this on-off-on AuNC-based fluorescent probe. Due to the coordinate interaction between Cu2+ and the amino acid residues on the BSA surface to induce the excited state of AuNC to lose its energy, the fluorescence of AuNC can be quenched by Cu2+ in the absence of bacteria.35 Yet in the presence of E. coli bacteria, Cu2+ can be quickly removed from AuNC surface and subsequently reduced by E. coli through their copper-binding and redox pathways, leading to bacteria-responsive fluorescence recovery of AuNC. Based on this unique bacteria-responsive fluorescence recovery phenomenon, rapid E. coli differentiation, detection and subsequent bactericide screening can be achieved. RESULTS AND DISCUSSION The AuNC consisting 25 gold atoms (Au25) was prepared by using BSA as a template to sequester and reduce the gold precursors in situ.36,37 As shown in Figure 1a, the as-prepared deep brown solution of AuNC emitted an intense red fluorescence under UV light irradiation, and possessed the characteristic UV absorption peak (at around 280 nm) of BSA. The AuNC exhibited excitation and emission peaks at around 500 and 700 nm, respectively, and its fluorescence quantum yield was calculated to be 6% using Rhodamine 6G as the reference


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(Figure 1a and Figure S1). From the transmission electron microscopy (TEM) image, the asprepared AuNC was found to have a uniform spherical structure with mean size of 2.29 ± 0.31 nm (Figure 1b). Subsequently, Cu2+-induced fluorescence quenching of AuNC and E. coliresponsive fluorescence recovery were investigated. We found that Cu2+ could efficiently quench the fluorescence of AuNC without interference from other common metal ions, which was consistent with the results of previous works (Figure 1c).38 More importantly, the fluorescence of AuNC-Cu2+ ensemble was nearly recovered after incubation with E. coli (106 colony-forming units (CFU)/mL) for 0.5 h (Figure 1d). This result demonstrates that the capability of E. coli for Cu2+ binding and reduction can indeed result in the recovery of AuNC fluorescence as we expected. Similar to Cu2+, the AuNC exhibited Hg2+ concentration-dependent fluorescence quenching, but no significant fluorescence increase was found after E. coli (106 CFU/mL) incubation possibly due to the poor capability of E. coli for Hg2+ binding and reduction (Figure S2). Moreover, to confirm the Cu2+ reduction-associated mechanism we proposed and investigate the reduction capability of blood components (for sepsis sensing purpose), the fluorescence spectra of AuNC-Cu2+ ensemble were investigated after incubation with the bare blood or the blood containing a reductant (sodium ascorbate). As shown in Figure S3, fluorescence recovery was only found for the AuNC-Cu2+ ensemble after incubation with the blood containing sodium ascorbate rather than the bare one. This result could confirm the Cu2+ reduction-associated mechanism of AuNC-Cu2+ fluorescence recovery, and the inability of bare blood to recover the fluorescence may relate to the weak reduction capability of bare blood components. The fluorescence recovery of AuNC-Cu2+ ensemble triggered by different species of bacteria was investigated. Here, 4 types of bacteria including Gram-negative E.coli and Pseudomonas


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aeruginosa (P. aeruginosa), and Gram-positive Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus) were employed. As shown in Figure 2a, the fluorescence of AuNC-Cu2+ ensemble was only recovered effectively after incubation with E. coli (106 CFU/mL) rather than the other 3 bacterial species with the same concentration (106 CFU/mL), indicating the outstanding capability of Cu2+ binding and reduction in E. coli bacteria. In addition, the recovery degree of AuNC fluorescence triggered by E. coli (106 CFU/mL) and other 3 species of bacteria with higher concentration (107 CFU/mL) was quantitatively analyzed, respectively. As shown in Figure 2b, the AuNC-Cu2+ ensemble exhibited obviously higher fluorescence recovery level after E. coli bacteria incubation compared to that of other 3 bacterial species tested with extremely higher concentration. Furthermore, E. coli-triggered fluorescence recovery of AuNC-Cu2+ ensemble in the mixture of 6 other bacterial stains was carried out. As shown in Figure S4, E. coli (106 CUF/mL) efficiently recovered the quenched fluorescence of AuNC-Cu2+ ensemble incubated with the mixture of 6 other bacterial stains (107 CUF/mL). These results demonstrate that the AuNC-Cu2+ ensemble can be used as an effective and selective probe for E. coli differentiation and detection based on its fluorescence recovery level after bacteria incubation. The interesting phenomenon of bacterial species-dependent fluorescence recovery of AuNC-Cu2+ ensemble may be closely related with the reduction ability of different bacterial species for Cu2+.39 Moreover, the fluorescence recovery of AuNC-Cu2+ ensemble triggered by E. coli in different types of medium was evaluated. As shown in Figure S5, obvious fluorescence recovery was observed for the AuNC-Cu2+ ensemble after incubation with E. coli in 3 different types of medium (drinking water, Dulbecco’s modified eagle medium (DMEM) and blood), respectively, indicating the flexibility of the AuNC-Cu2+ ensemble with regards to selective E. coli detection from different sample sources.


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The E. coli-responsive fluorescence recovery of AuNC-Cu2+ ensemble lays a solid foundation for the construction of on-off-on AuNC-based fluorescent probe for rapid E. coli differentiation and detection. To investigate this feasibility, the AuNC was incubated with Cu2+ and E. coli with various concentrations from 102 to 106 CFU/mL for 0.5 h, and its fluorescence was systematically analyzed. As shown in Figure 3a and 3b, the fluorescence of AuNC gradually increased with bacterial concentration, and there was a good linear relationship (R2 = 0.99) between the change of AuNC fluorescence intensity (I/I0) and the concentration of E. coli bacteria in the range of 103-106 CFU/mL. This result indicates that the on-off-on AuNC-based fluorescent probe can be used for rapid detection of E. coli. In addition, it was found that the value of I/I0 was little higher than 0.1 (the threshold to eliminate the influence of bacterial sample medium on AuNC-Cu2+ ensemble’s fluorescence (Figure S6)) in the condition of 102 CFU/mL E. coli (Figure 3b). The limit of detection (LOD) of the AuNC-based on-off-on fluorescent probe was calculated to be 89 CFU/mL (Signal/Noise = 3), showing outstanding capability for the detection of E. coli with trace concentration. Furthermore, artificial contaminated water sample containing around 100 CFU/mL of E. coli (as depicted by the agar plate result shown in Figure 3c), was analyzed based on our on-off-on AuNC-based fluorescent probe. As shown in Figure 3d, compared to the AuNC-Cu2+ ensemble with fully quenched fluorescence, obvious red fluorescence signal was observed in the AuNC-Cu2+ ensemble incubated with the artificial contaminated sample. Although the sensitivity is not comparable with the conventional bacterial culture methods and PCR-based assays, our on-off-on fluorescent probe-based strategy do not need tedious and time-consuming procedures, as well as the complicated and expensive instruments, and can enable rapid E. coli differentiation and detection just by naked eyes with the assistance of a UV lamp. This feature makes this on-off-on


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fluorescent probe-based strategy perfectly suitable for rapid point-of-care detection of pathogenic E. coli bacteria in environmental monitoring and clinical bedside diagnosis. Furthermore, the AuNC-Cu2+ ensemble can be easily coupled with bacterial enrichment methods,40,41 making the highly sensitive point-of-care detection down to trace concentrations of pathogenic E. coli bacteria (around 1-10 CFU/mL) become possible. The capability of this on-off-on AuNC-based fluorescent probe for the differentiation of different strains of E. coli was evaluated. Unfortunately, for the two strains (E. coli without drug resistance, and multidrug-resistant E. coli) tested, no distinguishable fluorescence recovery level was found for the AuNC-Cu2+ ensemble after bacteria incubation (Figure S7). However, this makes the operation of bactericide screening for pathogenic E. coli especially the drug-resistant ones become possible, which is critical in clinical settings to choose effective bactericide to combat infectious diseases. Therefore, the feasibility of our on-off-on AuNC-based fluorescent probe for rapid bactericide screening was performed. Here NDM-1 E. coli bacteria showing resistance to conventional β-lactam antibiotics, was chosen as the model bacterium due to its rapid emergence and serious damage in both hospital and community settings.42,43 Three kinds of antibiotics including two β-lactam antibiotics (penicillin-G and ampicillin) and the vancomycin with effective anti-NDM-1 bacteria capability,44 were tested. To perform the antibiotic screening, the NDM-1 E. coli-spiked blood sample was first incubated with the three antibiotics for 1.5 h respectively, and then mixed with AuNC and Cu2+ for 0.5 h. The final antibiotic susceptibility result could be obtained based on the fluorescence state of AuNC. As we expected, only the vancomycin-treated blood sample was unable to restore the fluorescence of AuNC in the presence of Cu2+ quencher (I/I0 < 0.1), confirming the capability of vancomycin for anti-NDM-1 E. coli applications (Figure 4a). This result was consistent with the fluorescence photographs


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(Figure 4b) and the culture-based agar plate assay (Figure 4c), demonstrating the potential of our on-off-on AuNC-based fluorescent probe for rapid bactericide screening test for multidrugresistant E. coli in sepsis blood sample. CONCLUSION In summary, we report an on-off-on AuNC-based fluorescent probe for selective E. coli differentiation and detection, by hijacking the unique copper-binding and redox pathways of E. coli bacteria to recover the fluorescence of AuNC from Cu2+-caused quenching. Based on this fluorescent probe, rapid detection of E. coli bacteria in artificial contaminated water sample with trace concentration of bacteria (around 100 CFU/mL) is successfully realized within 0.5 h, showing great potential for rapid point-of-care detection of pathogenic E. coli bacteria in environmental monitoring and clinical bedside diagnosis. Furthermore, this on-off-on AuNCbased fluorescent probe can enable rapid and accurate bactericide screening test for multidrugresistant E. coli in sepsis blood sample, which is greatly helpful to accelerate the pace of treatment and reduce the mortality of infectious diseases.


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Scheme 1.

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Schematic illustration of the working principle of the on-off-on AuNC-based

fluorescent probe for rapid E. coli differentiation, detection and bactericide screening.


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Figure 1. (a) Absorption and fluorescence emission (λex = 500 nm) spectra of AuNC solution. The inset shows the photographs of AuNC solution under daylight (1) and UV light (2) irradiation. (b) TEM image of AuNC. (c) The fluorescence change of AuNC (1 µM) before and after incubation with different metal ions (4 µM) for 10 min. Here, “I0” and “I” represents the integrated fluorescence intensity of AuNC before and after incubation with metal ion, respectively. The I/I0 values represent the mean of three independent measurements and the error bars indicate the standard deviation (SD) from the mean. (d) Fluorescence emission spectra of AuNC (1 µM) and AuNC-Cu2+ ensemble (1 µM of AuNC, 4 µM of Cu2+) with and without E. coli (106 CFU/mL) incubation.


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Figure 2.

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(a) The photographs of AuNC-Cu2+ ensemble after incubation with 4 different

bacterial species with the same concentration (106 CFU/mL) for 0.5 h under UV light irradiation. (b) The recovery degree of AuNC fluorescence in the AuNC-Cu2+ ensemble with different bacterial species incubation. The fluorescence recovery values represent the mean of three independent measurements and the error bars indicate the SD from the mean. ***P

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