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A Novel Strategy for Tracking the Microbial Degradation of Azo Dyes with Different Polarity in Living Cells Fei Liu, Meiying Xu, Xingjuan Chen, Yonggang Yang, Haiji Wang, and Guoping Sun Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02003 • Publication Date (Web): 10 Sep 2015 Downloaded from http://pubs.acs.org on September 18, 2015

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Environmental Science & Technology

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A Novel Strategy for Tracking the Microbial

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Degradation of Azo Dyes with Different Polarity in

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Living Cells

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Fei Liu, Meiying Xu*, Xingjuan Chen, Yonggang Yang, Haiji Wang, and Guoping Sun

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State Key Laboratory of Applied Microbiology Southern China, Guangdong Institute of

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Microbiology.100 Central Xianlie Road, Guangzhou 510070, P.R. China ， E-mail:

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[email protected]

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KEYWORDS: microbial degradation, azo dyes, FRET, fluorescence tracking, degradation

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pathways

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ABSTRACT: Direct visualization evidence is important for understanding the microbial

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degradation mechanisms. To track the microbial degradation pathways of azo dyes with different

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polar characterizations, sensors based on the FRET (fluorescence resonance energy transfer)

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from 1, 8-naphthalimide to azo dyes were synthesized, in which the quenched fluorescence will

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recover when the azo bond was cleaved. In living cells, the sensor tracking experiment showed

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that the low polar and hydrophobic azo dye can be taken up into the cells and reduced inside the

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cells, whereas the highly polar and hydrophilic azo dye can be reduced only outside the cells

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because of the selective permeability of the cell membranes. These results indicated that there

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were two different bacterial degradation pathways available for different polarity azo dyes. To

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our knowledge, no fluorescent sensor has yet been designed for illuminating the microbial

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degradation mechanisms of organic pollutants with different characteristics.

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INTRODUCTION

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Microorganisms can detoxify or remove many environmental pollutants because of their

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diverse metabolic capabilities, providing noninvasive and cost-effective methods to protect us

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from pollution.1 The traditional strategies for understanding microbial degradation mechanisms

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usually relied on genetic engineering and protein separation and purification technologies,

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complicating the monitoring of the mode of action and the transfer process of pollutants in vivo.2-

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4

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remain in the conjectural stage, notably for those organic pollutants that are anthropogenic

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sources and are only difficultly or partially degraded.5-8 Describing the metabolic reductive

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processes will assist the understanding of microbial degradation mechanisms. Thus, substantial

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attention has been focused on developing new methods to reveal the details of microbial

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degradation, notably visualization methods that provide direct evidence.

Because of the lack of strong evidence, most studies of microbial degradation mechanisms

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During the past decade, a substantial number of fluorescent chemosensors have been

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published and have attracted attention because of their simplicity, high selectivity and sensitivity

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in fluorescent assays. These sensors can be conveniently used as a tool to analyze guest species

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and sense biologically important species in vitro and in vivo to clarify their function in living

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systems.9-11 However, examples of fluorescent sensors applied in microbial systems to study

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microbial degradation are rare. This study provides a fluorescence method to track the microbial

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degradation processes of azo dyes, which are commonly used in a number of industries and have


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been identified as a dominant toxic organic pollutant around the world, to better understand

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microbial degradation mechanisms.12

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Azo dyes are compounds bearing a functional group with an azo bond (R–N=N–R). Over the

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last few years, the carcinogenicity and other toxic effects of azo dyes have raised significant

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concerns.13-16 A prolonged intake of azo dyes can result in the formation of tumors, allergies,

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respiratory problems and birth defects.17-18 To date, many azo dye degrading bacteria have been

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found, and select studies have implied that the ability of bacteria to reduce azo dyes is mostly

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attributed to enzymes (azoreductases) that cleave the azo bond inside or outside the cells. These

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enzymes might be related to the characteristics of the azo dyes.19-25 However, no direct

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visualization evidence has been provided. To understand the microbial degradation mechanisms

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of the azo dyes, we designed a new fluorescence method utilizing the different polarity to track

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the mode of action and transfer processes of azo dyes during microbial degradation.

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Materials and methods

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1. Strains and growth conditions:

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Shewanella decolorationis strain S12T (CCTCCM203093T = IAM 15094T) was isolated from

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activated sludge from a textile-printing wastewater treatment plant and preserved in our

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laboratory.26-27 Strain S12-22 was a mutant of strain S12 constructed by transposon insertion in

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ccmA 170 bp downstream of the initiation codon, resulting in a deficiency in producing mature c-

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type cytochromes (Fig S5, S6).28

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Strain S12 and S12-22 were grown aerobically in standard luria-bertani medium (LB) or

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anaerobically inlactate medium (LM) containing 2.0 g/l lactate, 2.0 g/l yeast extract, 12.8 g/l


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Na2HPO4·7H2O, 3 g/l KH2PO4, 0.5 g/l NaCl, and 1.0 g/l NH4Cl at 33°C (asdescribed

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previously).28 The defined medium could be supplemented with lactate or 1 mM NADH.

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2. Analytical procedures:

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Decolorizing reactions were conducted in an anaerobic station at 33°C for each assay in

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triplicate. An NADH or lactate and sensors were added to the defined medium, which was then

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purged with pure nitrogen gas for more than 5 min. Intact cells of strain S12 and S12-22 reduced

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0.05 mM NA-MR and NA-OG (Scheme 1) within 7 h when NADH or lactate were existed in

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the defined medium.

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3. HPLC conditions:

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The degradation products of NA-MR and NA-OG were analyzed by HPLC. Ethanol was

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added to the supernatants, and the suspensions were filtered through 0.22-µm pore-sized acetyl

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cellulose membranes. The HPLC system employed an Athena C18-WP-100 V column (4.6 mm /

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3150 mm); a detection wavelength of 280 nm; a flow rate of 3 ml/min; and water: methanol ratio

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of 9:91(v/v) as the eluent.

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4. Absorption and Fluorescence Measurements.

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Absorption spectra were measured on a Thermo Scientific MULTISKAN GO. Fluorescence

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spectra were obtained with a Perkin Elmer LS 45 Fluorescence Spectrometer. The spectra were

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obtained every 2 h (0-6 h) in the recovered supernatants. Cells were removed by centrifugation,

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and the supernatant was recovered by filtering through 0.22-µm pore-sized acetyl cellulose

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membranes.

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5. Fluorescence imaging:


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The fluorescence imaging of NA-MR and NA-OG in cells were obtained with a spectral

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CLSM (Confocal Laser Scanning Microscopy, LSM 700, Zeiss). Before the CLSM analysis, the

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cells were separated from the culture by centrifugation (6000 g) for 2 min and then washed twice

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with a sterilized phosphate buffer solution (PBS) to remove residual nutrients. The probes (50

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µM) were loaded into LM medium (1 mM NADH for NA-MR) with strain S12, S12-22 or

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Pseudomonas stutzeri cells and inoculated for 2, 4 and 6 h, respectively.

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6. Synthesis of azo dye Sulfo-red and sensor NA-MR, NA-OG:

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Synthesis of Sulfo-red:

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Diluted HCl (5 mL of conc. HCl, 58 mmol) was added slowly to p-aminobenzoic acid (1 g,

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7.24 mmol) in water at 0oC and the mixture was stirred for 15 min. A solution of NaNO2 (0.75g,

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11 mmol) in water is dropped into the slurry mixture. The resulting clear mixture was added

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dropwise to a solution of N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (1.8 g, 6 mmol).

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After stirring for 2 h at 0-5 °C, it was stirred at room temperature for 24 h. Heat the mixture to 70

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°C for 3 minutes to dissolve most of the precipitate. When all the dye is dissolved, add 4 g of

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sodium chloride, and cool the mixture in an ice bath. The precipitate was filtered and washed

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with water to give compound Sulfo-red as an orange solid (1.2 g) in 46.8 % yield.

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Synthesis of sensor NA-MR and NA-OG:

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Compound b (shown in the Scheme S1.) (0.17 mL, 0.58 mmol) and (Benzotriazol-1-yl-

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oxytripyrrolidinophosphonium hexafluorophosphate) PyBOP (0.56 g, 0.32 mmol) were added to

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the solution of TEA (0.22 mL, 0.58 mmol) and Sulfo-red (0.50 g, 0.29 mmol) in an hydrous

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dimethylformamide (5 mL). The mixture was stirred at room temperature for 2 days. The

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mixture was poured into ice water and filtered off the precipitate. The precipitate was washed

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with ethanol. The crude product was purified by silica gel column chromatography eluting with

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dichloromethane/ethanol (6/4) to give NA-MR as a red solid (109 mg, 65%). With the same


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experiment method, we got the probe NA-OG.

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7. Test method of octanol-water partition coefficient (Log KOW)

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The octanol-water partitioning coefficients of the four azo dyes were measured at 25 ˚C

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following the related literatures.29-30 10 mL of deionized water containing 100 µg of azo dyes

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were mixed with 10 mL of n-octanol in ten glass bottles of 20 mL. The bottles were sealed with

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rubber plug, and shaken for 5 min. After standing for 24 hours and phase separation, the

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concentrations of azo dyes in two aqueous phases were measured by HPLC.

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Results and discussion

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Azo reduction and spectrum response.

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Because azo dyes are non-fluorescent and quenching dyes, when a new fluorophore is

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designed to connect with the azo dye by an alkyl chain, the fluorescence of the molecule will be

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quenched. Nevertheless, the fluorescence will recover when the azo moiety is degraded by

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microorganisms. This reduction is examined here using strain S12 with lactate as the electron

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donor (in LM medium) and the sensors as the electron acceptor.31 To track the azo dye

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degradation, new fluorescent sensors NA-MR and NA-OG (Scheme 1) were developed based on

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the FRET mechanism by linking fluorescent moieties with low polar azo-moieties and highly

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polar sulfonated azo-moieties, respectively.

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Scheme 1. Chemical structures of the sensors NA-MR and NA-OG and microbial reduction.

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Decolorizing reactions were conducted in an anaerobic station for each assay. The bacterial

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metabolism of azo dyes is initiated in most cases by a reductive cleavage of the azo bond,

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resulting in the formation of colorless aromatic amines. The azo bond is an important targeted

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position; under anaerobic conditions, the decolorizing reaction works in conjunction with a direct

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enzymatic reaction involving the azoreductase. However, if the extracellular environment is

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aerobic, then this reduction mechanism will be inhibited by oxygen because of the preferential

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oxidation of the reduced redox mediator by oxygen rather than by the azo dye.32-33 The proposed

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fluorescence sensing mode of NA-MR and NA-OG for the decolorizing tracking experiment is

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displayed in Scheme 1. The generation of the fluorescence is mainly because of the azo bond

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reduced by the microorganism. In solution, after reduction by the cells of strain S12, a

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naphthalimide derivative was the main product and fluorescence appeared.

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As illustrated in Figure. 1, NA-MR and NA-OG have an absorption peak at 420 nm in the

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recovered supernatants. Intact cells of strain S12 has high azoreduction efficiency when lactate

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or NADH is existed in the defined medium. In total, 0.05 mM of the probes NA-MR and NA-

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OG are completely reduced within 7 h, and the absorptions band of NA-MR and NA-OG

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gradually decrease with an apparent color change from dark yellow to bright yellow. The

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fluorescence changed from none to yellow at 520 nm (Fig 1e). With the degradation of the

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probes in the solution, the main fluorescence signal was produced by the generation of the

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naphthalimide derivative, which is mainly produced in this degradation process. These results

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indicated that the fluorescence is mainly because of the azo bond being reduced by the enzymes

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in strain S12, showing a good selection to the azo bond and that no other chemical bond is

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broken in the sensors.


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a)

0.35

Absorption intensity


0.30 0.25 0.20 0.15 0.10 0.05 0.00 360

149

400

440

480

520

400

320

240

160

80

0 480

560

c)

0.5

Absorption intensity

0.4

520

560

600

640

680

640

680

Wavelength(nm)

Wavelength(nm)

Fluorescence Intensity

d) 300

0.3

0.2

0.1

0.0 360

150

Fluorescence intensity

b)

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420

480

Wavelength(nm)

540

600

250 200 150 100 50 0 480

520

560

600

Wavelength(nm)

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Figure 1. Changes in the absorption spectra of NA-MR (a) and NA-OG (c) (50µM) with strain

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S12 in the standard LM medium (1 mM NADH for NA-MR) and the fluorescence spectra of

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NA-MR (b) and NA-OG (d). λex = 420 nm. e) Photographs of two probes in solution after 0, 4,

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and 6 h. The visible absorption and fluorescence observed in recovered supernatants.

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In order to further scrutinize the relationship between the molecular property and the tracking

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degradation mechanisms, the n-octanol-water partition coefficient (log KOW) of four azo dyes,

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sulfo-red, methyl-red (chemical structures shown in scheme S1), including NA-OG and NA-MR,

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were measured (Table S1). The log KOW is a key parameter of hydrophobicity which is an

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intrinsic property that relates to a chemical’s tendency to partition between a polar and a

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nonpolar phase. Based on the results shown in Table S1, the azo dyes and their corresponding


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fluorescent sensors contained similar hydrophilic natures although the log KOW values of the

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corresponding fluorescent sensors were higher than their parent azo dyes. The partition

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coefficient Log KOW of sulfo-red and NA-OG were 0.25 and 1.16 at 25 °C, which explained the

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predominantly hydrophilic nature of high polar azo dyes, and the Log KOW of methyl-red and

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NA-MR were 4.62 and 6.05 which shown good hydrophobicity. Compared to the high polar azo

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dyes, the log KOW of low polar azo dyes methyl-red and NA-MR is at least 4-5orders of

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magnitude higher, suggesting that methyl-red and NA-MR were more powerful than sulfo-red

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and NA-OG to penetrate through the live cell membranes barriers. We also found that the

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degradation rates for strain S12 decreased when the azo dyes (methyl-red and sulfo-red) were

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connected

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molecular size and structure may influence on the affinities of the involved enzymes to the

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targeted compounds and the degradation rates of the azo dyes will be reduced when their

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molecular size increased.

the

fluorophore

(shown

in

Fig

S1),

which

indicated

that

the

176 177

Degradation products analysis

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NA-MR and NA-OG degradation products were analyzed by HPLC (Fig. 2). The HPLC and

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MS analysis of the test solutions confirmed that the reduction products of NA-MR and NA-OG

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with strain S12 in an anaerobic station were NA-M and NA-O (chemical structures shown in

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scheme 1). As shown in Figure 2a, we monitored the degradation of NA-MR in the supernatants

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just after inoculation using HPLC, and a single peak was shown at 5.7 min corresponding to

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NA-MR. After 3 h of culturing with NA-MR, a sharp peak at 2.2 min was apparent. Finally, the

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probe NA-MR completely disappeared, and the NA-M was left as the only production in

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solution after 6 h. To confirm the identity of the peaks, we used an ESI-TOF-MS analysis to


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detect the mass at m/z 443 and 574, which represent NA-MR and NA-M, respectively (Fig.

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S2a). In Figure 2b, the degradation products were analyzed by HPLC for the probe NA-OG, and

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a single peak was detected at 1.1 min. After 3 h of culturing, the peak with a retention time of

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2.7 min appeared, but the peaks at 1.1 min remained. After 6 h, some NA-OG probes were still

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present in the solution, and a slower degradation rate was noted for NA-OG than for NA-MR.

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Two peaks were isolated and analyzed by ESI-TOF-MS, and these peaks were confirmed to be

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NA-OG and NA-O (Fig. S2b). Compared with previous research,28 degradation product NA-M

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and NA-O were fluorophore, which could track the microbial degradation processes of azo dye

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using the fluorescence. In this process, we did not find another portion of the molecule;

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therefore, we predicted that the remaining portions of the probe molecules were presumably

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further biodegraded and used as carbon or energy sources by the bacteria.

197 198

Figure 2.HPLC chromatograms of the culture supernatant of probes NA-MR and NA-OG, a)

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NA-MR (50 µM) was incubated with S12 cells for 0 h, 3 h and 6 h in the standard LM


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medium(1 mM NADH for NA-MR); b) NA-OG (50 µM) with S12 in the standard LM

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medium(1 mM NADH for NA-MR) for 0 h, 3 h and 6 h.

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Fluorescence tracking imaging

203 204

The potential utility of NA-MR for tracing azoreduction in living cells of strain S12 was

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then investigated by confocal microscopy. Strain S12 was grown anaerobically in the standard

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LM medium. We monitored the change of the fluorescence in the cells of strain S12 over 6

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hours. Those cells are almost non-fluorescent at the beginning, but the fluorescence gradually

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increased as time progressed. Two hours later, a fluorescence signal in the green channels could

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be detected (Fig. S7a). After 6 hours, the confocal microscopic images exhibited intense

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fluorescence in the green channel, corresponding to NA-M (Fig. 3a-d), and the fluorescence

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image intensity did not disappear as the time progressed. We speculate that the NA-MR would

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continuously enter the cells and that the NA-M diffuses away from the cells, which is congruent

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with the fluorescence intensity increasing in the supernatant with a balanced concentration

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occurring in the cells. Moreover, there are a lot of reductases in the endoplasm or cell membrane.

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So we think, it is very likely the partitioning and accumulation of fluorescent degradation

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moieties of low polar and hydrophobic azo dye will occur in the cell membrane and endoplasm.

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This result demonstrated that NA-MR can be efficiently degraded to NA-M by strain S12 and

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displays a good stain in the cell.


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Figure 3. Degradation in the living cells of strain S12 imaged with NA-MR (a-d) and NA-OG

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(e-h). The two probes (50 µM) were loaded into LM medium (1 mM NADH for NA-MR) with

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the cells of strain S12 for 6 h. The images of the NA-MR (a) and NA-OG (e) with the cells of

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strain S12 after 6 h incubation; b) the green image merged with bright-field; c) enlarged image of

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the cells in (b); d) bright-field image merged with (c); f) DAPI-staining is shown in blue; h) the

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bright-field image merged with the green (e) and blue (f) channel.

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In Fig 3 (e-h), the probe NA-OG was used to track the azoreduction in living S12 cells and

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assessed using confocal microscopy. To investigate the reduction process and imaging of the

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probe NA-OG, DAPI staining (a commercially available nucleic acid sensor) was performed.

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DAPI was mainly used to determine the presence of the cells. Initially, we monitored the change

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of the fluorescence in the S12 cells over the first 2 hours; however, almost no fluorescence was

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generated (Fig S7d). Over the next 4 h, some fluorescence emerged, but the signal remained

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weak. Simultaneously, the supernatants displayed a distinct color change related to the

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degradation of azo dyes by strain S12 (Fig 1e). Therefore, the reducing activity for the probe is

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not dependent on the intracellular uptake of the probe, but this activity occurs outside the cells.


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The sulphonate group is unlikely to pass through the cell membranes of strain S12, and the

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electron transport components, such as OmpA and OmpB (outer membrane protein A and B),

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must be localized in the outer membrane of the bacterial cells, in which these components can

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directly transmit electrons to the probes at the cell surface.34-36 In this process, several

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degradation products, such as NA-O, could diffuse into the cells and produce a low fluorescence

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in the cells. This indicated that the NA-OG dye is likely not degraded in the cell because of its

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highly polar and hydrophilic characteristics, and this dye cannot migrate inside the cells through

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the cell membrane barriers. As shown in Fig 3e, over the 6 h, only little weak fluorescence

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emerged. Simultaneously, the supernatants displayed a distinct color change related to the

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degradation of azo dyes by strain S12. Therefore, the reducing activity for the probe is not

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dependent on the intracellular uptake of the probe, but this activity occurs outside the cells. If the

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degradation occurred in the cell membrane, degradation product NA-O may accumulate

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in the cell membrane and will appear bright fluorescence as Fig 3c. This result provided direct

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evidence that the chemical molecular polarity interaction with the lipid bacterial cell membrane

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prevents azo dyes with high polarity from penetrating bacteria cell membranes; therefore, the

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high polarity azo dyes are reduced outside the cell. By contrast, the low polarity azo dyes can

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penetrate the cell membrane, and the azo bond reduction can occur inside the cell.


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Figure 4. Degradation in the living cells of strain S12-22 imaged with NA-MR (a-d) and NA-

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OG (e-h). The two probes (50 µM) were loaded into LM medium (1 mM NADH for NA-MR)

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with the cells of strain S12-22 for 6 h. The image of the S12-22 cells incubated with NA-MR (a)

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and NA-OG (e) for 6 h; b) the green channel merged with bright-field image; c) enlarged image

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of the cells; d) bright-field image merged with (c); f) fluorescence image of DAPI compared with

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NA-OG; h) bright-field image (g) merged with(e) and (f).

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To further discuss the characteristics of the two probes, we investigated the application of the

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sensors in strain S12-22. Strain S12-22 is a mutant of strain S12 with a transposon insertion in

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ccmA with a result in a deficiency in producing mature c-type cytochromes (Fig S5, S6). Mature

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c-type cytochromes are reported to be essential electron mediators for the extracellular

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azoreduction of intact cells and defects of ccmA restricted the azoreduction outside the cells after

265

employing genetic engineering or protein separation and purification technologies.28

266 267

In this study, strain S12-22 displays a good azoreduction for NA-MR. The absorption band of

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NA-MR gradually decreases, and all cells exhibited fluorescence increases at 6 hours (Fig. S3a,

269

S8). Confocal microscopic images showed a strong and clear fluorescent staining, and this result


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is congruent with the in vitro tests (Figure. 5a). Meanwhile, strain S12-22 was not able to reduce

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NA-OG in 6 hours, as seen in Figure 5b. Almost no fluorescence change in the spectrum was

272

noted. The wild-type strain S12 was able to reduce NA-OG in the defined medium supplemented

273

with lactate or NADH, but the mutant strain S12-22 did not reduce NA-OG well in the identical

274

conditions. When cultivating the probe anaerobically in the standard LM medium, no

275

fluorescence was emitted from the S12-22 cells (Figure 4e). These results indicated that the

276

highly polar sulfonated azo dye NA-OG could not penetrate the cell membrane barriers and be

277

reduced by the mutant strain S12-22. Therefore, no apparent affects were noted in the aqueous

278

solution and cell images. The lowly polar azo dye NA-MR, which could penetrate through the

279

cell membrane barriers into the cell, was not influenced by the deficiency in the mature c-type

280

cytochromes and was reduced inside the cells of the mutant strain S12-22 (Figure 4a). Another

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bacterial strain (Pseudomonas stutzeri, which cannot use LM medium for azoreduction in vivo)

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was used to further test the two sensors. When the probes of NA-OG and NA-MR were used to

283

study the azoreduction of Pseudomonas stutzeri (Figure 5 and S4), no color change in the

284

aqueous solution and cell images was noted over 6 hours’ incubation (Figure S9).


a)

500 control NA-MR

400

300

200

100

0 S12

S12-22

pseudomonas

b)

300


270

250

control NA-OG

200 150 100 50 0 S12

S12-22

pseudomonas

285 286

Figure 5. The recovered supernatants of the maximum-fluorescence changes in spectra of NA-

287

MR (a) and NA-OG (b) (50 µM) in the cultures with 3 different bacteria in the standard LM


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medium within 7 hours.

289

In summary, the oxidoreductase catalyzed the reductive degradation of azo dyes depend on the

290

structure of the compounds. The two sensors showed a greater sensitivity towards the bacterial

291

reduction, which breaks the N=N double bond structure, and displayed a dramatic fluorescence

292

enhancement as the degradation of the azo moiety in vitro. The chemical structures of the sensors

293

NA-OG and NA-MR are distinct because of the sulfonic acid substituents on the dyes. NA-MR

294

(with no sulfonic acid substituent) is minimally polar, highly permeable and able to penetrate the

295

cell membranes, enabling the reduction of this compound inside the cells of the ccmA mutant

296

S12-22. However, NA-OG is a highly polar sulfonated azo compound with a low permeability

297

through the cell membranes. After removing c-type cytochromes, the bacterial membranes

298

restrict the transfer of electrons from the cytoplasm to the sulphonated sensor NA-OG. These

299

results indicate that the design of NA-OG and NA-MR may be a useful in investigating the

300

reduction processes of azo dyes with different characterizations in different bacteria. This study

301

may establish a new research model for microbial degradation and transformation mechanisms

302

based on a chemical fluorescent labeling technology.

303 304

ASSOCIATED CONTENT

305

Supporting Information. Synthesis, experimental details, additional spectroscopic data and

306

images. This material is available free of charge via the Internet at http://pubs.acs.org.

307

AUTHOR INFORMATION

308

Corresponding Author

309

*Tel.: +86 20 87684471. Fax: +86 20 87684587. E-mail: [email protected].


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Author Contributions

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F.L., M.X., and G.S. designed the experiments. F.L.,X. C., Y. Y., and H. W. performed the

312

experiments. F.L. and M.X. analyzed the data and wrote the manuscript.

313

Author Contributions

314

The manuscript was written through contributions by all authors. All authors have given

315

approval to the final version of the manuscript.

316

Notes

317

The authors declare no competing financial interest.

318

ACKNOWLEDGMENTS

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This research was supported by the National Natural Science Foundation (21307016); the

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Natural Science Foundation of Guangdong, China (2014A030308019; S2013040014438); the

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National Science Foundation for Excellent Young Scholars of China (51422803); Special

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Foundation for the Science and Technology Innovation Leaders of Guangdong Province

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(2014TX01Z038); the Special Fund for Agro-scientific Research in the Public Interest

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(201503108); and the Guangdong Provincial Innovative Development of Marine Economy

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Regional Demonstration Projects (GD2012-D01-002)

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Insert Table of Contents Graphic and Synopsis Here

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The sensors NA-MR and NA-OG were synthesized based on the FRET (fluorescence

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resonance energy transfer) from 1, 8-naphthalimide to the azo dye, in which the quenched

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fluorescence will recover when the azo bond is cleaved by the bacteria. In living cells, the sensor

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tracking experiment showed that the lowly polar and hydrophobic azo dye can be taken up into

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the cells and be reduced inside the cells, whereas the highly polar and hydrophilic azo dye can be

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reduced only outside the cells because of the permeability of the cell membranes.


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Novel Strategy for Tracking the Microbial ... - ACS Publications

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