optical features of the fluorophore azotobactin: applications for iron sensing in biological fluids

Research Article

Optical features of the fluorophoreazotobactin: Applications for iron sensingin biological fluids

Siderophores are bio-organic ligands secreted by microbes to chelate and assim-ilate iron to meet their metabolic requirements. Siderophores and their analogshave tremendous therapeutic and analytical potential including the use as Fe (III)biosensors; however, only few practical applications have been realized. The aimof this study was the optical and biophysical characterization of the siderophoreazotobactin (Az) secreted by the nitrogen-fixing bacteria Azotobacter vinelandii.The peptide exhibited fluorescence in the visible range. Quantum yield and life-time in excited state were measured to ascertain the sensitivity of the molecule as afluorescent marker in biochemical assays. Its high affinity toward iron in the ferricstate was demonstrated through fluorescence emission quenching studies. Theaccuracy of azotobactin as biosensing tool was determined by analyzing the levelsof iron in biological fluids, particularly in human serum. Furthermore, it wasdemonstrated that it can be encapsulated in sol–gel matrices without significantloss of its fluorescence signal, thus enabling it suitable for adaptation to opticalbiosensor for Fe (III).

Keywords: Azotobacter vinelandii / Azotobactin / Biosensor / Fluorescence / Serum iron

Received: March 12, 2010; revised: April 26, 2010; accepted: May 21, 2010

DOI: 10.1002/elsc.201000038

1 Introduction

To circumvent the low bio-availability of iron in nature, bothphysiological and environmental, many microbes secrete bio-organic ligands called siderophores [1]. These are solublemolecules (MW 400–2000 Da) with high affinity (Kf 5

1020–1052) for aqueous ferric iron [2, 3], which allows suchorganisms to chelate and assimilate this essential nutrient fromtheir oxidizing environments, to meet their metabolicrequirements. Siderophores form strong, hexa-coordinatecomplexes with iron (III) via six coordination sites, which areusually taken up via membrane receptors [4].

Siderophores and their analogs have tremendous ther-apeutic and analytical potential; however, few practical appli-cations of siderophores have been realized. Desferrioxamine, anatural siderophore, is widely used for iron chelation therapyto off-load excess iron in patients who receive repeated bloodtransfusions as in beta-thalassemia and certain forms ofcancer [5]. Hider and coworkers [6, 7] have been engaged in

pioneering studies on the synthesis of bio-mimetic analoguesof siderophores for iron chelation therapy. Another potentialtherapeutic application is to use the iron transport abilities ofsiderophores to carry drugs into cells by preparation ofconjugates between siderophores and antimicrobial agents [8].The analytical application of siderophores comes as fluorescentchemical sensors. Palanche et al. [2] were the first to suggestthe use of siderophores as chemical sensors for detection oftrace levels of iron (III).

The analytical methods available for iron estimation includeUV/visible spectrophotometry, colorimetry, atomic absorptionspectrometry, inductively coupled plasma mass spectrometryand superconducting quantum interference device suscept-ometers. Barring the first two techniques, all other instrumentsare expensive and technically demanding to operate. Fluor-escent metallosensors provide an alternate means to detect ironin biological samples that is versatile, economical, sensitive andof a high-throughput nature. Fluorescent materials are nowroutinely used for wide variety of sensing applications. Theprincipal advantages of fluorimetric sensing are its highsensitivity, which allows easy measurement of low analyteconcentrations, and its selectivity, due to the excitationand emission wavelengths of each fluorescent species. Keepingin view this scenario, fluorescence spectrometric investigationof highly fluorescent iron-chelating molecules, such as naturally

Manisha Sharma

Nivedita Karmakar Gohil

Centre for Biomedical

Engineering, Indian Institute

of Technology Delhi, New

Delhi, India

Abbreviation: Az, azotobactin

Correspondence: Dr. Nivedita Karmakar Gohil ([email protected],

[email protected]), Centre for Biomedical Engineering, Indian

Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.els-journal.com

304 Eng. Life Sci. 2010, 10, No. 4, 304–310

occurring siderophores or synthetic siderophore-based mole-cules, is relevant for the development of new fluorescent bio/chemosensors for detection of trace iron concentrations.

In this context, the aim was to investigate in detail thebiophysical characteristics of the siderophore Az, which is a1.3-kDa pyoverdin-type siderophore produced under ironlimiting conditions by the nitrogen-fixing bacteria Azotobactervinelandii. Structure elucidation of azotobactin has shown that itconsists of a fluorescent chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline, bound via a carboxylic acidgroup to the N terminus of an oligopeptide of ten amino acidsproducing a yellow-green fluorescence [9–12]. Quenching of thisfluorescence as a result of Fe(III) complexation is taken as theanalytical signal for it. The fluorescence and biophysical char-acterization of Az and its application as a metallo-biosensor inhuman serum is reported. Further, the feasibility of employing itfor optical bio-sensing using sol–gel matrix is demonstrated.

2 Material and methods

2.1 Reagents

Reagents and solvents commercially purchased were of analy-tical grade and used without further purification. To eliminateiron contamination, all flasks and glassware used were acidwashed (4 N HCl), soaked in 50 mmol/L EDTA, followed byrinsing with doubly deionized Milli-Q water.

2.2 Bacterial strain and growth conditions

Azotobactin was isolated and purified from a mutant strain ofA. vinelandii (F-196).The iron-limited minimal media used forthe growth of the culture is composed of the following:sucrose, 10 g; ammonium acetate, 1.1 g; magnesium sulfateheptahydrate, 0.2 g; sodium molybdate, 0.24 mg; calciumchloride, 8.5 mg in 5.0 mM phosphate buffer (pH 7.1).Cultures were grown at 281C in Erlenmeyer flask and subjectedto mechanical agitation (200 rpm) for 3–5 days.

2.2.1 Measurement of growth and azotobactinsynthesis

Bacterial growth was monitored turbidimetrically at 620 nmwith a UV/visible spectrophotometer (Lambda 25, PerkinElmer). The concentration of fluorescent pigment in culturemedia was determined from the absorbance of the supernatantat 380 nm; measurements were converted to weight basis usingthe known absorption coefficient (e5 23500 mol L�1 cm�1) [9].

2.3 Isolation and purification of azotobactin

F-196 cultures were removed from incubation when azoto-bactin concentration reached �100 mg/L as determined withA380 measurements. The supernatant from pooled cultures,containing the azotobactin fraction, was filtered (0.22 mm,

Millipore) and loaded on to 19.0� 2.0 cm reverse phaseoctadecyl silane (RP-18; 47–60 mm particle size, Princeton, NJ,USA) preparative column using 0.05 mol/L acetate buffer, pH5.0. Az was eluted with acetonitrile and buffer at an averageflow rate of 1 mL/min. The fractions collected were scanned at380 nm, pooled and loaded on to Sephadex G-25 column withthe same buffer, flushed and then the purified Az was elutedwith linear gradient of buffer. These fractions were pooled,dried under nitrogen and re-suspended in doubly deionizedMilli-Q water (18.2 mO/cm), lyophilized and stored at �201C.The purity was checked by running through a HPLC column(ODS 5 m, 250� 4.6 mm RP-18 column) with acetonitrile-acetate buffer (0.05 mol/L) as the mobile phase on a WatersHPLC system. The compound was eluted following gradientelution and detected at 380 nm.

2.4 Characterization of purified azotobactin

2.4.1 Molecular weight analysis by MALDI-TOF

Az samples were mixed 1:1 with a-cyano-4-hydroxycinnamicacid in 50% ACN/0.1% formic acid and spotted on 100-wellstainless steel probes. MALDI-TOF analysis was performedusing a Voyager DE-STR (Applied Biosystems) in positive ionlinear mode.

2.4.2 Differential scanning calorimetry

Calorimetric measurements of Az (5.0 mg sample size) werecarried out on differential scanning calorimeter (Perkin Elmer,Pyris 6 DSC) in the temperature range of 0–3001C. Thecalorimetric enthalpy DH was determined by integrating thearea under the transition peak.

2.5 Spectra

Absorption spectra were determined on a UV/visible spectro-photometer (100Bio, Cary). Fluorescence analysis of purifiedazotobactin was done on a Perkin Elmer LS 50B LuminescenceSpectrometer. A pre-scan was run for the full range of wave-length (200–900 nm) and then a synchronous scan wasperformed between 200 and 600 nm.

2.5.1 Effect of pH on fluorescence properties ofazotobactin

The effect of pH on fluorescence emission spectra of Az wasdetermined by using different buffers, with pH varying in therange of 1.0–10.0 and scanning fluorescence emission intensityfrom 400 to 600 nm with lexc at 380 nm.

2.5.2 Fluorescence quantum yield

The relative quantum yield (U) of Az was measured with8-hydroxypyrene-1, 3, 6-trisulfonic acid as the standard, using

Eng. Life Sci. 2010, 10, No. 4, 304–310 Metallosensing by azotobactin in biological fluids 305


the formula Fu 5Fs X Fu qs As/Fs qu As [13] where, F is therelative fluorescence determined by integrating the areabeneath the corrected fluorescence spectrum, q the relativephoton output of the source at the excitation wavelength(taken directly from the curve) and A the absorbance (u isunknown, s is standard, Us 5 0.96 8-hydroxypyrene-1, 3,6-trisulfonic acid in buffer, pH 2.0).

2.5.3 Fluorescence lifetime measurements

The fluorescence lifetime measurements were performed in thetime-resolved spectrofluorimeter (Model FL900CDT, Edin-burgh Analytical Instruments, UK). Hydrogen gas filled flashlamp (0.47 bar) was the excitation source. The data acquisitionwas based on time correlated single photon counting techni-que. The instrument response function for this study wasobtained from scattering solution barium sulfate. Fluorescencelife times were calculated from the intensity decay of samplesfitting with sum of exponentials It 5 IoSAi exp (�t/ti).where It is the intensity at time ‘‘t’’ and Ai is the pre expo-nential factor representing the relative contribution of eachdecay components (ti).

2.5.4 Fluorescence quenching studies

A series of aliquots of Fe(III) (10–100 mL) were freshlyprepared from an aqueous stock solution of Fe(III) (Ironstandard, E-Merck, Germany). These were interacted with1.0 mmol/L azotobactin solution in 0.05 mol/L acetate buffer.The fluorescence emission spectrum was scanned at an exci-tation wavelength of 380 nm and the fluorescence intensity atmaximum emission lmax was recorded both in the absence andpresence of Fe(III) for each concentration. All measurementswere carried out at a room temperature of 25711C.

2.6 Estimation of Fe(III) in biological samples byazotobactin

Blood samples were collected from normal healthy volunteersand serum was separated. Serum proteins were removed byultrafiltration. To the serum ultrafiltrate, standard iron solu-tion varying in concentration from 0.1 to 2.0 mmol/L wasadded and was allowed to equilibrate for an hour at 371C. Theiron induced in the serum was then estimated by azotobactinsolution (4.0 mmol/L) in acetate buffer. Fluorescence emissionspectrum was scanned with lexc 5 380 nm and lem 5 490 nm.

2.7 Preparation of sol–gel matrix and encapsulationof azotobactin

Sol–gel matrix was prepared following the well-describedprocedure [14]. Briefly Milli-Q water and the precursortetraethyl orthosilicate in the molar ratio of 4 and 15 weremixed along with 0.1 M HCl as catalyst in a glass vial. Themixture was sonicated in ultrasonic sonicator 200 R (Hielscher,

Germany) for a period of 1 h at regular intervals of 15 min inan ice bath. Sonication was continued till a clear homogenoussolution was obtained. To the prepared sol, acetate buffercontaining 10 mM azotobactin in 1:1 volume ratio was addedand sonicated further for 2–3 min. Disposable Rinzl plasticmicro slides were used as a substrate for the formation ofsol–gel thin film. The microslides were cut into appropriatesize (15� 30 mm) to fit into fluorescent cuvettes at an angle of451. The microslides were first cleaned with laboratory gradeliquid detergent, followed by ethanol, and then washedextensively with Milli-Q water. Sol–gel thin film was preparedby dip coating technique, where substrates were withdrawnfrom low viscosity sols containing the biomolecule at aconstant speed [15]. For smoothness and crack-free film, 50 mLof surfactant Triton X-100 (10%) was also added. Triton Xenables better adhesion of the sol–gel coating to the basesubstrate. Films were dip coated at a constant speed of 1 cm/min, dried and stored at room temperature (28711C). Spec-trofluorimetric measurements were made on a Perkin ElmerLS 50B Luminescence Spectrometer.

2.8 Statistical analysis

All data have been analyzed as mean7SEM.

3 Results and discussion

The mutant strain of A. vinelandii F-196 showed exclusiveoverexpression of azotobactin, facilitating its production asreported earlier [16]. Growth of A. vinelandii in culture wasmonitored by measuring absorbance at 620 nm at specific timeintervals. The production of siderophore was monitored bymeasuring the absorbance at 380 nm (Fig. 1). The growth ofthe cultures was accompanied with extracellular secretion of Azwithin first 4 days of culture inoculation, which induced afluorescent yellow-green color to the culture medium. Maxi-mum production of Az occurred in the exponential phase,after which a plateau was observed (Fig. 1). The total yield ofAz after 4 days was 100–200 mg/L (143.2771.33). Purity of theextraction was established with an absorption maximum at380 nm, which is specific for azotobactin [9] and the HPLCspectra, which consisted of a single narrow peak at 3.2 min.The molecular mass analysis of this fraction by MALDI-TOF,displayed a dominant peak at m/z 1412.06. An additional peakat m/z 1394.81 indicates the lactonized form. In solution, thetwo forms are in equilibrium causing no effect on the physi-cochemical properties of azotobactin (Fig. 2A). Figure 2Bdepicts the thermogram of azotobactin. The scan of the nativemolecule exhibited a single sharp endothermic transition peakwith Tm of 55.681C and DH of 191.40 J/g. Tm indicates themidpoint transition temperature at which folded and unfoldedstates of protein molecules are equal. Further heating resultedin heat denaturation with Td (denaturation temperature) of122.71C and DH of 277.52 J/g.

Detailed fluorescence spectroscopic studies (steady stateand lifetime) of this purified fraction provided several opticalfeatures of azotobactin. The synchronous excitation/emission

306 M. Sharma and N. K. Gohil Eng. Life Sci. 2010, 10, No. 4, 304–310


scan confirmed that Az derived from the A. vinelandii mutantstrain F-196 when excited maximally at a wavelength of380 nm emits maximally at 490 nm. Furthermore, fluorescencewas retained till 2 wk of storage in buffer solution underrefrigeration. This indicates that the fluorophore is unalteredin the molecule derived from different strains. The previousstudy was done on A. vinelandii strain D [2].

The emission spectrum of Az solution in different bufferswith pH varying from 1.0 to 10.0 showed that fluorescenceemission maxima shifted under the influence of pH (Fig. 3). In

the acidic pH range 1–5, the maximum emission was observedat 490 nm with a peak at pH 5. As the pH was altered towardneutral and alkaline, a progressive blue shift was observed inthe peak, with predominant emission peak appearing at470 nm in the pH range of 6–8. As the pH became furtheralkaline upto 10, the main emission peak was at 465 nm. Thisblue shift may be attributed to the many ionizable groupspresent in the molecule. Apart from the five protonation sitesinvolved in the binding of Fe(III) [11], there are presentadditionally a carboxyl and an amino in the peptide chain andan imidazole group in the chromophore of azotobactinmolecule. Interestingly, at pH 7, the emission intensity at allthe three wavelengths is the same and this most likely corre-sponds to the imidazole group in the chromophore. The pKa

value of this group ranges between 6.5 and 7.4, giving themaximal buffering action around neutral pH. We obtained twoabsorption maxima as a function of pH at 380 and 408 nmcorresponding to the di-protonated and mono-protonatedforms, respectively. The maximum fluorescence intensity ofazotobactin was observed between pH 4 and 5 at 490 nm,which was selected for future studies.

The fluorescence quantum yield is defined as the ratio ofthe number of photons emitted to the number of photonsabsorbed and varies in a scale of 0–1. In practice, the quantumyields are usually determined by comparing the fluorescenceemission of the species of interest with a standard having aknown quantum yield. Since the quantum yield is proportionalto the area under fluorescence band in the spectrum, relativequantum yields can be obtained by comparing the peak areas(or heights) under identical excitation conditions. The meanquantum yield of azotobactin was calculated to be 0.2870.05at pH 2.0. This is a fairly high yield, making the moleculesuitable as a label in fluorescence quenching studies andestablishes its potential as a chemosensing molecule.

Fluorescence lifetime measurement provides more detailedinformation on fluorescent molecules as well as their excitedstate relaxation dynamics of different conformers in solvent[13]. It refers to the time the molecule stays in its excited statebefore emitting a photon. The fluorescence decay profile of Azin acetate buffer in absence and presence of quencher Fe(III)

Figure 1. Growth curve correlating azotobactin (Az) production;growth (� turbidity at 620 nm), Az production (& absorbanceat 380 nm, lmax of chromophore). Each value representsmean7SEM (n 5 3).

Figure 2. (A) Positive-ion MALDI-TOF mass spectrum ofazotobactin from mutant strain F-196 (B) Thermogram ofazotobactin.

Figure 3. Fluorescence emission spectrum of azotobactin as afunction of pH.



was found to be bi-exponential, with two exponential decaycomponents (life time values t1 and t2). The average lifetime,tf 5 t1A11t2A2 (where A1 and A2 are the % contributions)may be considered as shown in Table 1. Moreover, in thepresence of quencher Fe(III), no significant changes in lifetimeof the fluorophore was observed and t0/t (t0 and t are thelifetimes in the absence and presence of quencher) wasequivalent to unity. This suggests that the complexed azoto-bactin is nonfluorescent and the observed fluorescence is fromthe free fluorophore.

The quenching effects of Fe(III) ions on fluorescence ofazotobactin were determined as a function of its concentration.The binding of the fluorophore and its ligand Fe(III) takesplace through the co-ordination complex formed between thethree different iron binding sites. We observed that thecorresponding Fe(III)-azotobactin complex exhibited a veryweak or no fluorescence intensity. In bimolecular liquidsystems, the fluorescence intensity is hindered due to severalmechanisms, such as static and dynamic quenching, excimerand exciplex formation and charge transfer process [17–19].The role of fluorescence quenching was studied experimentallyby determining quenching rate parameters using Stern–Volmer(S–V) plots, drawn in accordance with the S–V equation:

F0=F ¼ 11KSV ½Q� and t0=t ¼ 11K 0SV ½Q�

where F0 and F are the fluorescence intensity and t0 and t arethe fluorescence lifetime, in the absence and presence of thequencher concentration [Q], respectively, while KSV (KSV ) isthe S–V constant.

A typical fluorescence emission spectrum of azotobactindecreases with increasing Fe(III) concentration. A significantquenching of its fluorescence intensity is observed in Fig. 4 and alinear S–V plot is indicative of the presence of a single class offluorophore all equally accessible to the quencher. It also indicatesa static quenching phenomenon in the present case, due to theformation of nonfluorescent ground state complex between thefluorophore and Fe(III). This is further supported by lifetimestudies discussed earlier, where there is no significant decrease inthe lifetime of the azotobactin on complexation with Fe(III).Experimental evidence of the formation of azotobactin–Fecomplex is provided by the characteristic change in the absorp-tion spectra of the azotobactin. The free ligand absorbs maximallyat 380 nm while its iron complex absorbs maximally at 412 nm asshown in Fig. 4 (inset). The results offer the basis of developingan analytical assay for estimating iron levels in biological fluids.

Application in the most routine biological specimen usedfor clinical testing is illustrated in human serum. Humanserum ultrafiltrate obtained from healthy volunteers was

spiked with aliquots of known iron concentration in the rangeof 0.1–2.0 mmol/L and estimated by azotobactin by recordingthe optical response in spectrofluorimeter. Accurate measure-ment of serum iron could be made (Fig. 5); the correspondingmeasured value for the lowest aliquot was found to be0.1470.018mmol/L (mean7SD, n 5 2).

Adaptability of the molecular probe to optical sensing hasbeen demonstrated in the sol–gel matrix. Sol–gel technology hasemerged out as a promising tool for encapsulation of biomole-cules, making it a compatible host for chemical as well asbiosensing [20]. The porosity of the matrix can be manipulatedby using acid as the catalyst. The porous nature allows thebiomolecule to be accessible to the analyte for interaction. At thesame time, its transparent physical character makes it suitable foroptical sensing [21]. Two varying compositions were used to givewater to precursor molar ratio (R) 4 and 15, which led to twodifferent porosities of the sol–gel thin film. The molar ratiobasically governs the condensation process during gelation and

Table 1. Fluorescence lifetime values of azotobactin free (Az)and bound to Fe(III) (Az-Fe); solvent; 0.05 M acetate buffer(pH 4.4).

Sample Emission at t1(Ai) t2(Ai) tf 5 t1A1

1t2A2

w2

Az 490 nm 4.3 ns (0.44) 6.7 ns (0.56) 5.64 ns 1.22

Az-Fe 490 nm 3.6 ns (0.33) 6.3 ns (0.67) 5.41 ns 1.08

Figure 4. Stern Volmer plot of F0/F-1 against Fe(III) concentra-tion as quencher. Inset (top right) represents the absorptionspectra of Azotobactin free ligand (left) and of its Fe(III)complex (right). Solvent: 0.05 M acetate buffer (pH 4.4).

Figure 5. Variation of the fluorescence intensity ratio F0/F as afunction of the Fe(III) concentration in normal human serum. Azconcentration 5 4.0mM, lEx 5 380 nm, lEm 5 490 nm.



thus determines the average pore size in the sol–gel matrix. Inboth cases, the fluorescence properties (lExc/lEm: 380 nm/490 nm) of Az remain unchanged (Fig. 6). The fluorescenceemission peak observed at 490 nm indicates that the structuralintegrity of Az bio-molecule is well preserved in the encapsulatedform. However, the intensity of emission as a function of storageindicates about 19.5% decrease in the fluorescence intensity ofentrapped Az, corresponding to R value 4, where as in case ofmatrix R 15, the percentage decrease was about 10.09% after 30days of storage (Fig. 6). These results when considered inconjunction with the leaching rates over a period of time indicatethat R 4 matrix may be more porous as compared to R 15matrix, leading to a higher percentage loss in fluorescenceintensity in the former (Fig. 7).

4 Concluding remarks

In conclusion, this study uses a combination of biological andbiophysical approach to see how well the optical properties ofazotobactin are suited for chemosensing of iron in its ferricstate. Through the detailed fluorescence analysis, we haveestablished the suitability of azotobactin secreted by A. vine-

landii (F-196 strain) as a molecular marker in fluorescencelabeling studies specifically for Fe(III). Its natural high affinityand specificity for Fe(III) further allows the detection to beachieved by a single molecular species with high precision.This is a bonus for its effective use in bio/chemo sensing ofiron. The high quantum yield of the fluorophore contributesto its bright emission and results in the increased sensitivity ofmeasurement of its interaction with the ligand. Further, wehave shown its efficacy in estimating Fe (III) in human serumwith high accuracy. Through the sol–gel entrapment studies, itis evident that azotobactin is amenable to encapsulation inchemically inert medium without significant loss in its opticalproperties making it suitable for an optical biosensor.

Acknowledgements

This study has been funded by Institutional grants; ManishaSharma is supported by Student Fellowship from IIT Delhi.Assistance from and discussions with N. K. Chaudhary in thefluorescence lifetime measurements is gratefully acknowledged.Generous donation of the mutant strain of A. vinelandii fromWilliam Page is appreciated.

Conflict of interest

The authors have declared no conflict of interest.

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optical features of the fluorophore azotobactin: applications for iron sensing in biological fluids

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