УДК 577.3


© 2012 M. S. Kadam, A. B. Chaudhari*, S. B. Chincholkar

School of Life Sciences, North Maharashtra University, Jalgaon-425 001, Maharashtra, India;

*e-mail: ambchasls@gmail.com Received May 13, 2011; in final form, August 16, 2011

Optical fluorescence-quenching-based biosensing cell is described and optimization of covalent binding of highly selective natural iron-chelating peptide secreted by bacteria is suggested. Pyoverdin biosynthesized by Pseudomonas monteilii and having 70% iron chelating activity was immobilized on amino alkylated controlled pore glass (CPG) and cross-linked with glutaraldehyde (2.5%, 28°C, 30 min). The pyoverdin—CPG immobilization was confirmed using fluorescence microscopic images (excitation range, 465—495 nm) for bright green fluorescence and by FTIR spectrum stretching at 3406.4 cm-1 for amino group. The pyoverdin loading capacity of activated CPG matrix was 25 mg g-1 of CPG and its rinsing analysis (leaking profile of the immobilized peptide vs. washing) detected negligible (2-3 p.g) pyoverdin in the second wash.

Keywords: protein immobilization, iron (III); covalent binding; rinsing analysis; pyoverdin; biosensor.

An increased sophistication in qualitative and quantitative molecular interaction of proteins or peptides with other biomolecules or metal ions has always directed to newer analytical approaches [1]. The resulting biosensor and its use for quantitative analysis has spurt impetus in the last decade as a bio-analytical device to provide a high selectivity and sensitivity with significant reduction in (i) sample preparation, (ii) assay time and (iii) reagent expenses compared to conventional analysis [2]. Among them, optical biosensors have been recognized as most versatile in application based on absorbance, reflectance, or fluorescence emission of ultraviolet (UV), visible, or near-infrared region of light [3]. Towards this objective, usual format in development of the optical biosensors encompasses efficient utilization of biological component immobilized on the surface of a transducer.

The biosensor based on immobilized protein has provided sensitivity as well as selectivity in determination of metal ions [4]. Most optical biosensors have been constructed using complex designs, either in terms of their reaction scheme or immobilization of protein and transduction procedures. Hence, appropriate design, preparation of inputs, and immobilization procedure constitute critical steps for the construction of a biosensor [5]. This mainly relies on optimal immobilization of protein and ease of coupling with the transducer for stable, sensitive and sustainable functional biosensor. This necessitates compatibility of biosensing element as well as analyte. Moreover, a suitable immobilization method offers continuous flow system, optimal quantitative analysis, and regen-erable biosensor [3]. On the contrary, ineffective immobilization and uneven distribution of biosensing el-

ement can cause loss in activity, alter configuration that fails to bind analyte, exhibit poor reproducibility, and reduce sensor response.

Basically, immobilization of protein encompasses methods of physical or chemical immobilization of biomolecules on solid support, either through covalent binding or simply adsorption on the matrix. Although covalent binding precludes protein diffusion and mass transfer, it causes protein inactivation and support rigidity problem. However, covalently immobilized protein in sensor bestows quick response during analysis and obviates problems experienced in covalent binding method.

This work describes the first step towards the development of biosensing element using pyoverdin, a fluorescent siderophore for the construction of optical biosensor. Optimal pyoverdin immobilization onto optically inert matrix, i.e., controlled porosity glass (CPG) and its spectrofluorometric assessment is reported.


Cell culture and pyoverdin production. Pyoverdin was biosynthesized by P. monteilii (NCBI accession number JF732909, obtained from lab repository, School of Life Sciences, North Maharashtra University, Jalgaon). The cells were inoculated into 500 ml Erlenmeyer flasks containing iron-restricted synthetic succinate medium (28°C, 48 h, 120 rpm) [6] and subsequently its iron chelation ability [7] and decoloriza-tion percent [8] was confirmed with Chrome azurol S (CAS) assay. The pyoverdin fractions were pooled from cell-free supernatant (15 min, 8000 rpm) through XAD-4 column [9]. Fractions eluted with

methanol were assayed on UV-spectrophotometer (ND 1000, Nano-drop Technologies, USA), and fractions with a peak absorbance at 404 nm were concentrated in rotary vacuum evaporator (BUCHI Ro-tavapor, R-124) at 50°C under vacuum (10—5 torr) and used for immobilization.

Immobilization of pyoverdin. For immobilization of pyoverdin, about 0.2 g of controlled porosity glass (CPG) with pore sizes of300 A (porosity 283 A, pore volume 1.25 cc/g, grain size 120—200 mesh, s.s. area 177 m2/g, bulk density 0.31g/cc) and of 500 A (porosity 487 A, pore volume 1.6 cc/g, grain size 120—200 mesh, s.s. area 134 m2/g, bulk density 0.23 g/cc), a porous, white color silica powder with free SiO2 functional groups, manufactured by 3 Prime, USA was boiled in 15 ml 10% nitric acid for 30 min and washed 10 times with deionized water to remove traces of acid and then oven-dried to remove residual water. Transparent CPG of each pore size was separately amino alkylated with 10% aqueous 3-amino-propyl-triethoxysilane (pH 3.5) in shaking water bath at 80°C for 150 min and washed with deionized water. The process was repeated five times to ensure complete aminoalkylation. After alky-lation, each CPG bead was activated in the absence of oxygen (with nitrogen purging in closed vessel) using 2.5% glutaraldehyde solution in Tris—HCl buffer (0.1 M, pH 7.0) at 28°C for 30 min. The activated CPG beads were stored at 4°C in closed vessels. Various amounts (2500—10000 ^g) of pyoverdin dissolved in bipthalate buffer (0.01 M, pH 6) were added to the activated CPG beads (0.2 g) and incubated at 4°C for 3 h in the absence of oxygen (with nitrogen purging in a closed vessel).

Optimization of amino alkylation. For this purpose, 1—5 cycles of amino alkylation were carried out to determine optimal procedure with respect to total unbound pyoverdin. The remaining (unimmobilized) pyoverdin concentration was used for rating the efficiency of CPG for alkylation process. Various amounts (2500—10000 ^g) of pyoverdin in aqueous solution (0.1 M phosphate buffer, pH 7.0) were added to the activated glass CPG (0.2 g) and incubated for 120— 150 min at 4°C for immobilization. The pyoverdin immobilized onto CPG was stored at 4°C till further analysis and use.

Microscopic and spectroscopic analysis of CPG-py-overdin composite. Microscopic observations of immobilized pyoverdin were carried out in fluorescence and phase contrast using inverted 50i Nikon fluorescence microscope equipped with 10x objective (Nikon, Japan). The samples were excited using FITC filter (excitation range, 465—495 nm) and observed for emission images of control CPG and immobilized py-overdin. Phase-contrast images of the same samples were also taken. Images were acquired using CCD camera (Nikon, Japan). The exposure time for image acquisition was kept the same to allow signal comparison. Similarly, shift in functional group was examined

through Fourier Transform Infrared (FTIR) spectros-copy. FTIR spectra of pyoverdin immobilized on CPG and of active CPG were obtained for wave number range of 4000—400 cm-1 with infrared spectrophotometer (FTIR 8400s, Shimadzu). Samples were prepared separately by mixing KBr with pyoverdin immobilized on CPG and active CPG as control. Each mixture was compressed into pellets. The major peak data were acquired by using IRsolution software [10].

Determination of loading capacity of pyoverdin. The

loading capacity of pyoverdin was determined by CPG-free buffer solution (pH 7.0) containing different concentrations of pyoverdin by UV absorption at 404 nm [11]. Unbound pyoverdin concentration with respect to concentration of pyoverdin immobilized on CPG of different pore size (300 and 500 A) was analyzed for the optimum loading capacity of active CPG. Optimal loading of pyoverdin on CPG was further evaluated for analysis of various parameters.

Rinsing analysis (leaking profile vs. washing). CPG

with pyoverdin immobilized was rinsed serially with deionized water and the washing-off samples were centrifuged at 7000 rpm for 10 min. Supernatant was collected and assayed for pyoverdin as described earlier [11]. The procedure was repeated five times to ensure the leaking profile as a function of wash.

Determination of iron sensing activity. Pyoverdin— CPG composites were analyzed for its biosensing ability at different concentrations (10, 25, 50, 75, 100 ^g/ml) of ferric iron (FeCl3, 7H2O). Changes in fluorescence intensity (excitation, 500 nm; emission, 550 nm) at various concentrations of iron on spectrof-luorimeter (Cary Eclipse, Fluorescence Spectrophotometer, Varain, US) was used for quantitative rating of biosensing activity [12]. The sensor cell was regenerated with 0.1 M HCl followed by washing with deionized water for further experimentation.


In response to iron starvation, some microorganisms secrete low-molecular weight iron-chelating ligands for acquisition of iron from insoluble forms present in neutral and oxidizing environments by mineralization and sequestration for their growth. Under iron starvation, Pseuodomonas monteilii secretes water-soluble yellow-green iron-chelating peptide called pyoverdin, a siderophore [13]. Generally, pyoverdins are chromopeptides consisting of 2,3-diamino-6,7-dihydroxyquinoline linked to a short peptide [12]. Binding of iron is mediated by catechol gr

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