Investigation of volatile metabolites during growth of Escherichia coli and Pseudomonas aeruginosa by needle trap-GC-MS

A new method for the growth-dependent headspace analysis of bacterial cultures by needle trap (NT)-gas chromatography-mass spectrometry (GC-MS) was established. NTs were used for the first time as enrichment technique for volatile organic compounds (VOCs) in the headspace of laboratory cultures. Reference strains of Escherichia coli and Pseudomonas aeruginosa were grown in different liquid culture media for 48 h at 36 °C. In the course of growth, bacterial culture headspace was analysed by NT-GC-MS. In parallel, the abiotic release of volatile organic compounds (VOC) from nutrient media was investigated by the same method. By examination of microbial headspace samples in comparison with those of uninoculated media, it could be clearly differentiated between products and compounds which serve as substrates. Specific microbial metabolites were detected and quantified during the stationary growth phase. P. aeruginosa produced dimethyl sulfide (max. 125 μg L^?1??1) and 2-nonanone (max. 200 μg L^?1), whereas E. coli produced carbon disulfide, butanal and indole (max. 149 mg L^?1). Both organisms produced isoprene. Graphical Abstract

MVOCs produced by P. aeruginosa and E. coli at T = 36 °C in autoclaved LB + TRP medium      

xMAP-based analysis of three most prevalent staphylococcal toxins in Staphylococcus aureus cultures

Detection of staphylococcal toxins presents a great interest for medical diagnostics. Screening of clinical samples for the presence of several types of staphylococcal toxins using traditional methods—biological tests on animals or cell cultures as well as ELISA—is laborious. Multiplex detection methods would simplify testing. We have designed an xMAP-based assay to detect three staphylococcal toxins—enterotoxins A and B (SEA and SEB) and toxic shock syndrome toxin (TSST)—in cultural supernatants obtained from different strains of Staphylococcus aureus. The limits of detection of SEA, SEB, and TSST multiplex detection in S. aureus growth medium were 10, 1,000, and 5 pg/mL, respectively. Fifty-nine samples of S. aureus cultural supernatants were tested with the xMAP assay. The developed assay has proved highly effective detection of the natural toxins in the samples obtained due to bacterial cells cultivation. In prospect, the developed test system can be used in clinical diagnostics and in monitoring of foodstuffs and environmental objects.     

Isolation,Purification, and Characterization of Antimicrobial Compound 6-[1,2-dimethyl-6-(2-methyl-allyloxy)-hexyl]-3-(2-methoxy-phenyl)-chromen-4-one from Penicillium sp. HT-28

A fungal culture (Penicillium sp., HT-28), isolated from soil has been evaluated for its bioactivity, which showed broad spectrum antimicrobial activity and was effective against methicillin-resistant Staphylococcus aureus (MRSA) also. Statistical optimization of the medium by response surface methodology (RSM) enhanced the antimicrobial activity up to 1.8-fold. Column chromatography was used to isolate the active compound (A), which was characterized to be 6-[1,2-dimethyl-6-(2-methyl-allyloxy)-hexyl]-3-(2-methoxy-phenyl)-chromen-4-one by various spectroscopic techniques such as infrared (IR), ¹H and ¹³C nuclear magnetic resonance (NMR) spectra, and mass spectroscopy. Minimum inhibitory concentration (MIC) of the active compound (A) ranged from 0.5 to 15 μg/mL. Viable cell count studies of the active compound (A) showed S. aureus, Escherichia coli, Staphylococcus epidermidis, and Salmonella typhimurium 1 to be the most sensitive. The compound retained its bioactivity after treating it at 100 °C for 1 h. Furthermore, the compound (A) when tested for its biosafety was found neither to be cytotoxic nor mutagenic. The study demonstrated that an apparently novel compound isolated from Penicillium sp. (HT-28) seems to be a stable and potent antimicrobial.     

Transparent poly(thiourethane-urethane)s based on dithiol chain extender

New segmented poly(thiourethane-urethane)s (PTU-Us) (with hard-segment content of 30–60 mass%) were synthesized by a one-step melt polymerization from poly(oxytetramethylene) diol of \( \overline{M}_{n} \)  = 1,000 g mol^?1 or \( \overline{M}_{n} \)  = 2,000 g mol^?1 or poly(hexamethylene carbonate) diol of \( \overline{M}_{n} \)  = 860 g mol^?1 as soft segments, 1,1′-methanediylbis(4-isocyanatocyclohexane) (Desmodur W ^®) and (methylenedi-1,4-phenylene)dimethanethiol as a chain extender. The PTU-Us were examined by FTIR, GPC, XRD, DSC, TG, Shore hardness, and tensile testing. Moreover, refractive index, transparency, adhesive properties, and resistance to bacteria and fungi were determined for selected polymers. The obtained high-molar-mass amorphous polymers showed elastomeric or plastic properties. Their T _gs were in the range from ?70 to 58 °C. The PTU-Us with the polycarbonate soft segments demonstrated a better segmental miscibility (higher T _gs), transparency as well as generally higher tensile strength and hardness than those with the polyether soft segments. All the synthesized PTU-Us showed a relatively good thermal stability. The temperature of 1 % mass loss of all PTU-Us was in the range of 236–255 °C. The introduction of thiourethane linkages to polyurethane chain caused increase of the adhesive strength on copper–polymer junction and refractive index values. From the microbial studies, it was found that the obtained polymers had delayed the growth of Gram-positive bacteria.     

Investigation on the Binding of Terazosin Hydrochloride and Naftopidil to an Immobilized α 1-Adrenoceptor by Zonal Elution

The interaction between drugs and receptors is particularly important in revealing the drug acting mechanism and developing new leads. In this work, α ₁-Adrenoceptor (α ₁-AR) from HEK293 cell line is purified and immobilized on the surface of macro-pore silica gel to prepare an high-performance affinity chromatography stationary phase for the pursuit of drug–receptor interactions by competition zonal elution. Naftopidil is found to have only one type of binding site to α ₁-AR with an association constant of 1.45 × 10⁶ M^?1 and a concentration of binding sites of 1.56 × 10^?6 M, while terazosin hydrochloride proves to present two kinds of binding site on the receptor at which the association constants are determined to be 1.61 × 10⁵ M^?1 and 2.06 × 10³ M^?1, and the corresponding concentrations of the binding sites are 1.56 × 10^?6 M and 1.11 × 10^?3 M, respectively. It is concluded that the stationary phase containing attached α ₁-AR can be used to realize the binding of a drug to the receptor.     

A fluorogenic heterogeneous immunoassay for cardiac muscle troponin cTnI on a digital microfluidic device

We describe a fluorogenic two-site noncompetitive heterogeneous immunoassay with magnetic beads on a low-voltage digital microfluidic platform using closed electrowetting-on-dielectric (EWOD). All the steps of an enzyme-linked immunosorbent assay (ELISA) were performed on the device using 9H-(1, 3-dichloro-9, 9-dimethylacridin-2-one-7-yl) phosphate as the fluorogenic substrate for the enzyme alkaline phosphatase. The performance of the system was demonstrated with cardiac marker Troponin I (cTnI) as a model analyte in phosphate-buffered saline samples. cTnI was detected within the diagnostically relevant range with a limit of detection of 2.0 ng/mL (CV?=?6.47 %). Washing of magnetic beads was achieved by movement through a narrow region of buffer bridging one drop to another with minimal fluid transfer. More than 90 % of the unbound reagents were removed after five washes. Further experiments testing human blood serum on the same platform demonstrated a sample-to-answer time at ～18.5 min detecting 6.79 ng/mL cTnI.     

Feasibility of in-house sterilization of laboratory plastic consumables using 2 MeV electron beam facility

In-house sterilization by electron beam (EB) radiation of various plastic consumables (plastic petridishes, micro centrifugal tubes and screw-capped vials) used routinely in the lab was studied by use of three microbiological cultures (S. aureus, Bacillus subtilis and Candida albicans). Current international standards (ISO 11137-Part 2 -ISO, 2011) recommend an irradiation dose of 25 kGy as a reference dose for terminal sterilization. All containers were exposed in an ILU-6, 2 MeV, 20 kW, Pulse EB accelerator located in our complex. Sterility test (S.T.) was performed and results revealed that 10⁶ and 10⁷ population of all strains passed whereas 10⁸ population failed S.T. for all micro-organisms indicating the potential of 2 MeV EB for commercial sterilization of plastic lab consumables for up to 10⁷ population of these micro-organisms.     

Biotechnological Production of Phenyllactic Acid and Biosurfactants from Trimming Vine Shoot Hydrolyzates by Microbial Coculture Fermentation

Coculture fermentations show advantages for producing food additives from agroindustrial wastes, considering that different specified microbial strains are combined to improve the consumption of mixed sugars obtained by hydrolysis. This technology dovetails with both the growing interest of consumers towards the use of natural food additives and with stricter legislations and concern in developed countries towards the management of wastes. The use of this technology allows valorization of both cellulosic and hemicellulosic fractions of trimming vine shoots for the production of lactic acid (LA), phenyllactic acid (PLA), and biosurfactants (BS). This work compares the study of the potential of hemicellulosic and cellulosic fractions of trimming vine shoots as cheaper and renewable carbon sources for PLA and BS production by independent or coculture fermentations. The highest LA and PLA concentrations, 43.0 g/L and 1.58 mM, respectively, were obtained after 144 h during the fermentation of hemicellulosic sugars and simultaneous saccharification and fermentation (SSF) carried out by cocultures of Lactobacillus plantarum and Lactobacillus pentosus. Additionally, cell-bond BS decreased the surface tension (ST) in 17.2 U; meanwhile, cell-free supernatants (CFS) showed antimicrobial activity against Salmonella enterica and Listeria monocytogenes with inhibition halos of 12.1?±?0.6 mm and 11.5?±?0.9 mm, respectively.     

Synthesis,characterization, crystal structure and cytotoxicity of 2,4-bis(selenomethyl)quinazoline

Organoselenium compounds have already been reported to be good anticarcinogenic candidates. A new selenoquinazoline derivative, 2,4-bis(selenomethyl)quinazoline (compound 1), has been synthesized, spectroscopically characterized and its crystal structure has been studied. An intermolecular coupling between C₂ and \( {\text{H}}_{5}^{\prime } \) in the Heteronuclear Multiple Bond Correlation (HMBC) experiment has been observed. Assuming that the head-to-tail overlap of parallel molecules (as identified by X-ray diffraction) remains in solution to give bimolecular entities, the π–π interaction enables heteronuclear coupling between the former atoms with a three-bond distance [C₂···(π–π)···\( {\text{C}}_{5}^{\prime } \)–\( {\text{H}}_{5}^{\prime } \)]. The crystal structure of compound 1 has been solved by X-ray diffraction. It crystallizes in triclinic system, space group P?1. Unit cell parameters are a = 7.4969(7) Å, b = 8.7008(8) Å, c = 10.1666(9) Å, α = 110.215(2)°, β = 90.354(2)°, γ = 115.017(1)°. Linear chains in crystals of compound 1 are generated by C–H···Se and Se···Se bonds between molecules. Furthermore, head-to-tail overlap of parallel molecules, in which π–π interactions can occur, is observed. Compound 1 exhibited a cytotoxic effect in all of the evaluated tumoral cell lines and showed a higher cytotoxic effect in colon and breast cancer cell lines than etoposide, which was used as a reference compound.     

Non-isothermal reduction of silica-supported nickel catalyst precursors in hydrogen atmosphere: a kinetic study and statistical interpretation

A series of silica-supported nickel catalyst precursors was synthesized with different SiO₂/Ni mole ratios (0.20, 0.80 and 1.15). Non-isothermal reduction of Ni catalyst precursors was investigated by temperature-programmed reduction at four different heating rates (2, 5, 10 and 20 °C min^?1), in a hydrogen atmosphere. Kinetic parameters (E _a, A) were determined using Friedman isoconversional method. It was found that for all mole ratios, apparent activation energy is practically constant in conversion range of α = 30–70 %. In considered conversion range, the following values of apparent activation energy were found: E _a = 129.5 kJ mol^?1 (SiO₂/Ni = 0.20), E _a = 133.8 kJ mol^?1 (SiO₂/Ni = 0.80) and E _a = 125.0 kJ mol^?1 (SiO₂/Ni = 1.15). Using two special functions (y(α) and z(α)), the kinetic model was determined. It was established that reduction of Ni catalyst precursors with different SiO₂/Ni mole ratios is a complex process and can be described by two-parameter ?esták–Berggren (SB) autocatalytic model. Based on established values of SB parameters for each mole ratio, the possible mechanism was discussed. It was found that for all investigated ratios, the Weibull distribution function fits very well the experimental data, in the wide range of conversions (α = 5–95 %). Based on obtained values of Weibull shape parameter (θ), it was found that experimentally evaluated density distribution functions of the apparent activation energies can be approximated by the unbalanced peaked normal distribution.     

Sustainable synthesis of Penicillium-derived highly conductive carbon film as superior binder-free electrode of lithium ion batteries

Fungi (Penicillium chrysogenum) were used as green and sustainable sources to fabricate free-standing binder-free carbon film through pyrolysis in inert atmosphere. The fungi before and after carbonization were characterized with scanning electron microscope (SEM), Fourier transformed infrared spectroscopy (FTIR), electron microprobe (EM), and Raman spectrum. The results showed that the fungi were composed of ultra-long microfibers around 3 μm in diameter, which can be readily transformed into membrane precursor. Abundant functional groups were detected on fungi. The carbon membrane from the pyrolysis of membrane precursor was constructed by the uniformly interconnected fibers. After carbonization, the functional groups disappeared, while the product was doped by O and N atoms. The conductivity of carbon film was as high as 29.4 S cm^?1. Moreover, the carbon film was successfully applied as low-cost electrode in lithium ion batteries (LIBs). The capacity of the LIBs maintained 207 mA h g^?1 with 89.6 % capacity retention after 80 cycles.     

Characterization of chitosan microparticles reinforced cellulose biocomposite sponges regenerated from ionic liquid

The chitosan-microparticles reinforced cellulose biocomposite sponges regenerated from ionic liquid were prepared and characterized. Fourier transform infrared (FTIR) spectroscopy confirmed that the cellulose dissolved in 1-allyl-3-methylimidazolium chloride without derivatization. Chitosan particles as reinforcement were incorporated into the cellulose matrix. FTIR spectra indicated hydrogen bonding between hydroxyl groups of cellulose and chitosan. The biocomposite sponges showed uniform three-dimensional interconnected porous structures. The breaking strength of the sponges increased significantly, from 0.09 to 0.32 MPa with the addition of 1.0 wt% chitosan. The sponges also demonstrated excellent antibacterial activity against S. aureus and E. coli with the average inhibition zone diameters >2 mm and the inhibition rate higher than 80 %. Furthermore, the biocomposite sponges exhibited good moisture penetrability and high porosity. The water uptake ability of the sponge was >25 times of its weight in water with a fast swelling. The chitosan/cellulose composite sponge is expected to be a promising material for potential applications as wound dressing.     

Quantitative characterization by asymmetrical flow field-flow fractionation of IgG thermal aggregation with and without polymer protective agents

Complexes formed between poly(acrylates) and polyclonal immunoglobulin G (IgG) in its native conformation and after heat stress were characterized using asymmetric flow field-flow fractionation (AF4) coupled with on-line UV-Vis spectroscopy and multi-angle light-scattering detection (MALS). Mixtures of IgG and poly(acrylates) of increasing structural complexity, sodium poly(acrylate) (PAA), a sodium poly(acrylate) bearing at random 3 mol % n-octadecyl groups, and a random copolymer of sodium acrylate (35 mol %), N-n-octylacrylamide (25 mol %) and N-isopropylacrylamide (40 mol %), were fractionated in a sodium phosphate buffer (0.02 M, pH 6.8) in the presence, or not, of 0.1 M NaCl. The AF4 protocol developed allowed the fractionation of solutions containing free poly(acrylates), native IgG monomer and dimer, poly(acrylates)/IgG complexes made up of one IgG molecule and a few polymer chains, and/or larger poly(acrylates)/IgG aggregates. The molar mass and recovery of the soluble analytes were obtained for mixed solutions of poly(acrylates) and native IgG and for the same solutions incubated at 65 °C for 10 min. From the combined AF4 results, we concluded that in solutions of low ionic strength, the presence of PAA increased the recovery ratio of IgG after thermal stress because of the formation of electrostatically-driven PAA/IgG complexes, but PAA had no protective effect in the presence of 0.1 M NaCl. Poly(acrylates) bearing hydrophobic groups significantly increased IgG recovery after stress, independently of NaCl concentration, because of the synergistic effect of hydrophobic and electrostatic interactions. The AF4 results corroborate conclusions drawn from a previous study combining four analytical techniques. This study demonstrates that AF4 is an efficient tool for the analysis of protein formulations subjected to stress, an important achievement given the anticipated important role of proteins in near-future human therapies. ?      

Interactions of collagen and cellulose in their blends with 1-ethyl-3-methylimidazolium acetate as solvent

Collagen/cellulose blended solutions with collagen/cellulose mass ratio (Col/Cel) of 0, 1/40, 1/20, 1/10 and 1/5 were prepared using [Emim]Ac as solvent. The interactions between the two polymers before and after regeneration were investigated. In steady shear flow, all of the experimental viscosity values were greater than those of the estimated values calculated from the log-additivity rule for each sample, suggesting interactions between the two polymers in solutions. All solutions exhibited shear thinning behavior and the flow curves could be described by Cross model. Zero shear viscosity (η ₀) versus Col/Cel was examined and a linear increase (from 8.73 to 16.39 Pa·s) can be observed for η ₀ as Col/Cel ≤ 1/10, while there was only a slight increase (from 16.39 to 18.42 Pa·s) in η ₀ as Col/Cel increased to 1/5. Dynamic rheology results suggested the existence of aggregates in solution with Col/Cel = 1/10. Furthermore, the activation energy of solution was 84.5 kJ mol^?1 as Col/Cel = 1/10, higher than that of cellulose solution (44.2 kJ mol^?1). Regenerated films were prepared and characterized to trace back the interactions between the two polymers in [Emim]Ac. Fourier transform infrared spectroscopy indicated the hydrogen-bond interaction between collagen and cellulose in films. The denaturation temperature of collagen in films with Col/Cel ≤ 1/10 could be improved, but it was decreased with the increase of collagen content, and finally was reduced to be close to that of collagen as Col/Cel = 1/5. The features of dynamic mechanical analysis for films were indicative of the lack of homogeneity between collagen and cellulose as Col/Cel = 1/5. Atomic force microscopy images further confirmed the phase-separation when Col/Cel = 1/5.     

Fluorescence imaging of macromolecule transport in high molecular weight cut-off microdialysis

When microdialysis (MD) membrane exceeds molecular weight cut-off (MWCO) of 100 kDa, the fluid mechanics are in the ultrafiltration regime. Consequently, fluidic mass transport of macromolecules in the perfusate over the membrane may reduce the biological relevance of the sampling and cause an inflammatory response in the test subject. Therefore, a method to investigate the molecular transport of high MWCO MD is presented. An in vitro test chamber was fabricated to facilitate the fluorescent imaging of the MD sampling process, using fluoresceinylisothiocyanate (FITC) dextran and fluorescence microscopy. Qualitative studies on dextran behavior inside and outside the membrane were performed. Semiquantitative results showed clear dextran leakage from both 40 and 250 kDa dextran when 100 kDa MWCO membranes were used. Dextran 40 kDa leaked out with an order of magnitude higher concentration and the leakage pattern resembled more of a convective flow pattern compared with dextran 250 kDa, where the leakage pattern was more diffusion based. No leakage was observed when dextran 500 kDa was used as a colloid osmotic agent. The results in this study suggest that fluorescence imaging could be used as a method for qualitative and semiquantitative molecular transport and fluid dynamics studies of MD membranes and other hollow fiber catheter membranes. Graphical Abstract

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High-level Expression of a Manganese Superoxide Dismutase (PoMn-SOD) from Pleurotus ostreatus in Pichia pastoris

The full-length cDNA of Pleurotus ostreatus superoxide dismutase (PoMn-SOD) was cloned and successfully expressed by using the pPIC9K vector under the control of alcohol oxidase 1 promoter with a secretion signal peptide (α-factor) in Pichia pastoris. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting demonstrated that recombinant PoMn-SOD, a 21.8 kDa protein, was secreted into the culture medium. Nondenaturing PAGE experiments confirmed that recombinant PoMn-SOD was secreted in a functionally active form and the expression system did not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. The maximum enzyme activity (156.9 U/mg) was observed under the following conditions: Initial medium pH was 6.0, induction time point was at the 6th day, and methanol concentration was 0.7 % (v/v). This was the first report on secretory expression of recombinant PoMn-SOD in P. pastoris, which might provide a reference for further practical applications.     

New EDTA determination method based on ion chromatography with suppressed conductimetric detection

A novel direct method for the determination of EDTA in alkaline radioactive evaporator residue solution was developed and validated based on ion chromatography with suppressed conductimetric detection and anion exchange columns (A Supp 4, 4 mm × 250 mm and A Supp 5, 4 mm × 150 mm). The yttrium-EDTA complex resulted one single chromatographic peak in the eluent and allowed the correct determination of EDTA in an alkaline, high concentration radioactive waste water. Depending on coexisting substances, suitable eluent is 10.0 mM carbonate buffer/pH 10.6 or 10.75 (t _R,Y–EDTA = 7.01 and 6.4 min, respectively). For 10.0 mM carbonate buffer/pH 10.6 and isocratic flow rate of 1.0 cm³/min, a linear calibration curve was obtained from 5 to 40 mg/dm³ (r > 0.999) EDTA. Good resolution was achieved from commonly coexisting anions (chloride, nitrite, nitrate, sulphate, phosphate, bromide and citrate). The developed simple ion chromatographic method was applied for the assay of EDTA in various radioactive alkaline solutions.     

Biosynthesis and Characterization of Pd and Pt Nanoparticles Using Piper betle L. Plant in a Photoreduction Method

An extremely rapid green approach that generates bulk quantities of nanocrystals of noble metals such as palladium (Pd) and platinum (Pt) nanoparticles (NPs) with a small sizes of 3.8 ± 0.2 and 2.1 ± 0.4 nm by using Piper betle L. (Piperaceae) leaf extract is described. The highly stable and monodispersed Pd and Pt NPs were obtained at 10 min of continuous sunlight exposure. The bio-reduced Pd and Pt NPs were further characterized by using UV–Visible spectroscopy, transmission electron microscopy, selected area electron diffraction, X-ray diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis and cyclic voltammetry measurements. The particles, although discrete, were predominately associated with the P. betle plant proteins, which makes them stable over long time periods. These synthesized biogenic Pd and Pt NPs were evaluated for their acute toxicity studies against aquatic organism, Daphnia magna.     

The Development and Validation of a Stability-Indicating UHPLC-DAD Method for Determination of Perindopril l-Arginine in Bulk Substance and Pharmaceutical Dosage Form

A stability-indicating ultra-high-performance liquid chromatography (UHPLC) method with a diode array detector was developed and validated for the determination of cis/trans isomers of perindopril l-arginine in bulk substance and pharmaceutical dosage form. The separation was achieved on a Poroshell 120 Hilic (4.6 × 150 mm, 2.7 µm) column using a mobile phase composed of acetonitrile–0.1 % formic acid (20:80 v/v) at a flow rate of 1 mL min^?1. The injection volume was 5.0 µL and the wavelength of detection was controlled at 230 nm. The selectivity of the UHPLC-DAD method was confirmed by determining perindopril l-arginine in the presence of degradation products formed during acid–base hydrolysis and oxidation as well as degradation in the solid state, at an increased relative air humidity and in dry air. The method’s linearity was investigated in the ranges 0.40–1.40 µg mL^?1 for isomer I and 0.40–2.40 µg mL^?1 for isomer II of perindopril l-arginine. The UHPLC-DAD method met the precision and accuracy criteria for the determination of the isomers of perindopril l-arginine. The limits of detection and quantitation were 0.1503 and 0.4555 µg mL^?1 for isomer I and 0.0356 and 0.1078 µg mL^?1 for isomer II, respectively.     

Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level

Monitoring of intracellular redox status in a bacterial cell provides vital information about the physiological status of the cell, which can be exploited in several applications such as metabolic engineering and computational modeling. Fluorescent protein-based genetically encoded sensors can be used to monitor intracellular oxidation/reduction status. This study reports the development of a redox sensor for intracellular measurements using fluorescent protein pairs and the phenomenon of Förster resonance energy transfer (FRET). For the development of the sensor, fluorescent proteins Citrine and Cerulean were genetically modified to carry reactive cysteine residues on the protein surface close to the chromophore and a constructed FRET pair was fused using a biotinylation domain as a linker. In oxidized state, the FRET pairs are in close proximity by labile disulfide bond formation resulting in higher FRET efficiency. In reducing environment, the FRET is diminished due to the increased distance between FRET pairs providing large dynamic measurement range to the sensor. Intracellular studies in Escherichia coli mutants revealed the capability of the sensor in detecting real-time redox variations at single cell level. The results were validated by intensity based and time resolved measurements. The functional immobilization of the fluorescent protein-based FRET sensor at solid surfaces for in vitro applications was also demonstrated. Graphical Abstract

Schematic representation of FRET-based redox sensor