Development of Ratiometric Fluorescence Sensors Based on CdSe/ZnS Quantum Dots for the Detection of Hydrogen Peroxide.

In this study, carboxyl group functionalized-CdSe/ZnS quantum dots (QDs) and aminofluorescein (AF)-encapsulated polymer particles were synthesized and immobilized to a sol-gel mixture of glycidoxypropyl trimethoxysilane (GPTMS) and aminopropyl trimethoxysilane (APTMS) for the fabrication of a hydrogen peroxide-sensing membrane. CdSe/ZnS QDs were used for the redox reaction of hydrogen peroxide (H2O2) via a reductive pathway by transferring electrons to the acceptor that led to fluorescence quenching of QDs, while AF was used as a reference dye. Herein, the ratiometric fluorescence intensity of CdSe/ZnS QDs and AF was proportional to the concentration of hydrogen peroxide. The fluorescence membrane (i.e., QD-AF membrane) could detect hydrogen peroxide in linear detection ranges from 0.1 to 1.0 mM with a detection limit (LOD) of 0.016 mM and from 1.0 to 10 mM with an LOD of 0.058 mM. The sensitivity of the QD-AF membrane was increased by immobilizing horseradish peroxidase (HRP) over the surface of the QD-AF membrane (i.e., HRP-QD-AF membrane). The HRP-QD-AF membrane had an LOD of 0.011 mM for 0.1-1 mM H2O2 and an LOD of 0.068 mM for 1-10 mM H2O2. It showed higher sensitivity than the QD-AF membrane only, although both membranes had good selectivity. The HRP-QD-AF membrane could be applied to determine the concentration of hydrogen peroxide in wastewater, while the QD-AF membrane could be employed for the detection of α-ketobutyrate.

Effects of CdSe and CdSe/ZnS Core/Shell Quantum Dots on Singlet Oxygen Production and Cell Toxicity.

Four types of quantum dots (QDs) with varying emission wavelengths were synthesized in this study. The dots included CdSe and CdSe/ZnS core/shell QDs with short emission wavelengths of 540 nm and 560 nm, respectively, as well as CdSe and CdSe/ZnS core/shell QDs with longer emission wavelengths of 585 nm and 595 nm, respectively. The ligands on the QD surfaces were exchanged with mercaptopropionic acid (MPA) to make them water-soluble. The efficiency of singlet oxygen (1O2) production from both CdSe and CdSe/ZnS core/shell QDs was highest at a QD concentration of 14 µg/ml Singlet oxygen production from the CdSe QDs was higher than that with the CdSe/ZnS core/shell QDs after 1 h of LED470 irradiation. However, the singlet oxygen production of CdSe/ZnS core/shell QDs was much higher than that of CdSe QDs at concentrations above 14 µg/ml. The cytotoxicities of both CdSe and CdSe/ZnS core/shell QDs were investigated using HeLa cells.

Development of Colorimetric and Ratiometric Fluorescence Membranes for Detection of Nitrate in the Presence of Aluminum-Containing Compounds.

In this study, a quantitative analysis of nitrate in aqueous solution was performed through the combination of an oxazine170 perchlorate⁻ethyl cellulose (O17-EC) membrane with aluminum-containing compounds. Aluminum of Devarda's alloy (DA) or a clay hydrotalcite (HT) was employed for the reduction of nitrate to produce ammonia, and the produced ammonia was detected by the O17-EC membrane. The method of combining the O17-EC membrane with aluminum compounds has showed a broad detection range of nitrate. That is, the DA was combined with the O17-EC membrane and showed the linear nitrate detection ranges of 1⁻10 mM and 10⁻100 mM, while the O17-EC membrane immobilized with the clay HT showed a linear detection range of 0.1⁻1 mM nitrate. The visual color transition of the nitrate-sensing membranes at different nitrate concentrations was clearly observed under sunlight or irradiation of a light-emitting diode (LED) at an excitation wavelength of 470 nm (LED470).

Development of Ratiometric Fluorescent Biosensors for the Determination of Creatine and Creatinine in Urine.

In this study, the oxazine 170 perchlorate (O17)-ethylcellulose (EC) membrane was successfully exploited for the fabrication of creatine- and creatinine-sensing membranes. The sensing membrane exhibited a double layer of O17-EC membrane and a layer of enzyme(s) entrapped in the EC and polyurethane hydrogel (PU) matrix. The sensing principle of the membranes was based on the hydrolytic catalysis of urea, creatine, and creatinine by the enzymes. The reaction end product, ammonia, reacted with O17-EC membrane, resulting in the change in fluorescence intensities at two emission wavelengths (λem = 565 and 625 nm). Data collected from the ratio of fluorescence intensities at λem = 565 and 625 nm were proportional to the concentrations of creatine or creatinine. Creatine- and creatinine-sensing membranes were very sensitive to creatine and creatinine at the concentration range of 0.1-1.0 mM, with a limit of detection (LOD) of 0.015 and 0.0325 mM, respectively. Furthermore, these sensing membranes showed good features in terms of response time, reversibility, and long-term stability. The interference study demonstrated that some components such as amino acids and salts had some negative effects on the analytical performance of the membranes. Thus, the simple and sensitive ratiometric fluorescent sensors provide a simple and comprehensive method for the determination of creatine and creatinine concentrations in urine.

Development of a Sequential Injection Analysis System for the Determination of Saccharin.

Saccharin is a powerfully sweet nonnutritive sweetener that has been approved for food-processing applications within the range of 100-1200 mg/kg. A simple, rapid, and cost-effective sequential injection analysis (SIA) technique was developed to determine the saccharin level. This method is based on the reaction of saccharin with p-chloranil in an ethanol medium with a hydrogen peroxide (H2O2) acceleration, and the resultant violet-red compound was detected using a UV-Vis spectrophotometer at λmax = 420 nm. To ascertain the optimal conditions for the SIA system, several parameters were investigated, including buffer flow rate and volume, p-chloranil concentration, and reactant volumes (saccharin, p-chloranil, and H2O2). The optimum setup of the SIA system was achieved with a buffer flow rate, buffer volume, and draw-up time of 1.2 mL/min, 2900 µL, and ~145 s, respectively. The optimal p-chloranil concentration is 30 mM, and the best reactant volumes, presented in an ordered sequence, are as follows: 30 µL of H2O2, 450 µL of saccharin, and 150 µL of p-chloranil. The optimized SIA configuration produced a good linear calibration curve with a correlation coefficient (R² = 0.9812) in the concentration range of 20-140 mg/L and with a detection limit of 19.69 mg/L. Analytical applications in different food categories also showed acceptable recovery values in the range of 93.1-111.5%. This simple and rapid SIA system offers great feasibility for the saccharin quality control in food-product processing.

CdSe/ZnS Core/Shell Quantum Dots in Cooperation with Other Materials for Direct and Indirect Production of Reactive Oxygen Species.

In this present work, CdSe/ZnS core/shell quantum dots (QDs) were exploited in the oxidation reactions of 5-aminolevulinic acid (ALA) and glutamate (GLU) for the production of reactive oxygen species (ROS). Fast and highly efficient oxidation reactions of ALA to produce the hydroxyl radicals (HO*) and of GLU to produce the superoxide anion (O2*-) were observed in the cooperation of mercaptopropionic acid (MPA) capped-CdSe/ZnS QDs (MPA-QDs) under LED irradiation. Whereas, binding between MPA-QDs and coumarin-derived dendrimer (CdD)-captured silica particles (SiCdDs) through sol-gel GA enhanced singlet oxygen production under LED irradiation by about 80% as compared to that achieved using QDs only. Confocal fluorescent microscopic images of the size and morphology of HeLa cells confirmed the ROS production from ALA, GLU in cooperation with CdSe/ZnS QDs or QDs-coated SiCdDs under LED irradiation.

Simple and sensitive progesterone detection in human serum using a CdSe/ZnS quantum dot-based direct binding assay.

In this study, we developed a CdSe/ZnS quantum dot (QD)-based immunoassay for use in determining the presence of progesterone (P4) in human serum. Hydrophilic QDs were conjugated to anti-progesterone antibody (P4Ab) via ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling reagents. After purification, the P4Ab-QD conjugates were immobilized onto the wells of a 96-well microtiter plate, and a direct-binding immunoassay based on the binding of P4 to immobilized P4Ab-QD conjugates had a detection limit of 0.21 ng/ml and a sensitivity of 1.37 ng/ml, with a linear range of 0.385 to 4.55 ng/ml. The proposed immunoassay was successfully used to determine the P4 concentration in real human serum, and the results showed a good correlation with the accredited radioimmunoassay (RIA).

Ratiometric Fluorescent Biosensors for Glucose and Lactate Using an Oxygen-Sensing Membrane.

In this study, ratiometric fluorescent glucose and lactate biosensors were developed using a ratiometric fluorescent oxygen-sensing membrane immobilized with glucose oxidase (GOD) or lactate oxidase (LOX). Herein, the ratiometric fluorescent oxygen-sensing membrane was fabricated with the ratio of two emission wavelengths of platinum meso-tetra (pentafluorophenyl) porphyrin (PtP) doped in polystyrene particles and coumarin 6 (C6) captured into silica particles. The operation mechanism of the sensing membranes was based on (i) the fluorescence quenching effect of the PtP dye by oxygen molecules, and (ii) the consumption of oxygen levels in the glucose or lactate oxidation reactions under the catalysis of GOD or LOX. The ratiometric fluorescent glucose-sensing membrane showed high sensitivity to glucose in the range of 0.1-2 mM, with a limit of detection (LOD) of 0.031 mM, whereas the ratiometric fluorescent lactate-sensing membrane showed the linear detection range of 0.1-0.8 mM, with an LOD of 0.06 mM. These sensing membranes also showed good selectivity, fast reversibility, and stability over long-term use. They were applied to detect glucose and lactate in artificial human serum, and they provided reliable measurement results.

Development of novel optical pH sensors based on coumarin 6 and nile blue A encapsulated in resin particles and specific support materials.

Inspired by traditional pH papers, two types of ratiometric fluorescent pH sensors for neutral and alkaline pH ranges were developed in this study by using two fluorescent dyes, coumarin 6 (C6) and nile blue A (NB). These dyes were encapsulated into melamine-formaldehyde (MF) resin particles, which were then incorporated with polymer Nafion (Nf) or polyurethane hydrogel (PU) to prepare pH-sensing membranes. MF-C6 particles immobilized into polymer Nafion (i.e., MF-C6-Nf membrane) showed a dynamic ratiometric pH detection range of 4.5-7.5 through shift in fluorescence emission spectra at acidic and neutral pH solutions, as well as distinct color transition under normal visual sense from pink to yellow color. By contrast, MF-NB particles immobilized into polyurethane hydrogel (i.e., MF-NB-PU membrane) displayed a dynamic ratiometric fluorescence detection range of pH 9 - pH 12 via change in ratiometric fluorescence intensity at two emission band edges. The membrane also showed a clear change from blue to purple color under sunlight at high pH values. These pH-sensing membranes also exhibited high sensitivity, good reversibility, and stability. They were then applied to measure pH values in real urine samples and fermentation media.

Development of a Ratiometric Fluorescent Glucose Sensor Using an Oxygen-Sensing Membrane Immobilized with Glucose Oxidase for the Detection of Glucose in Tears.

Glucose concentration is an important parameter in biomedicine since glucose is involved in many metabolic pathways in organisms. Many methods for glucose detection have been developed for use in various applications, particularly in the field of healthcare in diabetics. In this study, ratiometric fluorescent glucose-sensing membranes were fabricated based on the oxygen levels consumed in the glucose oxidation reaction under the catalysis of glucose oxidase (GOD). The oxygen concentration was measured through the fluorescence quenching effect of an oxygen-sensitive fluorescent dye like platinum meso-tetra (pentafluorophenyl) porphyrin (PtP) by oxygen molecules. Coumarin 6 (C6) was used as a reference dye in the ratiometric fluorescence measurements. The glucose-sensing membrane consisted of two layers: The first layer was the oxygen-sensing membrane containing polystyrene particles (PS) doped with PtP and C6 (e.g., PS@C6^PtP) in a sol-gel matrix of aminopropyltrimethoxysilane and glycidoxypropyltrimethoxysilane (GA). The second layer was made by immobilizing GOD onto one of three supporting polymers over the first layer. These glucose-sensing membranes were characterized in terms of their response, reversibility, interferences, and stability. They showed a wide detection range to glucose concentration in the range of 0.1 to 10 mM, but high sensitivity with a linear detection range of 0.1 to 2 mM glucose. This stable and sensitive ratiometric fluorescent glucose biosensor provides a reliable way to determine low glucose concentrations in blood serum by measuring tear glucose.

Ratiometric fluorescent l-arginine and l-asparagine biosensors based on the oxazine 170 perchlorate-ethyl cellulose membrane.

Ratiometric fluorescent l-arginine (Arg) and l-asparagine (Asn) biosensors were developed using an oxazine 170 perchlorate (O17) ethyl cellulose (EC) membrane and the enzymes entrapped into the matrix of EC and hydrogel polyurethane. The sensing principles were based on the hydrolysis reactions of urea and l-Arg under the catalysis of the urease and arginase to produce ammonia in the case of an l-Arg-sensing membrane and also on the hydrolysis reaction of l-Asn under the catalysis of asparaginase in the case of an l-Asn-sensing membrane. The O17-EC membrane reacted with the ammonia produced from the hydrolysis reactions and changed the fluorescence intensities at λ em = 565 and 625 nm. The ratio of the fluorescence intensities at λ em = 565 and 625 nm was proportional to the concentrations of l-Arg or l-Asn in the range of 0.1-10 mM. The LOD of the l-Arg- and l-Asn-sensing membranes was 0.082 ± 0.0014 and 0.074 ± 0.0023 mM, respectively. The sensing membranes also showed good quality in terms of response time, reversibility, and stability. The interference study demonstrated that some components such as amino acids had little negative effects on the performance of the sensing membranes for the detection of l-Arg and l-Asn. These simple and sensitive ratiometric fluorescent sensing membranes provide a basic or comprehensive method for detecting l-Arg and l-Asn in blood and urine samples as well as in the fermentation processes.

Optimization of extracellular production of recombinant human bone morphogenetic protein-7 (rhBMP-7) with Bacillus subtilis.

Extracellular production of recombinant human bone morphogenetic protein-7 (rhBMP-7) was carried out through the fermentation of Bacillus subtilis. Three significant fermentation conditions and medium components were selected and optimized to enhance the rhBMP-7 production by using the response surface methodology (RSM). The optimum values of the three variables for the maximum extracellular production of rhBMP-7 were found to be 2.93 g/l starch, 5.18 g/l lactose, and a fermentation time of 34.57 h. The statistical optimization model was validated with a few fermentations of B. subtilis in shake flasks under optimized and unoptimized conditions. A 3-L jar fermenter using the shake-flask optimized conditions resulted in a higher production (413 pg/ml of culture medium) of rhBMP-7 than in a shake flask (289.1 pg/ml), which could be attributed to the pH being controlled at 6.0 and constant agitation of 400 rpm with aeration of 1 vvm.

Sequential injection immunoassay for human bone morphogenic protein-7 using an immunoreactor immobilized with anti-human bone morphogenic protein-7 antibody--CdSe/ZnS quantum dot conjugates.

The detection of human bone morphogenic protein-7 (BMP-7) was achieved using a sequential injection immunoassay (SIIA) system. The SIIA system is based on the binding between BMP-7 and anti-human BMP-7 (AbBMP7)-CdSe/ZnS quantum dot (QD) conjugates immobilized onto a glass disk or an optical fiber, using fluorescence detection at excitation and emission wavelengths of 470 nm and 580 nm, respectively. The AbBMP7-QD conjugates were prepared by conjugating anti-human BMP-7 antibody (AbBMP7) to hydrophilic CdSe/ZnS core/shell quantum dots (QDs). The SIIA system was fully automated using software written in the LabVIEW™ development environment. The analytical performance of the SIIA system was characterized with a number of variables such as carrier flow rate and elution buffer. Under partially optimized operating conditions, the SIIA system had a linear calibration graph at up to 10.0 ng mL(-1) BMP-7 (R(2)≥0.975) and a sample frequency of two samples per hour. The SIIA system with an optical fiber immunosensor was used to detect and quantify BMP-7 in spiked real samples obtained from a biological process with recoveries in the range of 95-102%.

Use of CdSe/ZnS luminescent quantum dots incorporated within sol-gel matrix for urea detection.

In this work, urea detection techniques based on the pH sensitivity of CdSe/ZnS QDs were developed using three types of sol-gel membranes: a QD-entrapped membrane, urease-immobilized membrane and double layer consisting of a QD-entrapped membrane and urease-immobilized membrane. The surface morphology of the sol-gel membranes deposited on the wells in a 24-well microtiter plate was investigated. The linear detection range of urea was in the range of 0-10mM with the three types of sol-gel membranes. The urea detection technique based on the double layer consisting of the QD-entrapped membrane and urease-immobilized membrane resulted in the highest sensitivity to urea due to the Michaelis-Menten kinetic parameters. That is, the Michaelis-Menten constant (K(m)=2.0745mM) of the free urease in the QD-entrapped membrane was about 4-fold higher than that (K(m)=0.549mM) of the immobilized urease in the urease-immobilized membrane and about 12-fold higher than that (K(m)=0.1698mM) of the immobilized urease in the double layer. The good stability of the three sol-gel membranes for urea sensing over 2 months showed that the use of sol-gel membranes immobilized with QDs or an enzyme is suitable for biomedical and environmental applications.

Encapsulation of tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) complex linked with dendrons in sol-gels: Stable optical sensing membranes for dissolved oxygen.

To circumvent the leaching problem of optical sensing membranes used for dissolved oxygen (DO) measurements, the encapsulation of Ru(II) complexes linked with bulky dendron(s) in a sol-gel matrix was investigated. A dendron, readily formed via chemical transformations such as amidation and catalytic reduction, was covalently incorporated into tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) complex, leading to an increase in the size and lipophilicity of dye molecules. Sol-gel-based sensing membranes encapsulating these Ru(II) complexes displayed a strong luminescence emission at 590 nm induced by radiation at 480 nm, and showed excellent DO sensing properties and stability for repeated measurements in aqueous solution. The encapsulation of the dendron-incorporated Ru(II) complexes in sol-gels prevented the dyes from leaching out of the membranes.

Use of CdSe/ZnS core-shell quantum dots as energy transfer donors in sensing glucose.

In the present work, CdSe/ZnS core-shell quantum dots were synthesized and conjugated with enzymes, glucose oxidase (GOD) and horseradish peroxidase (HRP). The complex of enzyme-conjugated QDs was used as QD-FRET-based probes to sense glucose. The QDs were used as an electron donor, whereas GOD and HRP were used as acceptors for the oxidation/reduction reactions involved in oxidizing glucose to gluconic acid. Electron transfer between the redox enzymes and the electrochemical reduction of H(2)O(2) (or O(2)) occurred rapidly, resulting in an increase of the turnover rate of the electron exchange between the substrates (e.g. glucose, H(2)O(2) and O(2)) and the enzymes (GOD, HRP), as well as between the QDs and the enzymes. The transfer of non-radiative energy from the QDs to the enzymes resulted in the fluorescence quenching of the QDs, corresponding to the increase in the concentration of glucose. The linear detection ranges of glucose concentrations were 0-5.0g/l (R=0.992) for the volume ratios of 10/5/5, 0.2-5.0g/l (R=0.985) for the volume ratios of 10/5/3 and 1.0-5.0g/l (R=0.982) for the volume ratios of 10/5/0. Temperature (29-37 degrees C), pH (6-10) and some ions (NH(4)(+), NO(3)(-), Na(+), Cl(-)) had no interference effect on the glucose measurement.

Preparation and characterization of sensing membranes for the detection of glucose, lactate and tyramine in microtiter plates.

In this work, sensing membranes for the detection of glucose, lactate and tyramine were successfully prepared by immobilizing enzymes and fluorophore on sol-gels. The membranes were fabricated on the bottom of the wells in a microtiter plate. Glucose oxidase (GOD), lactate oxidase (LOD) and tyramine oxidase (TOD) were immobilized on individual sol-gels or a mixture of different sol-gels (3-glycidoxypropyl-trimethoxysilane (GPTMS), methyl-triethoxysilane (MTES), aminopropyl-trimethoxysilane (APTMS)). The oxidation of the analytes specifically catalyzed by the enzymes resulted in the reduction of the oxygen concentration, which changed the fluorescence intensity (FI) of the oxygen sensitive ruthenium complex acting as the transducer. The linear calibration graphs were in the ranges of 0.0-5.0g/l for glucose, 0.0-9.0mg/l for lactate and 0.0-100mg/l for tyramine. The values of the detection limit were found to be 0.10-0.52g/l for glucose, 7.77mg/l for lactate and 6.30-8.73mg/l for tyramine. The covalent binding between the epoxy and amine groups of the sol-gels and enzymes, respectively, prevented the enzymes from being washed out and preserved the high stability of the sensing membranes. The different ratios of silanes in the sol-gels, which were used as the supporting matrix for the immobilization of the enzymes led to different responses of the sensing membranes to various concentrations of glucose, lactate and tyramine. The kinetic parameters of the enzymatic reactions, and the stability and other parameters for the sensing membranes were also investigated.

Flow injection analysis system for monitoring of succinic acid in biotechnological processes.

In this study, a flow injection analysis (FIA) system using a cartridge of immobilized isocitrate lyase (ICL) and isocitrate dehydrogenase (ICDH) was developed to monitor the concentrations of succinic acid in biotechnological processes. The ICL and ICDH immobilized on VA-Epoxy Biosynth E3-carrier had a good operational lifetime (up to 24h) and storage stability (up to 30 days). The FIA system with the immobilized ICL/ICDH cartridge was characterized with respect to the factors affecting the activity of the immobilized enzymes, such as pH of carrier solution, temperature, sample matrix, etc. Optimal pH value of the immobilized enzymes was slightly shifted in the alkaline range, i.e. 9.0. Some components such as 10gl(-1) lactose, 3gl(-1) malate and 3gl(-1) oxaloacetate in sample solution had significant activating effects (more than 10%) on the response of the FIA system. But the activity of the immobilized ICL and ICDH was not largely influenced by some components like imidazole (1mM), sodium azide (10mM) and semicarbazide (2gl(-1)) added to carrier buffer solution. The FIA system with an enzyme cartridge was applied to on-line monitor the concentrations of succinic acid in a continuously stirred reactor and a fermentation process of immobilized Escherichia coli, and showed good sensitivity and reliability of the FIA system developed in this work.

Sequential injection analysis system for on-line monitoring of l-cysteine concentration in biological processes.

A sequential injection analysis (SIA) system was developed to monitor the concentration of l-cysteine in biological processes on-line. It is based on the redox reaction of l-cysteine with iron(III) in the presence of 1,10-phenanthroline (phen) and the detection of the red-iron(II)-phen complex with a spectrophotometry. The system was fully automated using software written in the LabVIEWtrade mark development environment. A number of system variables such as the flow rate of the carrier buffer solution, the volume ratio of the sample to the reagents, and the reaction coil length, etc., were evaluated to increase the sensitivity and performance of the SIA system. Under partially optimized operating conditions the performance of the SIA system was linear up to a concentration of l-cysteine of 1mM (R(2)=0.998) with a detection limit of 0.005mM and a sample frequency of 15hr(-1). The SIA system was employed to monitor the concentration of l-cysteine on-line in a continuously stirred reactor and a fermentation process of Saccharomyces cerevisiae. The on-line monitored data were in good agreement with the off-line data measured by a HPLC with a fluorescence detector (n=15, R(2)=09899).

High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization.

The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris. The phospholipase C (PLC) when expressed in P. pastoris was fused to the alpha-factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium. Recombinant P. pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg(-1) protein which was more than a recombinant P. pastoris GS115 (552 U mg(-1) protein) or KM71H (539 U mg(-1) protein). The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg(-1) protein by P. pastoris GS115, 1176 U mg(-1) protein by P. pastoris KM71H and 1522 U mg(-1) protein by P. pastoris X-33. The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g. stable at 75 degrees C) than the wild-type PLC from B. cereus . Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co(2+) and Mn(2+) etc., also influenced the activity of the recombinant PLCs.