Respiration activity of Escherichia coli entrapped in a cone-shaped microwell and cylindrical micropore monitored by scanning electrochemical microscopy (SECM)

The metabolic activity of E. coli cells embedded in collagen gel microstructures in a cone-shaped well and in a cylindrical micropore was investigated using scanning electrochemical microscopy (SECM), based on the oxygen consumption rate and the conversion rate from ferrocyanide to ferricyanide. The analysis of the concentration profiles for oxygen and ferrocyanide afforded the oxygen consumption rate and the ferrocyanide production rate. A comparison indicated that the ferrocyanide production rates were larger than the oxygen consumption rate, and also that the rates observed in the cylindrical micropore were larger than those observed in the cone-shaped well. The ferrocyanide production rate of a single E. coli cell was calculated to be (5.4 +/- 2.6) x 10(-19) mol s(-1), using a cylindrical micropore system.     

Synergistic effect of Ni(II) and Co(II) ions on the sulfite induced autoxidation of Cu(II)/tetraglycine complex

The synergistic effect of Ni(II) and Co(II) on the sulfite induced autoxidation of Cu(II)/tetraglycine was investigated spectrophotometrically at 25.0 degrees C, pH = 9.0, 1 x 10(-5) mol dm(-3) < or = [S(IV)] < or = 8 x 10(-5) mol dm(-3), [Cu(II)]= 1 x 10(-3) mol dm(-3), 1 x 10(-6) mol dm(-3) < or = [Ni(II)] or [Co(II)] < or = 1 x 10(-4) mol dm(-3), [O2] approximately 2.5 x 10(-4) mol dm(-3), and 0.1 mol dm(-3) ionic strength. In the absence of added nickel(II) or cobalt(II), the kinetic traces of Cu(III)G4 formation show a large induction period (about 3 h). The addition of trace amounts of Ni(II) or Co(II) increases the reaction rate significantly and the induction period drastically decreases (less than 0.5 s). The effectiveness of Cu(III)G4 formation becomes much higher. The metal ion in the trivalent oxidation state rapidly oxidizes SO3(2-) to SO3*-, which reacts with oxygen to produce SO5*-. The strongly generated oxidants oxidize Cu(II)G4 to Cu(III).     

Scanning electrochemical microscopy in combination with piezoelectric quartz crystal impedance analysis for studying the growth and electrochemistry as well as microetching of poly(o-phenylenediamine) thin films

The combination of scanning electrochemical microscopy (SECM) with piezoelectric quartz crystal impedance (PQCI) analysis was proposed as a novel multiparameter method for investigating the cyclic voltammetric growth of poly(o-phenylenediamine) (PoPD) thin films at Au electrodes in aqueous solutions of various pH values and the potentiostatic microetching (localized degradation) of these films in 0.10 mol/L aqueous H2SO4 for comparative examinations on polymer porosity and stability. Two potential-sweep ranges, -0.4 to 0.9 (I) and 0 to 0.9 (II) V versus SCE, and four solutions, acidic (A, 0.20 mol/L H2SO4 + 0.10 mol/L Na2SO4; B, 0.10 mol/L H2SO4 + 0.20 mol/L Na2SO4), neutral (C, 0.10 mol/L PBS + 0.20 mol/L Na2SO4, pH 7.2), and alkaline (D, 0.20 mol/L NaOH + 0.20 mol/L Na2SO4) aqueous solutions, were selected for PoPD growth. The pH increase for the polymerization solution increased the molar percentage of polyaniline-like chains in PoPD, as quantified from the current peaks at approximately 0.6 V versus a saturated calomel electrode (SCE) for the oxidation of -NH2 groups in as-prepared PoPD (grown from solutions C and D) during their redox switching in 0.10 mol/L aqueous H2SO4 for the first time. The unusual PQCI responses observed at negative potentials (potential range I) in the first several potential cycles during the cyclic voltammetric growth of PoPD in acidic and neutral solutions have been reasonably explained as being due to the precipitation/dissolution of the poorly soluble phenazinehydrine charge-transfer complexes developed during redox switching of oligomers for the first time, which brought about much less compact PoPD films and their higher degradability than those grown in the same solution but over potential range II. SECM, scanning electron microscopy (SEM), and piezoelectric quartz crystal (PQC) frequency were used to estimate the sizes of etched microscale spots. In addition, the x-, y-, or z-axis movement of a Pt microelectrode of 25-mum diameter near the PQC electrode was found to influence negligibly the PQCI responses in 1.0 mol/L aqueous Na2SO4 containing K4Fe(CN)6 up to 0.10 mol/L, and a new protocol of dynamically electrodepositing silver microwires via the chemical-lens method was proposed for examining the local mass-sensitivity distribution on the PQC surface.     

Electrochemiluminescence of terbium (III)-two fluoroquinolones-sodium sulfite system in aqueous solution

The electrochemiluminescence (ECL) of Tb3+-enoxacin-Na2SO3 system (ENX system) and Tb3+-ofloxacin-Na2SO3 system (OFLX system) in aqueous solution is reported. ECL is generated by the oxidation of Na2SO3, which is enhanced by Tb3+-fluoroquinolone (FQ) complex. The ECL intensity peak versus potential corresponds to oxidation of Na2SO3, and the ECL emission spectra (the peaks are at 490, 545, 585 and 620 nm) match the characteristic emission spectrum of Tb3+, indicating that the emission is from the excited state of Tb3+. The mechanism of ECL is proposed and the difference of ECL intensity between ENX system and OFLX system is explained. Conditions for ECL emission were optimized. The linear range of ECL intensity versus concentrations of pharmaceuticals is 2.0 x 10(-10) -8.0 x 10(-7)mol l(-1) for ENX and 6.0 x 10(-10) -6.0 x 10(-7)mol l(-1) for OFLX, respectively. A theoretical limit of detection is 5.4 x 10(-11)mol l(-1) for ENX and 1.6 x 10(-10)mol l(-1) for OFLX, respectively. The ECL was satisfactorily applied to the determination of the two FQs in dosage form and urine sample.     

Stoichiometries and thermodynamic stabilities for aqueous sulfate complexes of U(VI)

The formation constants of UO2SO4 (aq), UO2(SO4)2(2-), and UO2(SO4)3(4-) were measured in aqueous solutions from 10 to 75 degrees C by time-resolved laser-induced fluorescence spectroscopy (TRLFS). A constant enthalpy of reaction approach was satisfactorily used to fit the thermodynamic parameters of stepwise complex formation reactions in a 0.1 M Na(+) ionic medium: log 10 K 1(25 degrees C) = 2.45 +/- 0.05, Delta r H1 = 29.1 +/- 4.0 kJ x mol(-1), log10 K2(25 degrees C) = 1.03 +/- 0.04, and Delta r H2 = 16.6 +/- 4.5 kJ x mol(-1). While the enthalpy of the UO2(SO4)2(2-) formation reaction is in good agreement with calorimetric data, that for UO2SO4 (aq) is higher than other values by a few kilojoules per mole. Incomplete knowledge of the speciation may have led to an underestimation of Delta r H1 in previous calorimetric studies. In fact, one of the published calorimetric determinations of Delta r H1 is here supported by the TRLFS results only when reinterpreted with a more correct equilibrium constant value, which shifts the fitted Delta r H1 value up by 9 kJ x mol(-1). UO2(SO 4) 3 (4-) was evidenced in a 3 M Na (+) ionic medium: log10 K3(25 degrees C) = 0.76 +/- 0.20 and Delta r H3 = 11 +/- 8 kJ x mol(-1) were obtained. The fluorescence features of the sulfate complexes were observed to depend on the ionic conditions. Changes in the coordination mode (mono- and bidentate) of the sulfate ligands may explain these observations, in line with recent structural data.     

A fast and simple method to fabricate circular microchannels in polydimethylsiloxane (PDMS)

A simple method to fabricate circular microchannels in polydimethylsiloxane (PDMS) is presented. A coating of liquid PDMS is applied on the walls of rectangular microchannels, fabricated using standard soft-lithography, by introducing a pressurized air stream inside the PDMS filled microchannels. Surface tension of the liquid PDMS forces the coating to take a circular cross-section which is preserved by baking the device to cure the coated layer. Diameters ranging from a few micrometres to a few hundreds of micrometres were achieved. The method was verified to work on microchannel networks as well as in straight channels. Different coating conditions were systematically tested. Design curves are reported for one to choose appropriate coating conditions for obtaining a desired diameter. A comparison between the performance of square and circular microchannels in trapping SiHa cells (cervical cancer cell line) is shown.     

High efficiency micellar electrokinetic chromatography of hydrophobic analytes on poly(dimethylsiloxane) microchips

This paper describes a simple method for the effective and rapid separation of hydrophobic molecules on polydimethylsiloxane (PDMS) microfluidic devices using Micellar Electrokinetic Chromatography (MEKC). For these separations the addition of sodium dodecyl sulfate (SDS) served two critical roles - it provided a dynamic coating on the channel wall surfaces and formed a pseudo-stationary chromatographic phase. The SDS coating generated an EOF of 7.1 x 10(-4) cm(2) V(-1) s(-1) (1.6% relative standard deviation (RSD), n = 5), and eliminated the absorption of Rhodamine B into the bulk PDMS. High efficiency separations of Rhodamine B, TAMRA (6-carboxytetramethylrhodamine, succinimidyl ester) labeled amino acids (AA), BODIPY FL CASE (N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester) labeled AA's, and AlexaFluor 488 labeled Escherichia coli bacterial homogenates on PDMS chips were performed using this method. Separations of Rhodamine B and TAMRA labeled AA's using 25 mM SDS, 20% acetonitrile, and 10 mM sodium tetraborate generated efficiencies > 100,000 plates (N) or 3.3 x 10(6) N m(-1) in <25 s with run-to-run migration time reproducibilities <1% RSD over 3 h. Microchips with 30 cm long serpentine separation channels were used to separate 17 BODIPY FL CASE labeled AA's yielding efficiencies of up to 837,000 plates or 3.0 x 10(6) N m(-1). Homogenates of E. coli yielded approximately 30 resolved peaks with separation efficiencies of up to 600,000 plates or 2.4 x 10(6) N m(-1) and run-to-run migration time reproducibilities of <1% RSD over 3 h.     

Determination of the pesticide carbaryl by chemical deoxygenation micellar-stabilized room temperature phosphorescence

This paper presents a convenient determination method for carbaryl in polluted water by micellar-stabilized room temperature phosphorescence with Na(2)SO(3) as oxygen scavenger. The effect of various experimental conditions on the determination of carbaryl is discussed in detail. The analytical curve of carbaryl gives a linear dynamic range of 2 x 10(-7)-6 x 10(-5) mol/l., and a detection limit of 2 x 10(-7) mol/l. A recovery of 90-100% was obtained for 0.05-0.1 ppm carbaryl.     

Speciation of Co(II), Co(III), and Cu(II) in ethylenediamine solutions by capillary electrophoresis

A capillary electrophoretic (CE) method for the speciation of Co(II), Co(III), and Cu(II) in electroless copper-plating baths containing ethylenediamine (En) has been developed. The method is based on the selective pre-capillary derivatization of Co(II) with 1,10-phenanthroline (Phen) followed by CE separation of stable [CoPhen(3)](2+), [CoEn(3)](3+), and [CuEn(2)](2+) chelates. The proposed derivatization procedure protects Co(II) from oxidation by dissolved oxygen and enables rapid determination of all three metal species within a single run. The optimized separations were carried out in a fused silica capillary (57 cmx75-microm I.D.) filled with an ethylenediamine sulfate electrolyte (20 mmol L(-1) H(2)SO(4), pH 7.0 with En, applied voltage +30 kV) using direct UV detection at 214 nm. The detection limits for a signal-to-noise ratio of 3 and 10 s, hydrodynamic injections were 5x10(-6) mol L(-1) for Cu(II), 1x10(-6) mol L(-1) for Co(III), and 4x10(-7) mol L(-1) for Co(II). Application of the method to the speciation of Co(II), Co(III), and Cu(II) in copper-plating bath samples is also demonstrated.     

聚四氟乙烯和聚二甲基硅烷的链尺寸和偶极矩

基于改进的旋转异构态方法,推导了对称双取代基高分子链的均方回转半径和均方偶极矩公式,用于研究聚四氟乙烯(PTFE)、聚二甲基硅烷(PDMS)和聚乙烯(PE)链构象依赖的性质.计算得到长链聚四氟乙烯PTFE4(四态模型)和PDMS的构型参数值分别为0.44·mol0.5·g-0.5和0.43·mol0.5·g-0.5,PDMS链的相应温度系数值-7.32×10-3K-1,它与PTFE和PE的值(约-1.00×10-3K-1)相差较大.得到长链PTFE和PDMS的均方电偶极矩特征比结果都很小,如PDMS链的特征比为0.27,说明虽然PTFE和PDMS的侧基键C—F和Si—CH3都有强极性,但其聚合物却是非极性的电介质,具有较好的电绝缘性能,可见高分子链的构象和结构也对聚合物极性和电学性质有很大的影响.得到短链PTFE和PDMS(重复单元x10)均方偶极矩表现出明显的端点效应,特征比随x的改变而呈现明显的波动,这与Flory等的计算结果一致.     

Separation via flotation, spectrophotometric speciation, and determination of vanadium(IV) in wastes of power stations.

1-(2-Hydroxy-4-methoxybenzophenone)-4-phenylthiosemicarbazone (HMBPT) was investigated as a new reagent for the flotation of vanadium(IV). At pH approximately 1.5, vanadium(IV) forms a 1:1 pale-violet complex with HMBPT in aqueous solution. An intense clear violet layer was formed after flotation, by adding an oleic acid (HOL) surfactant. The composition of the float was 1:1 [V(IV)]:[HMBPT]. A highly selective and sensitive spectrophotometric procedure was proposed for the determination of microamounts of V(IV) as its floated complex. The molar absorptivities of the V(IV)-HMBPT and V(IV)-HMBPT-HOL systems were 0.4 x 10(4) and 0.12 x 10(5) L mol(-1) cm(-1) at 560 nm, respectively. The formation constants of the species formed in the presence and absence of HOL were 4.6 x 10(7) and 8.7 x 10(5) L mol(-1), respectively. Beer's law was obeyed up to 1 x 10(-4) mol L(-1) in the aqueous layer as well as in the oleic acid layer. The HMBPT-V(IV) complexes formed in the aqueous solution and scum layer were characterized by elemental analysis, infrared and UV spectrophotometric studies. The mode of chelation between V(IV) and HMBPT is proposed to be due to a reaction between the protonated bidentate HMBPT ligand and V(IV) through the S=C and N=C groups. Interferences from various foreign ions were avoided by adding excess HMBPT and/or Na2S2O3 as a masking agent. The proposed flotation method was successfully applied to the analysis of V(IV) in synthetic mixtures, wastes of power stations, simulated samples and in real ores. The separation mechanism is discussed.     

Micro gas analysis system for measurement of atmospheric hydrogen sulfide and sulfur dioxide

A honeycomb structure microchannel scrubber was developed to achieve efficient and stable gas collection. A thin porous membrane was pasted on a microchannel by the adhesive force of a fresh polydimethylsiloxane surface. The microchannel scrubber achieved much more efficient gas collection than conventional impingers and diffusion scrubbers. Two sets of the microchannel scrubbers and detectors were integrated in a 10 cm x 9 cm plastic board to create a micro gas analysis system (microGAS) for simultaneous measurements of H2S and SO2. The whole system including a battery was incorporated in a carrying case 34 cm W x 16 cm D x 17 cm H for use in the field. Liquid flows at 30 microl min(-1) were obtained by bimetal micropumps. The estimated detection limits were 0.1 ppbv for H2S and 1 ppbv for SO2. The system was demonstrated for real atmospheric gas analysis, and the results agreed well with data concurrently obtained by ion chromatography coupled with a cylindrical diffusion scrubber. The system we developed allowed automated continuous analyses in the field and achieved a much higher time resolution compared to those by ion chromatographic analysis.     

Uptake of SO2 on HOBr-ice surfaces

The uptake of SO2 on HOBr-treated ice surfaces has been studied using a flow reactor coupled with a differentially pumped quadrupole mass spectrometer at 190-240 K. The initial uptake coefficient was determined as a function of HOBr surface coverage, theta(HOBr), on the ice. The uptake coefficients increase as the HOBr coverage increases. The uptake coefficient can be expressed as gamma(t) = k(h)theta(HOBr), where k(h) = 1.5 x 10(-19) molecules(-1) cm(-2) at 191 K and k(h) = 6.4 x 10(-21) molecules(-1) cm(-2) at 210 K and theta(HOBr) is in the range of 8 x 10(13) to 1.2 x 10(15) molecules cm(-2). The effects of temperature and film thickness on the uptake coefficients of SO2 by the HOBr-treated ice films were also studied. The activation energy E(a) of SO(2) on HOBr-ice surfaces is approximately -81 +/- 8 kJ/mol in the 190-215 K range. Kinetic results were interpreted in terms of the Eley-Rideal mechanism. This study suggests that the uptake of SO2 on ice/snow surfaces is enhanced by the presence of HOBr near the ice surface. The implication for atmospheric chemistry is that HOBr-ice surfaces may not provide a significant pathway to oxide S(IV) in the boundary layer due to both lower uptake coefficient and smaller HOBr surface coverage at T > 220 K.     

Electrokinetic control of fluid flow in native poly(dimethylsiloxane) capillary electrophoresis devices

Capillary zone electrophoresis (CZE) devices fabricated in poly(dimethylsiloxane) (PDMS) require continuous voltage control of all intersecting channels in the fluidic network in order to avoid catastrophic leakage at the intersections. This contrasts with the behavior of similar flow channel designs fabricated in glass substrates. When the injection plugs are shaped by voltage control and leakage from side channels is controlled by the application of pushback voltages during separation, fluorescein samples give 64 200 theoretical plates (7000 V separation voltage, E = 1340 V/cm). Native PDMS devices exhibit stable retention times (+/- 8.6% RSD) over a period of five days when filled with water. Contact angles were unchanged (+/- 1.9% RSD) over a period of 16 weeks of dry storage, in contrast to the known behavior of plasma-oxidized PDMS surfaces. Electroosmotic flow (EOF) was observed in the direction of the cathode for the buffer systems studied (phosphate, pH 3-10.5), in the presence or absence of hydrophobic ions such as tetrabutylammonium or dodecyl sulfate. Electroosmotic mobilities of 1.49 x 10(-5) and 5.84 x 10(-4) cm2/Vs were observed on average at pH 3 and 10.5, respectively, the variation strongly suggesting that silica fillers in the polymer dominate the zeta potential of the material. Hydrophobic compounds such as dodecyl sulfate and BODIPY 493/503 adsorbed strongly to the PDMS, indicating the hydrophobicity of the channel walls is clearly problematic for CZE analysis of hydrophobic analytes. A method to stack multiple channel layers in PDMS is also described.     

Electrochemical detector for microchip electrophoresis of poly(dimethylsiloxane) with a three-dimensional adjustor

This paper presents an electrochemical detector for poly(dimethylsiloxane) (PDMS) microchip CE with a three-dimensional adjustor which makes it possible to accurately align a separate working electrode that can be easily fabricated in laboratory to the uncertain PDMS microchannel outlet. The substantial influence of the electrode-PDMS microchannel distance was investigated. The optimal electrode-outlet distance was found to be 10 microm for the PDMS microchannel with the width of 50 microm due to its relatively slow electroosmotic flow. Adrenaline and catechol were well separated, with a linear response range from 20 microM to 1 mM, and a detection limit of 2 microM for catechol, using carbon disk electrode (diameter of 300 microm). Furthermore, arginine and histidine can be well separated and detected directly in the PDMS microchannel using a Cu disk electrode (diameter of 150 microm).     

Diffusion of buffer layer assisted grown gold nanoclusters on Ru(100) and p(1 x 2)-O/Ru(100) surfaces

Patterning of metallic clusters on surfaces is demonstrated by utilizing a buffer layer assisted laser patterning technique (BLALP). This method has been employed in order to measure the diffusion of AFM and STM characterized size selected gold nanoclusters (5-10 nm diameter), over Ru(100) and p(1 x 2)-O/Ru(100) surfaces. Optical linear diffraction from gold cluster coverage gratings was utilized for the macroscopic diffusion measurements. The clusters were found to diffuse on the surface intact without significant coalescence or sintering. The barrier for metastable gold nanocluster diffusion on the surface is thought to be lower than the energy required for surface wetting. The apparent activation energy for diffusion was found to depend on the cluster size, increasing from 6.2 +/- 0.4 kcal/mol for 5 nm clusters to 10.6 +/- 0.5 kcal/mol for 9 nm clusters. The macroscopic diffusion of gold nanoclusters has been studied on the p(1 x 2)-O/Ru(100) surface as well, where surface diffusion was found to be rather insensitive to the clusters size with activation energy of 5.5 +/- 1 kcal/mol. The difference between the two surfaces is discussed in terms of a better commensurability (higher level of friction) of the gold facets at the contact area with the clean Ru(100) than in the case of the oxidized surface.     

Spectral properties of (5-phenyl-1,3,4-oxadiazol-2-yl)-7-hydroxycoumarin (POHC)

The electronic absorption, emission and excitation spectra of POHC were measured in different solvents and are affected by solvent polarity. The fluorescence quantum yield of POHC decreases with increasing Richardt and Dimorth solvent parameter (E(T)) value of the solvent. In dilute solutions POHC is almost totally present in its protonated nitrogen tautomer form. The deprotonation is a reversible process. A shoulder in the absorption spectra at approximately 473 nm indicates the presence of a portion of the tautomer (s) that disappears on lowering the temperature. Molecular oxygen acts as a quencher with quenching rate constant of 1.8 x 10(10) M(-1) s(-1) in DMF. Energy transfer from POHC to rhodamine 6G in ethanol was also studied. POHC is relatively photostable in ethanol (phic approximately 1.7 x 10(-4)). Quantum chemical calculations were carried out and correlated to experimental observations.     

Miniaturized multichannel electrospray ionization emitters on poly(dimethylsiloxane) microfluidic devices.

A multichannel electrospray ionization (ESI) emitter was fabricated as part of a poly(dimethylsiloxane) (PDMS) microfluidic device using a three-layer photoresist process which also produces a self-alignment system to make a bonding between the top and bottom PDMS parts. The prototype device (2 cm high x 5 cm wide x 5 cm long) had 16-channels (30 microm wide x 50 microm deep) with emitters of 1 mm length and 60 degrees point angle. The PDMS emitter tips enabled interfacing the device to ESI-mass spectrometry; a stable electrospray from the tips was performed with limits of detection under 1 microM for reference peptides (adrenocorticotropic hormone fragment 1-17, angiotensin I and III).     

Grafting epoxy-modified hydrophilic polymers onto poly(dimethylsiloxane) microfluidic chip to resist nonspecific protein adsorption

In order to achieve a simple covalent hydrophilic polymer coating on poly(dimethylsiloxane) (PDMS) microfluidic chip, epoxy modified hydrophilic polymers were synthesized in aqueous solution with a persulfate radical initiation system, and crosslinked onto PDMS pretreated by oxygen plasma and silanized with 3-aminopropyl-triethoxysilanes (APTES). Glycidyl methacrylate (GMA) was copolymerized with acrylamide (poly(AAM-co-GMA)) or dimethylacrylamide (poly(DAM-co-GMA)), and graft polymerized with polyvinylpyrrolidone (PVP-g-GMA) or polyvinylalcohol (PVA-g-GMA). The epoxy groups in the polymers were determined by UV spectra after derivation with benzylamine. Reflection absorption infrared spectroscopy (RAIRS) confirmed covalent grafting of GMA-modified polymers onto PDMS surface. Electroosmotic flow (EOF) in the polymer grafted microchannel was strongly suppressed within the range pH 3-11. Surface adsorption of lysozyme and bovine serum albumin (BSA) was reduced to less than 10% relative to that on the native PDMS surface. On the GMA-modified polymer coated PDMS microchip, basic proteins, peptides, and sodium dodecyl sulfate (SDS) denatured proteins were separated successfully.     

Influence of Cu(II) on the interaction between sulfite and horseradish peroxidase in vitro

This paper discussed the quantitative influence of Cu(II) on the interaction between horseradish peroxidase (HRP) and sulfite (SO3(2-)), which is a derivate of sulfite dioxide in human bodies, by using fluorescence spectrum and ultraviolet (UV) absorption spectrometry in vitro. The results show that under the conditions of physiological pH and room-temperature, Cu(II) can bind strongly with both the protein part and the ferroporphyrin part in HRP at a low concentration (10(-4) mol L(-1)), and the combination constants are 2.047 x 10(3) and 7.66 x 10(2) L mol(-1), respectively. Under the same conditions, SO3(2-) at low concentrations (<0.15 mol L(-1)) has little quenching for the fluorescence of HRP at 330 nm, and the combination constant is 0.108 L mol(-1). While the fluorescence intensity at 440 nm enhance gradually with the increased concentration of SO3(2-) (<0.1 mol L(-1)), and the combination constant is 8.219 L mol(-1). These indicate that SO3(2-) at low concentration has little reaction with the enzyme protein part in HRP but obvious reaction with the ferroporphyrin part in HRP. After SO3(2-) at low concentrations is added into the HRP-Cu(II) binary system, the reaction constants between SO3(2-) and the enzyme protein part in HRP increase rapidly. Compared with the absence of Cu(II), the combination constant of SO3(2-) with the enzyme protein part in HRP increases nearly 70 times with a certain Cu(II) concentration (5.0 x 10(-4) mol L(-1)) in the system. However, the presence of Cu(II) in the system has little effect on the reaction constants between SO3(2-) and the ferroporphyrin part in HRP.