Inorganic phosphate assay with malachite green: An improvement and evaluation

An improvement over existing procedures for the determination of nanomole quantities of inorganic phosphate (P_i) is described. The protocol is simplified, and the effective concentration range of P_i in which the assay may be used is increased to 60 nmol/ml. Many of the substances commonly used in association with P_i assays (i.e. phosphohydrolase studies) are shown not to interface with the measurement of P_i by this method. The effects of detergents and protein on the assay also were investigated, and methods for avoiding interferences by them are described.     

Properties of the phosphate and phosphite transport systems of Phytophthora palmivora

Germlings of Phytophthora palmivora possess at least two systems for the uptake of inorganic phosphate (P_i). The first is synthesized on germination in medium containing 50 M P_i and has a K_m of approx. 30 M (V_max=7–9 nmol P_i/h·10⁶ cells). The second is synthesized under conditions of P_i-deprivation and has a higher affinity for P_i (K_m=1–2 M), but a lower V_max (0.5–2 nmol P_i/h·10⁶ cells). The fungicide phosphite likewise enters the germlings via two different transport systems, the synthesis of which also depends on the concentration of P_i in the medium. The K_m of the lower affinity system is 3 mM (V_max=20 nmol phosphite/h·10⁶ cells) and that of the higher affinity system is 0.6 mM (V_max=12 nmol/h·10⁶ cells). P_i and phosphite are competitive inhibitors for each other's transport in both systems. However, whereas mM concentrations of phosphite are necessary to inhibit P_i transport, only M concentrations of P_i are required to inhibit phosphite transport. A third system of uptake for P_i also exists, since when phosphate-deprived cells are presented with mM concentrations of P_i, they transport the anion at a very high rate (around 100 nmol/h·10⁶ cells). High rates of transport of phosphite are also observed when these cells are presented with mM concentrations of this anion.     

A sensitive and precise isotopic assay of ATPase activity

A manual ATPase assay which measures the release of ³²P_i from [γ-³²P]ATP is described. Sodium dodecyl sulfate is used to terminate the enzyme reaction and extraction of the phophomolybdate complex into xylene: isobutanol is used to separate ³²P_i from [γ-³²P]ATP for quantitation by scintillation counting. The three-step assay is rapid (75–90 samples/h) and minimizes hydrolysis of ATP due to exposure to acidie conditions. The extraction procedure separates 10⁻¹⁵ to 10⁻⁷ mol of ³²P_i from aqueous solution with an efficiency of 100,7 ± 0.62%. Less than 1% of unhydrolyzed [γ-³²P]ATP is extracted. Extraction efficiency is not affected by protein or salts commonly present in enzyme incubation mixtures. Results obtained with this assay are precise, with an intraassay coefficient of variation of 0.6% and an interassay coefficient of variation of 1.8%. The results are comparable to results obtained with a spectrophotometric assay, with a correlation coefficient of 0,996, though assay performance and sensitivity are greatly improved with the isotopic assay.     

Isolation of a highly active H+-ATPase from beef heart mitochondria

The lysolecithin extraction procedure originally described by Sadleret al. (1974) has been modified to yield a H⁺-ATPase with high levels of P_i-ATP exchange activity (400–600 nmol × min^–1 × mg^–1). This activity is further enhanced (1400–1600 nmol × min^–1 × mg^–1) following sucrose density gradient centrifugation in the presence of asolectin. This enhancement results in part from a lipid-dependent activation and in part from removal of inactive complexes. The H⁺ translocating activity of the complex has been determined spectrophotometrically using binding of oxonol VI as an indicator of membrane potential. P_i-ATP exchange, ATP hydrolysis, and oxonol binding are sensitive to energy-transfer inhibitors (oligomycin, rutamycin) and/or uncouplers (DNP, FCCP).     

Butylated hydroxytoluene and inorganic phosphate plus Ca2+ increase mitochondrial permeability via mutually exclusive mechanisms

Mitochondria undergo a permeability transition (PT), i.e., become nonselectively permeable to small solutes, in response to a wide range of conditions/compounds. In general, opening of the permeability transition pore (PTP) is Ca²⁺- and P_i-dependent and is blocked by cyclosporin A (CsA), trifluoperazine (TFP), ADP, and butylated hydroxytoluene (BHT). Gudz and coworkers have reported [7th European Bioenergetics Conference, EBEC Short Reports (1992)7, 125], however, that, under some conditions, BHT increases mitochondrial permeability via a process that may not share all of these characteristics. Specifically, they determined that the BHT-induced permeability transition was independent of Ca²⁺ and was insensitive to CsA. We have used mitochondrial swelling to compare in greater detail the changes in permeability induced by BHT and by Ca²⁺ plus P_i with the following results. (1) The dependence of permeability on BHT concentration is triphasic: there is a threshold BHT concentration (ca. 60 nmol BHT/ mg mitochondrial protein) below which no increase occurs; BHT enhances permeability in an intermediate concentration range; and at high BHT concentrations (> 120 nmol/mg) permeability is again reduced. (2) The effects of BHT depend on the ratio of BHT to mitochondrial protein. (3) Concentrations of BHT too low to induce swelling block the PT induced by Ca²⁺ and P_i. (4) The dependence of the Ca²⁺-triggered PT on P_i concentration is biphasic. Below a threshold of 50–100 M, no swelling occurs. Above this threshold swelling increases rapidly. (5) P_i levels too low to support the Ca²⁺-induced PT inhibit BHT-induced swelling. (6) Swelling induced by BHT can bestimulated by agents and treatments that block the PT induced by Ca²⁺ plus P_i. These data suggest that BHT and Ca²⁺ plus P_i, increase mitochondrial permeability via two mutually exclusive mechanisms.     

Phosphate concentration and transport in Ehrlich ascites tumor cells: Effect of sodium

The effects of extracellular P_i and Na⁺ on cellular P_i concentration and transport were studied. Steady-state P_i exchange flux was measured by ³²P uptake in the presence and absence of Na⁺. Model experiments were also conducted to assess the possibility that hydrolysis of organic phosphate esters contributes to the chemically measured intracellular P_i concentration of Ehrlich ascites tumor cells. The results of these experiments indicate that hydroloysis of labile organic phosphate esters does not contribute to the measured intracellular pool of P_i. The P_i transport system exhibits an apparent K_s of 0.115 mM P_i and a maximal flux of 1.73 mmole min^?1 (kg dry wt)^?1. When incubated in a phosphate-buffered choline chloride medium (5 mM P_i) the intracellular P_i and the P_i influx fall by 65 and 88%, respectively. At 5 mM extracellular P_i, the Na⁺-dependent component of P_i transport fits Michaelis-Menten kinetics with the maximal flux equal to 2.46 mmole min^?1 (kg dry wt)^?1 and an apparent K_s of 35.4 mM Na⁺. In addition, a Na⁺-independent component of P_i transport, comprising about 12% of the total P_i flux, was identified. The data support the hypothesis that a P_i transport system, dependent on Na⁺, plays a principal role in the maintenance of intracellular P_i concentration.     

Transport of neutral, cationic and anionic amino acids by systems B, b, XAG, and ASC in swine small intestine

Amino acid influx across the brush border membrane of the intact pig ileal epithelium was studied. It was examine whether in addition to system B, systems ASC and b^o,+ were involved in transport of bipolar amino acids. The kinetics of interactions between lysine and leucine demonstrates that system b^o,+ is present and accessible also to -glutamine. -aspartate (K_1/2 0.3 mM) and -glutamate (K_i 0.5 mM) share a high affinity transporter with a maximum rate of 1.3 μmol cm⁻² h⁻¹, while only -glutamate with a K_1/2 of 14.4 mM uses a low affinity transporter with a maximum rate of 2.7 μmol cm⁻² h⁻¹, system ASC, against which serine has a K_i of 1.6 mM. In the presence of 100 mM lysine, -glutamine (A), leucine (B), and methionine (C) fulfilled the criteria of the ABC test for transport by one and the same transporter. However, serine inhibits not only transport of -glutamate but also of glutamine (K_i 0.5 mM), and -glutamate inhibits part of the transport of glutamine. The test does, therefore, only indicate that the three bipolar amino acids have similar affinities for transport by systems B and ASC. Further study of the function of system B must be carried out under full inhibition by lysine and glutamate.     

Standard electromotive force of the H2---AgCl;Ag cell in 30, 40, and 50 mass% dimethyl sulfoxide/water from −20 to 25 °C: pK2 and pH values for a standard “Bicine” buffer solution at subzero temperatures

The establishment of the pH (designated pH*) of a standard buffer solution suitable as a pH reference in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H₂O mixtures at temperatures in the range −20 to 0 °C is reported. The buffer material selected was the ampholyte Bicine (N,N-bis(2-hydroxyethyl)glycine), and the reference standard consists of equal molal quantities of Bicine and its sodium salt. The assignment of pH* values rests on measurements of the emf of cells without liquid junction, Pt;H₂(g, 1 atm) ¦Bicine, Na Bicinate, NaCl ¦AgCl;Ag, and the pH* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Bicine) ^± (Bicinate)⁻ + H⁺. The standard emf in the DMSO/H₂O solvents at subzero temperatures was determined from emf measurements of the cell with solutions of HCl replacing the buffer-chloride mixture.     

Influence of low plasma progesterone concentrations on the release of prostaglandin F2α during luteolysis and oestrus in heifers

A study was conducted to determine the effect of suprabasal plasma concentrations of progesterone on the release of prostaglandin F_2α (PGF_2α) at luteolysis and oestrus. Heifers received silicone implants containing 2.5 (n = 4), 5 (n = 4), 6 (n = 3), 7.5 (n = 3), 10 (n = 4), or 15 (n = 3) g of progesterone, or an empty implant (controls, n = 4) between Days 8 and 25 post ovulation. Blood was collected frequently between Days 14 and 28 and assayed for progesterone and 15-ketodihydroprostaglandin F_2α. Basal progesterone concentrations in control heifers did not differ from those in heifers with 2.5- or 5-g implants and remained around 0.4−0.5 nmol l⁻¹ until ovulation in all three groups. In the heifers treated with 6–15 g of progesterone, basal concentrations were maintained at higher (P < 0.05) levels compared with those in the controls, ranging from 0.8 to 1.6 nmol 1⁻¹. The effect of these elevated progesterone levels was to delay ovulation by prolonging the growth of the ovulatory follicle, which continued growing until the implant was removed. In all experimental groups, the first significant increase of the PGF_2α metabolite occurred between Days 15.3 and 16.3 (P > 0.05) and was associated with the onset of a decrease in progesterone concentrations, which had reached levels below 3 nmol 1⁻¹ by Days 17.4−19.1. PGF_2α metabolite peaks associated with luteolysis were frequent until Day 20. In the period from Day 20 until implant removal, sporadic peaks were observed, ranging in number from 1.0 ± 1.2 (mean ± SEM) in the control group to 3.0 ± 1.4 peaks in the heifers treated with 7.5 g of progesterone (P > 0.05). The number of PGF_2α metabolite peaks during that period was higher (P < 0.05) in heifers treated with 10 and 15 g than in controls. A positive correlation was found between the basal concentration of progesterone and the number of PGF_2α peaks after luteolysis (r = 0.54; P < 0.01). Plasma progesterone concentrations above approximately 1.4 nmol l⁻¹ were able to maintain the release of PGF_2α until the progesterone implants were removed and plasma levels decreased to basal values. These heifers had a preovulatory PGF_2α release pattern resembling that found in repeat breeder heifers.     

Proton-coupled high-affinity phosphate transport revealed from heterologous characterization in Xenopus of barley-root plasma membrane transporter, HvPHT1;1

High‐affinity phosphate transporters mediate uptake of inorganic phosphate (P_i) from soil solution under low P_i conditions. The electrophysiological properties of any plant high‐affinity P_i transporter have not been described yet. Here, we report the detailed characterization of electrophysiological properties of the barley P_i transporter, HvPHT1;1 in Xenopus laevis oocytes. A very low K_m value (1.9 µm ) for phosphate transport was observed in HvPHT1;1, which falls within the concentration range observed for barley roots. Inward currents at negative membrane potentials were identified as nH⁺:P_i^‐ (n > 1) co‐transport based on simultaneous P_i radiotracer uptake, oocyte voltage clamping and pH dependence. HvPHT1;1 showed preferential selectivity for P_i and arsenate, but no transport of the other oxyanions SO₄^2? and NO₃^‐. In addition, HvPHT1;1 locates to the plasma membrane when expressed in onion (Allium cepa L.) epidermal cells, and is highly expressed in root segments with dense hairs. The electrophysiological properties, plasma membrane localization and cell‐specific expression pattern of HvPHT1;1 support its role in the uptake of P_i under low P_i conditions.     

The determination of inorganic orthophosphate in biological systems

A simple and rapid method is described for determining P_i by spectrophotometric measurement of a soluble complex of phosphomolybdic acid and Cirrasol ALN-WF, a non-ionic detergent formerly known as Lubrol W. The measured complex has a molar extinction coefficient of 4.59 · 10³ at 390 nm and little interference is found with relatively high concentrations of chelating agents, salts, and other compounds which interfere with most other P_i assays. Linearity is observed in the range 0–1.2 μmoles P_i and developed assay samples are stable for 8 h at 20 °C or 24 h at 4 °C. The method is suitable for use in the presence of moderate concentrations of protein or ATP.After suitable modification the assay can be used at pH 4.0. Sensitivity is reduced at this pH (ε_{M, 390nm} = 2.79 · 10³) but linearity is maintained up to 1 μmole P_i and the coloured complex is stable for 4 h at 20 °C. The pH-4 procedure is suitable for measurement of P_i in the presence of very labile phosphate esters such as creatine phosphate.The phosphomolybdic acid-Cirrasol complex can be reduced at ambient temperature in both the above systems. A blue complex results with ε_{M, 820nm} of 9.9 · 10³ at pH 4.0, and 1.8 · 10⁴ under more acidic conditions.     

Characterization of Two Inducible Phosphate Transport Systems in Rhizobium tropici

Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (P_i). To better understand phosphorus movement between the bacteroid and the host plant, P_i transport was characterized in R. tropici. We observed two P_i transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The K_m and V_max values for the low-affinity system were estimated to be 34 ± 3 μM P_i and 118 ± 8 nmol of P_i · min⁻¹ · mg (dry weight) of cells⁻¹, respectively, and the K_m and V_max values for the high-affinity system were 0.45 ± 0.01 μM P_i and 86 ± 5 nmol of P_i · min⁻¹ · mg (dry weight) of cells⁻¹, respectively. Both systems were inducible by P_i starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but P_i transport through both systems was eliminated by the ATPase inhibitor N,N′-dicyclohexylcarbodiimide; the P_i transport rate was correlated with the intracellular ATP concentration. Also, P_i movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P_i transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.     

Microfluorometric analysis of Cl permeability and its relation to oscillatory Ca signalling in glucose-stimulated pancreatic β-cells

The cytoplasmic concentrations of Cl⁻([Cl⁻]_i) and Ca²⁺ ([Ca²⁺]_i) were measured with the fluorescent indicators N-(ethoxycarbonylmethyl)-6-methoxyquinilinum bromide (MQAE) and fura-2 in pancreatic β-cells isolated from ob/ob mice. Steady-state [Cl⁻]_i in unstimulated β-cells was 34 mM, which is higher than expected from a passive distribution. Increase of the glucose concentration from 3 to 20 mM resulted in an accelerated entry of Cl⁻ into β-cells depleted of this ion. The exposure to 20 mM glucose did not affect steady-state [Cl⁻]_i either in the absence or presence of furosemide inhibition of Na⁺, K⁺, 2 Cl⁻ co-transport. Glucose-induced oscillations of [Ca²⁺]_i were transformed into sustained elevation in the presence of 4,4′ diisothiocyanato-dihydrostilbene-2,2′-disulfonic acid (H₂DIDS). A similar effect was noted when replacing 25% of extracellular Cl⁻ with the more easily permeating anions SCN⁻, I⁻, NO₃⁻ or Br⁻. It is concluded that glucose stimulation of the β-cells is coupled to an increase in their Cl⁻ permeability and that the oscillatory Ca²⁺ signalling is critically dependent on transmembrane Cl⁻ fluxes.     

Permeability of bilayer lipid membranes for superoxide (O2) radicals

Egg yolk phosphatidylcholine monolamellar liposomes (1000 Å in diameter) loaded with cytochrome c were placed into an external solution, in which superoxide radicals, O₂⁻, were generated by a xanthine-xanthine oxidase system. The penetration of the superoxide radicals across the liposomal membrane was detected by cytochrome c reduction in the inner liposome compartment. The effects of modifiers and temperature on this process were studied. The permeability of liposomal membrane for O₂⁻(P′_O2⁻ = (7.6 ± 0.3) · 10^-8 cm/s), or HO₂^• (P′_HO2⁻ = 4.9 · 10^-4 cm/s) were determined. The effect of the transmembrane electric potential (K⁺ concentration gradient, valinomycin) on the permeability of liposomal membranes for O₂⁻ were investigated. It was found that O₂⁻ can penetrate across liposomal membrane in an uncharged form. The feasibility of penetration of superoxide radicals through liposomal membrane, predominantly via anionic channels, was demonstrated by the use of an intramolecular cholesterol-amphotericin B complex.     

Effect of renoguanylin on hydrogen/bicarbonate ion transport in rat renal tubules

Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 μM and 10 μM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 μM concentrations. Comparing control and REN concentration of 1 μM, JHCO₃⁻, nmol cm^− 2 s^− 1 − 1,76 ± 0,11_control × 1,29 ± 0,08_{REN 10 μM}; P < 0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 μM (JHCO₃⁻, nmol cm^− 2 s⁻ 1 − 0.80 ± 0.07_control × 0.60 ± 0.06_{REN 1 μM}; P < 0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na⁺/H⁺exchanger and H⁺-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway.     

A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase

A new and convenient method for the determination of P_i was developed. Phosphomolybdate is measured colorimetrically, without reduction to molybdenum blue, by dissolving the whole assay mixture in acetone, where phosphomolybdate is bright yellow. The hydrolysis of acid-labile phosphates (e.g., creatine phosphate) causes no problems, because extra molybdate is complexed with citrate immediately after the color has been developed. Strong reductants and SH compounds which interfere, if present in high concentrations, are eliminated by adding H₂O₂. Detergents, organic bases, protein, and sucrose do not interfere. The assay is as sensitive as most modifications of the Fiske-SubbaRow method. In the routine procedure the useful range is 50–1500 nmol of P_i. The application of the method to the assay of inorganic pyrophosphatase in the cells of Escherichia coli is described.     

Thiopurine methyl transferase activity: new extraction conditions for high-performance liquid chromatographic assay

A new liquid–liquid extraction is described for thiopurine methyl transferase (TPMT, EC 2.1.1.67) activity determination: the use of a pH 9.5 NH₄Cl buffer solution, before adding the solvent mixture, allows more rapid extraction, avoiding a centrifugation step, and reduces the global cost of analysis. After the extraction step, 6-methylmercaptopurine, synthesised during the enzymatic reaction, is determined by a liquid chromatographic assay. Analytical performance of the assay was tested on spiked erythrocyte lysates. The linear concentration range was 5–250 ng ml ⁻¹ (r≥0.997, slope=1.497, intercept=−0.367). The recoveries were 82.8, 89.9 and 82.2% for 75, 125 and 225 ng ml⁻¹, respectively. The coefficients of variation were ≤6.1% for within-day assay (n=6) and ≤9.5% for between-day assay precision (n=6; 14 days). TPMT activity was determined in a French adult Caucasian population (n=70). The results ranged from 7.8 to 27.8 nmol h⁻¹ ml⁻¹ packed red blood cells and the frequency distribution histogram is similar to that previously published.     

Asymptotic theory for analyzing dose-response survival data with application to the low-dose extrapolation problem

A model of the form P(t, d)=1−;exp{-(t)σ^K_i=0q_idⁱ}, q_i 0, is proposed for analyzing dose-response survival data with right censoring. The q_i's in the dose polynomial are estimated by maximizing the Cox partial likelihood, and given these estimates. Λ(t) is estimated nonparametrically by an estimator proposed by Breslow. Large-sample properties of these estimators are established. Estimates and related large-sample properties are provided for the “virtually safe dose” and other parameters for assessing low-dose carcinogenic risk as a function of age, using data from animal carcinogenesis experiments.     

Determination of the optimum operating time for batch isothermal performance of enzyme-catalyzed multisubstrate reactions

This communication consists of a mathematical analysis encompassing the maximization of the average rate of monomer production in a batch reactor performing an enzymatic reaction in a system consisting of a multiplicity of polymeric substrates which compete with one another for the active site of a soluble enzyme, under the assumption that the form of the rate expression is consistent with the Michaelis-Menten mechanism. The general form for the functional dependence of the various substrate concentrations on time is obtained in dimensionless form using matrix terminology; the optimum batch time is found for a simpler situation and the effect of various process and system variables thereon is discussed. The reasoning developed here emphasizes, in a quantitative fashion, the fact that the commonly used lumped substrate approaches lead to nonconservative decisions in industrial practice, and hence should be avoided when searching for trustworthy estimates of optimum operation.List of Symbols O 1/s row vector of zeros - a 1/s row vector of rate constants k _i(i = 2,...,N) - A 1/s matrix of rate constants k _i and k_–i (i=2,...,N) - b 1/s row vector of rate constant k ₂ and zeros - C mol/m³ molar concentration of S - C mol/m³ vector of molar concentrations of C _i (i=0, 1, 2, ..., N) - C ₀ mol/m³ column vector of initial molar concentrations of C _i(i=0, 1, 2,.., N) - C _–01 mol/m³ column vector of initial molar concentrations of C _i(i=2,..., N) - C _{E, tot} mol/m³ total molar concentration of enzyme molecules - C _i mol/m³ molar concentration of S _i (i=0,1,2,...,N) - C _{i, o} mol/m³ initial molar concentration of S _i(i=0, 1, 2, ..., N) - E enzyme molecule - I identity matrix - K 1/s matrix of lumped rate constants - k _i 1/s pseudo-first order lumped rate constant associated with the formation of S _i -1 (i=1, 2, ...,N) - k _{cat, i} 1/s first order rate constant associated with the formation of S _i-1 (i=1, 2, ..., N) - K _m mol/m³ Michaelis-Menten constant - L number of distinct eigenvalues - M _i multiplicity of the i-th eigenvalue - N maximum number of monomer residues in a single polymeric molecule - r ₁ mol/m³ s rate of formation of S ₀ - r _i mol/m³ s rate of release of S _i -1 - r _opt maximum average dimensionless rate of production of monomer S₀ - S lumped, pseudo substrate - S₁ inert moiety - S _i substrate containing i monomer residues, each labile to detachment as - S₀ by enzymatic action (i=1,2,...,N) - t s time elapsed since startup of batch reaction - t _lag s time interval required for cleaning, loading, and unloading the batch reactor - t _opt s time interval leading to the maximum average rate of monomer production - v _ij s^1-j eigenvectors associated with eigenvalue imi (i=1, 2, ..., L; j =1, 2, ..., M_i) Greek Symbols _ij mol/m³ arbitrary constant associated with eigenvalue _i (i=1, 2, ..., L; j=1, 2, ..., M _i ) - 1/s generic eigenvalue - _i 1/s i-th eigenvalue     

A Rapid Surface-Enhanced Raman Scattering Method for the Determination of Trace Hg2+ Using Rhodamine 6G-Aggregated Nanosilver as Probe

In pH 6.0 Na₂HPO₄-NaH₂PO₄ buffer solution and in the presence of cetyltrimethyl ammonium bromide, nanosilver particles were aggregated to a stable suspension. Therein, rhodamine 6G (Rh6G) exhibited three strong surface-enhanced Raman scattering (SERS) peaks at 613, 1,363, and 1,510 cm^?1, and their SERS intensities were enhanced when the concentration of Rh6G increased. In the presence of Hg²⁺, the SERS intensity decreased greatly owing to formation of stable Rh6G-HgBr ₄ ^2? ternary association complex molecules as well as its particles. In the optimal condition, the decreased SERS intensity at 613 cm^?1 responds linearly with the concentration of Hg²⁺ over 25–2,000 nmol/L. Thus, a new sensitive SERS method has been proposed for the determination of trace Hg²⁺ in the water sample, with satisfactory results.