A new and sensitive catalytic resonance scattering spectral assay for the detection of laccase using guaiacol as substrate

In pH 4.0 succinic acid-sodium hydroxide buffer solution, laccase catalyzed the oxidization of guaiacol substrate to form red particles, which exhibited a strong resonance scattering (RS) peak at 590 nm. Under the chosen conditions, as the laccase increased, the RS intensity (ΔI) increased linearly. The ΔI was proportional to laccase activity in the range of 0.10-1.2 U/mL, with a regression equation of ΔI = 734.0 U(laccase) - 9.7, and a detection limit of 0.05 U/mL. This RS method was applied to the detection of laccase activity in real samples, and the results were agreement with those from spectrophotometry.     

Assembly of horseradish peroxidase within supported cationic bilayers

The interaction between horseradish peroxidase (HRP) and dioctadecyldimethylammonium bromide (DODAB) bilayers supported on polystyrene microspheres (PSS) or on flat silicon wafers was evaluated from the following techniques: (1) dynamic light-scattering for determining size distributions, zeta-potentials and polydispersities for dispersions; (2) spectrophotometric determination of HRP concentration in supernatants of centrifuged mixtures; (3) in situ ellipsometry for mean thickness of deposited layers on wafers; (4) kinetics of product appearance for oxidation of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid by H(2) O(2) in presence of free or immobilized enzyme. HRP incorporation (3.0 mg/m(2)) did not alter mean diameter and zeta-potential of PSS/DODAB particles but reduced enzyme activity by 50%, though activity persisted after several rinsing steps. In situ ellipsometry could not detect any HRP layer on top of the DODAB bilayer. HRP insertion in the bilayer core explained all results for both systems. Useful biotechnological applications are anticipated for such assemblies.     

Resonance scattering spectral detection of ultratrace Hg(II) using herring sperm DNA modified nanogold probe as catalyst

Herring sperm DNA (hsDNA) was used to modify 10 nm nanogold to obtain a resonance scattering (RS) probe (AuhsDNA) for detection of Hg²⁺. In the presence of salt, Hg²⁺ interacts with AuhsDNA to form stable Hg²⁺–hsDNA complexes, and releases nanogold particles to form larger nanogold clusters that can be removed by membrane filtration. The excess AuhsDNA in the filtrate solution exhibits a catalytic effect on the reaction between hydroxylamine (NH₂OH) and Cu(II). The excess AuhsDNA decreased with the addition of Hg²⁺, which led the RS intensity at 602 nm to decrease. The decreased RS intensity (Δl_{602 nm}) had a linear response to Hg²⁺ concentration in the range of 0.4–400 nM, with a detection limit of 0.2 nM Hg²⁺. This RS method was applied for the detection of Hg²⁺ in water samples, with sensitivity, selectivity and simplicity. Copyright © 2009 John Wiley & Sons, Ltd.     

Catalytic effect of ReAu nanoalloy on the Te particle reaction and its application to resonance scattering spectral assay of CA125

ReAu nanoparticles with a molar ratio of 2:8 Re and Te nanoparticles were prepared by NaBH₄ reduction. In HCl medium at 65°C, ultratrace Re, Te and ReAu bimetallic nanoparticles strongly catalyzed the slow reaction between Sn(II) and Te(VI) to form Te particles, which exhibited the strongest resonance scattering (RS) peak at 782 nm. As the amount of nanocatalyst increased, the RS intensity at 782 nm (I_{782 nm}) increased linearly, and the increase in intensity ΔI_{782 nm} was linear to the ReAu, Re and Te concentrations in the ranges 0.07–9.0, 0.01–4.5 and 30–1200 nm , respectively. As a model, a ReAu immunonanoprobe catalytic Te–particle resonance scattering spectral (RSS) method was established for detection of CA125, using ReAu nanoparticle labeling CA125 antibody (CA125Ab) to obtain an immunonanoprobe (ReAuCA125Ab) for CA125. In pH 7.6 citric acid–Na₂HPO₄ buffer solution, ReAuCA125Ab aggregated nonspecifically. Upon addition of CA125, the immunonanoprobe reacted with it to form ReAuCA125Ab–CA125 dispersive immunocomplex in the solution. After the centrifugation, the supernatant containing the immunocomplex was used to catalyze the reaction of Te(VI)–Sn(II) to produce the Te particles that resulted in the I_{782 nm} increasing. The ΔI_{782 nm} was linear to CA125 concentration (C_CA125) in the range 0.1–240 mU/mL. The regression equation, correlation coefficient and detection limit were ΔI_{782 nm} = 1.61 C_CA125 + 1.5, 0.9978 and 0.02 mU/mL, respectively. The proposed method was applied to detect CA125 in serum samples, with satisfactory results. Copyright © 2010 John Wiley & Sons, Ltd.     

Spectrofluorimetric determination of vitamin B1 using horseradish peroxidase as catalyst in the presence of hydrogen peroxide

In this work a new spectrofluorimetric method for the determination of vitamin B₁, based on the catalytic activity of horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H₂O₂), has been developed. Non‐fluorescent vitamin B₁ was easily converted through catalytic oxidation in alkaline medium into a fluorescent compound, even without exposure to light. The linear range for vitamin B₁ observed was 0.026–16.83 µg/mL (RSD = 1.75%). The correlation coefficient for the calibration curve and limit of detection were found to be 0.9964 and 0.015 µg/mL, respectively. The developed method is practical, simple, sensitive and relatively free from interference by coexisting substances and has been successfully applied for the determination of vitamin B₁ in pharmaceutical preparations. Copyright © 2008 John Wiley & Sons, Ltd.     

Direct determination of polymyxin B sulfate using resonance Rayleigh scattering and resonance non‐linear scattering methods with hexatungstate

At pH 1.3–1.6, tungstate WO₄^2–, can be converted to hexatungstate W₆O₁₉^2–, which can react with positively charged polymyxin B sulfate (PMB) to result in enhancement of resonance Rayleigh scattering (RRS) and resonance non‐linear scattering, including second order scattering and frequency doubling scattering. Linear relationships can be established between enhanced scattering intensity and PMB concentration. The detection limits (3σ) were 5.5 ng/mL (RRS), 10.1 ng/mL (second order scattering) and 34.6 ng/mL (frequency doubling scattering). The optimum reaction conditions, influencing factors and related analytical properties were tested. The interaction mechanism was investigated via absorption spectrum, circular dichroism spectra and atomic force microscopy imaging. The basis of scattering enhancement is discussed. PMB in eardrops, human serum and urine, were quantified satisfactorily by RRS. Copyright © 2013 John Wiley & Sons, Ltd.     

A simple,real-time assay of horseradish peroxidase using biolayer interferometry

ABSTRACT

Horseradish peroxidase (HRP) isoenzyme C1a is one of the most widely used enzymes for various analytical methods in bioscience research and medical fields. In these fields, real-time monitoring of HRP activity is highly desirable because the utility of HRP as a reporter enzyme would be expanded. In this study, we developed a simple assay system enabling real-time monitoring of HRP activity by using biolayer interferometry (BLI). The HRP activity was quantitatively detected on a BLI sensor chip by tracing a binding response of tyramide, a substrate of HRP, onto an immobilized protein. This system could be applied to analyses related to oxidase activity, as well as to the functional analysis of recombinant HRP.     

Resonance Raman spectroscopy study of change of iron spin state in horseradish peroxidase C induced by removal of calcium

Resonance Raman spectroscopy is used to probe the effect of calcium depletion on the heme group of horseradish peroxidase C at pH 8. Polarized Raman spectra are recorded with an argon ion laser at eight different wavelengths to provide a sound database for a reliable spectral decomposition. Upon calcium depletion, the spectrum is indicative of a predominantly pentacoordinated high spin state of the heme iron coexisting with small fractions of hexacoordinated high and low spin states. The dominant quantum mixed spin state of native ferric horseradish peroxidase, which is characteristic for class III peroxidases, is not detectable in the spectrum of the enzyme with partial distal Ca(2+) depletion. The quenching of the quantum mixed spin state and the predominance of the pentacoordinated high spin state are likely to arise from distortions induced by distal calcium depletion, which translates into a weaker Fe-N(epsilon)(His) bond and a more tilted imidazole. A correlation is proposed between the lower enzyme activity and the elimination of the pentacoordinated quantum mixed state upon Ca(2+) depletion.     

Effects of the interaction between Na2Wo4‐6‐benzylaminopurine anionic chelate and rhodamine 6G on the resonance Rayleigh scattering and fluorescence spectra and their analytical applications

In pH 4.99‐6.06 Britton‐Robinson (BR) buffer medium, 6‐benzylaminopurine (6‐BA) reacted with Na₂WO₄ to form 1:1 anionic chelate (6‐BA·WO₄)^2‐, which further reacted with rhodamine 6G to form ternary ion complexes at room temperature. This resulted in a significant enhancement of resonance Rayleigh scattering (RRS) with a maximum RRS wavelength of 316 nm. Meanwhile, the fluorescence of the solution was quenched and excitation (λ_ex) and emission (λ_em) wavelengths of the fluorescence were 290 and 559 nm, respectively. Intensities of RRS enhancing (ΔI_RRS) and fluorescence quenching (ΔI_F) were directly proportional to concentrations of 6‐BA. As a result, RRS and fluorescence quenching for determination of trace amounts of 6‐BA were developed. Under optimal conditions, linear ranges and detection limits of the two methods were 0.05‐15.00 µg/mL and 8.2 ng/mL (RRS), 0.50‐15.00 µg/mL and 17.0 ng/mL, respectively. It was found that the RRS method was superior to fluorescence quenching. The influence of these methods were investigated and results showed that RRS had good selectivity. RRS was applied to determine 6‐BA in vegetable samples with satisfactory results. Furthermore, the reaction mechanisms of the ternary ion‐association system are discussed. In addition, the polarization experiment revealed that the resonance light scattering (RLS) peak of Na₂WO₄‐6‐BA‐R6G consisted mainly of depolarized resonance fluorescence and resonance scattering. It was speculated that light emission fluorescence energy (E_L) transformed into resonance light scattering energy (E_RLS), which was a key reason for enhancement of RRS. Copyright © 2012 John Wiley & Sons, Ltd.     

Resonance Rayleigh‐scattering spectral method for the determination of some aminoglycoside antibiotics using CdTe quantum dots as a probe

A novel method is used for the determination of some aminoglycoside antibiotics (AGs) such as etimicin (ETM), isepamicin (ISP) and amikacin (AMK). It is based on the resonance Rayleigh scattering (RRS) intensities enhanced by AGs‐induced CdTe quantum dots aggregation. Under the optimum conditions, the increments in RRS intensity were directly proportional to the concentration of AGs in certain ranges. At the same time, the second‐order scattering, the frequency‐doubling scattering and the frequency‐trebling scattering intensities were also enhanced and their increments were proportional to the concentration of AGs. Among them, the RRS method had the highest sensitivity; the linear ranges and detection limits for ETM, ISP and AMK were 0.085–7.2, 0.0067–1.2, 0.017–6.0 and 0.025, 0.0051, 0.0020 μg mL^?1. This method was applied to the measurement of AGs in human serum and urine with satisfactory results. In addition, the reaction mechanism and the reasons for the enhancement of RRS are discussed using fluorescence, RRS, transmission electron microscope technology and quantum chemistry method. Copyright © 2009 John Wiley & Sons, Ltd.     

Catalytic solid substrate–room temperature phosphorimetry for the determination of residual perphenazine based on the electronic effect of rhodamine 6G

The rhodamine 6G⁺‐perphenazine (Rhod 6G⁺–PPH) compound is formed in the ester‐exchange reaction between ‐OH of PPH and ‐COOC₂H₅ of Rhod 6G⁺. PPH was oxidized to a red compound (PPH') in the presence of K₂S₂O₈. Interestingly, the room temperature phosphorescence (RTP) of Rhod 6G⁺ was quenched because the ‐OH of PPH′ reacted with ‐COOC₂H₅ of Rhod 6G⁺–PPH to form Rhod 6G⁺–PPH’ and PPH, which decreased the π‐electron density (δ) of the carbon atom in the Rhod 6G⁺–PPH’ conjugated system and enhanced the nonradiation energy loss of the excited Rhod 6G⁺ of the triplet state. The PPH content was directly proportional to the ΔI_p of the system. Thus, a new catalytic solid‐substrate room temperature phosphorimetry (SSRTP) method was established for the determination of PPH. The method had high sensitivity (the limit of detection was 0.019 fg/spot, corresponding to a concentration of 4.8 × 10^–14 g/mL; the sampling quantity was 0.40 μL/spot), good selectivity, convenience and speed. The analytical results were in accordance with those of high‐performance liquid chromatography (HPLC). The structures of Rhod 6G⁺, PPH and Rhod 6G⁺–PPH were characterized by infrared spectra. The reaction mechanism by which PPH was determined is discussed. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of doxepin hydrochloride in spiked human plasma and capsule after derivatization with eosin Y using resonance Rayleigh scattering,second-order scattering and frequency doubling scattering methods

Doxepin hydrochloride (DOX) is a tricyclic antidepressant drug. Three sensitive spectrofluorimetric methods, namely resonance Rayleigh scattering (RRS), frequency doubling scattering (FDS) and second-order scattering (SOS), were developed and validated for their estimation of doxepin in spiked human plasma and formulation using liquid–liquid extraction method through the formation of an ion pair complex with eosin Y at a pH of 4.5. Various factors affecting fluorescence intensity were optimized, and the reaction kinetics was determined using the Arrhenius equation method. Different scattering methods such as RRS, FDS and SOS were developed at maximum scattering wavelengths λ_ex/λ_em = 567/567 nm for RRS, 720/360 nm for SOS and 260/520 nm for FDS, respectively. The methods exhibited high sensitivities, and the detection limits for DOX were found to be 0.82, 1.20 and 1.03 ng/ml for RRS, FDS and SOS methods, respectively. The FDS method exhibited the highest sensitivity. The methods were validated using the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines and applied to determine DOX in capsule and spiked human plasma samples.     

Improved method for the routine determination of acetylcholine and choline in brain microdialysate using a horseradish peroxidase column as the immobilized enzyme reactor

A modified microbore high-performance liquid chromatography-immobilized enzyme reactor-electrochemical detection system for acetylcholine (ACh) and choline (Ch) was developed. The system used the horseradish peroxidase and a solution mediator ferrocene to convert the analyte into an oxidized ferrocene species which was detected electrochemically by reduction at 0 mV. There was an excellent linear relationship between the concentration of ACh/Ch and the peak height over the range of 1-5000 nmol/l. The limit of detection for ACh was 2 fmol/5 microl (S/N=3:1). Compared with the common method recommended by Bioanalytical System Inc. (BAS), this method exhibits a 200-fold improvement in the detection limit. The ACh and Ch levels in rat brain microdialysate were examined.     

Comparison of the structure of ribosomal 5S RNA from E. coli and from rat liver using X-ray scattering and dynamic light scattering

The structures of eukaryotic ribosomal 5S RNA from rat liver and of prokaryotic 5S RNA from E. coli (A-conformer) have been investigated by scattering methods. For both molecules, a molar mass of 44,500±4,000 was determined from small angle X-ray scattering as well as from dynamic light scattering. The shape parameters of the two rRNAs, volume V _c, surface O _c, radius of gyration R _s, maximum dimension of the molecule L, thickness D, and cross section radius of gyration R _sq, agree within the experimental error limits. The mean values are V _c=57±3 nm³, O _c=165±10 nm², R _s=3.37±0.05 nm, L=10.8±0.7 nm, D=1.57±0.07 nm, R _sa=0.92±0.01 nm.Identical structures for the E. coli 5S rRNA and the rat liver 5S rRNA at a resolution of 1 nm can be deduced from this agreement and from the comparison of experimental X-ray scattering curves and of experimental electron distance distribution function. The flat shape model derived for prokaryotic and eukaryotic 5S rRNA shows a compact region and two protruding arms. Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm. Combining the shape information obtained from X-ray scattering with the information about the frictional behaviour of the molecules, deduced from the diffusion coefficients D _20,w ⁰ =(5.9±0.2)·10^-7 cm²s^-1 and (6.2±0.2)·10^-7 cm²s^-1 for rat liver 5S rRNA and E. coli 5S rRNA, respectively, a solvation shell of about 0.3 nm thickness around both molecules is determined. This structural similarity and the consensus secondary structure pattern derived from comparative sequence analyses suggest that all 5S rRNAs may indeed have conserved essentially the same type of folding of their polynucleotide strands during evolution, despite having very different sequences.     

A nanogold resonance Rayleigh scattering method for determination of trace As based on the hydride nanoreaction

In H₂SO₄ solution, As(III) was reduced to arsine (AsH₃) by NaBH₄, and was absorbed in HAuCl₄ solution to form nanogold particles (NGs) that exhibited a resonance Rayleigh scattering (RRS) effect at 370 nm. Under the selected conditions, when the As(III) concentration increased the RRS peak also increased due to the formation of more NGs. There was a linear correlation between RRS intensity and As(III) concentration in the range 6–1000 ng/mL, with a detection limit of 3 ng/mL. This new hydride generation–nanogold reaction RRS (HG–NG RRS) method was applied to determine trace amounts of As in milk samples, with satisfactory results. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of bismuth in pharmaceutical products using phosphoric acid as molecular probe by resonance light scattering

A novel method for the sensitive determination of bismuth(III) in pharmaceutical products using phosphoric acid as a molecular probe by resonance light scattering (RLS) is discussed. In 0.5 mol/L phosphoric acid (H₃PO₄) medium, bismuth(III) reacted with PO₄^3? to form an ion association compound, which resulted in the significant enhancement of RLS intensity and the appearance of the corresponding RLS spectral characteristics. The maximum scattering peak of the system existed at 364 nm. Under optimal conditions, there was linear relationship between the relative intensity of RLS and concentration of bismuth(III) in the range of 0.06–10.0 µg/mL for the system. A low detection limit for bismuth(III) of 3.22 ng/mL was achieved. The relative standard deviations (RSD) for the determination of 0.40 and 0.80 µg/mL bismuth(III) were 2.1% and 1.1%, respectively, for five determinations. Based on this fact, a simple, rapid, and sensitive method was developed for the determination of bismuth(III) at nanogram level by RLS technique with a common spectrofluorimeter. This analytical system was successfully applied to determine the trace amounts of bismuth(III) in pharmaceutical products, which was in good agreement with the results obtained by atomic absorption spectrometry (AAS). Copyright © 2011 John Wiley & Sons, Ltd.     

A sensitive and selective immuno‐nanogold resonance‐scattering spectral method for the determination of trace penicillin G

A sensitive and selective immuno‐nanogold resonance scattering spectral assay was developed for the determination of trace hapten penicillin G, based on the resonance scattering (RS) effect of the nanogold at 560 nm, and the nanogold‐labelled immunoreaction took place in pH 5.4 phosphate citric acid buffer solutions and in the presence of polythylene glycol (PEG). The nanogold‐labelled immunocomplex formed more and more with addition of penicillin G. The enhanced RS intensity at 560 nm ΔI_RS was linear to the penicillin G concentration in the range 7.5–1700 ng/mL, with a detection limit of 0.78 ng/mL. The results indicate that the immunonanogold‐labelled RS spectral assay has a high specificity and sensitivity for quantitative determination of penicillin G in raw milk samples. Copyright © 2008 John Wiley & Sons, Ltd.     

Direct spectroscopic determination of functional sulphydryl groups on intact cell surfaces by surface-enhanced resonance Raman scattering

Semi-quantitative and direct determination of labelled sulphydryl groups on the surface of intact erythrocytes has been accomplished for the first time with surface-enhanced resonance Raman scattering (SERRS). The method, which involves the use of citrate-reduced silver colloids, is sensitive and selective. A 10^–8 M effective concentration of picomole quantities of sulphydryl groups was determined in the presence of the normally overwhelming signal from haemoglobin. This seminal study suggests that SERRS may be applied to other in situ, site-directed labelling experiments. Correspondence to: W.E. Smith     

Study on interactions of aminoglycoside antibiotics with calf thymus DNA and determination of calf thymus DNA via the resonance Rayleigh scattering technique

A simple and sensitive resonance Rayleigh scattering (RRS) spectra method was developed for the determination of calf thymus DNA (ctDNA). The enhanced RRS signals were based on the interactions between ctDNA and aminoglycoside antibiotics (AGs) including kanamycin (KANA), tobramycin (TOB), gentamicin (GEN) and neomycin (NEO) in a weakly acidic medium (pH 3.3–5.7). Parameters influencing the method were investigated. Under optimum conditions, increments in the scattering intensity (?I) were directly proportional to the concentration of ctDNA over certain ranges. The detection limit ranged from 12.2 to 16.9 ng/mL. Spectroscopic methods, including RRS spectra, absorption spectra and circular dichroism (CD) spectroscopy, coupled with thermo‐denaturation experiments were used to study the interactions, indicating that the interaction between AGs with ctDNA was electrostatic binding mode. Copyright © 2015 John Wiley & Sons, Ltd.     

A rapid and sensitive resonance Rayleigh scattering spectra method for the determination of quinolones in human urine and pharmaceutical preparation

A new method based on resonance Rayleigh scattering (RRS) was proposed for the determination of quinolones (QNS) at the nanogram level. In pH 3.3–4.4 Britton–Robinson buffer medium, quinolones such as ciprofloxacin, pipemidic acid (PIP), lomefloxacin (LOM), norfloxacin (NOR) and sarafloxacin (SAR) were protonated and reacted with methyl orange (MO) to form an ion‐pair complex, which then further formed a six‐membered ring chelate with Pd(II). As a result, new RRS spectra appeared and the RRS intensities were enhanced greatly. RRS spectral characteristics of the MO–QNS–Pd(II) systems, the optimum conditions for the reaction, and the influencing factors were investigated. Under optimum conditions, the scattering intensity (∆I) increments were directly proportional to the concentration of QNS with in certain ranges. The method had high sensitivity, and the detection limits (3σ) ranged from 6.8 to 12.6 ng/mL. The proposed method had been successfully applied for the determination of QNS in pharmaceutical formulations and human urine samples. In addition, the mechanism of the reaction system was discussed based on IR, absorption and fluorescence spectral studies. The reasons for the enhancement of scattering spectra were discussed in terms of fluorescence‐scattering resonance energy transfer, hydrophobicity and molecular size. Copyright © 2014 John Wiley & Sons, Ltd.