Direct quantification of γ-globulin in human blood serum by resonance light scattering techniques without separation of human serum albumin

We describe the preparation and characterization of functionalized nano-PbS. The functionalized nanoparticles are water-soluble. Reaction of mercaptoacetic acid functionalized nano-PbS with γ-globulin (γ-IgG) results in an enhanced resonance light scattering (RLS) at 385 nm. Based on this, a new direct quantitative method for γ-globulin in blood serum samples without separation of human serum albumin is established. Under optimal conditions, the enhanced RLS intensity is in proportion to the γ-IgG concentration in the range 10-500 ng ml⁻¹. The 3σ limit of detection is 2.75 ng ml⁻¹. The contents of γ-IgG in blood serum samples were determined with a recovery of 97-104% and R.S.D. of 1.5-2.1% (n=6). This method proved to be very sensitive, rapid, simple and tolerant of most interfering substances.     

Ferric nanoparticle-based resonance light scattering determination of DNA at nanogram levels

A novel assay of DNA has been proposed by using ferric nanoparticles as probes coupled with resonance light scattering (RLS) detection. At pH 7.40, the RLS intensity of ferric nanoparticles can be greatly enhanced by the aggregation of positively charged ferric nanoparticles through electrostatic interaction with negatively charged DNA. The enhanced intensity of RLS at 452 nm is proportional to the concentration of DNA in the range of 0.01-0.8 μg ml⁻¹ for calf thymus and salmon sperm DNA and in the range of 0.005-0.3 μg ml⁻¹ for E. coli K12 genomic DNA. Detection limits are 3.6 ng ml⁻¹ for calf thymus DNA, 4.4 ng ml⁻¹ for salmon sperm DNA, and 1.9 ng ml⁻¹ for E. coli K12 genomic DNA, respectively. Compared with the chromophores previously used in RLS assay, the ferric nanoparticles have offered several advantages in easy preparation, good photostability and high sensitivity without being modified or functionalized.     

Determination of proteins with Alizarin Red S by Rayleigh light scattering technique

A new protein determination method by enhanced Rayleigh light scattering (RLS) technique has been developed. In acid condition (pH=3.60), RLS of 1,2-dihydroxyanthraquinone-3-sulfonate (Alizarin Red S) can be greatly enhanced by addition of proteins, resulting in two characteristic peaks, 360 and 505 nm, respectively. The new protein assay is based on the RLS enhancement and spectrum change. The optimum condition for the reaction was investigated. The linear range is 0.20-24.9 μg ml⁻¹ for BSA and 0.20-15.5 μg ml⁻¹ for HSA. The detection limits (S/N=3) are 9.59 ng ml⁻¹ for BSA and 9.51 ng ml⁻¹ for HSA. The results of determination for human serum samples were comparable to those obtained by Bradford method. The binding stoichiometry was determined.     

Study of the interaction of Azur B with DNA and the determination of DNA based on resonance light scattering measurements

Based on the measurements of molecular absorption and resonance light scattering (RLS), the aggregation of Azur B (AB) was in a medium of pH ranging from 1.98 to 2.56 and ionic strength <0.12 M. The presence of double stranded DNA prompts the aggregation, resulting in enhanced RLS signals. Linear relationships were achieved between the enhanced RLS intensity at 359.7 nm and DNA concentration in the range of 0-4.5 μg ml⁻¹ for both calf thymus DNA (ctDNA) and fish sperm DNA (fsDNA) if 3.0×10⁻⁵ M AB was employed. The 3σ limits of detection were 9.3 and 8.9 ng ml⁻¹ for ctDNA and fsDNA, respectively. Five synthetic samples were analysed satisfactorily.     

Rapid and sensitive determination of proteins by enhanced resonance light scattering spectroscopy of sodium lauroyl glutamate

A rapid and sensitive method for the determination of proteins is proposed based on the measurements of the enhanced resonance light scattering (RLS) spectroscopy of sodium lauroyl glutamate (SLG). Under the optimum conditions, the interaction between SLG and proteins occurred rapidly, resulting in greatly enhanced RLS intensity with the maximum peak located at 394 nm. It was found that the enhanced RLS intensities were in proportion to the concentrations of proteins in the range of 0.01-3.1 μg ml⁻¹ depending on the kind of proteins. The detection limits were below 6 ng ml⁻¹. Compared with some other methods for the determination of proteins, this method shows high sensitivity, low detection limit and simplicity. This is an inexpensive, simple and fast one-step procedure which requires only measuring the RLS intensities. Human serum samples were determined with satisfactory results.     

Immunoassay by detecting enhanced resonance light scattering signals of immunocomplex using a common spectrofluorometer

A sensitive, highly specific immunoassay method has been developed by measuring the enhanced resonance light scattering (RLS) signals of immunoreactions with simultaneously scanning both the excitation and the emission monochromators of a common spectrofluorometer. For a given content of antibody (Ab), the RLS signals of an immunoreaction follow Gaussian distribution with antigen (Ag) concentration. The central position of the Gaussian curve represents the concentration of given Ab, and the half bandwidth has proved to be a characteristic constant of a given Ab-Ag immunoreaction. With the RLS signals, the limit of detection for human immunoglobulin G (HIgG) in serum samples could reach 10 ng ml⁻¹, and the concentration of HIgG in blood serum samples could be detected with the recovery of 90.2-107.7% and R.S.D. of 0.8-2.7%. The results of determination for three human serum samples are identical to those obtained by immunoturbidimetry.     

Synchronous fluorescence determination of protein with functionalized CdS nanoparticles as a fluorescence probe

Nanometer-sized fluorescent particles have been successfully synthesized. A synchronous fluorescence method, with high sensitivity and selectivity, has been developed for rapid determination of protein with functionalized CdS as a fluorescence probe. When Δλ=260 nm, maximum synchronous fluorescence is produced at 274 nm at pH 7.0. Under optimal conditions, the calibration graphs are linear over the range 0.1-3.0 μg ml⁻¹ for bovine serum albumin (BSA), 0.1-11.0 μg ml⁻¹ for γ-globulin (γ-G) and 0.1-1.4 μg ml⁻¹ for human serum albumin (HSA), respectively. Limits of determination were 0.01 μg ml⁻¹ for BSA, 0.019 μg ml⁻¹ for γ-G and 0.021 μg ml⁻¹ for HSA, respectively. The relative standard deviations of seven replicate measurements were 1.8% for 1.0 μg ml⁻¹ BSA, 2.2% for 1.0 μg ml⁻¹ γ-G and 2.3% for 1.0 μg ml⁻¹ HSA.     

Quantitative determination of proteins by the Rayleigh light-scattering technique after optimization of the derivation reaction with Arsenazo-DBS

This paper researched the determination of proteins with 2-(2-arsonophehenylazo)-7-[(2,6-dibromo-4-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid (Arsenazo-DBS) by Rayleigh light-scattering (RLS). The reaction parameters, such as acidity, volume of buffer solution and concentration of Arsenazo-DBS, were examined by orthogonal array design (OAD). Under optimal conditions, the weak RLS of Arsenazo-DBS and BSA can be enhanced greatly and two enhanced RLS signals were produced at 340-350 and 400-420 nm. Based on this reaction, a new quantitative determination method for proteins has been developed. This method is proved to be very sensitive (the determination limits are 0.077 μg ml⁻¹ for bovine serum albumen (BSA) and 0.074 μg ml⁻¹ for human serum albumen (HSA)), rapid (<2 min), simple (one step) and tolerance of most interfering substances. The effects of different surfactants were also examined. The amount of proteins in human serum samples was determined and the maximum relative error was no more than 2% and the recovery was between 95 and 105%.     

Determination of lead in environmental water by a backward light scattering technique

An optical fiber assembly developed in our laboratory, which is based on detecting backward light scattering (BLS) signals, is now applied to detect the lead content in environmental samples. Due to effectively eliminating the interference of reflected light, this BLS signals based detection assembly can be used to determine analyte directly. In HAc-NaAc buffer medium (pH 4.8), the interaction of lead and sodium tetraphenylboron (TPB) in the presence of polyethylene glycol (PEG) yields large particles of ternary complex, resulting in strong enhanced backward light scattering (BLS) signals characterized at 371 nm. By measuring the BLS signals with the homemade optical fiber assembly coupled with a common spectrofluorometer, we found that the enhanced BLS intensity is proportional to lead content over the range of 0.03-1.0 μg ml⁻¹ with the limit of determination (LOD) of 2.6 ng ml⁻¹. Three artificial water samples containing various coexistent substances were detected with the recovery of 90.1-107.5%. Standard addition method was used to detect the lead content in drink tap water, and found that the lead is hardly to detect due to too low content. Prior enrichment should be made in order to detect river water samples, and it was found that the content of lead in Jialing River at Bebei Dock is about 14 ng ml⁻¹, identical to the results using inductively coupled plasma atomic emission spectrometry (ICP-AES).     

A dual-wavelength resonance light scattering ratiometry of biopolymer by its electrostatic interaction with surfactant

A dual-wavelength resonance lighting scattering (DW-RLS) ratiometry is developed to detect anion biopolymer based on their bindings with cation surfactant. Using the interaction of Hyamine 1622 (HM) with fish sperm DNA (fsDNA) as an example, a dual-wavelength resonance light scattering (DW-RLS) ratiometric method of DNA was constructed. In Britton-Robinson buffer controlled medium, fish sperm DNA (fsDNA) could interact with Hyamine 1622 (HM), displaying significantly enhanced RLS signals. By measuring the RLS signals characterized at 300.0 nm (I_300.0) and the RLS intensity ratio (I_276.0/I_294.0), respectively, fsDNA over a wide dynamic range of content could be detected. Typically, when HM concentration is kept at 6.0 × 10⁻⁵ mol l⁻¹, using I_300.0 could detect fsDNA over the range of 50-2000 ng ml⁻¹ with the limit of 3.0 ng ml⁻¹, while using I_276.0/I_294.0 could detect fsDNA over the range of 0.5-2500 ng ml⁻¹ with the limit of 0.05 ng ml⁻¹. Thus the latter so-called DW-RLS ratiometry is obviously superior to the former one. Based on the measurements of I_300.0 and I_276.0/I_294.0 data, a Scatchard plot concerning the interaction between HM and fsDNA could be constructed and thus the binding number (n) and binding constant (K) could be available with the values of 13.5 and 1.35 × 10⁵ mol⁻¹ l, and 11.9 and 1.65 × 10⁵ mol⁻¹ l, respectively.     

Backscattering light detection of nucleic acids with tetraphenylporphyrin-Al(III)-nucleic acids at liquid/liquid interface

A backscattering light (BSL) detection assembly is constructed and applied to the determination of nucleic acids with high sensitivity and selectivity based on the measurements of BSL signals at water/tetrachloromethane (H₂O/CCl₄) interface. In aqueous medium of pH 3, the binary complex of of Al(III)-DNAs could be formed by the interaction of Al(III) with the phosphate group of DNAs, which then could interact with tetraphenylporphyrin (TPP) in tetrachloromethane through liquid/liquid interaction, forming a ternary complex of TPP-Al(III)-DNAs at the interface. It was observed that greatly enhanced BSL signals occurred with maximum peak at 469 nm when the ternary complex of TPP-Al(III)-DNAs were absorbed to the liquid/liquid interface. The enhanced backscattering light intensity (I_BSL) is in proportion to the concentration of calf thymus DNA (ctDNA) and fish sperm DNA (fsDNA) in the range of 0.6-1200 ng ml⁻¹ and 1.1-1200 ng ml⁻¹, respectively. The limits of determination (3σ) are 60 pg ml⁻¹ and 110 pg ml⁻¹, correspondingly. Artificial samples with highly interference backgrounds were determined with the recovery ranging from 94.5 to 106.7%, and relative standard deviation (R.S.D.) less than 2.40%.     

On the factors affecting the enhanced resonance light scattering signals of the interactions between proteins and multiply negatively charged chromophores using water blue as an example

Electrostatic interactions of proteins, including bovine serum albumin (BSA), human serum albumin (HSA), γ-globulin (γ-IgG), α-chymotrypsin (Chy), lysozyme (Lys) and cellulase (Cel), with multiply negatively charged chromophores were investigated based on the measurements of the enhanced resonance light scattering (RLS) signals. Using triply negatively charged water blue (WB) as an example, the factors were discussed that affect the enhanced resonance light scattering signals of the interactions between proteins and the negatively charged chromophores. It was found that the enhanced RLS signals with the maximum light scattering peak at 346.0 nm in these interacting systems are strongly dependent on the isoelectric points of proteins and show adverse linear relationships with increasing ionic strength depending on the positive charges of the inorganic metal ions used to control the ionic strength of the medium, sufficiently disclosing that the electrostatic attraction performs an important role in the combination of proteins with WB. Linear responses were discovered between the enhanced RLS signals and the protein molecular weights (M_w), displaying the dimensions of scattered particles formed by proteins and WB make a key contribution to the RLS enhancements. An empirical equation is proposed which possibly displays the factors affecting the enhanced RLS signals of the interactions between proteins with negatively charged chromophores.     

Resonance scattering method for the ultrasensitive determination of peptides using semiconductor nanocrystals

Resonance light scattering (RLS) of the functionalized ZnS nanocrystals-peptides system and its analytical application have been studied. The RLS intensity can be efficiently enhanced when various peptides were added. The mechanism of the RLS enhancement of ZnS nanocrystals was discussed. The change of RLS intensity was in proportion to the concentration of peptides. The limits of detection were in range of 2.8-5.7 ng mL⁻¹. Application results to synthetic samples showed simplicity, rapidity, high sensitivity and satisfactory reproducibility of the presented method. Measurements of real samples also give satisfactory results which were in good agreement with those obtained using high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrography (LC-MS) methods.     

Application of generalized artificial neural networks coupled with an orthogonal design to optimization of a system for the kinetic spectrophotometric determination of Hg(II)

A simple and sensitive kinetic method for the determination of traces of mercury (70-760 ng ml⁻¹) based on its inhibitory effect on the addition reaction between methyl green and sulfite ion is proposed. The reaction was monitored spectrophotometrically by measuring the decrease in absorbance of methyl green at 596 nm between 2 and 4 min using a fixed time method. Artificial neural networks with back propagation algorithm coupled with an orthogonal array design were applied to the modeling of the proposed kinetic system and optimization of experimental conditions. An orthogonal design was utilized to design the experimental protocol, in which pH, concentration of sulfite, temperature, and concentration of methyl green were varied simultaneously. Optimum experimental conditions in term of sensitivity were generated by using ANNs. The rate of decrease in absorbance is inversely proportional to the concentration of Hg(II) over entire concentration range tested (100-550 ng ml⁻¹) with a detection limit of 45 ng ml⁻¹ and a relative standard deviation at 200-400 ng ml⁻¹ Hg(II) of 3.2% (n=5). A simple preconcentration step improved the limit of detection and linear dynamic range of the method to about 8 and 12-760 ng ml⁻¹, respectively, by about 10 times enrichment of mercury between 12 and 75 ng ml⁻¹. The method was based on enrichment of Hg(II) from dilute samples on an anionic ion exchanger fixed on a plastic strip and was applied to the determination of Hg(II) in environmental samples with satisfactory results.     

Near-infrared fluorimetric determination of nucleic acids by shifting the ion-association equilibrium between heptamethylene cyanine and Alcian blue 8GX

A new method based on near-infrared (near-IR) fluorescence recovery, employing a two-reagent system which is composed of an anionic heptamethylene cyanine (HMC) and a polycationic phthalocyanine dye, Alcian blue 8GX, is presented for the determination of nucleic acids. With a maximum excitation wavelength at 766 nm and a maximum emission wavelength at 796 nm, the fluorescence recovery is linear with the concentration of nucleic acids added. Factors including the acidity of the medium, the reaction time, the optimal ratio of the two reagents, as well as the influence of foreign substance were all investigated. Meanwhile, the mechanism of fluorescence recovery was also studied. Under the optimal conditions, the linear ranges of the calibration curves were 10-250 ng ml⁻¹ for calf thymus DNA (CT DNA) and 10-200 ng ml⁻¹ for yeast RNA. The detection limits were 6.8 ng ml⁻¹ for CT DNA and 6.3 ng ml⁻¹ for yeast RNA, respectively. The method has been applied to the analysis of practical samples and the recovery results were satisfactory.     

Room-temperature luminescence optosensings based on immobilized active principles actives: Application to nafronyl and naproxen determination in pharmaceutical preparations and biological fluids

This paper presents two easy and selective methods for determining the active principles nafronyl (NFL) and naproxen (NAP), using a flow-through fluorescence optosensor based on the on-line immobilization on a nonionic-exchanger (Silica Gel, Davisil™ and Amberlite XAD 7, respectively) solid support. The determination was performed in 5×10⁻³ M HAc/NaAc buffer solution at pH 5 for NFL and 15×10⁻³ M glycine/HCl buffer solution at pH 2.5 for NAP at a working temperature of 20 °C. The fluorescence intensities were measured at λ_ex/em=294/336 nm and λ_ex/em=332/354 nm for NFL and NAP, respectively. The response time for these optosensors were practically instant, obtaining a linear concentration range between 0 and 700.0 ng ml⁻¹ with a detection limit of 20.8 ng ml⁻¹, an analytical sensitivity of 10.1 ng ml⁻¹ and a standard deviation of 1.27% at a 500 ng ml⁻¹ concentration level for NFL and a linear concentration range between 0 and 200.0 ng ml⁻¹ with the detection limit of 13.3 ng ml⁻¹, an analytical sensitivity of 6.0 ng ml⁻¹ and a standard deviation of 3.52% at a 100 ng ml⁻¹ concentration level for NAP. The proposed methods were satisfactorily applied to real samples (three commercial formulations and urine samples). The effects of the possible interferences were evaluated in all cases.     

Development of functionalized terbium fluorescent nanoparticles for antibody labeling and time-resolved fluoroimmunoassay application

Silica-based functionalized terbium fluorescent nanoparticles were prepared, characterized and developed as a fluorescence probe for antibody labeling and time-resolved fluoroimmunoassay. The nanoparticles were prepared in a water-in-oil (W/O) microemulsion containing a strongly fluorescent Tb³⁺ chelate, N,N,N¹,N¹-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)phenylpyridine] tetrakis(acetate)-Tb³⁺ (BPTA-Tb³⁺), Triton X-100, octanol, and cyclohexane by controlling copolymerization of tetraethyl orthosilicate (TEOS) and 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (AEPS) with ammonia water. The characterizations by transmission electron microscopy and fluorometric methods show that the nanoparticles are spherical and uniform in size, 45 ± 3 nm in diameter, strongly fluorescent with fluorescence quantum yield of 10% and a long fluorescence lifetime of 2.0 ms. The amino groups directly introduced to the nanoparticle’s surface by using AEPS in the preparation made the surface modification and bioconjugation of the nanoparticles easier. The nanoparticle-labeled anti-human α-fetoprotein antibody was prepared and used for time-resolved fluoroimmunoassay of α-fetoprotein (AFP) in human serum samples. The assay response is linear from 0.10 ng ml⁻¹ to about 100 ng ml⁻¹ with the detection limit of 0.10 ng ml⁻¹. The coefficient variations (CVs) of the method are less than 9.0%, and the recoveries are in the range of 84-98% for human serum sample measurements.     

Partial least squares multicomponent fluorimetric determination of fluoroquinolones in human urine samples

Ternary mixtures of fluoroquinolones, with a 7-piperazinyl substituted group have been simultaneously determined in human urine samples by application of a multivariate calibration partial least squares (PLS) model. The calibration set was designed with 15 urine samples containing different concentrations of the three fluoroquinolones and 16 blank urine samples. The concentration range for the fluoroquinolones were up to 25 ng ml⁻¹ for norfloxacin (NOR), 80 ng ml⁻¹ for ofloxacin (OFLO) and 300 ng ml⁻¹ for enoxacin (ENO). The method is based on the native fluorescence emission of these compounds in sodium dodecyl sulfate (SDS) medium, at pH 4.0, when exciting at 277 nm. A selection of the emission wavelength range used for the analysis was made for each component. Intraday and interday precision values were determined. Figures of merit as selectivity, sensitivity, limit of detection (LOD) and analytical sensitivity were also calculated. Using the standard addition methodology, five urine samples from five different persons, fortified with three concentration levels of the fluoroquinolones, were analyzed. The limits of detection in urine were 10.0, 0.5 and 0.8 ng ml⁻¹ for ENO, NOR and OFLO, respectively.     

High-performance liquid chromatographic method for the simultaneous determination of cyclosporine A and its four major metabolites in whole blood

This study aimed to develop a simple and efficient optimized high-performance liquid chromatograph (HPLC) method for simultaneous determination of cyclosporine A (CyA) and its major, partly active metabolites AM1, AM9, AM4N, and AM19 in whole blood from transplant patients using cyclosporine D (CyD) as internal standard. The method used a CN analytical column maintained at 60 °C with hexan-isopropanol (93:7, v/v) as mobile phase; detection was at 212 nm. Linearity for all five compounds was tested in the range of 31-1500 ng ml⁻¹ for CyA and of 31-1000 ng ml⁻¹ for metabolites. The limit of detection was found to be 15 ng ml⁻¹ for all compounds.This modified, inexpensive method is also suitable for measuring cyclosporine A and metabolite concentrations in routine monitoring of patients undergoing treatment with CyA.     

Spectrofluorimetric determination of human serum albumin using a doxycycline-europium probe

A new spectrofluorimetric method is proposed for determination of human serum albumin (HSA) with the limit of detection at ng levels. Using doxycycline (DC)-europium (Eu³⁺) as a fluorescent probe, in a buffer solution of pH 10.2, HSA can remarkably enhance the fluorescence intensity of the DC-Eu³⁺ complex at 612 nm and the enhanced fluorescence intensity of Eu³⁺ is proportional to the concentration of HSA. Optimum conditions for the determination of HSA are also investigated. The linear ranges for HSA are 0-9.2 and 9.2-34.5 μg ml⁻¹ with limits of detection of 64 and 115 ng ml⁻¹, respectively. This method is simple, practical and relatively free of interference from coexisting substances, as well as much more sensitive than most of the existing assays. The determination results for human serum and urine samples are identical to those by the AOAO method, with relative standard deviations of five determinations of 1.1-3.6%. By the Rosenthal graphic method, the binding number and association constant of human serum albumin with the probe are 1.8 and 3.71×10⁵ l mol⁻¹, respectively.