Using Ho3+ Fluorescence Enhancement as a Novel Probe in Monitoring of Human Serum Albumin

Abstract

In this work, for the first time, the fluorescence enhancement of Ho³⁺ ions is introduced as a novel probe for human serum albumin (HSA) determination in aqueous solution. Lanthanides express very specific luminescence emissions, due to their unfilled 4fⁿ electronic orbital, and thus, the fluorescence of the lanthanide ions can be radically improved when they are coordinated with the appropriate organic molecules. By applying this method, HSA can be determined up to 0.066 mg L^?1.     

A novel immunoassay strategy based on combination of chitosan and a gold nanoparticle label

A novel immunoassay strategy based on combination of chitosan (CHIT) and a gold nanoparticle (GNP) label has been developed. The susceptibility of CHIT to further chemical modifications due to the abundant amino groups is explored in order to covalently immobilize antibody (Ab) onto the (3-aminopropyl) triethoxysilane derivatized glass slide by cross-linking with glutaraldehyde (GA). After incubating in antigen (Ag) solution, the obtained substrate is immersed in GNP labeled antibody solution for signal generation. The two steps were repeated alternatively for three times, forming multilayer of gold nanoparticles via antigen-antibody specific reaction. Ultraviolet-visible (UV-vis) absorption spectrum is recorded to obtain quantitative information about the specific antigen. The presented immunoassay strategy is applied for determination of human serum albumin (HSA) as a model analyte. The immunoassay of HSA is specific. Compared to previous correlative work, the proposed immunosensing strategy shows some advantages, such as improved sensitivity as much more gold nanoparticles can be coupled to the functionalized surface making use of the abundant amino groups of CHIT. Moreover, a significantly extended linear detection range of 8.0-512.0 μg/mL is gained under the optimized experimental conditions. In particular, the presented biosensing method shows low cost and simplicity, and only a conventional UV-vis detector is involved.     

An ultrasensitive streptavidin-functionalized carbon nanotubes platform for chemiluminescent immunoassay

An ultrasensitive chemiluminescent (CL) immunoassay system was developed for the detection of tumor marker. This sandwich CL assay method was for the first time designed based on a highly efficient streptavidin-functionalized multi-walled carbon nanotubes (MWCNTs) platform. The glass slide was firstly silylanized with 3-gycidoxypropyltrimethoxysilane (GPTMS) to generate surface epoxy group functionality. Subsequently, the MWCNTs/chitosan solution was mixed with streptavidin solution, and a certain amount of the resulting suspension was dropped on the surface of the epoxy-activated glass substrate to form a firm streptavidin-functionalized MWCNTs platform. The biofunctionalized-MWCNTs platform shows large reactive surface area and excellent biocompatibility. The capture antibody can be efficiently immobilized on the biosensing platform surface based on the highly selective recognition of streptavidin to biotinylated antibody. Using α-fetoprotein (AFP) as model analyte, the proposed method exhibits wide linear range of 0.001–0.1 ng mL⁻¹ with a low detection limit down to 0.52 pg mL⁻¹. The CL immunoassay system displays 7.9-fold increase in the detection sensitivity compared to the immunosensor without using MWCNTs. Moreover, the resulting immunosensor demonstrates excellent specificity, good reproducibility, and acceptable stability. This streptavidin-functionalized MWCNTs platform opens a novel and promising avenue for fabricating ultrasensitive CL immunoassay system.     

Fluorescence immunoassay of α-fetoprotein with iron(III) tetrasulfonatophthalocyanine as a mimetic enzyme labeling reagent

A new fluorimetric immunoassay for α-fetoprotein (AFP) has been developed using a novel promising mimetic peroxidase, iron(III) tetrasulfonatophthalocyanine (FeTSPc), as a labeling reagent to catalyze the fluorescence reaction of P- hydroxyphenylacetic acid (P-HPA) and hydrogen peroxide (H₂O₂). In the competitive immunoassay, anti-AFP antibody was coated on a 96-well plate (polystyrene) and a constant amount of FeTSPc-labeled AFP and a known amount of test solution were added. Non-labeled and FeTSPc-labeled AFP compete for binding to the plate-bound antibody. After the immunoreaction, the immunochemically adsorbed FeTSPc–AFP conjugate moiety was determined by measuring the fluorescence produced in a solution containing P-HPA and H₂O₂. AFP can be determined in the concentration range of 1–300 ng mL^–1 with a detection limit of 0.5 ng mL^–1. Received: 14 November 2000 / Revised: 29 December 2000 / Accepted: 15 January 2001     

Polarographic immunoassay coupled with catalysis of non-radioactive multiple iodine label

A now polarographic immunoassay was developed In this assay,human serum albumin (HSA) as the model antigen was covalently labeled with organic compound erythrosin B(EB) containing four non-radioactive iodides through Ⅰ step chemical reaction The labeling procedure is simple and the conditions needed are moderate.The molar labeling ratio of KB HSA was 12 Ⅰ The content of iodine in the conjugate obtained by the proposed procedure is ninth higher than that by the other existing methods.A heterogeneous competitive immunoassay was established by compling the catalysis of the conjugate to substrate As(Ⅲ)-Ce(Ⅳ) reaction with the linear-sweep polarographic detec-tion of As(Ⅲ) amount HSA can be determined in the HSA concentration range from 1 to 200μg/mL,with the de-tection hum of 0 66μg/ml.     

Immunosensor for the Measurement of Human Serum Albumin in Urine Based on the Spreeta Surface Plasmon Resonance Sensor

The application of SPR for measurement of the concentration of human serum albumin (HSA) in urine was studied using the compact integrated SPR sensing system Spreeta. HSA was immobilized via cystamine and glutaraldehyde onto the gold sensing area and a competitive assay for HSA was developed using a limited amount of the monoclonal antibody AL-01 in solution. Measurements were carried out in the flow-through mode and the interaction between immobilized HSA and antibody was observed in real time. To obtain reproducible results, different conditions of the measurement (method of immobilization of HSA, data evaluation, concentration of antibody, regeneration procedure) were tested. The calibration curve for clinically relevant concentrations of HSA in urine samples was constructed using 300-times diluted antibody in the form of ascites fluid. The measuring range was between 0.1 and 5 mg/l of HSA, the sensing surface was successfully regenerated and suitable for more than 20 assays. The developed method was tested on real samples of urine; to overcome the non-specific adsorption of urine components, the differential approach was adopted and the measured signal was corrected by subtraction of the response observed in the absence of the antibody.     

A novel approach for a non competitive capillary electrophoresis immunoassay with laser-induced fluorescence detection for the determination of human serum albumin

A new non competitive capillary electrophoresis immunoassay format based on a separation into a capillary modified by analyte immobilisation is described. The injection of an excess of labelled antibody off-line preincubated with the analyte allows the surface capture of the free antibody and the immunocomplex detection. It was developed using the human serum albumin (HSA) as analyte, two FITC-labelled antibodies and a HSA covalently linked capillary. Two calibration curves with good run-to-run reproducibility and LOD--respectively 14.0 nM for the FITC-polyclonal antibody and 9.0 nM for the FITC-monoclonal antibody--were achieved. The assay was applied to HSA determination in spiked samples of human urine with acceptable recoveries.     

Liquid crystal-based immunoassay for detecting human serum albumin

Human serum albumin (HSA) is the most abundant protein in human blood plasma. It is commonly used as a biomarker in urine samples to identify the chronic kidney disease caused by high blood pressure or diabetes. In our research, a thin layer of liquid crystals (LCs) is used as a readout system for developing an immunoassay that reports the presence of HSA in the aqueous solution with optical signals. The detection principle of this assay is based on the variation of surface density of protein upon the specific binding of HSA on an anti-HSA immobilized surface, which leads a dark-to-bright transition of LC images under cross-polarizers. Our results show that the LC-based immunoassay can detect HSA at a concentration of 50 μg/mL. By using the slide with immobilized anti-HSA in array format, the concentration level of HSA can be simply determined by the number of LC spot shown on the slide.     

Spectral and fluorescent kinetics features of Nd3+ ion in Nb2O5, Ta2O5 and La2O3 mixed lithium zirconium silicate glasses

A sensitive competitive flow injection chemiluminescence (CL-FIA) immunoassay for immunoglobulin G (IgG) was developed using gold nanoparticle as CL label. In the configuration, anti-IgG antibody was immobilized on a glass capillary column surface by 3-(aminopropyl)-triethoxysilane and glutaraldehyde to form immunoaffinity column. Analyte IgG and gold nanoparticle labeled IgG were passed through the immunoaffinity column mounted in a flow system and competed for the surface-confined anti-IgG antibody. CL emission was generated from the reaction between luminol and hydrogen peroxide in the presence of Au (III), generated from chemically oxidative dissolution of gold nanoparticle by an injection of 0.10 mol L⁻¹ HCl–0.10 mol L⁻¹ NaCl solution containing 0.10 mmol L⁻¹ Br₂. The concentration of analyte IgG was inversely related to the amount of bound gold nanoparticle labeled IgG and the CL intensity was linear with the concentration of analyte IgG from 1.0 ng mL⁻¹ to 40 ng mL⁻¹ with a detection limit of 5.2 × 10⁻¹⁰ g mL⁻¹. The whole assay time including the injections and washing steps was only 30 min for one sample, which was competitive with CL immunoassays based on a gold nanoparticle label and magnetic separation. This work demonstrates that the CL immunoassay incorporation of nanoparticle label and flow injection is promising for clinical assay with sensitivity and high-speed.     

Homogeneous immunoassay based on gold nanoparticles and visible absorption detection

A sensitive homogeneous immunoassay, using human serum albumin (HSA) as a model analyte coupled with simple visible absorption detection, has been developed. The new assay is based on the use of gold nanoparticles functionalized with the target protein, which compete with the analyte for the binding of a specific polyclonal antibody. The binding of antibodies to the functionalized nanoparticles determines a shift of the visible absorption maximum of the gold colloid, and quantification of the analyte could be obtained as the competitive inhibition of the binding of antibodies to the nanoparticles. The proposed immunoassay has been optimized and successfully applied to measuring HSA in human urine samples, in which results agreed well with those obtained by a nephelometric reference method.     

A catalytic immuno-reactor for the amperometric determination of human serum albumin

A catalytic immuno-reactor for the determination of human serum albumin (HSA) was constructed by using immobilized antibody and an amperometric detector. A sandwich assay with hemin-labeled antibody to catalyze the decomposition of hydrogen peroxide was used, the catalytic activity of the hemin-antibody conjugate being determined by measuring the decrease in hydrogen peroxide concentration. The reaction of hemin-labeled antibody with antigen was complete within 30 min and the current decrease was correlated with the HSA concentration. The relative standard deviation was about 9% at an HSA concentration of 1 mg ml^?1.     

In situ monitoring of gold-surface adsorption and acidic denaturation of human serum albumin by an isolation-capacitance-adopted electrochemical quartz crystal impedance system

Combined measurements of piezoelectric quartz crystal impedance (PQCI) and electrochemical impedance spectrum (EIS) using a suitable isolation capacitance is reported for the first time to monitor in situ adsorption and acidic denaturation of human serum albumin (HSA) on gold electrodes in Britton-Robinson (B-R) buffers. This method provides simultaneously mutual-interference-free and accurate parameters of EIS and PQCI. Effects of surface thiol-modification, electrode-potential and solution pH on HSA adsorption were examined and discussed. Comparative experiments of HSA adsorption in a B-R buffer of pH 6.42 on bare, cysteine- and 1-dodecanethiol-modified gold electrodes revealed that HSA adsorption is more significant on a hydrophobic (1-dodecanethiol-modified) surface. Insignificant electrode-potential effect implied minor electrostatic effects on HSA adsorption. The adsorption amount of HSA at pH 3.28 was found to be notably greater than those at pH 4.84 and 6.42. To characterize HSA adsorption, electrode standard rate constants (k_s) of the Fe(CN)₆³⁻/Fe(CN)₆⁴⁻ couple were measured before and after HSA adsorption. The k_s-pH curves on an HSA-modified Au electrode revealed that k_s increased abruptly with the decrease of solution pH below pH ∼4. Moreover, pH-dependent responses of the resonant frequency, the motional resistance, the double-layer capacitance, the capacitance of adsorbed HSA layer and the peak absorbance of HSA solutions at 278 nm all exhibited an inflexion change at pH ∼4, and these findings have been explained on the basis of acidic denaturation of HSA and electrical charges carried by HSA molecules.     

Chromatographic study of the adsorption kinetics of albumin on monoclonal and polyclonal immunoadsorbents

Summary High-performance liquid chromatography (HPLC) has been used to study the adsorption kinetics of proteins on immunoadsorbents. The adsorption rate constant of human serum albumin (HSA) on monoclonal and polyclonal anti-HSA antibodies immobilized on a silica HPLC support was determined by saturating the column with repeated pulse injections. Studies on polyclonal immunodsorbents of different capacities enable evaluation of the contribution of transport to the binding sites. The adsorption properties of two different monoclonal anti-HSA antibodies immobilized on a chromatographic support were characterized by different approaches. The location of the epitope on the HSA molecule was determined by enzyme-linked immunoassay (ELISA) with albumin fragments. The chromatographic method was used to determine the column capacity and the adsorption rate constant of HSA on the immunoadsorbent. To compare the affinity of the antibodies for the antigen, an indirect ELISA method was used to determine the equilibrium constant of antigen-antibody association in solution Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th september, 1996.     

A novel immunoassay based on the dissociation of immunocomplex and fluorescence quenching by gold nanoparticles

This study reports a novel, simple and sensitive immunoassay using fluorescence quenching caused by gold nanoparticles coated with antibody. The method is based on a non-competitive heterogeneous immunoassay of human IgG conducted by the typical procedure of sandwich immunocomplex formation. Goat anti-human IgG was first adsorbed on polystyrene microwells, and human IgG analyte was captured by the primary antibody and then sandwiched by antibody labeled with gold nanoparticles. The sandwich-type immunocomplex was subsequently dissociated by the mixed solution of sodium hydroxide and trisodium citrate, the solution obtained, which contains gold nanoparticles coated with antibody, was used to quench fluorescence. The fluorescence intensity of fluorescein at 517 nm was inversely proportional to the logarithm of the concentration of human IgG in the dynamic range of 10-5000 ng mL⁻¹ with a detection limit of 4.7 ng mL⁻¹. The electrochemical experiments and the UV-vis measurements were applied to demonstrate whether the immunoglod was dissociated completely and whether the gold nanoparticles aggregated after being dissociated, respectively. The proposed system can be extended to detect target molecules such as other kinds of antigen and DNA strands, and has broad potential applications in disease diagnosis.     

NMR relaxation studies of GdDTPA in human serum albumin solution

The water relaxation enhancement behavior of GdDTPA in human serum albumin (HSA) solution has been studied The results indicate that GdDTPA can integrate noncovalently with HSAmainly in forms of (GdDTPA) HSA and (GdDTPA)₂HSA for which the apparent equilibrium constants are 005 mM ⁻¹ and 002 mM ⁻²respectively.     

Micro-plate chemiluminescence enzyme immunoassay for aflatoxin B1 in agricultural products

In this work, a micro-plate chemiluminescence enzyme immunoassay by antibody-coated for the determination of aflatoxin B1 (AFB1) in agricultural products has been established. Aflatoxin B1 antibody (AFB1-Ab) was adsorbed physically on polystyrene micro-plate hole as solid phase antibody, which took place immunity-reaction between antigen and antibody with AFB1 standard solution or samples by direct competition. Luminol-hydrogen peroxide chemiluminescence system catalyzed by horseradish peroxidase (HRP) with p-iodophenol enhancement was used as signal detecting system. The effects of several factors, including composition and pH of coating solution, dilution ratio and amount of antibody and enzyme labeled antigen, time of antibody-coating, incubation and chemiluminescence reaction, and other relevant variables upon the immunoasaay were studied and optimized. The linear range of proposed method for AFB1 was 0.05-10.0 ng g⁻¹ with a correlative coefficient of −0.9997. The sensitivity of the proposed method was 0.01 ng g⁻¹. The RSDs of intra- and inter-assay were less than 12.2% and 10.0%, respectively. This method has been successfully applied to the evaluation of AFB1 in agricultural products with recoveries of 79.8%, 101.9% and 115.4% for low, middle and high concentration samples, respectively. It shows a good correlation with the commercial available ELISA kit for AFB1 with correlative coefficient of 0.9098 indicating that the established CLEIA method can be used to determine AFB1 in real samples.     

Time-resolved luminescent lateral flow assay technology

We here report a detection technology that integrates highly sensitive time-resolved luminescence technique into lateral flow assay platform to achieve excellent detection performance with low cost. We have developed very bright, surface-functionalized and mono-dispersed phosphorescent nanoparticles of long lifetime under ambient conditions. The phosphorescent nanoparticles have been used to conjugate with monoclonal antibody for C-reactive protein (CRP), an inflammatory biomarker. Lateral flow immunoassay devices have been developed using the conjugate for highly sensitive detection of CRP. The CRP assay can achieve a detection sensitivity of <0.2 ng mL⁻¹ in serum with a linear response from 0.2 to 200 ng mL⁻¹ CRP. We have also developed a low cost time-resolved luminescence reader for the lateral flow immunoassay (LFIA) devices. The reader does not use expensive band pass filter and still provide very low detection background and high detection sensitivity on solid substrates such as nitrocellulose membranes. The reader can detect less than 2.5 ng phosphorescent particles captured on a nitrocellulose membrane strip with more than three orders of magnitude linear detection dynamic range. The technology should find a number of applications, ranging from clinical diagnostics, detection of chemical and biological warfare agents, to food and environmental monitoring.     

Capillary electrophoresis immunoassay based on an on-column immunological reaction

An on-column immunological reaction was employed to achieve simple and rapid analysis in an immunoassay based on capillary electrophoresis using semiconductor laser-induced fluorescence detection. Human serum albumin (HSA) labeled with sulfoindocyanine succinimidyl ester (Cy5), a fluorescent compound with an absorption maximum at 649 nm, was used as a fluorescent probe for the immunoassay. In a binding assay, with anti-HSA as the analyte molecule, Cy5-HSA was injected in a capillary column followed by the injection of anti-HSA so as to form individual zones. By applying a potential, the anti-HSA reacted with Cy5-HSA at the boundary between Cy5-HSA and anti-HSA zones, since anti-HSA has a higher electrophoretic mobility than Cy5-HSA. Furthermore, the on-column method enhances the sensitivity by injecting a large volume of the sample. Free Cy5-HSA and its immunocomplex with anti-HSA were separated with less degradation in resolution than that predicted from the injection time of anti-HSA, even when the injection time for anti-HSA was increased. The ratio of the peak area of the complex to that of the total Cy5-HSA (free Cy5-HSA and the complex) increased in proportion to the injection time of anti-HSA. As a result, the detection limit was improved up to eight-fold (the concentration detection limit, 0.007 mg mL(-1), for an injection time of 240 s, compared to that obtained using an off-column sample preparation. Furthermore, the on-column reaction method was applicable to an immunoassay to determine native HSA, in which native HSA and Cy5-HSA react with anti-HSA stepwise. The detection limit in the stepwise reaction immunoassay was 0.005 mg mL(-1), which is 14 times lower than that in an off-column method, with the analysis time less than 10 min as the result of increasing the injection time of native HSA. In addition, the present on-column immunoassay was applied to the sample containing a high concentration of salts for investigating the effect of salts in the sample solution.     

Development of an immunomagnetic bead-based time-resolved fluorescence immunoassay for rapid determination of levels of carcinoembryonic antigen in human serum

A novel immunoassay for the determination of tumor markers in human serum was established by combining a time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a sandwich-type immunoassay format, analytes in samples were captured by magnetic beads coated with one monoclonal antibody and “sandwiched” by another monoclonal antibody labeled with europium chelates. The immunocomplex was separated and washed by exposure to a magnetic field and treatment with enhancement solution; fluorescence was then measured according to the number of europium ions dissociated. Levels of the model analyte, carcinoembryonic antigen (CEA), were determined in a linear range (1–1000 ng mL⁻¹) with a limit of detection of 0.5 ng mL⁻¹ under optimal conditions. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. To evaluate this novel assay for clinical applications, 239 serum samples were evaluated. Compared with the conventional TRFIA and chemiluminescence immunoassay (CLIA), the correlation coefficients of the developed immunoassay were 0.985 and 0.975, respectively. These results showed good correlation and confirmed that our method is feasible and could be used for the clinical determination of CEA (or other tumor antigens) in human serum.     

A Direct Competitive Homogeneous Immunoassay for Progesterone – the Redox Quenching Immunoassay

A direct competitive amperometric immunoassay format for the detection of haptens and proteins was developed. The method is based on the quenching of electroactivity of ferrocenium, which is coupled to the antigen and used as the primary reporter, upon binding to a monoclonal anti‐ferrocenium antibody, which is coupled to the detection antibody and used as a secondary reporter. A separation‐free progesterone immunoassay with a lower detection limit of 1 ng mL^?1 (3.18 nmol L^?1) in 1 : 2 diluted blood serum was realised by combining two bifunctional conjugates, a ferrocenium‐PEG‐progesterone tracer and a bioconjugate of one anti‐progesterone and one anti‐ferrocenium antibody. The immune complex is formed within 30 s upon addition of progesterone, resulting in a total analysis time of 1.5 min.