Competitive quenching fluorescence immunoassay for chlorophenols based on laser-induced fluorescence detection in microdroplets

An improved biomonitoring system for the analysis of 2,4,6-trichlorophenol (TCP) in urine samples has been developed. The principle of the biosensor device is the detection of laser-induced fluorescence (LIF) in single microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay occurs in microdroplets (d = 58,4 microm) produced by a piezoelectric generator system with 10-microm-diameter orifice. A continuous Ar ion laser (488 nm) excites the fluorescent tracer; its fluorescence is detected by a spectrometer attached to a 512 x 512 cooled, charge-coupled device camera. Fluorescence is quenched by specific binding of TCP polyclonal antibodies to the fluorescent tracer (hapten A-fluorescein); the quenching effect is diminished by the presence of the analyte. Thus, an increase in the signal is produced in a positive dose-dependent manner when TCP is present in the sample. In 10 mM PBS buffer, the IC50 of the LIF-microdroplet QFIA is 0.45 microg L(-1) reaching a LOD of 0.04 microg L(-1). The QFIA with the same reagents performed in microtiter plate format achieved a LOD of 0.36 microg L(-1) in buffer solution. Performance in human urine was similar to that observed in the buffer. A LOD of 1.6 ,g L(-1), with a dynamic range between 4 and 149.5 microg L(-1) in urine, was obtained without any sample treatment other than dilution with the assay buffer. The detectability achieved is sufficient for occupational exposure risk assessment.     

Microdialysis sampling combined with electron spin resonance for superoxide radical detection in microliter samples

Quantitation of superoxide radical (O2.-) production at the site of radical generation remains challenging. Microdialysis sampling is an advantageous tool for sampling from localized environments. It is difficult to combine electron spin resonance (ESR) spin traps with microdialysis because O2.- adducts with common nitrone spin traps have shorter half-lives than typical microdialysis collection times. Furthermore, typical dialysate samples (5-15 microL) suffer significant sensitivity loss when diluted for detection in a conventional ESR flat cell (200 microL). To overcome these difficulties, a cyclic hydroxylamine, 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H), which produces a stable nitroxide radical (PP.) product upon reaction with O2.- was employed. Capillary cells (1.4 microL effective volume) coupled with a loop-gap resonator were utilized to measure PP. in microliter microdialysis samples (LOD 0.36 pmol). A xanthine/xanthine oxidase (X/XO) model system provided sustained O2.- production. When PP-H was included in the X/XO medium external to the microdialysis probe, a relative recovery of 22.1 +/- 1.1 and 57.2 +/- 5.7% for PP. was achieved at perfusion fluid flow rates of 0.5 and 1.0 microL/min, respectively. The respiratory burst in interferon-gamma and zymosan-stimulated RAW 264.7 macrophages was also investigated.     

Ligand and protein fishing with heat shock protein 90 coated magnetic beads

Heat shock protein 90alpha (Hsp90alpha) is a molecular chaperone that has been targeted for the development of new anticancer therapies. To date, co-immunoprecipitation (IP) has been primarily used to identify novel client proteins. We now report an alternative approach in which Hsp90alpha has been immobilized onto the surface of silica-based magnetic beads. The beads were used to isolate known Hsp90alpha ligands from a mixture containing ligands and nonligands. In addition, they were also used to isolated proteins from a mixture of proteins, as well as a cellular extract. The results indicate that the Hsp90alpha coated magnetic beads can be used to "fish" from complex chemical and biological mixtures for new lead drug candidates and client proteins.     

Magnetic beads based immunoaffinity capillary electrophoresis of total serum IgE with laser-induced fluorescence detection

A magnetic beads based immunoaffinity capillary electrophoresis method for total Immunoglobulin E quantification in serum has been developed. The method combines speed, automation ability, and minimal sample consumption. Only 1 microL of serum is required while the whole immunoaffinity capillary electrophoresis method is performed in less than 50 min. The concomitant use of online immunocapture, transient isotachophoresis, and laser-induced fluorescence detection provides a sensitivity in the low picomolar range and a highly linear fluorescence response over 4 orders of magnitude (IgE concentration ranging from 2.4 to 2400 ng/mL). After validation with a reference material, the method has been successfully applied to the quantification of total IgEs in patient sera. The results compared well with classical ImmunoCap data.     

Rotating sample system: an equivalent of a rotating electrode for microliter samples

Voltammetric experiments in sample droplets of 20 microL volume were carried out with gold ring electrodes microfabricated on Pyrex substrates. The droplet studied was centered and kept in place by a hydrophobic ring deposited on the substrate around the ring electrode, which also ensured that each sample assume a semispherical shape. A small hole in the center of the substrate filled with an agar gel membrane served as a junction. A mild jet of humidified nitrogen gas directed tangentially at the droplet caused it to rotate at a high rate. Cyclic voltammetry and constant-potential electrolysis of potassium ferricyanide were used to characterize this rotating sample system and to calculate diffusion layer thickness. The results clearly demonstrate that rotating of a semispherical microsample placed above a stationary ring electrode with a mild gas jet can be as effective as a rotating electrode system. As a practical example, voltammetric stripping analysis of 20-120 pmol mercuric nitrate samples was performed with a calibration of good linearity (r2 = 0.988) and a time constant for exhaustive electrolysis of approximately 2 min.     

Tailored magnetic nanoparticles for direct and sensitive detection of biomolecules in biological samples

We developed nanoparticles with tailored magnetic properties for direct and sensitive detection of biomolecules in biological samples in a single step. Thermally blocked nanoparticles obtained by thermal hydrolysis, functionalized with specific ligands, are mixed with sample solutions, and the variation of the magnetic relaxation due to surface binding is used to detect the presence of biomolecules. The binding significantly increases the hydrodynamic volume of nanoparticles, thus changing their Brownian relaxation frequency which is measured by a specifically developed AC susceptometer. The system was tested for the presence of Brucella antibodies, a dangerous pathogen causing brucellosis with severe effects both on humans and animals, in serum samples from infected cows and the surface of the nanoparticles was functionalized with lipopolysaccharides (LPS) from Brucella abortus. The hydrodynamic volume of LPS-functionalized particles increased by 25-35% as a result of the binding of the antibodies, measured by changes in the susceptibility in an alternating magnetic field. The method has shown high sensitivity, with detection limit of 0.05 microg x mL(-1) of antibody in the biological samples without any pretreatment. This magnetic-based assay is very sensitive, cost-efficient, and versatile, giving a direct indication whether the animal is infected or not, making it suitable for point-of-care applications. The functionalization of tailored magnetic nanoparticles can be modified to suit numerous homogeneous assays for a wide range of applications.     

Monosize magnetic hydrophobic beads for lysozyme purification under magnetic field

Monosize and magnetic poly(glycidyl methacrylate-N-methacryloyl-(l)-tryptophan) [mPGMATrp] beads (1.6 µm in diameter) were used for hydrophobic affinity capture of lysozyme from chicken egg-white. N-methacryloyl-(l)-tryptophan (MATrp), which gives hydrophobicity to the resulting polymer, was synthesized by reacting methacryloyl chloride and l-tryptophan methyl ester then characterized by Nuclear Magnetic Resonance (NMR). mPGMATrp beads were produced by dispersion polymerization in the presence of magnetite nano-powder. mPGMATrp beads were characterized by means of swelling studies, elemental analysis, Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM). Lysozyme adsorption experiments were performed under different experimental conditions (i.e., lysozyme concentration, temperature, and ionic strength) in magnetically stabilized fluidized bed system, (MSFB). Maximum adsorption capacity was 263.9 mg/g. It was observed that mPGMATrp beads can be used without significant loss in lysozyme adsorption capacity after 25 adsorption-elution cycle.     

Encoded silica colloidal crystal beads as supports for potential multiplex immunoassay

We developed a new kind of suspension array for multiplexed immunoassays using silica colloidal crystal beads (SCCBs) as coding carriers. The monodisperse and size-controlled SCCBs were fabricated by a microfluidic device. Calcination was employed to improve the mechanical stability and lower the fluorescent background of the SCCBs. Immobilization of protein molecules on the surface of the SCCBs through chemical bonds was studied, and the modification condition was optimized to increase the detection sensitivity. Results indicated that the SCCBs as supports were more sensitive (0.92 ng/mL IgG) than the glass beads (27 ng/mL IgG) and the planar carriers (140 ng/mL IgG). A multiplex immunoassay showed the flexibility and feasibility of SCCBs array in clinical applications.     

An immunoassay for small analytes with theoretical detection limits

A flow-based immunoassay that uses microspheres as the solid phase accomplished the theoretical limit of detectability achievable with the antibody. An equilibrated mixture of anti-estriol monoclonal antibody and estriol was briefly exposed to a bead pack containing immobilized estriol in a flow cell. A small portion of free antibody was separated rapidly from the mixture by binding it to immobilized hormone, but the antibody-hormone complex was kinetically excluded from binding. This rapid separation prevented shift in the equilibrium of the liquid phase binding. Signals were generated by labeling the separated antibodies on the beads with a Cy5-conjugated antispecies secondary antibody. By labeling after the separation step, perturbing the liquid-phase or solid-phase binding was prevented. This assay allowed the reduction of the concentration of primary antibody by continuously accumulating free antibody onto the beads prior to quantification and, thus, offered ideal conditions to achieve theoretical limits of detectability. The optimum achievable dynamic range of this immunoassay was 4-300 pM. Because the proportion of free anti-estriol antibody in the mixture was controlled by the Kd of the antibody-estriol interaction, when the concentration of the antibody was below the Kd, the smallest detectable estriol concentration approached the theoretical limit of detectability achievable with this antibody.     

Microfabricated on-chip-type electrochemical flow immunoassay system for the detection of histamine released in whole blood samples

This paper describes an on-chip-type electrochemical flow immunoassay system with a multichanneled matrix column. The multichanneled matrix column was functionally coated with cation-exchange resin and used for separation of proteins. Antihistamine immunoglobulin G (IgG) antibody conjugated with ferrocenemonocarboxylic acid (Fc) was also prepared and used as a novel analytical reagent. Antibody-antigen complexes were separated from free Fc-conjugated IgG antibody (Fc-IgG) on the basis of differences in isoelectric point (pI) using the multichanneled matrix column coated with cation-exchange resin. The assay yields a good relationship between current and histamine concentration in the range of 200-2000 ng/mL. This simple technique enables the assay of histamine released in whole blood within 2 min. Furthermore, a good correlation was found between the response of the electrochemical immunoassay described in this paper and the conventional RIA (radioimmunoassay). This on-chip-type electrochemical flow immunoassay requires only minute quantities of whole blood samples and generates highly reproducible results.     

Capillary electrophoresis with lamp-based wavelength-resolved fluorescence detection for the probing of protein conformational changes

Native protein fluorescence spectra encompass information on protein conformation. In this study, capillary electrophoresis (CE) combined with lamp-based wavelength-resolved fluorescence detection (wrFlu) is presented as a novel tool for the analysis of protein mixtures and the monitoring of protein unfolding. The CE-wrFlu system provides three-dimensional data (time, emission wavelength, intensity) from which electropherograms and accurate emission spectra of separated proteins can be extracted. For model proteins, linear detector responses (peak height vs concentration) were obtained (R(2) > 0.96) with detection limits (LODs) in the 6-32 nM range. The minimum protein concentration required for precise determination of the maximum emission wavelength by CE-wrFlu was about 15 times the LOD. Unfolding of various model proteins was induced by protein incubation and analysis in background electrolyte (BGE) containing 7.0 M urea. CE-wrFlu of the unfolded species revealed peaks with clear red-shifted spectra, which adequately corresponded to reference spectra obtained on a standard spectrophotometer. Moreover, unfolded proteins showed a significant decrease in effective electrophoretic mobility (after correction for BGE viscosity) due to the increase of their molecular hydrodynamic radii. It is concluded that the CE-wrFlu system provides two independent indicators for changes in protein folding and will allow the simultaneous assessment of protein purity and conformation.     

Evaluation of 2-methacryloyloxyethyl phosphorylcholine polymeric nanoparticle for immunoassay of C-reactive protein detection

To prepare novel 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymeric nanoparticle (MPC-PNP), water-soluble amphiphilic phospholipid polymer, poly [MPC-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP) (PMBN)], which has active ester groups for bioconjugation on the side chains, was synthesized. MPC-PNP was prepared by a solvent evaporation technique where the poly(l-lactic acid) was used as core and PMBN was applied as an emulsifier and a surface modifier under systematical design of well-arranged phospholipids polar groups in its surface. Characteristics for MPC-PNP were thoroughly investigated with dynamic light scattering, electrophoresis light scattering, X-ray photoelectron spectroscopy, and field emission scanning electron microscopy measurements. Through a protein adsorption test, the phosphorylcholine group on the surface of MPC-PNPs, which had their active ester groups substituted by glycine, were shown to suppress the nonspecific adsorption of bovine serum albumin. These particles were used for C-reactive protein (CRP) detection, where anti-CRP monoclonal antibodies were immobilized on the MPC-PNP using the active ester group, while the remaining active ester groups were thoroughly reacted with glycine. The detection limit about serum-free CRP in the calibration curve was shown to extend from 0.01 to 10 mg/dL when anti-CRP antibody immobilized MPC-PNP was used for serum-free CRP detection. This compares favorably with measurement using polystyrene nanoparticles that were shown to detect from 0.1 to 10 mg/dL by an immunoagglutination technique. Also, for the detection of CRP in serum, MPC-PNP was shown to give the same calibration curve explained by the efficient suppression of nonspecific binding. Furthermore, denaturation of immobilizing anti-CRP antibody on the MPC-PNP hardly occurred despite increasing the temperature. It is concluded that MPC-PNP is unique due to the design of its interfacial properties, also it will perform well in a diagnostic immunoassay because of its optimized material properties.     

Detection of C-reactive protein based on immunoassay using antibody-conjugated magnetic nanoparticles

We report a detection method for C-reactive protein (CRP) based on competitive immunoassay using magnetic nanoparticles under magnetic fields. Functional magnetic nanoparticles were prepared and conjugated with anti-CRP for immunoassay. Magnetic nanoparticles labeled with anti-CRP were flowed through a separation channel to form depositions for selective capture of CRP under magnetic fields. Free CRP and a fixed number of CRP-labeled particles were used to compete for a limited number of anti-CRP binding sites on the magnetic nanoparticles. The deposited percentages of CRP-labeled particles at various concentrations of free CRP were determined and used as a reference plot. The determination of CRP in the unknown sample was deduced from the reference plot using the deposited percentages. The running time was less than 10 min. The CRP concentration of serum sample was linearly over the range of 1.2-310 microg/mL for deposited percentages of CRP-labeled particles. The detection limit of this method was 0.12 microg/mL which was approximately 8-fold lower than the typical clinical cutoff concentration (1 microug/mL). This method can provide a fast, simple, and sensitive way for protein detection based on competitive immunoassay using magnetic nanoparticles under magnetic fields.     

Enzymatically amplified time-resolved fluorescence immunoassay with terbium chelates.

We report an ultrasensitive, enzymatically amplified, time-resolved fluorescence immunoassay with a terbium chelate as the detectable moiety. In this immunoassay, the primary label is the enzyme alkaline phosphatase (ALP). ALP cleaves phosphate out of a fluorogenic substrate, 5-fluorosalicyl phosphate, to produce 5-fluorosalicylic acid (FSA). 5-Fluorosalicylic acid can then form a highly fluorescent ternary complex of the form FSA-Tb(3+)-EDTA, which can be quantified by measuring the Tb3+ fluorescence in a time-resolved mode. In this assay, exceptional sensitivity is achieved because of the enzymatic amplification introduced by ALP and the quantification by laser-induced microsecond time-resolved fluorometry. Time-resolved fluorometry is applicable because of the long fluorescence lifetime of the Tb3+ complexes. It is shown that in a model AFP assay 10(6) or 1.5 x 10(5) molecules can be detected (final assay volume, 100 microL) by using monoclonal or polyclonal detection antibodies, respectively. The assay demonstrates excellent precision (approximately 4%), and it seems to be highly suited for automated, sensitive, and rapid immunoassays.     

Aptamer-based detection of plasma proteins by an electrochemical assay coupled to magnetic beads

The DNA thrombin aptamer has been extensively investigated, and the coupling of this aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. The goal of this work was to design an aptamer-based sandwich assay with electrochemical detection for thrombin analysis in complex matrixes, using a simple target capturing step by aptamer-functionalized magnetic beads. The conditions for the aptamer immobilization and for the protein binding have been first optimized by surface plasmon resonance, and then transferred to the electrochemical-based assay performed onto screen-printed electrodes. The assay was then applied to the analysis of thrombin in buffer, spiked serum, and plasma and high sensitivity and specificity were found. Moreover, thrombin was generated in situ in plasma by the conversion of its precursor prothrombin, and the formation of thrombin was followed at different times. The concentrations detected by the electrochemical assay were in agreement with a simulation software that mimics the formation of thrombin over time (thrombogram). The proposed work demonstrates that the high specificity of aptamers together with the use of magnetic beads are the key features for aptamer-based analysis in complex matrixes, opening the possibility of a real application to diagnostics or medical investigation.     

Single ultrasmall Mn2+-doped NaNdF4 nanocrystals as multimodal nanoprobes for magnetic resonance and second near-infrared fluorescence imaging

Nano Research - Multimodal imaging probes have attracted wide attention and have potential to diagnose diseases accurately because of the complementary advantages of multiple imaging modalities....     

Sampling-resolution strategy for one-way multiplexed immunoassay with sequential chemiluminescent detection

A sampling-resolution strategy was designed by using a multichannel flow-injection technique for rapid one-way multiplexed immunoassay. The multichannel sampling combined an incubation process in batch with a simple magnetic collection. After incubation for 6 min, free enzyme conjugates could be separated from the formed enzyme-labeled sandwich immunocomplexes with a magnet for simultaneously stopping the immunoreaction. With the help of two valves, the chemiluminescence (CL) substrate was then sequentially mixed with the immunocomplexes in different channels for sequentially triggering the CL reaction in a time interval of 15 s. After triggering for 5 min, the mixtures were sequentially injected into a one-way detection channel in the same interval to form the analyte zones separated with HCl solution and washing buffer for avoiding cross talk. With the use of alpha-fetoprotein, carcinoma antigen 125, carcinoma antigen 199, and carcinoembryonic antigen as proof-of-principle analytes, the sequential CL detection could be completed within 1 min with the linear calibration ranges of 1.0-80 microg/L, 1.0-60 kU/L, 1.0-120 kU/L, and 1.0-100 microg/L, respectively. This system showed acceptable detection and fabrication reproducibility, and the assay results were in acceptable agreement with those from single-analyte tests of clinical sera, showing a promise of automated clinical application.