An electrochemical metalloimmunoassay based on a colloidal gold label

A novel, sensitive electrochemical immunoassay has been developed using a colloidal gold label that, after oxidative gold metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a single-use carbon-based screen-printed electrode (SPE). The use of disposable electrodes allows for simplified measurement in 35 microL of solution. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) and a concentration as low as 3 x 10(-12) M was determined, which is competitive with colorimetric ELISA or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is related to the sensitive ASV determination of gold(III) at a SPE (detection limit of 5 x 10(-9) M) and to the release of a large number of gold(III) ions from each gold particle anchored on the immunocomplex (e.g., 1.7 x 10(5) gold atoms are theoretically contained in a 18-nm spherical gold particle).     

Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using signal DNA probe modified with Au and CuS nanoparticles

A novel and sensitive flow injection chemiluminescence assay for sequence-specific DNA detection based on signal amplification with nanoparticles (NPs) is reported in the present work. The "sandwich-type" DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe labeled with CuS NPs and Au NPs on the 3'- and 5'-terminus, respectively. The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu(2+) after the cupric ions were dissolved from the hybrids. We demonstrated that the incorporation of Au NPs in this sensor design significantly enhanced the sensitivity and the selectivity because a single Au NP can be loaded with hundreds of signal DNA probe strands, which were modified with CuS NPs. The ratios of Au NPs, signal DNA probes, and CuS NPs modified on the gold electrode were approximately 1/101/103. A preconcentration process of cupric ions performed by anodic stripping voltammetry technology further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar target DNA and exhibited excellent selectivity against two-base mismatched DNA. Under the optimum conditions, the CL intensity was increased with the increase of the concentration of target DNA in the range of 2.0 x 10(-14)-2.0 x 10(-12) M. A detection limit of 4.8 x 10(-15) M target DNA was achieved.     

Magnetic bead-sensing-platform-based chemiluminescence resonance energy transfer and its immunoassay application

A competitive immunoassay based on chemiluminescence resonance energy transfer (CRET) on the magnetic beads (MBs) is developed for the detection of human immunoglobulin G (IgG). In this protocol, carboxyl-modified MBs were conjugated with horseradish peroxidase (HRP)-labeled goat antihuman IgG (HRP-anti-IgG) and incubated with a limited amount of fluorescein isothiocyanate (FITC)-labeled human IgG to immobilize the antibody-antigen immune complex on the surface of the MBs, which was further incubated with the target analyte (human IgG) for competitive immunoreaction and separated magnetically to remove the supernatant. The chemiluminescence (CL) buffer (containing luminol and H(2)O(2)) was then added, and the CRET from donor luminol to acceptor FITC in the immunocomplex on the surface of MBs occured immediately. The present protocol was evaluated for the competitive immunoassay of human IgG, and a linear relationship between CL intensity ratio (R = I(425)/I(525)) and human IgG concentration in the range of 0.2-4.0 nM was obtained with a correlation coefficient of 0.9965. The regression equation was expressed as R = 1.9871C + 2.4616, and a detection limit of 2.9 × 10(-11) M was obtained. The present method was successfully applied for the detection of IgG in human serum. The results indicate that the present protocol is quite promising for the application of CRET in immunoassays. It could also be developed for detection of other antigen-antibody immune complexes by using the corresponding antigens and respective antibodies.     

A renewable electrochemical magnetic immunosensor based on gold nanoparticle labels

A particle-based renewable electrochemical magnetic immunosensor was developed by using magnetic beads and gold nanoparticle labels. Anti-IgG antibody-modified magnetic beads were attached to a renewable carbon paste transducer surface by magnet that was fixed inside the sensor. Gold nanoparticle labels were capsulated to the surface of magnetic beads by sandwich immunoassay. Highly sensitive electrochemical stripping analysis offers a simple and fast method to quantify the capatured gold nanoparticle tracers and avoid the use of an enzyme label and substrate. The stripping signal of gold nanoparticles is related to the concentration of target IgG in the sample solution. A transmission electron microscopy image shows that the gold nanoparticles were successfully capsulated to the surface of magnetic beads through sandwich immunoreaction events. The parameters of immunoassay, including the loading of magnetic beads, the amount of gold nanoparticle conjugate, and the immunoreaction time, were optimized. The detection limit of 0.02 microg ml(-1) of IgG was obtained under optimum experimental conditions. Such particle-based electrochemical magnetic immunosensors could be readily used for simultaneous parallel detection of multiple proteins by using multiple inorganic metal nanoparticle tracers and are expected to open new opportunities for disease diagnostics and biosecurity.     

Metal nanoparticle-based electrochemical stripping potentiometric detection of DNA hybridization

A new nanoparticle-based electrical detection of DNA hybridization, based on electrochemical stripping detection of the colloidal gold tag, is described. In this protocol, the hybridization of a target oligonucleotide to magnetic bead-linked oligonucleotide probes is followed by binding of the streptavidin-coated metal nanoparticles to the captured DNA, dissolution of the nanometer-sized gold tag, and potentiometric stripping measurements of the dissolved metal tag at single-use thick-film carbon electrodes. An advanced magnetic processing technique is used to isolate the DNA duplex and to provide low-volume mixing. The influence of relevant experimental variables, including the amounts of the gold nanoparticles and the magnetic beads, the duration of the hybridization and gold dissolution steps, and the parameters of the potentiometric stripping operation upon the hybridization signal, is examined and optimized. Transmission electron microscopy micrographs indicate that the hybridization event leads to the bridging of the gold nanoparticles to the magnetic beads. Further signal amplification, and lowering of the detection limits to the nanomolar and picomolar domains, are achieved by precipitating gold or silver, respectively, onto the colloidal gold label. The new electrochemical stripping metallogenomagnetic protocol couples the inherent signal amplification of stripping metal analysis with discrimination against nonhybridized DNA, the use of microliter sample volumes, and disposable transducers and, hence, offers great promise for decentralized genetic testing.     

Sequential determination of two proteins by temperature-triggered homogeneous chemiluminescent immunoassay

A novel protocol for performing a sequential dual-protein immunoassay, based on a temperature-triggered separation/mixing process and HRP-catalyzed chemiluminescence (CL) detection, is described. In contrast to current multilabel-based detection techniques, a single HRP label is employed in this proposed method. Herein we introduce poly(N-isopropylacrylamide) (PNIP) and magnetic beads as bimolecular immobilizing carriers to separate different targets by taking advantage of thermal response, as demonstrated by sequential detection of human IgG and IgA. PNIP is known to aggregate and precipitate out of water when the temperature is raised above the lower critical solution temperature (LCST) of 31 degrees C; thus, it can be separated from supernatant by centrifugation. Besides, magnetic beads can be separated from PNIP by magnetic force as the temperature is lower than LCST. A homogeneous noncompetitive ELISA was employed, formed by primary antibodies immobilized onto the surface of magnetic beads and PNIP, antigen as IgG and IgA in the sample, and HRP-labeled second antibodies. Moreover, highly sensitive CL detection of HRP was applied, and the detection limits of IgG and IgA were as low as 2.0 and 1.5 ng/mL, respectively. Within the calibrated amount, the protocol had excellent precision within 11% for each target and was comparable in performance to commercial single-analyte ELISAs. Furthermore, the proposed method has been successfully applied to the determination of dual analyte in real samples without cross-reaction, and a good correlation was achieved after comparison with the conventional assay for IgG and IgA in 40 human serum samples.     

Sub-femtomolar electrochemical detection of DNA hybridization based on latex/gold nanoparticle-assisted signal amplification

We report a relatively simple electrostatic method for modifying submicrometer-size latex spheres with gold nanoparticles (AuNPs) based on layer-by-layer modification of the latex by polyelectrolytes. The AuNP coverages for 343- and 501-nm-diameter spheres were 4.0 x 10 (10) +/- 1.3 x 10 (10) and 8.2 x 10 (10) +/- 2.7 x 10 (10) particles cm (-2), respectively, which is an increase of 1 order of magnitude on the previously reported coverage at latex-AuNPs using streptavidin-biotin binding (Kawde, A.N.; Wang, J. Electroanalysis 2004, 16, 101-107). Due to the fact that the AuNPs used here are also of a larger size (mean diameter 15.5 +/- 1.6 nm, cf. 5 nm), this represents an increase of 2 orders of magnitude in the number of Au atoms delivered per sphere. The spheres were attached to DNA probes specific to E. coli and used to detect probe hybridization by dissolution of the AuNPs, followed by measurement of Au (3+) ions by anodic stripping voltammetry (ASV). Use of differential pulse voltammetry for the stripping step, along with optimization of the ASV conditions, enabled a detection limit of 0.5 fM, which is, to the best of our knowledge, equal or lower than previous voltammetric nanoparticle methods for detection of DNA hybridization.     

Determination of carcinoembryonic antigen in human sera by integrated bead-bed immunoassay in a microchip for cancer diagnosis

A bead-bed immunoassay system was structured on a microchip and applied to determine carcinoembryonic antigen (CEA), which is a commonly used marker of colon cancer. Polystyrene beads precoated with anti-CEA antibody were introduced into a microchannel, and then a serum sample containing CEA, the first antibody, and the second antibody conjugated with colloidal gold were reacted successively. The resulting antigen-antibodies complex, fixed on the bead surface, was detected using a thermal lens microscope (TLM). A highly selective and sensitive determination of an ultratrace amount of CEA in human sera was made possible by a sandwich immunoassay system that needs three antibodies for an assay. A detection limit dozens of times lower than the conventional ELISA was achieved. Moreover, when serum samples for 13 patients were assayed with this system, there was a high correlation (r = 0.917) with the conventional ELISA. The integration reduced the time necessary for the antigen-antibody reaction to approximately 1%, thus shortening the overall analysis time from 45 h to 35 min. Moreover, troublesome operations required for conventional heterogeneous immunoassays could be much simplified. This microchip-based diagnosis system is the first microchip-based system that is practically useful for clinical diagnoses with short analysis time, high sensitivity, and easy procedures.     

Gold-coated magnetic particles for solid-phase immunoassays: enhancing immobilized antibody binding efficiency and analytical performance

The preparation and characterization of gold-coated magnetic particles are described for use as more efficient solid-phase materials in immunoassay development. A thin gold coating on commercial tosylated magnetic polystyrene particles (4.5 microm) is achieved via an electroless plating method involving initial reaction of the particles with Sn(II), followed by redox deposition of Ag0, that serves as a catalytic site for the subsequent reduction of Na3Au(SO3)2 in the presence of formaldehyde to yield the adhered gold layer. Scanning electron microscopy, energy-dispersive X-ray analysis, and X-ray photoelectron spectroscopy indicate the presence of the desired Au0 outer layer. To characterize the improved yield of antibody binding sites on such gold-coated phases, the modified particles are reacted with the free thiols of Fab' fragments of an anti-alkaline phosphatase (ALP) antibody to orient all the antigenic binding sites in a favorable direction. After equilibration with ALP, the amount of ALP bound to the surface of such particles is nearly 2.5-fold greater than on non-gold-coated particles possessing the same amount of immobilized anti-ALP Fab', but oriented randomly on the surface. The new gold-coated magnetic particles are further used as a solid phase for developing a sandwich-type enzyme immunoassay to detect C-reactive protein (CRP) using horseradish peroxidase as the enzyme label. The gold-coated magnetic particles with anti-CRP monoclonal Fab' reagents provide assays with enhanced assay slope (1.8-fold), lower nonspecific adsorption, and a detection limit improvement of nearly 10-fold (0.14 vs 1.9 ng/mL) compared to the same Fab' anti-CRP immobilized on the initial tosylated polystyrene magnetic particles. The improved assay performance is attributed to the more favorable binding orientation of the self-assembled monolayer of Fab' fragments on the gold-coated particles compared to the random orientation on the non-gold-coated surfaces.     

Colloidal gold-modified optical fiber for chemical and biochemical sensing

A novel class of fiber-optic evanescent-wave sensor was constructed on the basis of modification of the unclad portion of an optical fiber with self-assembled gold colloids. The optical properties and, hence, the attenuated total reflection spectrum of self-assembled gold colloids on the optical fiber changes with different refractive index of the environment near the colloidal gold surface. With sucrose solutions of increasing refractive index, the sensor response decreases linearly. The colloidal gold surface was also functionalized with glycine, succinic acid, or biotin to enhance the selectivity of the sensor. Results show that the sensor response decreases linearly with increasing concentration of each analyte. When the colloidal gold surface was functionalized with biotin, the detection limit of the sensor for streptavidin was 9.8 x 10(-11) M. Using this approach, we demonstrate proof-of-concept of a class of refractive index sensor that is sensitive to the refractive index of the environment near the colloidal gold surface and, hence, is suitable for label-free detection of molecular or biomolecular binding at the surface of gold colloids.     

Sensitive chemiluminescence immunoassay by capillary electrophoresis with gold nanoparticles

This technical note describes a new chemiluminescence immunoassay hyphenated to capillary electrophoresis (CE-based CL-IA) with gold nanoparticles (AuNPs) technique for biological molecules determination. AuNPs were used as a protein label reagent in the light of its excellent catalytic effect to the CL reaction of luminol and hydrogen peroxide. AuNPs conjugate with antibody (Ab) to form tagged antibody (Ab*), and then Ab* link to antigen (Ag) to produce an Ab*-Ag complex by a noncompetitive immunoreaction. The mixture of the excess Ab* and the Ab*-Ag complex was baseline separated and detected within 5 min under the optimized conditions. This new protocol was evaluated with human immunoglobulin G (IgG) as the target molecule. The calibration curve of IgG was in the range of 0.008-5 μg/mL with a correlation coefficient of 0.995. The detection limit (S/N = 3) of IgG was 1.14 × 10(-3) μg/mL (7.1 pmol/L, 0.39 amol). The proposed AuNPs enhanced CE-based CL-IA method was successfully applied for the quantification of IgG in human sera from patients. It proves that the present method could be developed into a new and sensitive biochemical analysis technique.     

Deposition method for preparing SERS-active gold nanoparticle substrates

Surface-enhanced Raman scattering or SERS, discovered some 20 years ago, has recently become a promising tool for routine biofluid assays in a clinical setting. Many attempts have been made to produce cheap and reproducible SERS-active substrates. In this study, we report on the fabrication of SERS-active substrates through the convective assembly of gold (Au) particles on electrostatically charged glass slides. We show that, by a proper control of the initial particle concentration in an evaporating Au suspension droplet, it is possible to obtain a closely packed colloidal film capable of generating SERS activity. Finally, AFM and SERS measurements of the resulting films reveal comparability in performance with previous silane-immobilized Au colloidal films. The minimum electromagnetic enhancement factor of our films is estimated to be about 2 x 10(4).     

Electrochemical coding for multiplexed immunoassays of proteins

An electrochemical immunoassay protocol for the simultaneous measurements of proteins, based on the use of different inorganic nanocrystal tracers is described. The multiprotein electrical detection capability is coupled to the amplification feature of electrochemical stripping transduction (to yield fmol detection limits) and with an efficient magnetic separation (to minimize nonspecific adsorption effects). The multianalyte electrical sandwich immunoassay involves a dual binding event, based on antibodies linked to the nanocrystal tags and magnetic beads. Carbamate linkage is used for conjugating the hydroxyl-terminated nanocrystals with the secondary antibodies. Each biorecognition event yields a distinct voltammetric peak, whose position and size reflects the identity and level, respectively, of the corresponding antigen. The concept is demonstrated for a simultaneous immunoassay of beta(2)-microglobulin, IgG, bovine serum albumin, and C-reactive protein in connection with ZnS, CdS, PbS, and CuS colloidal crystals, respectively. These nanocrystal labels exhibit similar sensitivity. Such electrochemical coding could be readily multiplexed and scaled up in multiwell microtiter plates to allow simultaneous parallel detection of numerous proteins or samples and is expected to open new opportunities for protein diagnostics and biosecurity.     

Electrochemical immunoassay of carcinoembryonic antigen based on a lead sulfide nanoparticle label

We describe a lead sulfide nanoparticle (PbS NP)-based electrochemical immunoassay to detect a tumor biomarker, carcinoembryonic antigen (CEA). Cubic PbS NPs were prepared and functionalized with thioglycolic acid (TGA), which stabilized the formed NPs and offered carboxyl groups to conjugate with CEA antibodies. PbS NP conjugated with monoclonal CEA antibody was used as a label in an immunorecognition event. After a complete sandwich immunoreaction among the primary CEA antibody (immobilized on the carboxyl-modified magnetic beads), CEA and the PbS-labeled secondary antibody (PbS-anti-CEA), PbS labels were captured to the magnetic-bead (MB) surface through the antibody-antigen immunocomplex. Electrochemical stripping analysis of the captured PbS was used to quantify the concentration of CEA after an acid-dissolution step. The MBs and the magnetic separation platform were used to integrate a facile antibody immobilization with immunoreactions and the isolation of immunocomplexes from reaction solutions in the immunoassay. The voltammetric response is highly linear over the range of 1-50?ng?ml(-1) CEA, and the limit of detection is estimated to be 0.5?ng?ml(-1). The performance of this nanoparticle-based electrochemical immunoassay was successfully evaluated with human serum spiked with CEA, indicating that this convenient and sensitive technique offers great promise for rapid, simple and cost-effective analysis of tumor biomarkers in biological fluids.     

Colloidal domain lithography for regularly arranged artificial magnetic out-of-plane monodomains in Au/Co/Au layers

Regularly arranged magnetic out-of-plane patterns in continuous and flat films are promising for applications in data storage technology (bit patterned media) or transport of individual magnetic particles. Whereas topographic magnetic structures are fabricated by standard lithographical techniques, the fabrication of regularly arranged artificial domains in topographically flat films is difficult, since the free energy minimization determines the existence, shape, and regularity of domains. Here we show that keV He(+) ion bombardment of Au/Co/Au layer systems through a colloidal mask of hexagonally arranged spherical polystyrene beads enables magnetic patterning of regularly arranged cylindrical magnetic monodomains with out-of-plane magnetization embedded in a ferromagnetic matrix with easy-plane anisotropy. This colloidal domain lithography creates artificial domains via periodic lateral anisotropy variations induced by periodic defect density modulations. Magnetization reversal of the layer system observed by magnetic force microscopy shows individual disc switching indicating monodomain states.     

Cell toxicity studies of albumin-based nanosized magnetic beads

The aim of this study was to prepare bovine serum albumin-based beads containing maghemite nanoparticles incorporated via ionic magnetic fluid and to evaluate the cell toxicity of this biocompatible system using the J774-A1 cell line. Transmission electron micrographs obtained from the magnetic fluid sample were used to estimate the average particle diameter around 7.6 nm and diameter dispersion of 0.22. The BSA-based magnetic beads were prepared using the heat protein denaturation route. The nanoparticle concentration in the magnetic fluid sample used for the synthesis of the magnetic beads was in the range of 1.2 x 10(16) to 2.3 x 10(17) particle/ml. The methodology used to investigate the cell toxicity of the magnetic beads was the classical MTT assay. Our observation showed that the toxicity against the J774-A1 cell line depends upon the amount of magnetic material incorporated into the magnetic nanobeads and was found to be 14, 11, 9, 5, and 3% for 2.3 x 10(17), 1.2 x 10(17), 4.6 x 10(16), 2.3 x 10(16), and 1.2 x 10(16) particle/ml, respectively.     

Disposable magnetic DNA sensors for the determination at the attomolar level of a specific enterobacteriaceae family gene

Disposable magnetic DNA sensors using an enzyme-amplified strategy for the specific detection of a gene related to the Enterobacteriaceae bacterial family, based on the coupling of streptavidin-peroxidase to biotinylated lacZ gene target sequences, has been developed. A biotinylated 25-mer capture probe was attached to streptavidin-modified magnetic beads and hybridization with the biotinylated target was allowed to proceed. Then, a streptavidin-peroxidase polymer was attached to the biotinylated target, and the resulting modified magnetic beads were captured by a magnetic field on the surface of tetrathiafulvalene (TTF) modified gold screen-printed electrodes (Au/SPEs). The amperometric response obtained at -0.15 V after the addition of hydrogen peroxide was used to detect the hybridization process. In order to improve the sensitivity of the determination and reduce the assay time, different variables of the assay protocol were optimized. A low detection limit (5.7 fmol) with good stability (RSD = 7.1%, n = 10) was obtained. The DNA nonspecific adsorption at the magnetic beads was negligible, the obtained results thus demonstrating the possibility to detect the hybridization event with great specificity and sensitivity. The developed method was used for the analysis of Escherichia coli DNA fragments (326 bases) in polymerase chain reaction (PCR) amplicons extracted from a cell culture. As low as 2.5 aM asymmetric PCR product could be detected with the developed methodology.     

Electrochemical magnetoimmunosensing strategy for the detection of pesticides residues

A novel electrochemical immunosensing strategy for the detection of atrazine based on magnetic beads is presented. Different coupling strategies for the modification of the magnetic beads with the specific anti-atrazine antibody have been developed. The immunological reaction for the detection of atrazine performed on the magnetic bead is based on a direct competitive assay using a peroxidase (HRP) tracer as the enzymatic label. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magnetosensor made of graphite-epoxy composite, which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate and mediator for the enzyme HRP. The electrochemical approach is also compared with a novel magneto-ELISA based on optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked real orange juice samples. The detection limit for atrazine using the competitive electrochemical magnetoimmunosensing strategy with anti-atrazine-specific antibody covalent coupled with tosyl-activated magnetic beads was found to be 6 x 10(-3) microg L(-1) (0.027 nmol L(-1)). This strategy offers great promise for rapid, simple, cost-effective, and on-site analysis of biological, food, and environmental samples.     

Electrogenerated chemiluminescence. 83. Immunoassay of human C-reactive protein by using Ru(bpy)3(2+)-encapsulated liposomes as labels

Liposomes ( approximately 100-nm diameter) containing Ru(bpy)32+ (bpy = 2,2'-bipyridine) were prepared as an electrogenerated chemiluminescent (ECL) tag for a sandwich-type immunoassay of human C-reactive protein (CRP). Polyclonal human CRP antibodies were introduced onto liposomes and magnetic beads through biotin-streptavidin interaction. The antigen-antibody conjugates formed on addition of a CRP-containing sample were separated from unreacted species magnetically. Addition of 0.1 M tri-n-propylamine and 0.1 M phosphate buffer (pH 7.6) containing 0.1 M NaCl and 1% (v/v) Triton X-100 caused liberation of the Ru(bpy)32+ from the liposome. ECL obtained in this medium showed a detection limit of 100 ng/mL for human CRP with good linearity of ECL intensity versus antigen concentration over the range 100 ng/mL-10 microg/mL.     

Preparation of highly stable oligo(ethylene glycol) derivatives-functionalized gold nanoparticles and their application in LSPR-based detection of PSA/ACT complex

A sandwich immunoassay for PSA/ACT complex detection based on gold nanoparticle aggregation using two probes was developed. The functionalized colloidal gold nanoparticles (AuNPs) showed highly stable not only in the presence of high ionic strength but also in a wide pH range. The functionalized AuNPs were tagged with PSA/ACT complex monoclonal antibody and goat PSA polyclonal antibody and served as the probes to induce aggregation of the colloidal particles. As a result, PSA/ACT complex was detected at concentrations as low as 1 ng/ml. This is the first time that a new aggregation sandwich-immunoassay technique using two gold probes has been used, and the results are generally applicable to other LSPR-based immunoassays.