The use of CdTe quantum dot fluorescent microspheres in fluoro-immunoassays and a microfluidic chip system.

Polystyrene fluorescent microspheres prepared by deposition of CdTe quantum dots (QDs) are used in an immunoassay in this study. CdTe QDs/polyelectrolyte multilayers on the surface of polystyrene microspheres have been formed by layer-by-layer self-assembly via electrostatic interactions. As a model antigen, rabbit IgG has been bound to the outermost layer of the fluorescent microspheres. The immunoreaction between fluorescent microspheres/rabbit IgG and the corresponding antibody was confirmed by change of the fluorescence spectrum and competitive immunoassay. This approach allowed detection of the antigen (rabbit IgG) in the range 1-500 mg/L, based on the change in the fluorescence intensity of the reporter (fluorescent microspheres/rabbit IgG). A novel microfluidic chip device with a laser-induced fluorescence system was established and used for the detection of fluorescent microspheres in this study.     

Investigating specific antigen/antibody binding with the atomic force microscope

The aim of this work is to detect immune complexes without any kind of labelling of each of the immunological species, with a view to create a very sensitive biosensor. This is achieved by using the atomic force microscopy. We have proceeded by imaging the antibody (anti-rabbit IgG) or anti-rabbit IgG moieties adsorbed onto mica surface, before and after incubation of two kinds of antigens: a specific (rabbit IgG) and a non-specific one (sheep IgG). The analysis using the height histograms reveals many interesting features. We propose a general framework for interpreting these analysis, which enables the discrimination between specific and non-specific complexes.     

Fluorimetric urease inhibition assay on a multilayer microfluidic chip with immunoaffinity immobilized enzyme reactors

We fabricated a three-layer polydimethylsiloxane (PDMS)-based microfluidic chip for realizing urease inhibition assay with sensitive fluorescence detection. Procedures such as sample prehandling, enzyme reaction, reagent mixing, fluorescence derivatization, and detection can be readily carried out. Urease reactors were prepared by adsorption of rabbit immunoglobulin G (IgG) and immunoreaction with urease-conjugated goat anti-rabbit IgG. Acetohydroxamic acid (AHA) as a competitive inhibitor of urease was tested on the chip. Microfluidically generated gradient concentrations of AHA with substrate (urea) were loaded into urease reactors. After incubation, the produced ammonia was transported out of reactors and then reacted with o-phthalaldehyde (OPA) to generate fluorescent products. Urease inhibition was indicated by a decrease in fluorescence signal detected by microplate reader. The IC₅₀ value of AHA was determined and showed good agreement with that obtained in microplate. The presented device combines several steps of the analytical process with advantages of low reagent consumption, reduced analysis time, and ease of manipulation. This microfluidic approach can be extended to the screening of inhibitory compounds in drug discovery.     

Fab fragments imprinted SPR biosensor for real-time human immunoglobulin G detection

F(ab) fragments imprinted surface plasmon resonance (SPR) chip was prepared for the real-time detection of human immunoglobulin G (IgG). In order to attach polymerization precursor on SPR chip, the SPR chip surface was modified with allyl mercaptan. F(ab) fragments of the IgG molecules were prepared by papain digestion procedure and collected by fast protein liquid chromatography (FPLC) system using Hi-Trap_r Protein A FF column. The collected F(ab) fragments were complexed with histidine containing specific monomer, N-methacryloyl-l-histidine methyl ester (MAH). Molecular imprinted polymeric nanofilm was prepared on SPR chip in the presence of ethylene glycol dimethacrylate and 2-hydroxyethylmethacrylate. The template molecules, F(ab) fragments, were removed from the polymeric nanofilm using 1M NaCl solution (pH: 7.4, phosphate buffer system). The molecular imprinted SPR chip was characterized by contact angle, atomic force microscopy and Fourier transform infrared spectroscopy. By the real-time IgG detection studies carried out using aqueous IgG solutions in different concentrations, the kinetics and isotherm parameters of the molecular imprinted SPR chip-IgG system were calculated. To show selectivity and specificity of the molecular imprinted SPR chip, competitive kinetic analyses were performed using bovine serum albumin (BSA), IgG, F(ab) and F(c) fragments in singular and competitive manner. As last step, IgG detection studies from human plasma were performed and the measured IgG concentrations were well matched with the results determined by enzyme-linked immunosorbent assay (ELISA). The results obtained with the molecular imprinted SPR chip were well fitted to Langmuir isotherm and the detection limit was found as 56 ng/mL. In the light of the results, we can conclude that the proposed molecular imprinted SPR chip can detect IgG molecules from both aqueous solutions and complex natural samples.     

Detection of dilute antigen in large sample volumes using antibody-coated polyester cloth

Summary A model antibody, goat anti-rabbit IgG antibody, was adsorbed onto a disk of polyester cloth and then fixed into a column apparatus. The macroporosity of the cloth allowed rapid immunoconcentration of a model antigen, rabbit IgG, by passing a large volume of the dilute antigen through the antibody-coated cloth. Such immunoconcentration permitted detection of the dilute antigen which otherwise would have gone undetected.     

A two-step antibody strategy for surface plasmon resonance spectroscopy detection of protein-DNA interactions in nuclear extracts

Surface plasmon resonance (SPR) spectroscopy has emerged as a powerful alternative to conventional biochemistry methods for studying protein-DNA interactions that involve recombinant proteins of known identity. There are, however, limited demonstrations of SPR detection of protein-DNA bindings in crude samples, e.g., cell extracts, where the challenge is to detect and identify specific DNA binding protein(s) among other protein components in a physiological setting. We have developed a two-step antibody approach for an SPR study of estrogen receptor α (ERα)-DNA interactions, in which nuclear extracts prepared from MCF-7 breast cancer cells were used as the source of ERα protein. Following the binding of nuclear extracts to surface-immobilized estrogen response elements, rabbit anti-ERα antibody followed by a secondary antibody (goat anti-rabbit IgG) were applied to recognize the bound ERα and amplify the signals, respectively. Through a series of experiments, we have demonstrated that the magnitude of the binding signals from the secondary antibody reflects the affinity by which ERα binds to different DNA sequences. The detection sensitivity is determined by the amount of nuclear extracts and the concentration of primary antibody used. The sequence specificity of the nuclear ERα measured using the two-step antibody approach is in agreement with that measured for recombinant ERα protein (using receptor binding signals).     

Antibody-based surface plasmon resonance detection of intact viral pathogen

Surface plasmon resonance (SPR) technique was used to directly detect an intact form of insect pathogen: the baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). An SPR sensor chip with three bio-functional layers was used to detect the intact AcMNPV: amine-reactive crosslinker with a disulfide bond that chemisorbs to gold film, Protein A, and a mouse IgG monoclonal antibody raised against a surface protein of the target viral pathogen. A two-channel (reference & test) micro-fluidic SPR system is used for reliable measurement. Bio-specific response to the AcMNPV is compared with the response for tobacco mosaic virus (TMV) as control. Successive exposure of the sensor chip to both viruses verifies a specific response to AcMNPV. This serves as a prerequisite to the development of a new type of viral pathogen detection sensors.     

Fabrication of protein microarrays using the electrospray deposition (ESD) method: Application of microfluidic chips in immunoassay

In this study, antibody-based protein microarrays for high-throughput immunoassay were fabricated on an aldehydemodified indium-tin oxide glass plate using the electrospray deposition (ESD) method and their characteristics were evaluated immunochemically. The microarrays were also integrated into microfluidic chips with a polydimethylsiloxane (PDMS) micro-channel to detect human cytokines, which were quantitatively analyzed with a high resolution chargecoupled device. Simultaneous detection of various antigens was performed using the microarrays with considerable sensitivity (ca. 100 pg/mL). The results of this study indicate that microfluidic chip comprising a protein microarray formed by the ESD method and a PDMS micro-channel could be easy to handle, and offers high-throughput detection of molecular biomarkers.     

Comparative study of surface plasmon resonance, electrochemical and electroassisted chemiluminescence methods based immunosensor for the determination of antibodies against human growth hormone

An indirect immunoassay format with human growth hormone (hGH) immobilized on the self-assembled monolayer (SAM) modified surface plasmon resonance (SPR) chip has been shown to detect specific anti-hGH antibodies using the combination of three different physical phenomena in the same channel of the SPR analyzer. For the enhancement of analytical signal and sensitivity of the immunosensor horseradish peroxidase (HRP) labeled secondary antibodies, specifically interacting with the formed immune complexes, were used. The electroassisted chemiluminescence (ECL) protocol offered the limit of detection (LOD) as low as 0.061 nM and this result was very similar to that obtained by SPR, which was 0.051 nM. In the case of anti-hGH detection using pulsed amperometry (PA) with 3,3',5,5'-tetramethylbenzidine (TMB) and H(2)O(2) in the electrochemical system the LOD was the lowest - 0.027 nm. Lower reproducibility of the analytical signal and higher limit of detection was observed using cyclic voltammetry (CV) where LOD was 0.056 nM. PA detection shows 1.89, 2.07 and 2.26 times higher sensitivity if compared with SPR, CV and ECL, respectively. This work demonstrates successful simultaneous exploitation of several techniques to detect the specific anti-hGH antibodies using indirect immunoassay format on the same area of the SPR-chip.     

Continuous flow immunosensor for highly selective and real-time detection of sub-ppb levels of 2-hydroxybiphenyl by using surface plasmon resonance imaging

A biosensor based on surface plasmon resonance (SPR) is developed for the detection of 2-hydroxybiphenyl (HBP). A monoclonal antibody against HBP (abbreviated hereafter as HBP-mAb) is developed and used for the detection of HBP by competitive SPR-based immunoassay and enzyme linked immunosorbent assay (ELISA) methods. A novel HBP-hapten compound, HBP-bovine serum albumin conjugate (HBP-BSA), derived by binding several HBP units with BSA by an aliphatic chain spacer is used in the development of antibody and for the functionalization of immunoprobes. HBP-BSA linked to the Au surface of the SPR sensor chip undergoes inhibitive immunoreaction with HBP-mAb in the presence of free HBP. The SPR-based immunoassay provides a rapid determination (response time: approximately 20 min) of the concentration of HBP in the range of 0.1-1000 ppb (ng/ml). Regeneration of the sensor chip is gained by treating the antibody-anchored SPR sensor chip with a pepsin solution (100 ppm (microg/ml); pH 2.0) for few minutes. The SPR sensor chip is reusable for the detection of HBP for more than 20 cycles with average loss of 0.35% reactivity per regeneration step. HBP concentration is determined as low as 0.1 and 3 ppb using the SPR sensor and ELISA measurements, respectively. The developed SPR sensor for HBP is free from interference by coexisting benzo[a]pyrene (BaP), 2,4-dichlorophenoxyacetic acid (2,4-D) and benz[a]anthracene; SPR angle shift obtained to the flow of HBP is almost same irrespective to the presence or absence of a same concentration of these carcinogenic polycyclic aromatic hydrocarbons together. The SPR sensor for HBP is proved to be applicable in simultaneous detection of HBP and BaP in parallel with another SPR sensor for BaP.     

Immune complex transfer enzyme immunoassays for anti-angiotensin I IgG in serum using angiotensin I conjugates prepared by two different methods

Anti-angiotensin I IgG in serum was measured by immune complex transfer enzyme immunoassays using angiotensin I conjugates prepared by two different methods. In the first method, angiotensin I was conjugated to dinitrophenyl bovine serum albumin and beta-D-galactosidase through covalent links. Anti-angiotensin I IgG in rabbit serum was reacted simultaneously with dinitrophenyl bovine serum albumin-angiotensin I conjugate and beta-D-galactosidase-angiotensin I conjugate, and the complex formed of the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls. After washing, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to goat (anti-rabbit IgG) IgG-coated polystyrene balls. Beta-D-Galactosidase activity bound to (anti-rabbit IgG) IgG-coated polystyrene balls was assayed by fluorometry. In the second method, biotinylated angiotensin I was coupled with dinitrophenyl bovine serum albumin-avidin conjugate and Beta-D-galactosidase-avidin conjugate and substituted for the two conjugates in the first method. The detection limits of anti-angiotensin I IgG in serum were 10-30 ng/liter (0.2-0.6 pg/assay). These methods were 330 to 1,000-fold more sensitive and much less affected by serum effect than the conventional enzyme immunoassay, in which an angiotensin I-bovine serum albumin-coated polystyrene ball was incubated with anti-angiotensin I IgG in serum and, after washing, with (anti-rabbit IgG) Fab'-peroxidase conjugate. The first method was more sensitive than the second method, but the second method may be superior in applicability to the first method.     

Development of a biosensor microarray towards food screening, using imaging surface plasmon resonance

In this study we examined the possibilities of implementing direct and competitive immunoassay formats for small and large molecule detection on a microarray, using IBIS imaging surface plasmon resonance (iSPR) system. First, IBIS iSPR optics performance was evaluated. Using a glycerol calibration curve on underivatized surface we observed high baseline variability, but uniform and robust sensitivity between hundred regions of interest. Further on, a direct immunoassay for bovine IgG detection and a competitive immunoassay for gentamicin and neomycin were developed. The direct immunoassay for bovine IgG detection in a microarray format showed poor sensitivity in comparison to the assay performed in Biacore 3000, due to low immobilization efficiency on spots. The competitive immunoassay for parallel gentamicin and neomycin detection in a microarray format displayed sensitivity in the ngmL(-1) range, comparable with the sensitivity achieved in Biacore 3000 and in the range of maximum residue limits in milk, established in the European Union. We expect that, utilization of the IBIS iSPR system for food analysis, by screening high and low molecular weight compounds, will allow rapid and simultaneous detection of various ingredients and contaminants, providing the end-user with a detailed food profile. However, assay transfer from conventional SPR biosensors to the imaging microarray platform also presents new challenges, such as sufficient immobilization on spots, that must be addressed in future studies.     

Signal enhancement of surface plasmon resonance immunoassay using enzyme precipitation-functionalized gold nanoparticles: a femto molar level measurement of anti-glutamic acid decarboxylase antibody

Colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance (SPR) biosensor. The AuNPs were synthesized and functionalized with HS-OEG3-COOH by self assembling technique. Thereafter, the HS-OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti IgG antibody to form an enzyme-immunogold complex. Characterizations were performed by several methods: UV-vis absorption, DLS, HR-TEM and FT-IR. The Au-anti IgG-HRP complex has been applied in enhancement of SPR immunoassay using a sensor chip constructed by 1:9 molar ratio of HS-OEG6-COOH and HS-OEG3-OH for detection of anti-GAD antibody. As a result, AuNPs showed their enhancement as being consistent with other previous studies while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. The limit of detection was found as low as 0.03 ng/ml of anti-GAD antibody (or 200 fM) which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.     

Directional surface plasmon-coupled emission: application for an immunoassay in whole blood

We present a new approach for performing fluorescence immunoassay in whole blood using fluorescently labeled anti-rabbit immunoglobulin G (IgG) on a silver surface. This approach, which is based on surface plasmon-coupled emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bioaffinity surface. This article describes the effect of an optically dense sample matrix, namely human whole blood and serum, on the intensity of the SPCE. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal was studied. It was demonstrated that the kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately two- and threefold, respectively, as compared with buffer, resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Excited fluorophores outside the 200-nm layer do not contribute to SPCE, and their free space emission is not transmitted through the opaque metallic film into the glass substrate. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood with no need for washing steps.     

A more sensitive enzyme immunoassay of anti-insulin IgG in guinea pig serum with less non-specific binding of normal guinea pig IgG

An enzyme immunoassay of anti-insulin IgG in guinea pig serum was improved in sensitivity by reducing the non-specific binding of normal guinea pig IgG and enhancing the specific binding of anti-insulin IgG. Silicone rubber pieces or polystyrene balls were coated with normal rabbit IgG, followed by coupling of insulin using glutaraldehyde. The insulin-normal rabbit IgG-coated silicone rubber pieces or polystyrene balls were incubated with normal rabbit IgG and then with diluted guinea pig anti-insulin serum in the presence of normal rabbit IgG at a lower temperature (20 degrees C). Finally, the solid phases were incubated with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate to measure the amount of guinea pig IgG bound. The detection limit of anti-insulin IgG in guinea pig serum was improved 10 to 100-fold compared to that of enzyme immunoassay performed by incubating insulin-bovine serum albumin-coated solid phases with diluted guinea pig anti-insulin serum at 37 degrees C and then with rabbit (anti-guinea pig IgG) Fab' conjugated to beta-D-galactosidase from Escherichia coli, according to a previous report (Kato, K., et al. (1978) J. Biochem. 84, 93-102).     

Immobilized chicken antibodies improve the detection of serum antigens with surface plasmon resonance (SPR)

Surface plasmon resonance (SPR) and other refractive index and mass sensitive methods are, due to complement activation by mouse monoclonal antibodies and with concomitant high background signal, only rarely used for the detection of antibody–antigen interactions in the blood serum milieu. In the present study chicken IgY and mouse IgG were immobilized to a sensor chip CM5 dextran matrix and compared for their background signal and detection of serum antigen. Ellipsometry with antibodies adsorbed to methylated silicon surfaces was used as a complementary detection method. As expected, fundamental differences in binding properties between the two kinds of antibodies were observed. Mouse antibodies bound large quantities of human serum. Human C1q was detected on mouse IgG and the complement system was activated, as seen from the rapid C3 and properdin depositions. Chicken antibodies bound low quantities of human serum and no human C1q. Moreover, C3 and properdin deposited only after prolonged serum incubations. Addition of EDTA to serum reduced the background signal modestly for both IgG and IgY. Serum samples with different concentrations of human C3 were injected over surfaces with immobilized chicken anti-C3, and the response was measured by SPR. Small concentration differences (<1.25 μg/ml) in a physiologically relevant range (1–40 μg/ml after 100 times dilution) could then be detected reproducibly. The SPR signal was totally obscured when a mouse monoclonal anti-C3 antibody was used for the detection.     

Colloidal Au replacement assay for highly sensitive quantification of low molecular weight analytes by surface plasmon resonance

A novel sensing method based on surface plasmon resonance (SPR) was developed for the highly sensitive quantification of low molecular weight (LMW) analytes (colloidal Au replacement assay). Gold nanoparticles (diameter = 20 nm) functionalized with lactosyl-poly(ethylene glycol) (PEG) were prepared and were specifically adsorbed onto a Ricinus communis agglutinin (RCA120)-immobilized SPR sensor chip surface. Subsequent injection of free d-galactose elicited the elution of the preadsorbed lactosyl-PEGylated gold nanoparticles in a manner proportional to the galactose concentration, achieving a substantial and quantitative analysis over a wide range of galactose concentrations (0.1-50 ppm). This method of d-galactose sensing through the substituted elution of preadsorbed nanoparticles from the sensor chip surface would be applicable for the highly sensitive SPR quantification of various LMW analytes, which are known to be difficult to detect by the conventional SPR sensing regime.     

Microfluidic immunosensor systems

Immunosensing microfluidic devices are reviewed. Devices are commonly fabricated in glass, silicon, and polymers, with polymers seeing greater attention in recent years. Methods have been developed to immobilize antibodies and other molecules and resist non-specific adsorption through surface modification. The most common detection method is fluorescence, followed by electrochemistry. Various microfluidic designs have been reported for immunoassay applications. The observed trends in microfluidic immunoassay applications closely resemble the trends of general immunoassays, where large molecules are detected principally through a sandwich procedure, while competitive assays are used to detect smaller molecules. The following future trends are suggested: more sensitive detection, increased integration and miniaturization, multianalyte analysis, more robust reagents and devices, and increased functionality of surface treatments.     

Enzyme Immunoassay for Cholecystokinin Octapeptide Sulfate and Its Application

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for cholecystokinin octapeptide sulfate (CCK-8S) has been developed using N-terminal specific antibody for CCK-8S. In this assay CCK-8S coupled with poly-L-Glu (CCK-poly-Glu), which is adsorbed on a solid phase, competes with CCK-8S for the binding sites of rabbit anti-CCK antibody, and the complex of the immobilized antibody and CCK-poly-Glu is measured using goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase. The total time for completion of the assay is less than 24 h. Near 50% bound levels, the intraassay coefficient of variation is 5.2-6.2% and the interassay coefficient of variation is 5.9-8.5%. This assay is sensitive enough to detect 9 pg of CCK-8S, and the data from rat brain regions using this ELISA are very similar to the data from those using radioimmunoassay (RIA). Therefore, this ELISA is simpler and more rapid in comparison with conventional RIA. In the preliminary experiments, we applied this method for determination of CCK content in the brain regions of adult rats treated with 6-hydroxy-dopamine or in newborn rats subjected to anoxia, and showed that this system is applicable to detection of changes of endogenous CCK content.     

Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes

Alkaline phosphatase from calf intestinal mucosa has been conjugated to a protein antigen, rabbit IgG. Such conjugates, prepared by glutardialdehyde, have been used in a competitive solid phase immunoassay. In this test native antigen inhibits the binding of the conjugate to homologous antibodies adsorbed to plastic tubes. Using this assay 1-100 ng/ml of the antigen could be determined.