Development of a low density colorimetric protein array for cardiac troponin I detection

This work presented a rapid, inexpensive, reliable, and flexible quantitative immunoassay for cardiac troponin I (cTnI). The assay was based on the concepts of one-step dual monoclonal antibody "sandwich" principle, the low density protein array, the nanogold probe, and the silver enhancement on the gold particles. The capture antibody (IgG1) coated supporting nitrocellulose membrane and the colloidal gold-labeled detection antibody (cAu-IgG2) were prepared before the detection. The detection procedure involved two steps, i.e., immunoreaction and silver amplification. The assay needs only small amounts of serum samples of patients, The whole detection procedure of the assay could be fulfilled within 40 min (much faster than the routine enzyme-linked immunosorbent assay (ELISA) that takes usually at least 3 hours for a turnaround test). The detection results could be easily imaged with a simple flatbed scanner or even observed with the naked eye. The assay showed good specific response to cTnI with very little cross-reactivity to the skeletal isoforms of troponin I (sTnl), cardiac troponin T (cTnT), and myoglobin (Mb). A cut-off value of 0.3 ng/ml was obtained from a reference control group (200 normal serum samples). 588 patients' serum samples were assayed simultaneously by routine ELISA and this colloidal gold method to test the validity of the method. The data were analyzed using the statistical package SPSS version 11.0 (SPSS Inc.) There was no significant difference between these two assays (P = 0.66 > 0.05). The agreement between this method (> or < 0.3 ng/ml) and ELISA was 86%.     

DNA-based hybridization chain reaction for amplified bioelectronic signal and ultrasensitive detection of proteins

This work reports a novel electrochemical immunoassay protocol with signal amplification for determination of proteins (human IgG here used as a model target analyte) at an ultralow concentration using DNA-based hybridization chain reaction (HCR). The immuno-HCR assay consists of magnetic immunosensing probes, nanogold-labeled signal probes conjugated with the DNA initiator strands, and two different hairpin DNA molecules. The signal is amplified by the labeled ferrocene on the hairpin probes. In the presence of target IgG, the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles. The carried DNA initiator strands open the hairpin DNA structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Numerous ferrocene molecules are formed on the neighboring probe, each of which produces an electrochemical signal within the applied potentials. Under optimal conditions, the immuno-HCR assay presents good electrochemical responses for determination of target IgG at a concentration as low as 0.1 fg mL(-1). Importantly, the methodology can be further extended to the detection of other proteins or biomarkers.     

Concentration gradient immunoassay. 1. An immunoassay based on interdiffusion and surface binding in a microchannel

We describe a novel microfluidic immunoassay method based on the diffusion of a small-molecule analyte into a parallel-flowing stream containing a cognate antibody. This interdiffusion results in a steady-state gradient of antibody binding site occupancy transverse to convective flow. In contrast to the diffusion immunoassay (Hatch, A.; Kamholz, A. E.; Hawkins, K. R.; Munson, M. S.; Schilling, E. A.; Weigl, B. H.; Yager, P. Nat. Biotechnol. 2001, 19, 461-465.), this antibody occupancy gradient is interrogated by a sensor surface coated with a functional analogue of the analyte. Antibodies with at least one unoccupied binding site may specifically bind to this functionalized surface, leading to a quantifiable change in surface coverage by the antibody. SPR imaging is used to probe the spatial distribution of antibody binding to the surface and, therefore, the outcome of the assay. We show that the pattern of antibody binding to the SPR sensing surface correlates with the concentration of a model analyte (phenytoin) in the sample stream. Using an inexpensive disposable microfluidic device, we demonstrate assays for phenytoin ranging in concentration from 75 to 1000 nM in phosphate buffer. At a total volumetric flow rate of 90 nL/s, the assays are complete within 10 min. Inclusion of an additional flow stream on the side of the antibody stream opposite to that of the sample enables simultaneous calibration of the assay. This assay method is suitable for rapid quantitative detection of low molecular weight analytes for point-of-care diagnostic instrumentation.     

Kinetic chromatographic sequential addition immunoassays using protein A affinity chromatography.

A new type of chromatographic immunoassay based on sequential addition is described. On a protein A column, the antibody, the sample containing the antigen, and then a known amount of antigen are sequentially injected. This assay is designed to shorten analysis times and reduce complexity of dual-column chromatographic immunoassays, circumvent desorption buffer interferences common to affinity chromatography, and eliminate the need for tagged molecules. This new technique is named kinetic immunochromatography sequential addition (KICQA). Because of its kinetic nature, flow rate will have a large effect on KICQA, and the impact of changing flow rate is studied extensively. By use of various amounts of antibody, the dynamic range of KICQA is shown to be selectable over 2.5 orders of magnitude. Finally, KICQA was used to determine transferrin and albumin in human serum. Both analytes show good agreement with their respective reference methods, and an albumin assay was performed in under 1 min.     

Automated detection of anti-double-stranded DNA antibody in systemic lupus erythematosus serum by flow immunoassay

We describe a novel automated flow immunoassay system for quantification of anti-double-stranded (ds) DNA autoimmune antibodies in the serum of patients suffering from systemic lupus erythematosus. dsDNA (360 bp) was covalently coupled with alkaline phosphatase (ALP) to form a novel analytical reagent (ALP-DNA). After immunoreaction, antibody-antigen complexes between ALP-DNA and anti-dsDNA monoclonal antibody were separated from unreacted ALP-DNA by an ion-exchange column on the basis of the difference in isoelectric point. Antibody-antigen complexes were subsequently quantified by luminescence following addition of 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane. The assay yielded a linear relationship between signal and concentration of anti-dsDNA monoclonal antibody in the range of 0-300 micrograms/mL. This simple technique permits the assay of anti-dsDNA autoimmune antibodies within 25 min. The ion-exchange column was simply regenerated by occasional elution with eluent (20 mM N-methylpiperazine, pH 5.5) supplemented with 0.5 M NaCl, to remove unreacted ALP-DNA.     

Development of a theoretical model for chromatographic-based competitive binding immunoassays with simultaneous injection of sample and label.

This study examined the theory and behavior of an HPLC-based chromatographic competitive binding immunoassay with the simultaneous injection of sample and a labeled analyte analogue. Equations based on nonlinear chromatographic theory were derived to describe the calibration curve for this assay in a system with adsorption-limited kinetics and homogeneous binding sites. These equations related the assay response (B/Bo) to the column's binding capacity, the moles of injected analyte or labeled analogue, and the flow rate/adsorption kinetics of the system. There was good agreement between the predicted theoretical response and experimental data obtained for the binding of human serum albumin (HSA) to an immobilized anti-HSA antibody column. This theory was also successful in describing the changes that occurred in the calibration curve when the flow rate or amount of labeled analogue applied to the column was varied. A comparison was made between the results of this study and previous theoretical work that examined the behavior of a related, sequential injection competitive binding method. On the basis of the results reported in this work, several general guidelines were developed for the design and optimization of simultaneous injection methods for use in such areas as clinical testing, pharmaceutical analysis, and environmental monitoring.     

Application of the biological conjugate between antibody and colloid Au nanoparticles as analyte to inductively coupled plasma mass spectrometry.

This paper describes the study of atomization of nanoparticles by inductively coupled plasma mass spectrometry (ICPMS) and developes a novel nonisotopic immunoassay by coupling sandwich-type immunoreaction to ICPMS. The goat-anti-rabbit immunoglobulin G (IgG) labeled with colloidal gold nanoparticles served as an analyte in ICPMS for the indirect measurement of rabbit-anti-human IgG. Matrix effect studies showed the gold signal was not sensitive to the organic matrix. A relatively good correlation (r2 = 0.9528) between the proposed method and enzyme-linked immunosorbent assay has been obtained. The method may have significant potential as an important ICPMS-based nonisotopic immnoassay method for the simultaneous determination of biologic analytes of interest by labeling different kinds of inorganic nanoparticles.     

Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer

A new signal amplification strategy based on thionine (TH)-doped magnetic gold nanospheres as labels and horseradish peroxidase (HRP) as enhancer holds promise to improve the sensitivity and detection limit of the immunoassay for carcinoembryonic antigen (CEA), as a model protein. This immunoassay system was fabricated on a carbon fiber microelectrode (CFME) covered with a well-ordered anti-CEA/protein A/nanogold architecture. The reverse micelle method was initially used for the preparation of TH-doped magnetic gold nanospheres (nanospheres), and the synthesized nanospheres were then labeled on HRP-bound anti-CEA as a secondary antibody (bionanospheres). Sandwich-type protocol was successfully introduced to develop a new high-efficiency electrochemical immunoassay with the labeled bionanospheres toward the reduction of H2O2. Under optimized conditions, the linear range of the proposed immunoassay without HRP as enhancer was 1.2-125 ng/mL CEA, whereas the assay sensitivity by using HRP as enhancer could be further increased to 0.01 ng/mL with the linear range from 0.01 to 160 ng/mL CEA. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method.     

Disposable electrochemical immunosensor diagnosis device based on nanoparticle probe and immunochromatographic strip

We describe a disposable electrochemical immunosensor diagnosis device that integrates the immunochromatographic strip technique with an electrochemical immunoassay and exploits quantum dot (QD, CdS@ZnS) as labels for amplifying signal output. The device takes advantage of the speed and low cost of the conventional immunochromatographic strip test and the high sensitivity of the nanoparticle-based electrochemical immunoassay. A sandwich immunoreaction was performed on the immunochromatographic strip, and the captured QD labels in the test zone were determined by highly sensitive stripping voltammetric measurement of the dissolved metallic component (cadmium) with a disposable screen-printed electrode, which is embedded underneath the membrane on the test zone. The new device coupled with a portable electrochemical analyzer shows great promise for in-field and point-of-care quantitative testing of disease-related protein biomarkers. The parameters (e.g., voltammetric measurement of QD labels, antibody immobilization, the loading amount of QD-antibody, and the immunoreaction time) that govern the sensitivity and reproducibility of the device were optimized with IgG model analyte. The voltammetric response of the optimized device is highly linear over the range of 0.1-10 ng mL(-1) IgG, and the limit of detection is estimated to be 30 pg mL(-1) in association with a 7-min immunoreaction time. The detection limit was improved to 10 pg mL(-1) using a 20-min immunoreaction time. The device has been successfully applied for the detection of prostate-specific antigen (PSA) in human serum sample with a detection limit of 20 pg mL(-1). The results were validated by using the commercial PSA enzyme-linked immunosorbent assay kit and showed high consistency. The new disposable electrochemical diagnosis device thus provides a rapid, clinically accurate, and quantitative tool for protein biomarker detection.     

Electrochemical enzyme immunoassays on microchip platforms.

A microfluidic device for conducting electrochemical enzyme immunoassays is described. The new "lab-on-a-chip" protocol integrates precolumn reactions of alkaline phosphatase-labeled antibody (anti-mouse IgG) with the antigen (mouse IgG), followed by electrophoretic separation of the free antibody and antibody-antigen complex. The separation is followed by a postcolumn reaction of the enzyme tracer with the 4-aminophenyl phosphate substrate and a downstream amperometric detection of the liberated 4-aminophenol product Factors influencing the reaction, separation, and detection processes were optimized, and the analytical performance was characterized. An applied field strength of 256 V/cm results in free antibody and antibody-antigen complex migration times of 125 and 340 s, respectively. A remarkably low detection limit of 2.5 x 10(-16) g/mL (1.7 x 10(-18) M) is obtained for the mouse IgG model analyte. Such combination of a complete integrated immunoassay, an attractive analytical performance, and the distinct miniaturization/portability advantages of electrochemical microsystems offers considerable promise for designing self-contained and disposable chips for decentralized clinical diagnostics or on-site environmental testing.     

Preparation of quantum dot-biotin conjugates and their use in immunochromatography assays

Biotinylated, highly luminescent CdSe-ZnS quantum dot (QD) conjugates were prepared and used in immunofiltration assays. Water-soluble quantum dot surfaces having a homogeneous negative charge density at basic pH were initially coated with a two-domain recombinant maltose-binding protein appended with a positively charged leucine zipper. Biotin functionalization of these electrostatically stabilized QD-protein complexes was then carried out using amine-reactive NHS biotin. These protein-coated biotin-functionalized quantum dot conjugates were incorporated into flow immunofiltration/displacement assays employing Affi-gel agarose resin for antibody immobilization, analyte capture, and immune complex formation with a second biotinylated antibody. A key component of the assay was the use of tetranitromethane-modified NeutrAvidin, used to link the biotinylated QDs to the immune complexes and facilitate their release at basic pH for subsequent quantification. This assay methodology was used to detect as little as 10 ng/mL staphylococcal enterotoxin type-B.     

Ultrasensitive multiplexed immunoassay with electrochemical stripping analysis of silver nanoparticles catalytically deposited by gold nanoparticles and enzymatic reaction

A novel ultrasensitive multiplexed immunoassay method was developed by combining alkaline phosphatase (ALP)-labeled antibody functionalized gold nanoparticles (ALP-Ab/Au NPs) and enzyme-Au NP catalyzed deposition of silver nanoparticles at a disposable immunosensor array. The immunosensor array was prepared by covalently immobilizing capture antibodies on chitosan modified screen-printed carbon electrodes. After sandwich-type immunoreactions, the ALP-Ab/Au NPs were captured on an immunosensor surface to catalyze the hydrolysis of 3-indoxyl phosphate, which produced an indoxyl intermediate to reduce Ag(+). The silver deposition process was catalyzed by both ALP and Au NPs, which amplified the detection signal. The deposited silver was then measured by anodic stripping analysis in KCl solution. Using human and mouse IgG as model analytes, this multiplexed immunoassay method showed wide linear ranges over 4 orders of magnitude with the detection limits down to 4.8 and 6.1 pg/mL, respectively. Acceptable assay results for practical samples could be obtained. The newly designed strategy avoided cross talk and the need of deoxygenation for the electrochemical immunoassay and, thus, provided a promising potential in clinical applications.     

In situ amplified electrochemical immunoassay for carcinoembryonic antigen using horseradish peroxidase-encapsulated nanogold hollow microspheres as labels

Methods based on sandwich-type electrochemical enzyme immunoassay protocol have been extensively developed for the detection of biomolecules, but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. In this study, we initially synthesized specially horseradish peroxidase-encapsulated nanogold hollow microspheres (HRP-GHS), and then the prepared HRP-GHS was conjugated to the secondary carcinoembryonic antibody (HRP-GHS- anti-CEA). Carcinoembryonic antigen (CEA), as a model protein, was monitored by using the electrochemical sandwich-type enzyme immunoassay format. Under optimized conditions, the linear range of the immunoassay by using single HRP-labeled anti-CEA (HRP- anti-CEA) as secondary antibodies is 2.5-120 ng/mL with a detection limit of 1.5 ng/mL CEA, while the assay sensitivity by using HRP-GHS- anti-CEA as secondary antibodies is further increased from 0.01 to 200 ng/mL with a lower detection limit of 1.5 pg/mL CEA. The intra- and interassay reproducibility is acceptable. The CEA concentrations of the clinical serum specimens assayed by the developed immunoassay show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay. This immunoassay system has many desirable merits including sensitivity, accuracy, and little required instrumentation. Significantly, the new protocol may be quite promising, with potentially broad applications for clinical immunoassays.     

Liposome flow injection immunoassay: implications for sensitivity, dynamic range, and antibody regeneration

We have developed a liposome-based flow injection immunoassay (FIIA) system for quantitation of a clinical analyte, theophylline. With very minor changes in assay format, this procedure can also be used for the quantitation of anti-theophylline. Automated sequential analyses were performed at room temperature with picomole sensitivity and a day-to-day coefficient of variation of less than 5% for aqueous solutions. The system components include liposomes that contain fluorophores in their aqueous centers and an immobilized-antibody reactor column. The immunoreactor was regenerated hundreds of times over 3 months of continuous use with no measurable loss of antibody activity. The two assay formats studied produced distinct dynamic ranges for their respective analytes. The special advantages of using flow injection analysis for immunoassays and of using liposomes in FIIA are discussed.     

Removal of copper(II) from aqueous phase by Purolite C100-MB cation exchange resin in fixed bed columns: modeling

The dynamic removal of copper by Purolite C100-MB cation exchange resin was studied in packed bed columns. The values of column parameters are predicted as a function of flow rate and bed height. Batch experiments were performed using the Na-form resin to determine equilibrium and kinetics of copper removal. The uptake of Cu(II) by this resin follows first-order kinetics. The effect of stirring speed and temperature on the removal kinetics was studied. The activation energy for the exchange reaction is 13.58kJmol(-1). The equilibrium data obtained in this study have been found to fit both the Langmuir and Freundlich isotherm equations. A series of column tests were performed to determine the breakthrough curves with varying bed heights and flow rates. To predict the breakthrough curves and to determine the characteristic parameters of the column useful for process design, four kinetic models; Bohart-Adams, Bed Depth Service Time (BDST), Clark and Wolborska models are applied to experimental data. All models are found suitable for describing the whole or a definite part of the dynamic behavior of the column with respect to flow rate and bed height. The simulation of the whole breakthrough curve is effective with the Bohart-Adams and the Clark models, but the Bohart-Adams model is better. The breakthrough is best predicted by the Wolborska model. The breakthrough data gave a good fit to the BDST model, resulting in a bed exchange capacity very close to the value determined in the batch process.     

Optical encoding of microbeads based on silica particle encapsulated quantum dots and its applications

A novel method concerning the coding technology of polystyrene beads with Si encapsulated quantum dot (QD) particles (Si@QDs particles) is studied in this paper. In the reverse microemulsion system containing tetraethoxysilane (TEOS), water-soluble QDs (emission peak at 600?nm) were enveloped within the silica shell, forming Si@QDs particles. The Si@QDs particles were characterized by TEM, showing good uniform size, with an average diameter of about 167.0?nm. In comparison with the pure water-soluble QDs, the encapsulation of water-soluble QDs in the silica shell led to an enhancement in anti-photobleaching by providing inert barriers for the QDs. Images presented by SEM and confocal laser scanning microscopy demonstrated that the Si@QDs particles were equably coated on the surface of carboxyl functionalized polystyrene (PS) beads. Then, with the assistance of ethyl-3-(dimethyl aminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), human IgG could be successfully crosslinked to Si@QDs particle coated PS-COOH beads. Furthermore, the Si@QDs coated PS-COOH beads with human IgG were examined in immunoassay experiments, and the results indicated that these beads could be applied in the specific recognition of goat-anti-human IgG in solution. This investigation is expected to provide a new route to bead coding in the field of suspension microarrays, based on the use of QDs.     

Graphene oxide sheet-mediated silver enhancement for application to electrochemical biosensors

Functionalized graphene oxide (GO) sheets coupled with a signal amplification method based on the nanomaterial-promoted reduction of silver ions for the sensitive and selective detection of bacteria. This paper aims to develop an electrochemical route combined with GO sheet-mediated Ag enhancement for biological/chemical analyte detection. A linear relationship between the stripping response and the logarithm of the bacterial concentration was obtained using an electrochemical technique for concentrations ranging from 1.8 × 10(2) to 1.8 × 10(8) cfu mL(-1), with a slope of 15.28 and a correlation coefficient of 0.995. Dot blot assay was used as a conventional immunoassay method for comparison with the electrochemical method, as well as to observe the quality of the anti-sulfate-reducing bacteria (SRB) antibody (Ab) used in the immunosensor. The GO sheet-mediated silver enhancement holds great potential for the rapid analysis of protein, DNA, and pathogens.     

Surface-enhanced Raman scattering immunoassays using a rotated capture substrate

A rapid, sensitive format for immunosorbent assays has been developed to meet the increasing levels of performance (i.e., reduction of incubation times and detection limits) demanded in the medical, veterinary, and bioterrorism prevention arenas. This paper introduces the concept of a rotating capture substrate as a facile means to increase the flux of antigen and label to the solid-phase surface and thereby reduce assay time. To this end, a sandwich-type assay is carried out that couples the specificity of antibody-antigen interactions with the high sensitivity of surface-enhanced Raman scattering detection. To investigate this strategy, polyclonal anti-rabbit IgG was immobilized on a gold capture substrate via a thiolate coupling agent. The capture substrate, capable of controlled rotation, was then immersed in a sample solution containing rabbit IgG, which served as a model analyte. After binding the target IgG, the substrates were immersed and rotated in an extrinsic Raman label (ERL) labeling solution, which is composed of gold nanoparticles (60 nm) coated with an aromatic moiety as the Raman scatterer and an antibody as the biospecific recognition element. The effect of substrate rotation on both the antigen binding and ERL labeling steps was investigated. Implementation of optimized rotation conditions resulted in the reduction of assay times from 24 h to 25 min and a 10-fold improvement in the limit of detection. Finally, the developed protocol was applied to the detection of rabbit IgG suspended in goat serum, which served to assess performance in a biological matrix.     

Detection of antistreptolysin O antibody: application of an initial rate method of latex piezoelectric immunoassay.

Latex plezoelectric immunoassay (LPEIA) is a new latex immunoassay using a plezoelectric quartz crystal (Kurosawa et al. Chem. Pharm. Bull. 1990, 38, 1117). This assay requires no immobilization of antigen or antibody on an electrode surface of a plezoelectric crystal, while the immobilization is indispensable for ordinary immunoassays using a plezoelectric crystal as a microbalance. The present paper improves a previous method (end-point analysis) by introducing the initial rate method using a batch cell; reduction of assay volume (1.2 mL) and shortening of assay time (2-3 min) were achieved. This assay was applied to the detection of antistreptolysin O antibody (ASO) in serum. The frequency change was proportional to the ASO concentration up to 1040 IU mL-1, and the method has good sensitivity for actual clinical application. The volume of serum required for the assay was 0.02 mL. Twenty-four clinical specimens were analyzed with this LPEIA, and the values obtained were compared with those obtained with a turbidimetric latex agglutination method. The correlation coefficient between these values was 0.950 (P < 0.01).     

Scanning electrochemical microscopy monitoring in microcantilever platforms

The deflection of cantilever systems may be performed by an indirect electrochemical method that consists of measuring the local cantilever activity and deflection in a feedback generation-collection configuration of the SECM. This is illustrated during the electrochemically assisted adsorption of Br onto a gold-coated cantilever, either in its pristine state or previously coated with a thin organic barrier. It is further extended to the adsorption of an antibody in a heterogeneous immunoassay at an allergen-coated microcantilever platform. In both reactions, the cantilever deflection is qualitatively detected from the SECM tip current measurement and a quantitative estimate is obtained through modeling. This electroanalytical strategy provides an alternative approach to standard optical detection. It can overcome some limitations of the optical method by allowing electrochemical characterization of nonconductive cantilevers and appropriate use for closed systems.