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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 carded 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.

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. Bioteehnol. 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.

A novel microfluidic surface-based competition immunoassay, termed the concentration gradient immunoassay (described in detail in a companion paper (Nelson, K.; Foley, J.; Yager, P. Anal Chem. 2007, 79, 3542-3548.) uses surface plasmon resonance (SPR) imaging to rapidly measure the concentration of small molecules. To conduct this assay, antibody and analyte are introduced into the two inlets of a T-sensor (Weigi, B. H.; Yager, P. Science 1999, 283, 346-347. Kamholz, A. E.; Weigl, B. H.; Finlayson, B. A.; Yager, P. Anal. Chem. 1999, 71, 5340-5347). Several millimeters downstream, antibody molecules with open binding sites can bind to a surface functionalized with immobilized antigen. This space- and time-dependent binding can be sensitively observed using SPR imaging. In this paper, we describe a complex three-dimensional finite element model developed to better understand the dynamic processes occurring with this assay. The model shows strong qualitative agreement with experimental results for small-molecule detection. The model confirms the experimental finding that the position within the microchannel at which the antibody binds to the immobilized analyte may be used to quantify the concentration of analyte in the sample. In addition, the model was used to explore the sensitivity of assay performance to parameters such as antibody and analyte concentrations, thereby giving insight into ways to optimize analysis speed and accuracy. Given the experimental verification of the computational results, this model serves as an efficient method to explore the influence of the flow rate, microchannel dimensions, and antibody concentration on the sensitivity of the assay.

Patulin is a toxic secondary metabolite of a number of fungal species belonging to the genera Penicillum and Aspergillus. It has been mainly isolated from apples and apple products contaminated with the common storage-rot fungus of apples, Penicillum expansum, but it has also been extracted from rotten fruits, moldy feeds, and stored cheese. Human exposure to patulin can lead to serious health problems, and according to a long-term investigation in rats, the World Health Organization has set a tolerable weekly intake of 7 ppb body weight. The content of patulin in foods has been restricted to 50 ppb in many countries. Conventional analytical detection methods involve chromatographic analyses, such as HPLC, GC, and, more recently, techniques such as LC/MS and GC/MS. However, extensive protocols of sample cleanup are required prior to the analysis, and to accomplish it, expensive analytical instrumentation is necessary. An immunochemical analytical method, based on highly specific antigen-antibody interactions, would be desirable, offering several advantages compared to conventional techniques, i.e., low cost per sample, high selectivity, high sensitivity, and high throughput. In this paper, the synthesis of two new derivatives of patulin is described, along with their conjugation to the bovine serum albumin for the production of polyclonal antibodies. Finally, a fluorescence competitive immunoassay was developed for the on-line detection of patulin.

A microchip-based, displacement immunoassay for the sensitive laser-induced fluorescence detection of staphylococcal enterotoxin B is presented. The glass microchip device consists of a microchannel that contains a double weir structure for supporting antibody-functionalized microbeads. After a 30-min sample preparation step, the displacement assay was performed without user intervention and produced quantitative results in an additional 20 min. Linear detection responses were observed over 6 orders of magnitude and provided detection limits down to 1 fM (28.5 fg/mL). The surprisingly low detection limits are hypothesized to arise from field-based enrichment analogous to field-amplified stacking, chromatographic effects, and limited diffusion lengths in the microbead bed. The assay was challenged with bovine serum albumin, casein, and milk sample matrixes. This system has the potential to provide highly sensitive detection capabilities for target biomolecules.

This paper reports a miniaturized immunosensor designed to determine a trace level cardiac marker, B-type natriuretic peptide (BNP), using a microfluidic device combined with a portable surface plasmon resonance (SPR) sensor system. Sample BNP solution was introduced into the microchannel after an immunoreaction with acetylcholine esterase-labeled antibody (conjugate), and only unbound conjugate was trapped on the BNP-immobilized surface in the flow channel. Then, the thiol compound generated by the enzymatic reaction with the trapped conjugate was accumulated on a gold thin film located downstream in the microchannel to monitor the real-time SPR angle shift. We achieved a detectable concentration range of 5 pg/mL-100 ng/mL by monitoring the SPR angle shift, which covers the required detection range for the BNP concentrations found in blood. This success resulted from the use of a T-shaped microfluidic device structure, which prevents the sample solution from flowing over the gold film used for SPR detection. We were able to measure trace levels of BNP peptide (15 fg) within 30 min since the procedure with our immunosensor is simpler than a multistep immunoassay through the simultaneous use of a labeled enzymatic reaction and the real-time monitoring of enzymatic product accumulation in the microfluidic device. We employed the procedure to detect serum BNP by using spiked samples in human serum and achieved satisfactory recovery for heat-treated samples to denature the esterase in the serum before the immunoreaction.

A plastic chip that can perform immunoassays using an enzyme as signal generator, i.e., ELISA-on-a-chip, was developed by incorporating an immunostrip into channels etched on the surfaces of the chip. To utilize an analytical concept of cross-flow chromatography, the chip consisted of two cross-flow channels in the horizontal and vertical directions. In the vertical channel, we placed a 2-mm-wide immunostrip for cardiac troponin I (cTnI), which was identical to a conventional rapid test kit except for the utilization of an enzyme, horseradish peroxidase (HRP), as tracer. An enzyme substrate supply channel and a horizontal flow absorption pad compartment were transversely arranged on each lateral side of the signal generation pad of the strip, respectively. Upon application of a sample containing cTnI, it migrated vertically through the membrane strip by capillary action, and antigen--antibody binding occurred. After 15 min, the horizontal flow was initiated by the addition of a chromogenic substrate solution for HRP into the supply channel and by partial superimposition of the horizontal flow absorption pad onto the signal generation pad. A color signal proportional to the analyte concentration was produced on this pad, measured after 5 min as optical densities using a digital camera-based detector, and quantified by integration of the densities under the peak after normalization. Its calibration curve indicated that the detection limit of the chip was ~0.1 ng/mL and its quantification limit was 0.25 ng/mL. In measuring blindly prepared samples, the chip performance correlated with that of a reference system, Beckman Coulter Access, within 2.5-fold discrepancy at the detection limit.

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 [micro]m) is achieved via an electroless plating method involving initial reaction of the particles with Sn(II), followed by redox deposition of [Ag.sup.0], that serves as a catalytic site for the subsequent reduction of [Na.sub.3]Au[(S[O.sub.3).sub.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 [Au.sup.0] 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.

A novel reagent for low-level detection in immunoadsorbent assays is described. The reagent consists of gold nanoparticles modified to integrate bioselective species (e.g., antibodies) with molecular labels for the generation of intense, biolyte-selective surface-enhanced Raman scattering (SERS) responses in immunoassays and other bioanalytical applications. The reagent is constructed by coating gold nanoparticles (30 nm) with a monolayer of an intrinsically strong Raman scatterer. These monolayer-level labels are bifunctional by design and contain disulfides for chemisorption to the nanoparticle surface and succinimides for coupling to the bioselective species. There are two important elements in this label design; it both minimizes the separation between label and particle surface and maximizes the number of labels on each particle. This approach to labeling also exploits several other advantages of SERS-based labels: narrow spectral bandwidth, resistance to photobleaching and quenching, and long-wavelength excitation of multiple labels with a single excitation source. The strengths of this strategy are demonstrated in the detection of free prostate-specific antigen (PSA) using a sandwich assay format based on monoclonal antibodies. Detection limits of ~1 pg/mL in human serum and ~4 pg/mL in bovine serum albumin have been achieved with a spectrometer readout time of 60 s. The extension of the method to multianalyte assays (e.g., the simultaneous determination of the many complexed forms of PSA) is discussed.

A novel strategy for immunoassay and the preparation of reagentless immunosensors was proposed. This strategy was based on the immobilization of antigen and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to an antibody. A reagentless immunosensor for carcinoma antigen-125 (CA 125) determination was developed. The immunosensor was prepared by immobilizing CA 125 with titania sol--gel on a glassy carbon electrode by the vapor deposition method. The incubation of the immunosensor in phosphate buffer solution (PBS) including HRP-labeled CA 125 antibody led to the formation of a HRP-modified surface. The immobilized HRP displayed its direct electrochemistry with a rate constant of 3.04 [+ or -] 1.21 [s.sup.-1]. With a competition mechanism, a differential pulse voltammetric determination method for CA 125 was established by the peak current decrease of the immobilized HRP. The current decrease resulted from the competitive binding of the CA 125 in sample solution and the immobilized CA 125 to the limited amount of HRP-labeled CA 125 antibody. Under optimal conditions, the current decrease was proportional to CA 125 concentration ranging from 2 to 14 units m[L.sup-1] with a detection limit of 1.29 units m[L.sup.-1] at a current decrease by 10%. The CA 125 immunosensor showed good accuracy and acceptable precision and fabrication reproducibility with intraassay CVs of 8.7 and 5.5% at 8 and 14 units m[L.sup.-1] CA 125 concentrations, respectively, and interassay CV of 19.8% at 8 units m[L.sup.-1]. The storage stability was acceptable in a pH 7.0 PBS at 4[degrees]C for 15 days. The proposed method provided a new promising platform for clinical immunoassay.

We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system is designed to provide early warning to civilians in the event of a terrorist attack. The final APDS will be completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. The system performance (current capabilities include aerosol collection, multiplexed immunoassays, sample archiving, data reporting, and alarming) was evaluated in a field test conducted in a Biosafety Level 3 facility, where the system was challenged with, and detected, a series of aerosolized releases containing two live, virulent biological threat agents (Bacillus anthracis and Yersinia pestis). Results presented here represent the first autonomous, simultaneous measurement of these agents.

Previously, immunological detection of a small hapten was only possible in competitive format, which needed a competitor antigen either labeled by a reporter or attached to a carrier protein. Recently, we proposed the open sandwich (OS) immunoassay, a simple immunoassay that can noncompetitively determine monovalent antigen concentration by measuring the antigen-dependent change in a heavy-chain variable region [V.sub.H]/light-chain variable region [V.sub.L] interaction of an antibody. However, there was a limitation in the assay that the antibody used should have a suitable property such that the [V.sub.H]/[V.sub.L] interaction would become fairly strong along with the addition of antigen. Here, we devised a phage-based 'split-Fv system' to rapidly evaluate and select antibody variable region (Fv) fragments that are suitable to OS immunoassay. When three antibodies raised against endocrine disruptor bisphenol A were tested with this system, all were more or less suitable to OS-ELISA. Among them, the best Fv selected was used to construct fusion proteins of [V.sub.H] tethered to an alkaline phosphatase and a tagged [V.sub.L] that can be site-specifically biotinylated to perform direct OS-ELISA. The results showed that the OS-ELISA detects bisphenol A with higher sensitivity than the corresponding competitive assay, also implying that many antibodies to small haptens have suitable properties for OS-ELISA.

Biotinylated, highly luminescent CdSe-ZnS quantum dot (QD) conjugates were prepared and used in immuno-filtration 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.

A self-contained, microelectrochemical immunoassay on the smallest volumes reported to date (1 [micro]L for the antigen, 1 [micro]L for the secondary antibody-enzyme conjugate, and 200 nL for the electrochemically detected species) has been developed using mouse IgG as a model system in a sandwich-type enzyme-linked immunosorbant assay, which takes less than 30 min to both complete the assembly of immunoassay components onto the antibody-modified surface and detect enzymatically generated species (excluding time for electrochemical cleaning of electrodes). These studies demonstrate the advantage of the close proximity of electrodes to modified surfaces and their application in the analysis of small volumes. Using a 50 [micro]m diameter x 8 [micro]m deep cavity with individually addressable electrodes on a microfabricated chip, the primary antibody was selectively and covalently attached at a gold, recessed microdisk (RMD) at the bottom of the microcavity to the free end of SAMs of either 11-mercaptoundecanoic acid or 11-mercapto-1-undecanol using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. Nonspecific adsorption to the surrounding material, polyimide, of the microcavity device was eliminated. Electrochemical desorption was used to confine the immunoassay activity at the RMD. Enzymatic conversion of the substrate p-aminophenyl phosphate to p-aminophenol is detectable in less than 30 s using cyclic voltammetry at a gold, tubular nanoband electrode, which is on the wall of the microcavity and immediately adjacent to the modified RMD. A third electrode, also within the region of the microcavity, served as the pseudoreference/auxiliary electrode. Calibration curves obtained for 1-[micro]L solutions of 5-100 ng/mL of IgG and for 200 nL-solutions of 5 [micro]M to 4 mM of PA[P.sub.R] gave detection limits of 4.4 nM (6.4 ng/mL) or 880 fmol (129 pg) for PA[P.sub.R] and 56 fM (9 pg/ mL) or 56 zmol (9 fg) for IgG. It is expected that the device may be suitable for analysis with volumes down to tens of picoliters.

Surface plasmon resonance (SPR) biosensing using colloidal Au enhancement is reported. Immobilization of [approximately] 11-nm-diameter colloidal Au to an evaporated Au film results in a large shift in plasmon angle, a broadened plasmon resonance, and an increase in minimum reflectance. The incorporation of colloidal Au into SPR biosensing results in increased SPR sensitivity to protein-protein interactions when a Au film-immobilized antibody and an antigen-colloidal Au conjugate comprise the binding pair. A highly specific particle-enhanced analogue of a sandwich immunoassay is also demonstrated by complexing the Au particle to a secondary antibody. A tremendous signal amplification is observed, as addition of the antibody-Au colloid conjugate results in a 25-fold larger signal than that due to addition of a free antibody solution that is 6 orders of magnitude more concentrated. Picomolar detection of human immunoglobulin G has been realized using particle enhancement, with the theoretical limits for the technique being much lower. Finally, a quasi-linear relationship between particle coverage and plasmon angle shift is presented, thereby providing for a direct correlation between plasmon shift and solution antigen concentration. Together, these results represent significant advances in the generality and sensitivity of SPR as it is applied to biosensing.

Surface plasmon resonance (SPR) biosensing using colloidal Au enhancement is reported. Immobilization of [approximately] 11-nm-diameter colloidal Au to an evaporated Au film results in a large shift in plasmon angle, a broadened plasmon resonance, and an increase in minimum reflectance. The incorporation of colloidal Au into SPR biosensing results in increased SPR sensitivity to protein-protein interactions when a Au film-immobilized antibody and an antigen-colloidal Au conjugate comprise the binding pair. A highly specific particle-enhanced analogue of a sandwich immunoassay is also demonstrated by complexing the Au particle to a secondary antibody. A tremendous signal amplification is observed, as addition of the antibody-Au colloid conjugate results in a 25-fold larger signal than that due to addition of a free antibody solution that is 6 orders of magnitude more concentrated. Picomolar detection of human immunoglobulin G has been realized using particle enhancement, with the theoretical limits for the technique being much lower. Finally, a quasi-linear relationship between particle coverage and plasmon angle shift is presented, thereby providing for a direct correlation between plasmon shift and solution antigen concentration. Together, these results represent significant advances in the generality and sensitivity of SPR as it is applied to biosensing.

This paper combines the topographic imaging capability of the atomic force microscope (AFM) with a compositionally patterned array of immobilized antigenic rabbit IgG on gold as an approach to performing immunoassays. The substrates are composed of micrometer-sized domains of IgG that are covalently linked to a photolithographically patterned array of a monolayer-based coupling agent. The immobilized coupling agent, which is prepared by the chemisorption of dithiobis(succinimidyl undecanoate) on gold, is separated by micrometer-sized grids of a monolayer formed from octadecanethiol (ODT). The strong hydrophobicity of the ODT adlayer, combined with the addition of the surfactant Tween 80 to the buffer solution that is used in forming the antibody-antigen pairs, minimizes the nonspecific adsorption of proteinaceous materials to the grid regions. This minimization allows the grids to function as a reference plane for the AFM detection of the height increase when a complementary antibody-antigen pair is formed. The advantageous features of this strategy, which include ease of sample preparation, an internal reference plane for the detection of topographic changes, and the potential for regeneration and reuse, are demonstrated using rabbit IgG as an immobilized antigen and goat anti-rabbit IgG as the complementary antibody. The prospects for further miniaturization are discussed.

A rapid immunoassay using a sulfated antibody for bound/free separation in a liquid-phase binding assay is described. A first anti-[Alpha]-fetoprotein monoclonal antibody was labeled with peroxidase (Fab[prime]-POD) and a second monoclonal antibody was conjugated with polysulfated tyrosine peptide (Fab[prime]-YS). The monoclonal antibodies and [Alpha]-fetoprotein (AFP) were mixed, incubated, and analyzed directly by anion-exchange column chromatography. The amount of POD activity in the column effluent was determined fluorophotometrically. The bound (Fab[prime]POD + AFP + Fab[prime] - YS) and free (Fab[prime] - POD) forms of the conjugate were clearly and easily separated by ionic charge, and the free sulfated antibody (Fab[prime] - YS) was not detectable fluorophotometrically. The elution position of the bound conjugate was adjusted by varying the length of the polysulfated tyrosine peptide. This method is convenient for antigen measurement because (1) only two modified antibodies are used in a buffer solution, (2) the concentration of antibodies and other assay conditions are easily set, (3) no solid phase is required, and (4) no washing is necessary.

The role of the solution environment for a light-scattering, latex-particle-enhanced, homogeneous immunoassay of C-reactive protein (CRP) has been investigated in order to assess and optimize the immunoagglutination response. Latex particles of 50-170-nm sizes were covalently coupled with an IgG polyclonal antibody and subjected to an extensive optimization regime. This consisted of conditions responsible, in different degrees, for the principal attractive/repulsive forces affecting both colloidal stability and the antibody/antigen interaction: particle size, antibody concentration, ionic strength and species, pH, and amino acid chemistry of the particle surface. Careful control of these parameters was found to be necessary to achieve the desired effects of balancing high colloidal stability in the absence of antigen but promoting a rapid, sensitive, and dose-dependent agglutination with pathological serum samples. In addition, the estimation of fundamental properties governing intermolecular interaction (i.e. the 'Hamaker' constant and critical coagulation concentration) was attempted to order to investigate a simple, practical means of defining a colloidal/immunoassay system under 'real conditions' as well as 'real time'. It is concluded that because each antibody system is unique, a similar optimization should be performed in diagnostic immunoassays of this type to maximize their clinical utility.

The influence of a variety and combination of both ionic surfactants and different chain lengths of the polyelectrolyte poly(ethylene glycol) (PEG) on the performance characteristics (with particular reference to signal response) of a homogeneous, latex agglutination immunoassay was investigated. The test analyte was human serum C-reactive protein (CRP), and the antibody reagent consisted of a sheep polyclonal anti-CRP IgG fraction covalently coupled to 50-nm-sized latex including a glycine-capped chloromethylstyrene shell. The amount and rate of immunoagglutination was monitored turbidimetrically after sample addition. It was found that 2.5 mmol/L concentrations of the small cationic surfactant tetramethylammonium hydroxide (TMH), when present alone, substantially increased both reaction rates and sensitivity in the lower clinical ranges of CRP concentration when compared to normally used assay conditions containing PEG and the anionic detergent Gafac. The nonspecific binding (NSB) was also found to be unchanged. Evidence is presented that the TMH enhances the actual antibody--antigen interaction as opposed to the known effects of other surfactants in immunocomplex dissociation or in maintenance of colloidal stability. We suggest that the enhancement seen with TMH Could be an alternative to PEG and may provide a new means of further extending detection limits. The utility of this type of immunoassay technology could therefore be increased whenever clinically required.