Enhancement of immunocomplex detection and application to assays for DNA adduct of benzo[a]pyrene

The stability of antibody and formation of immunocomplexes are essential to high-sensitivity capillary electrophoresis immunoassays (CEIA). However, little attention has been paid to enhancing or maintaining immunocomplex formation and antibody stability to improve the performance of CEIA. We report here the use of nonspecific proteins, such as bovine serum albumin (BSA) and rabbit immunoglobulin (rIgG), to enhance immunocomplex formation and to stabilize antibodies and immunocomplexes for immunoassays. Complexes between DNA adducts of benzo[a]pyrenediol epoxide (BPDE) and their antibodies were examined using capillary electrophoresis with laser-induced fluorescence detection (CE-HF). A tetramethylrhodamine (TMR)-labeled single-stranded oligonucleotide (16-mer) containing a single BPDE adduct was used as a fluorescent probe to study its immunocomplexes with a monoclonal antibody (8E11). To examine the formation of larger complexes, a TMR-labeled secondary antibody (anti-mouse), a primary antibody (mouse monoclonal antibody 5D11), and BPDE adducts in cellular DNA were used. We demonstrate that the use of nonspecific proteins stabilized the antibody and greatly enhanced the formation and stability of the immunocomplexes, resulting in substantial improvements in the detection limit (10-fold) and the reproducibility of the analysis. Another advantageous consequence of the stabilization was a 150-fold reduction of the concentration of the antibody needed for the immunoassay, resulting in reduced background and cost. We successfully applied this technique to the determination of DNA adducts of BPDE using a competitive immunoassay. The results from both small complexes (between a primary antibody and an oligonucleotide) and larger complexes (among a secondary antibody, a primary antibody, and cellular DNA) indicate that the technique can be extended to other immunoassays. We suggest that nonspecific proteins may assist the formation and stabilization of antibody-antigen complexes by maintaining the correct conformation of the antibody and antigen for optimum binding.     

Solid-phase immobilized tripod for fluorescent renewable immunoassay. A concept for continuous monitoring of an immunoassay including a regeneration of the solid phase

A new concept of immunoassay based on the use of a trifunctional reagent (tripod) and fluorescence resonance energy transfer (FRET) phenomenon is described. This procedure involves differential steps: (1) the tripod bearing (i) a fluorophore, (ii) a molecule structurally close to the target, and (iii) a linker reacts with the solid phase; (2) the solid phase is further activated with an anti-target antibody labeled with a quencher molecule, generating the decrease of the fluorophore emission via FRET; (3) FRET being distance dependent, the presence of the target by competing with the tripod for binding the quencher-labeled antibody leads to a rise of the fluorescence signal; (4) the solid phase is reactivated simply, by adding the quencher-labeled antibody. This method was evaluated in microtiter plates using the susbtance P as model while fluorescein and TAMRA were used as donor and acceptor, respectively. Results clearly illustrated the interest of the method, by allowing (i) a simple regeneration procedure, without requiring any drastic treatment, (ii) a direct fluorescence measurement onto the solid support, leading to a localized and cumulative signal, (iii) an increase of the signal when detecting the target, unlike classical competitive immunoassays, and (iv) a real-time monitoring of the competition and regeneration steps.     

Upconversion fluorescence resonance energy transfer in a homogeneous immunoassay for estradiol

We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17beta-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches.     

Reversed-phase capillary liquid chromatography coupled on-line to capillary electrophoresis immunoassays

Capillary reversed-phase liquid chromatography (RPLC) was coupled on-line to competitive capillary electrophoresis immunoassay (CEIA) to improve concentration sensitivity of the competitive CEIA and to provide a means for detecting multiple species that cross-react with antibody. A competitive CEIA for glucagon was used for demonstration of this technique. Five-microliter samples were injected onto a 4-cm-long by 50-micron-i.d. RPLC column. Sample was desorbed by gradient elution, mixed on-line with fluorescently labeled glucagon and anti-glucagon, incubated in a continuous-flow reaction capillary, and analyzed by capillary electrophoresis with flow-gated injection and laser-induced fluorescence detection. Electrophoretic analysis of the reactor stream was performed every 1.5 s, allowing nearly continuous monitoring of the RPLC separation. Preconcentration achieved by RPLC allowed improvement in the detection limit from 760 to 20 pM. Addition of the RPLC column also allowed multiple cross-reactive species to be differentiated by first separating them chromatographically and then detecting them with the immunoassay. The technique was used to measure glucagon secretion from single islets of Langerhans and to differentiate cross-reactive forms of glucagon with one assay.     

Use of excess solid-phase capacity in immunoassays: advantages for semicontinuous, near-real-time measurements and for analysis of matrix effects

A flow-based immunoassay system using solid-phase particles with high binding capacity was used for semicontinuous, near-real-time, measurement of 17beta-estradiol (E2). The high binding capacity of the solid phase was exploited to enable (i) a quantitative determination of E2 concentration, based on rate of accumulation of fluorescently labeled anti-E2 antibody on the solid phase, and (ii) the use of a single solid phase for more than a dozen competitive binding measurements. The high binding capacity of the solid phase also permitted the immobilization of a second capture antigen. Biotin was immobilized as a second antigen and used to evaluate a biotin anti-biotin system as a control for matrix effects in the E2 immunoassay. In phosphate-buffered saline, E2 could be quantified (in the range of 10-1000 pM) by using either the summation or ratio of the signals from the labeled anti-E2 and anti-biotin antibody in the presence of biotin at a constant concentration. The same referencing system was applied to estimate the matrix effects in selected environmental samples. Matrix effects that inhibited the binding of the anti-E2 antibody to the solid phase led to false positive responses, but these matrix effects could be identified and partially corrected using the response from the anti-biotin antibody.     

On-chip native gel electrophoresis-based immunoassays for tetanus antibody and toxin

By integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device, we have developed a microanalytical platform for performing electrophoresis-based immunoassays. The microfluidic immunoassays are performed by gel electrophoretic separation and quantitation of bound and unbound antibody or antigen. To retain biological activity of proteins and maintain intact immune complexes, nondenaturing polyacrylamide gel electrophoresis conditions were investigated. Both direct (noncompetitive) and competitive immunoassay formats are demonstrated in microchips. A direct immunoassay was developed for detection of tetanus antibodies in buffer as well as diluted serum samples. After an off-chip incubation step, the immunoassay was completed in less than 3 min and the sigmoidal dose-response curve spanned an antibody concentration range from 0.17 to 260 nM. The minimum detectable antibody concentration was 0.68 nM. A competitive immunoassay was also developed for tetanus toxin C-fragment by allowing unlabeled and fluorescently labeled tetanus toxin C-fragment compete to bind to a limited fixed concentration of tetanus antibody. The immunoassay technique described in this work shows promise as a component of an integrated microfluidic device amenable to automation and relevant to development of clinical diagnostic devices.     

Miniaturization of a homogeneous fluorescence immunoassay based on energy transfer using nanotiter plates as high-density sample carriers.

The miniaturization of a homogeneous competitive immunoassay to a final assay volume of 70 nL is described. As the sample carrier, disposable plastic nanotiter plates (NTP) with dimensions of 2 x 2 cm2 containing 25 x 25 wells, corresponding to approximately 15,000 wells on a traditional 96-well microtiter plate footprint, were used. Sample handling was accomplished by a piezoelectrically actuated micropipet. To reduce evaporation while pipetting the assays, the NTP was handled in a closed humid chamber and cooled to the point of condensation. To avoid washing steps, a homogeneous assay was developed that was based on energy-transfer (ET). As a model system, an antibody-based assay for the detection of the environmentally relevant compound, simazine, in drinking water was chosen. Antibodies were labeled with the long-wavelength-excitable sulfoindocyanine dye Cy5 (donor), and a tracer was synthesized by labeling BSA with a triazine derivative and the acceptor dye Cy5.5. At low analyte concentrations, the tracer was preferably bound to the antibody binding sites. As a result of the close proximity of Cy5.5 and Cy5, an efficient quenching of the Cy5 fluorescence occurred. Higher analyte concentrations led to a progressive binding of the analyte to the antibody binding sites. The increased Cy5 fluorescence was determined by using a scanning laser-induced fluorescence detector. The limit of detection (LOD), using an antibody concentration of 20 nM, was 0.32 microg/L, or 1.11 x 10(-16) mol of simazine. In comparison, the LOD of the 96-well microtiter-plate-based ET immunoassay (micro-ETIA) was 0.15 microg/L, or 1.87 x 10(-13) mol. The LOD of the optimized micro-ETIA at 1 nM IgG, was 0.01 microg/L.     

Online preconcentration by transient isotachophoresis in linear polymer on a poly(methyl methacrylate) microchip for separation of human serum albumin immunoassay mixtures

Online preconcentration of human serum albumin (HSA) and its immunocomplex with a monoclonal antibody by on-chip transient isotachophoresis is reported. An 800-fold signal enhancement was achieved following the preconcentration on standard cross-channel microchips made of poly (methyl methacrylate). Sample injection, preconcentration, and separation were done continuously and controlled solely by a sequential voltage switching program. The preconcentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution. The method was applied to microchip electrophoresis immunoassay of HSA. Baseline separation of HSA and its immunocomplex was achieved in 25 s in the first 1 cm of the microchannel. In a direct immunoassay, the minimum detectable concentration of fluorescent labeled HSA by laser-induced fluorescence detection was 7.5 pM.     

Immunoassay for phenylurea herbicides: application of molecular modeling and quantitative structure-activity relationship analysis on an antigen-antibody interaction study

An indirect competitive enzyme-linked immunosorbent assay (icELISA) for 12 phenylurea herbicides (PUHs) was established with the half-maximum inhibition concentration (IC(50)) of 1.7-920.7 μg L(-1). A method of computer-aided molecular modeling was established in quantitative structure-activity relationship (QSAR) studies to obtain a deeper insight into the PUHs' antibody interactions on how and which molecular properties of the analytes quantitatively affect the antibody recognition. A two-dimensional (2D)-QSAR model based on the Hansch equation and a hologram QSAR (HQSAR) model were constructed, and both showed highly predictive abilities with cross-validation q(2) values of 0.820 and 0.752, respectively. It was revealed that the most important impact factor of the antibody recognition was the PUHs' hydrophobicity (log P), which provided a quadratic correlation to the antibody recognition. Hapten-carrier linking groups were less exposed to antibodies during immunization; thus, groups of the analytes in the same position were generally considered to be less contributive to antibody recognition during immunoassay. But the results of substructure-level analysis showed that these groups played an important role in the antigen-antibody interaction. In addition, the frontier-orbital energy parameter E(LUMO) was also demonstrated as a related determinant for this reaction. In short, the result demonstrated that the hydrophobicity and the lowest unoccupied molecular orbital energy (E(LUMO)) of PUH molecules were mainly responsible for antibody recognition.     

Magnetic electrochemical sensing platform for biomonitoring of exposure to organophosphorus pesticides and nerve agents based on simultaneous measurement of total enzyme amount and enzyme activity

We report a new approach for electrochemical quantification of enzymatic inhibition and phosphorylation for biomonitoring of exposure to organophosphorus (OP) pesticides and nerve agents based on a magnetic bead (MB) immunosensing platform. The principle of this approach is based on the combination of MB immunocapture-based enzyme activity assay and competitive immunoassay of the total amount of enzyme for simultaneous detection of enzyme inhibition and phosphorylation in biological fluids. Butyrylcholinesterase (BChE) was chosen as a model enzyme. In competitive immunoassay, the target BChE in a sample competes with the BChE immobilized on the MBs to bind to the limited sites of anti-BChE antibody labeled with quantum dots (QD-anti-BChE), followed by stripping voltammetric analysis of the bound QD conjugate on the MBs. This assay shows a linear response over the total BChE concentration range of 0.1-20 nM. Simultaneous real time BChE activity was measured on an electrochemical carbon nanotube-based sensor coupled with a microflow injection system after immunocapture by the MB-anti-BChE conjugate. Therefore, the formed phosphorylated BChE adduct (OP-BChE) can be estimated by the difference values of the total amount of BChE (including active and OP-inhibited) and active BChE from established calibration curves. This approach not only eliminates the difficulty in screening of low-dose OP exposure (less than 20% inhibition of BChE) because of individual variation of BChE values but also avoids the drawback of the scarce availability of OP-BChE antibody. It is sensitive enough to detect 0.5 nM OP-BChE, which is less than 2% BChE inhibition. This method offers a new method for rapid, accurate, selective, and inexpensive quantification of OP-BChE and enzyme inhibition for biomonitoring of OP and nerve agent exposures.     

A general method to perform a noncompetitive immunoassay for small molecules.

A new general method to perform a noncompetitive immunoassay for low-molecular-mass analytes (less than 6000 Da) is described and checked using cortisol as a model system. The method is based on the use of a "polydentate ligand" (cortisol-poly(L-lysine) conjugate) able to block the antibody sites unoccupied by the analyte, followed by the replacement of an antibody-bound analyte by an enzyme-labeled analyte (cortisol-horseradish peroxidase), and permits the direct measurement of the analyte bound sites. The observed signal shows a near-linear correlation with the analyte concentration. The characteristics of interactions between the analyte and polydentate ligand with the specific antibody were studied to perform a preliminary evaluation of the noncompetitive immunoassay for cortisol. The noncompetitive assay was compared with a competitive immunoassay obtained under the same conditions and using the same reagents. The results of the experiments showed a lower detection limit for the noncompetitive model (0.15 ng mL-1 rather than 0.72 ng mL-1), emphasizing that the model is successful. Moreover, as the polydentate ligand is prepared from the same hapten used for the immunogen synthesis, this type of noncompetitive immunoassay appears generally applicable to all small molecules for which antibodies have been obtained.     

Homogeneous immunoassay based on two-photon excitation fluorescence resonance energy transfer

A two-photon excitable small organic molecule (abbreviated as TP-NH 2) with large two-photon absorption cross section and competitive fluorescence quantum yield was prepared, which emitted fluorescence in the visible region upon excitation at 800 nm. Using the TP-NH 2 molecule as an energy donor, a two-photon excitation fluorescence resonance energy-transfer (TPE-FRET) based homogeneous immunoassay method was proposed. The donor and the acceptor (DABS-Cl, a dark quencher) were labeled to bovine serum albumin (BSA) separately, and anti-BSA protein was determined by employing an antibody bridging assay scheme. Rabbit anti-BSA serum containing other biomolecules was intentionally used as the sample to introduce interference. A parallel assay was performed using the traditional one-photon excitation FRET model, which failed to carry out quantitative determination due to the serious background luminescence arising from those biomolecules in the sample. The TPE-FRET model showed its strong ability to overcome the problem of autofluorescence and provided satisfying analytical performance. Quite good sensitivity and wide linear range (0.05-2.5 nM) for anti-BSA protein was obtained. The results of this work suggest that TPE-FRET could be a promising technique for homogeneous assays excluding separation steps, especially in complicated biological sample matrixes.     

Genetically engineered elastin-protein A fusion as a universal platform for homogeneous, phase-separation immunoassay

A simple and universal platform for competitive phase-separation immunoassay is reported based on a fusion protein composed of a temperature-responsive elastin-like polypeptide (ELP) and the antibody-binding staphylococcal protein A (SpA). The basic principle is to take advantage of the ability of SpA to bind a variety of antibodies with high affinity, allowing simple separation of antigen-antibody complex by thermal precipitation. The resulting ELP-SpA fusion was shown to preserve the ability to reversibly precipitate as well as its high affinity toward different IgGs and IgMs. As a model system, a competitive phase-separation immunoassay based on the ELP-SpA format was established for paclitaxel (taxol) with IC(50) (20.18 nM) and the lower detection limit (2.94 nM) very similar to those reported for the ELISA format. Unlike the heterogeneous interaction in ELISA, which decreases the antibody-binding activity, the reported homogeneous immunoassay not only alleviates this problem but also enables the potential for high-throughput automation. We believe that the reported ELP-SpA fusion will find applications not only as a powerful diagnostic tool for diverse analytes but also a potential useful tool for purification and immobilization of antibody.     

Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants.

A competitive fluorescence microplate assay based on a red-shifted green fluorescent protein (rsGFP) and a blue fluorescent protein (BFP) was developed for the detection of two model peptides in the same sample. The assay employed gene fusion to prepare the fluorescently labeled peptide conjugates. Specifically, plasmids were constructed in which the genes encoding for the two small peptides (less than 12 amino acids in length) were fused to either the gene of the rsGFP or the BFP, as desired. The newly constructed plasmids were transformed into E. coli for expression of the fusion proteins. By employing the technique of gene fusion, one-to-one homogeneous populations of peptide-rsGFP or -BFP conjugates were produced. These peptide-GFP mutant conjugates exhibited the same excitation and emission spectral characteristics as the unmodified proteins. The naturally fluorescent proteins act as labels to provide sensitive dual detection of the two selected small peptides in a competitive assay format. To our knowledge, this is the first time that mutants of GFP, such as the rsGFP and BFP, have been used as quantitative labels for the development of a dual-analyte fluorescence immunoassay.     

Liquid-phase binding assay of alpha-fetoprotein using DNA-coupled antibody and capillary chip electrophoresis

An immunoassay using DNA-coupled antibody for bound/free separation in a liquid-phase binding assay format is described. Anti-alpha-fetoprotein monoclonal antibody was conjugated with DNA, mixed with alpha-fetoprotein (AFP), and incubated, and then 1 muL of the mixture was applied to capillary electrophoresis on a microchip. The DNA molecule of the antibody-DNA conjugate and the DNA-conjugated immune complex peak were detectable fluorophotometrically using intercalator dye within 90 s, whereas the Alexa-labeled antibody was detected as a broad and slower migrating peak. The electrophoretic mobility of the immune complex could be optimized for resolution and sharpness by changing the length of the DNA coupled to the antibody. The detection limit of AFP was approximately 300 pM in a sample. This immunoassay method utilizing a liquid-phase binding assay format is simple and convenient for antigen measurements on microchips.     

Quantum dot/carrier-protein/haptens conjugate as a detection nanobioprobe for FRET-based immunoassay of small analytes with all-fiber microfluidic biosensing platform

This study demonstrates the use of carrier-protein/haptens conjugate (e.g., BSA/2,4-dichlorophenoxyacetic acid, 2,4-D-BSA) for biological modification of quantum dots (QDs) for the detection of small analytes. Bioconjugated QDs, which are used as a detection nanoimmunoprobe, were prepared through conjugating carboxyl QDs with 2,4-D-BSA conjugate. Based on the principle of quantum dot-fluorescence resonance energy transfer (QD-FRET), an all-fiber microfluidic biosensing platform has been developed for investigating FRET efficiency, immunoassay mechanism and format, and binding kinetics between QD immunoprobe and fluorescence labeled anti-2,4-D monoclonal antibody. The structure of multiplex-haptens/BSA conjugate coupling to QD greatly improves the FRET efficiency and the sensitivity of the nanosensor. With a competitive detection mode, samples containing different concentrations of 2,4-D were incubated with a given concentration of QD immunoprobe and fluorescence-labeled antibody, and then detected by the all-fiber microfluidic biosensing platform. A higher concentration of 2,4-D led to less fluorescence-labeled anti-2,4-D antibody bound to the QD immunoprobe surface and, thus, a lower fluorescence signal. The quantification of 2,4-D over concentration ranges from 0.5 nM to 3 μM with a detection limit determined as 0.5 nM. The performance of the nanosensor with spiked real water samples showed good recovery, precision, and accuracy, indicating that it was less suspectable to water matrix effects. With the use of different QD nanobioprobes modified by other carrier-protein/haptens conjugates, this biosensing protocol based on QD-FRET can be potentially applied for on-site, real-time, inexpensive, and easy-to-use monitoring of other trace analytes.     

Reagentless amperometric immunosensors based on direct electrochemistry of horseradish peroxidase for determination of carcinoma antigen-125

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 +/- 1.21 s(-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 mL(-1) with a detection limit of 1.29 units mL(-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 mL(-1) CA 125 concentrations, respectively, and interassay CV of 19.8% at 8 units mL(-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.     

Microarray immunoassay for phenoxybenzoic acid using polymer encapsulated Eu:Gd2O3 nanoparticles as fluorescent labels

Currently, detection in microarray bioanalysis is based mainly on the use of organic dyes. To overcome photobleaching and spectral overlaps we applied a new type of fluorophore, crystalline europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles, as labels in immunoassay microarrays. The Eu:Gd2O3 nanoparticles synthesized by spray pyrolysis offer narrow red emission, large Stokes shift, photostable laser-induced fluorescence with a long lifetime (1 ms). The amino functionalization of the particles was achieved by poly(L-lysine) (PL) encapsulation. The formation of a stable PL shell was confirmed by TEM analysis, colloidal stability studies, and quantification of the surface reactive amino groups. The PL-encapsulated particles were covalently conjugated to antibodies and successfully applied as reporters in a competitive fluorescence microimmunoassay for phenoxybenzoic acid (PBA), a generic biomarker of human exposure to pyrethroid insecticides. Microarrays were fabricated by microcontact printing of BSA-PBA in line patterns (10 x 10 microm). Confocal fluorescence microscopy combined with internal standard (fluorescein) calibration was used for quantitative measurements. The microarray immunoassay demonstrated a limit of detection of 1.4 microg L(-1) PBA. This work suggests the potential application of lanthanide oxide nanoparticles as fluorescent probes in microarray and biosensor technology, immunodiagnostics, and high-throughput screening.     

New immunoassay platform utilizing yeast surface display and direct cell counting

In this study, we report a new immunoassay platform using yeast cell surface display. This method holds promise for very low limit of detection (LOD) and is suitable for 2-Plex antibody recognition. Instead of adopting a conventional enzyme linked immunosorbent assay (ELISA) protocol by detecting the enzymatic activities or other physicochemical properties of the labeled analytes, this approach determines the quantity of an antibody analyte by directly counting the amount of "modified" yeast cells bound with antibody on the cell surface. c-myc and hemagglutinin (HA) tags were employed as an epitope model to demonstrate our approach. This yeast surface display based cell counting immunoassay (abbreviated as YSD-CCI) for anti-c-myc has a detection limit of 0.2 ng/mL, which is about 80 times higher than that of a conventional yeast ELISA under a similar condition. Moreover, the YSD-CCI's capability for 2-Plex antibody detection was demonstrated by simultaneous detection of anti-c-myc and anti-HA using engineered yeast cells expressing intracellular enhanced green fluorescent protein (EGFP) and mCherry, respectively. This proof-of-concept study paves the way for a new ultrasensitive multiplexed immunoassay method for diagnostic applications.     

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.