Detection of pesticide residues using an immunodevice based on negative dielectrophoresis

The detection of atrazine using a novel optical immunosensing technique based on negative dielectrophoresis (n-DEP) in microfluidic channels is described. Atrazine is a toxic triazine herbicide within the most frequently used. Polystyrene microparticles (6 microm diameters) modified with bovine serum albumin conjugated with atrazine (atrazine-BSA) were manipulated and captured when subjected to intense n-DEP electric fields. Specifically, particles were trapped when AC voltages with amplitudes of 10 V(peak) and frequencies over 1 MHz were applied to the electrodes. The immunological reaction occurring on the particles for detecting atrazine is based on an indirect competitive assay using a secondary anti-mouse immunogloburin G (IgG) antibody labeled with fluorescein isothiocyanate. The microfluidic device, with three-dimensional microelectrodes, was fabricated comprising two caged areas, allowing two simultaneous measurements inside the same microfluidic channel. The performance of this n-DEP immunosensing technique was evaluated using wine samples. The immunodevice showed a limit of detection for atrazine in buffer samples of 0.11 microgL(-1) and in pre-treated wine samples of 6.8 microg L(-1); these detection limits are lower than the maximum residue level (MRL) established by the European Community for residues of this herbicide in wine (50 microg L(-1)). This methodology offers great promise for rapid, simple, cost effective, and on-site analysis of biological, foods and beverages, and environmental samples.     

Rapid and separation-free sandwich immunosensing based on accumulation of microbeads by negative-dielectrophoresis

We report here a rapid and separation-free immunoassay using a dielectrophoresis (DEP) device consisting of an interdigitated microarray (IDA) electrode and a polydimethylsiloxane (PDMS) substrate. On applying an AC voltage to the IDA in a negative-DEP (n-DEP) frequency region, goat anti-mouse immunoglobulin (anti-mouse IgG)-immobilized microbeads moved to the surface of the PDMS substrate placed above the IDA. The microbeads accumulated at designated areas of the PDMS surface that had been precoated with anti-mouse IgG. When the fluorescence microbeads bearing anti-mouse IgG were suspended in an analyte (mouse IgG) solution, the microbeads trapped the analyte to form immunocomplexes on microbeads. The microbeads reacted with mouse IgG accumulated and were captured at the designated areas of the PDMS surface via an antibody-antigen-antibody (sandwich) reaction. The captured microbeads were detected selectively by fluorescence measurements at the focused designated areas, regardless of the presence of uncaptured microbeads suspended in solution. Thus, the separation and washing-out steps usually required for conventional immunoassay are eliminated in the presented procedure. Since the formation of the sandwich structures was accelerated significantly by n-DEP, a period as short as 30s was sufficient to detect the immunoreaction at the surface. The fluorescence intensity of the captured microbeads at the designated areas increased with analyte concentration in the range 0.01-10ng/mL. The present procedure therefore yields a rapid, sensitive, and separation-free immunoassay in a simple device.     

Immunoassay Using Microfluid Filters Constructed by Deep X-Ray Lithography

Microfluid filters were fabricated, which possessed 2,100 cylindrical through-bores (φ40 μm) in 200 μm-thickness polymethylmethacrylate (PMMA) sheets (φ3 mm), by deep X-ray lithography using synchrotron radiation. To evaluate the microfluid filters as a device for an immunoassay, we bound the goat anti-mouse immunogloblin G (IgG) antibody to the surface of the filters, and set the filters between reaction vessels stacked vertically in a microreactor. An enzyme-linked immunosorbent assay (ELISA) of mouse IgG using the goat anti-mouse IgG/horseradish-peroxidase (HRP) conjugate indicated that mouse IgG could be quantitatively detected in the range of 0–100 ng/ml, demonstrating the applicability of vertical microfluidic operation to the immunoassay.     

Immunoassay using microfluid filters constructed by deep X-ray lithography

Microfluid filters were fabricated, which possessed 2,100 cylindrical through-bores (psi 40 microm) in 200 microm-thickness polymethylmethacrylate (PMMA) sheets (psi 3 mm), by deep X-ray lithography using synchrotron radiation. To evaluate the microfluid filters as a device for an immunoassay, we bound the goat anti-mouse immunoglobulin G (IgG) antibody to the surface of the filters, and set the filters between reaction vessels stacked vertically in a microreactor. An enzyme-linked immunosorbent assay (ELISA) of mouse IgG using the goat anti-mouse IgG/horseradish-peroxidase (HRP) conjugate indicated that mouse IgG could be quantitatively detected in the range of 0-100 ng/ml, demonstrating the applicability of vertical microfluidic operation to the immunoassay.     

Highly sensitive enzyme immunoassay for beta-nerve growth factor (NGF): a tool for measurement of NGF level in rat serum

A sensitive two-site enzyme immunoassay (EIA) system was established for mouse beta nerve growth factor (NGF) isolated from mouse submaxillary gland. Our EIA system is based on the sandwiching of antigen between anti-mouse beta NGF antibody IgG coated on a polystyrene plate and biotinylated anti-mouse beta NGF antibody IgG. The bound antibody complex was quantified with streptavidin linked-beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). With this system NGF concentrations as low as 0.02 pg/well (corresponding to 8 x 10(-19) mol) could be measured reproducibly. The sensitivity of this EIA system permitted the quantification of endogenous immunoreactive beta NGF in rat serum. The mean level in serum of male rats (153.2 pg/ml) was found to be almost the same as that of female rats (127.6 pg/ml).     

Microfluidic heavy metal immunoassay based on absorbance measurement

A simple and rapid flow-based multioperation immunoassay for heavy metals using a microfluidic device was developed. The antigen-immobilized microparticles in a sub-channel were introduced as the solid phase into a main channel structures through a channel flow mechanism and packed into a detection area enclosed by dam-like structures in the microfluidic device. A mixture of a heavy metal and a gold nanoparticle-labeled antibody was made to flow toward the corresponding metal through the main channel and make brief contact with the solid phase. A small portion of the free antibody was captured and accumulated on the packed solid phase. The measured absorbance of the gold label was proportional to the free antibody portion and, thus, to the metal concentration. Each of the monoclonal antibodies specific for cadmium-EDTA, chromium-EDTA, or lead-DTPA was applied to the single-channel microfluidic device. Under optimized conditions of flow rate, volume, and antibody concentration, the theoretical (antibody K(d)-limited) detection levels of the three heavy metal species were achieved within only 7 min. The dynamic range for cadmium, chromium, and lead was 0.57-60.06 ppb, 0.03-0.97 ppb, and 0.04-5.28 ppb, respectively. An integrated microchannel device for simultaneous multiflow was also successfully developed and evaluated. The multiplex cadmium immunoassay of four samples was completed within 8 min for a dynamic range of 0.42-37.48 ppb. Present microfluidic heavy metal immunoassays satisfied the Japanese environmental standard for cadmium, chromium and, lead, which provided in the soil contamination countermeasures act.     

Latex immunoagglutination assay for a vasculitis marker in a microfluidic device using static light scattering detection

We have developed a microfluidic immunoassay device using fiber optics to detect static light scattering (SLS) of latex microsphere agglutination. A 400-mum silica fiber was used to deliver blue light emitting diode (LED) or red laser light sources. A miniature, portable spectrometer was used to measure forward light scattering intensity collected by the same type of multi-mode fiber. To first show feasibility, anti-mouse IgG were used as target biomolecules and highly carboxylated polystyrene latex microspheres (510 nm) coated with mouse IgG were used as probes. Next, we tested for the vasculitis marker, anti-PR3, using the same type of microspheres coated with PR3 proteins. No false negatives or positives were observed. A limit of detection (LOD) of 50 ng mL(-1) was demonstrated for the vasculitis marker, anti-PR3. (Plasma samples from patients with vasculitis exhibited anti-PR3 at a median level of 380 ng mL(-1).) The optical detection system works without any fluorescence or chemiluminescence markers. The entire system proposed here is cost effective, small in size, and re-usable with simple rinsing. This may eventually lead to a portable, low-cost, re-useable, microfluidic, point of care immunoassay device.     

Sensitivity enhancement of surface plasmon resonance biosensor with colloidal gold

We enhanced the sensitivity of surface plasmon resonance biosensor by the conversion of the real-time direct binding immunoassay into the sandwich immunoassay, in which colloidal gold particles coated with anti-mouse IgG was used. By the immobilization of anti-mouse IgG onto the carboxymethyl dextran surface of thin gold film, the direct binding of analyte (mouse IgG) onto the sensor chip, and the injection of colloidal gold particles coated with antimouse IgG, about 100 times of sensitivity enhancement was obtained. This result suggests that nanoparticles, which has a high refractive index, homogeneous ultrafine structure and capability of size control, would be applicable for the detection of very small quantity of biomaterial.     

A highly sensitive enzyme immunoassay (EIA) system for mouse epidermal growth factor (mEGF)

A highly sensitive two-site enzyme immunoassay system for mouse epidermal growth factor (mEGF) was developed, based on the sandwiching of an antigen between anti-mouse EGF IgG antibody-coated on a polystyrene bead and anti-mouse EGF Fab' antibody-linked peroxidase (horseradish peroxidase, EC. 1.11.1.7). The procedure is simple and rapid compared to a bioassay. Also, the Fab' antibody-peroxidase complex is more stable than the 125I-labeled antibody. Purified mEGF is detectable at a concentration as low as 3 pg/ml. The detection range was 0.3 to 680 pg/sample with 0.1 ml samples. Levels of immunoreactive mEGF in extracts from adult male mice well agreed with those determined by a radioimmunoassay and a radioreceptor assay. The submaxillary gland contained an extremely high concentration of EGF, while other tissues had low levels of EGF.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments

The newly developed immobilized enzyme reactors (IMERs) with proteolytic enzymes chymotrypsin, trypsin or papain were used for specific fragmentation of high molecular-mass and heterogeneous glycoproteins immunoglobulin G (IgG) and crystallizable fragment of IgG (Fc). The efficiency of splitting or digestion were controlled by RP-HPLC. The specificity of digestion by trypsin reactor was controlled by MS. IMERs (trypsin immobilized on magnetic microparticles focused in a channel of magnetically active microfluidic device) was used for digestion of the whole IgG molecule. The sufficient conditions for IgG digestion in microfluidic device (flow rate, ratio S:E, pH, temperature) were optimized. It was confirmed that the combination of IMERs with microfluidic device enables efficient digestion of highly heterogeneous glycoproteins such as IgG in extremely short time and minimal reaction volume.     

Microfluidic systems integrated with two-dimensional surface plasmon resonance phase imaging systems for microarray immunoassay

This study reports a microfluidic chip integrated with an arrayed immunoassay for surface plasmon resonance (SPR) phase imaging of specific bio-samples. The SPR phase imaging system uses a surface-sensitive optical technique to detect two-dimensional (2D) spatial phase variation caused by rabbit immunoglobulin G (IgG) adsorbed on an anti-rabbit IgG film. The microfluidic chip was fabricated by using micro-electro-mechanical-systems (MEMS) technology on glass and polydimethylsiloxane (PDMS) substrates to facilitate well-controlled and reproducible sample delivery and detection. Since SPR detection is very sensitive to temperature variation, a micromachine-based temperature control module comprising micro-heaters and temperature sensors was used to maintain a uniform temperature distribution inside the arrayed detection area with a variation of less than 0.3 degrees C. A self-assembled monolayer (SAM) technique was used to pattern the surface chemistry on a gold layer to immobilize anti-rabbit IgG on the modified substrates. The microfluidic chip is capable of transporting a precise amount of IgG solution by using micropumps/valves to the arrayed detection area such that highly sensitive, highly specific bio-sensing can be achieved. The developed microfluidic chips, which employed SPR phase imaging for immunoassay analysis, could successfully detect the interaction of anti-rabbit IgG and IgG. The interactions between immobilized anti-rabbit IgG and IgG with various concentrations have been measured. The detection limit is experimentally found to be 1 x 10(-4)mg/ml (0.67 nM). The specificity of the arrayed immunoassay was also explored. Experimental data show that only the rabbit IgG can be detected and the porcine IgG cannot be adsorbed. The developed microfluidic system is promising for various applications including medical diagnostics, microarray detection and observing protein-protein interactions.     

Modeling micropatterned antigen-antibody binding kinetics in a microfluidic chip

The reaction kinetics of antigen-antibody binding in the electrokinetically controlled microfluidic heterogeneous immunoassays has been investigated by numerical simulations. A two-dimensional computational model was employed to include the mass transport (convection and diffusion) and binding reaction between the antigen in the bulk flow and the immobilized antibody at the channel surface. The influence of the bulk velocity, the concentrations of the antibody and antigen, and the geometry of the microchips was studied for a variation of conditions and the guidance for designing of microfluidic immunoassay was provided. The model also shows that electrokinetically driven immunoassays have better reaction kinetics than pressure-driven ones, resulting from the plug-like velocity profile. Finally, a multi-patch immunoassay chip was analyzed and the reaction kinetics was optimized by rearranging the reaction patches at the channel surfaces.     

High sensitivity immunoassays using particulate fluorescent labels.

The use of polystyrene fluorescent microspheres as sensitive labels in direct-detection (not enzymatically amplified) heterogeneous equilibrium "sandwich" immunoassays in 96-well plates is described. With mouse IgG as a model antigen, a fluorescent particulate label is more sensitive than a corresponding soluble reporter. The limit of detection of mouse IgG in the multiparametrically optimized assay was 0.2 ng/ml (7.6 x 10(8) antigens/ml) for the particulate reporter and 50 ng/ml (1.9 x 10(11) antigens/ml) for the soluble reporter. The sensitivities of assays using the particulate label were dependent on the surface densities of the capture and reporter antibodies and the concentration of reporter beads. Sensitivity was improved by adding the preformed reporter antibody/fluorescent microsphere complex to trapped antigen on the well surfaces instead of sequentially adding the reporter antibody and then the fluorescent microspheres. Maximal (equilibrium) binding of the particulate reporter to captured antigen occurred after 20 h with a concentration of 1.4 x 10(9) reporter beads/ml. Thus, particulate fluorescent labels provide high sensitivity in direct-detection immunoassays.     

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.     

Monoclonal alkaline phosphatase-anti-alkaline phosphatase (APAAP) complex: production of antibody, optimization of activity, and use in immunostaining

A mouse monoclonal antibody, FMC55 (an IgG1), to alkaline phosphatase was prepared and evaluated in immunostaining. Clones producing antibody to alkaline phosphatase were selected using a micro-ELISA which identified antibodies forming active soluble complexes (APAAP) with the enzyme. Conditions that influenced the formation of the complex were investigated by using a quantitative assay in which the complex was captured by a bridging anti-mouse antibody. The ratio of FMC55 to enzyme had a major influence on the activity of the complex. Although all complexes had some activity, those that contained excess antibody had reduced ability to bind to anti-mouse antibody because of competition with excess unlabeled antibody. The optimal complex was formed with 3 micrograms of FMC55 per unit of enzyme. This complex contained neither free enzyme nor free antibody. The molecular weight by gel permeation chromatography was 600,000, giving a composition of two enzyme and two antibody molecules or one enzyme and three antibody molecules. The size of the complex was not altered by adding excess antibody or excess enzyme. Immunoblotting showed that FMC55 bound only to the Mr 140,000 homodimeric form of alkaline phosphatase. The APAAP complex was used in combination with biotin-streptavidin-peroxidase reagent to detect two antigens labeled with two different mouse monoclonal antibodies in the same tissue preparation.     

Effect of antibody modifications on its biomolecular binding as determined by surface plasmon resonance

A surface plasmon resonance (SPR)-based procedure was developed to determine the effect of antibody modifications on its biomolecular binding behavior. Mouse immunoglobulin G (IgG) was immobilized on a protein A-functionalized gold-coated SPR chip. Goat anti-mouse IgG and its various commercially available modifications (i.e., conjugated with atto 550, atto 647, tetramethylrhodamine isothiocyanate [TRITC], horseradish peroxidase [HRP], or biotin) were employed in exactly the same concentration for the detection of mouse IgG. The various modifications of goat anti-mouse IgG decreased its biomolecular binding to mouse IgG in the order of unmodified>HRP-labeled>atto 550-labeled>biotinylated>TRITC-labeled>atto 647-labeled.     

Immunoblotting of mite aeroallergens collected with an indoor Burkard air sampler

We examined the kinetics of airborne levels of mite allergen particles in a house by combined use of an indoor Burkard air sampler and immunoblotting. Airborne mite allergens collected on the Burkard sampling tape were transferred onto a nitrocellulose membrane, reacted with mouse monoclonal anti-mite allergen (Der pI) antibody, then treated with alkaline phosphatase conjugated anti-mouse IgG. Finally, the blotted allergen on the membrane was reacted with BCIP/NBT phosphatase, and purple spots visible by the naked eye were produced. The shape of the spots was observed under a microscope, and the spot area was measured by an image processor. This technique might be useful for analyzing the behavior of airborne allergen particles in indoor environments.     

Patterned Immobilization of Antibodies within Roll-to-Roll Hot Embossed Polymeric Microfluidic Channels

This paper describes a method for the patterned immobilization of capture antibodies into a microfluidic platform fabricated by roll-to-roll (R2R) hot embossing on poly (methyl methacrylate) (PMMA). Covalent attachment of antibodies was achieved by two sequential inkjet printing steps. First, a polyethyleneimine (PEI) layer was deposited onto oxygen plasma activated PMMA foil and further cross-linked with glutaraldehyde (GA) to provide an amine-reactive aldehyde surface (PEI-GA). This step was followed by a second deposition of antibody by overprinting on the PEI-GA patterned PMMA foil. The PEI polymer ink was first formulated to ensure stable drop formation in inkjet printing and the printed films were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Anti-CRP antibody was patterned on PMMA foil by the developed method and bonded permanently with R2R hot embossed PMMA microchannels by solvent bonding lamination. The functionality of the immobilized antibody inside the microfluidic channel was evaluated by fluorescence-based sandwich immunoassay for detection of C-reactive protein (CRP). The antibody-antigen assay exhibited a good level of linearity over the range of 10 ng/ml to 500 ng/ml (R² = 0.991) with a calculated detection limit of 5.2 ng/ml. The developed patterning method is straightforward, rapid and provides a versatile approach for creating multiple protein patterns in a single microfluidic channel for multiplexed immunoassays.     

Generation and evaluation of anti-mouse IgG IgY as secondary antibody

Abstract

In order to evaluate the possibility of using IgY as the secondary antibody in immunoassay, specific IgY (1: 128,000) was generated by immunizing hens with mouse serum IgG purified by protein A column. IgY was extracted from egg yolk by polyethylene glycol 6000 (PEG-6000), and further purified using protein M affinity chromatography column. The purified IgY was conjugated with horseradish peroxidase (HRP) and fluorescein?isothiocyanate (FITC), in that order. The reactivity of conjugated antibodies was evaluated by ELISA, Western blot and Immunofluorescence, demonstrating that the obtained IgY was able to conjugate with enzymes, react with mouse primary IgG antibody, and subsequently amplify the antigen-antibody signals in different immune reaction conditions, in a comparable secondary effect to conventional goat anti-mouse IgG antibody. The obtained conjugated antibodies showed high stability in broad pH ranges (4–10; >70%) and high thermostability at 37?°C for 84?h (>85%). Despite the need to further consider and evaluate the industrial standardization and production process, our data provided the primary evidence that conjugated IgY antibodies can be used as a secondary antibody for broad immunological analysis.