A hydrogel-based versatile screening platform for specific biomolecular recognition in a well plate format

Precise determination of biomolecular interactions in high throughput crucially depends on a surface coating technique that allows immobilization of a variety of interaction partners in a non-interacting environment. We present a one-step hydrogel coating system based on isocyanate functional six-arm poly(ethylene oxide)-based star polymers for commercially available 96-well microtiter plates that combines a straightforward and robust coating application with versatile bio-functionalization. This system generates resistance to unspecific protein adsorption and cell adhesion, as demonstrated with fluorescently labeled bovine serum albumin and primary human dermal fibroblasts (HDF), and high specificity for the assessment of biomolecular recognition processes when ligands are immobilized on this surface. One particular advantage is the wide range of biomolecules that can be immobilized and convert the per se inert coating into a specifically interacting surface. We here demonstrate the immobilization and quantification of a broad range of biochemically important ligands, such as peptide sequences GRGDS and GRGDSK-biotin, the broadly applicable coupler molecule biocytin, the protein fibronectin, and the carbohydrates N-acetylglucosamine and N-acetyllactosamine. A simplified protocol for an enzyme-linked immunosorbent assay was established for the detection and quantification of ligands on the coating surface. Cell adhesion on the peptide and protein-modified surfaces was assessed using HDF. All coatings were applied using a one-step preparation technique, including bioactivation, which makes the system suitable for high-throughput screening in a format that is compatible with the most routinely used testing systems.     

Stability and Reusability of Enzyme-Linked Immunosorbent Assay (ELISA) Plates

Abstract

Due to the concern of stripping immobilized enzymes and antibodies from microtiter plates by the washing process, enzymelinked immunosorbent assay (ELISA) has been traditionally run on single-use, disposable units. This paper discusses the concept of stabilized, reusable ELISA plates. Antibody immobilized microtiter plates with three different modified polystyrene immobilizing surfaces were stored in Dulbecco's phosphate buffered saline/1% BSA/0.1% Thimerosal (DBT) and DBT with 3% sucrose (DBTS) for one month at 4°C. After treatment with the chaotrope and storage in buffer, the plates can be reused up to 4 times with 90% or greater antigenic capacity remaining. High and low binding microtiter plates were studied for reusability with anti-rabbit IgG (aRIgG) as a primary antibody. In each case, the procedure allowed repeated use of the same antibody immobilized plates for ELISA without re-immobilizing the antibody or loss of antigenicity. Immobilized ELISA plates were stable for one month if stored in DBTS at 4°C.     

Direct hapten-linked competitive inhibition enzyme-linked immunosorbent assay (CIELISA) for the detection of O-pinacolyl methylphosphonic acid

Immunoassay detection of O-pinacolyl methylphosphonic acid (PMPA) employing direct coating of N-2-aminoethyl-O-pinacolyl methylphosphonate (hapten B) on microtiter plates is reported. Coating was achieved by covalently linking hapten B to a glutaraldehyde (GA) polymer network directly bound to the polystyrene (PS) surface of a standard 96-well microtiter plate. 4-(2-(O-Pinacolylmethylphosphoryl amino)ethyl amino)-4-oxobutanoic acid (hapten A)-ovalbumin (OVA) conjugate served as the coating antigen for comparison with direct hapten B-coated plates in the CIELISA format. The developed assay employing direct hapten B coated plates demonstrated enhanced sensitivity with the IC(50) value for PMPA being 0.027 μg mL(-1). The assay could detect PMPA even at the concentration of 0.006 μg mL(-1). The mean recovery of standard PMPA (spiked in water) was found to be 83.7%.     

Combinatorial Low‐Volume Synthesis of Well‐Defined Polymers by Enzyme Degassing

The synthesis of well‐defined polymers in a low‐volume, combinatorial fashion has long been a goal in polymer chemistry. Here, we report the preparation of a wide range of highly controlled homo and block co‐polymers by Enz‐RAFT (enzyme‐assisted reversible addition–fragmentation chain transfer) polymerization in microtiter plates in the open atmosphere. The addition of 1 μm glucose oxidase (GOx) to water/solvent mixtures enables polymerization reactions to proceed in extremely low volumes (40 μL) and low radical concentrations. This procedure provides excellent control and high conversions across a range of monomer families and molecular weights, thus avoiding the need to purify for screening applications. This simple technique enables combinatorial polymer synthesis in microtiter plates on the benchtop without the need of highly specialized synthesizers and at much lower volumes than is currently possible by any other technique.     

Synthesis of sugar arrays in microtiter plate

1,3-Dipolar cycloadditions between azides and alkynes were exploited to attach oligosaccharides to a C(14) hydrocarbon chain that noncovalently binds to the microtiter well surface. Synthesis of sugar arrays was performed on a micromolar scale in situ in the microtiter plate. As a model study, the beta-galactosyllipid 5 was displayed on a 4-micromol scale. Formation of product was confirmed via ESI-MS, and the yield was determined via chemical and biological assays. Several complex carbohydrates (6-16) were also displayed in microtiter plates and successfully screened with various lectins. Moreover, sialyl Lewis x (17) was synthesized via the enzymatic fucosylation of a precursor displayed in the plate. Studies on inhibition of this biotransformation have been carried out, and the IC(50) value found for the known inhibitor 20 was consistent with previous studies in solution.     

Saccharide display on microtiter plates

New insight into the importance of carbohydrates in biological systems underscores the need for rapid synthetic and screening procedures for them. Development of an organic synthesis-compatible linker that would attach saccharides to microtiter plates was therefore undertaken to facilitate research in glycobiology. Galactosyllipids containing small, hydrophobic groups at the anomeric position were screened for noncovalent binding to microtiter plates. When the lipid component was a saturated hydrocarbon between 13 and 15 carbons in length, the monosaccharide showed complete retention after aqueous washing and could be utilized in biological assays. This alkyl chain was also successfully employed with more complex oligosaccharides in biological assays. In light of these findings, this method of attachment of oligosaccharides to microtiter plates should be highly efficacious to high-throughput synthesis and analyses of carbohydrates in biological assays.     

Inverted open microwells for cell trapping, cell aggregate formation and parallel recovery of live cells

The inverted open microwell is a novel microstructure supporting isolation and trapping of cells, analysis of cell-cell and cell-molecule interactions and functional cell sorting. This work introduces the inverted open microwell concept, demonstrating successful isolation of K562 cells in 75 μm microwells fabricated on a flexible printed circuit board substrate, and recovery of viable cells onto standard microtiter plates after analysis and manipulation. Dielectrophoresis (DEP) was used during the delivery phase to control cell access to the microwell and force the formation of cell aggregates so as to ensure cell-cell contact and interaction. Cells were trapped at the air-fluid interface at the bottom edge of the open microwell. Once trapped, cells were retained on the meniscus even after DEP de-activation and fluid was exchanged to enable perfusion of nutrients and delivery of molecules to the microwell, as demonstrated by a calcein-staining protocol performed in the microsystem. Finally, cell viability was assessed on trapped cells by a calcein release assay and cell proliferation was demonstrated after multiple cells had been recovered in parallel onto standard microtiter plates.     

一种新型分子印迹聚合物基的化学发光阵列传感器

建立了一种分子印迹-化学发光阵列传感器测定甘氨酸的新方法. 该方法以甘氨酸为模板分子, 合成了分子印迹聚合物微球, 将该聚合物微球固定在96孔板上, 用它来识别丹磺酰氯标记的甘氨酸(Dns-Gly). 最后加入化学发光试剂(TCPO-H2O2-咪唑), 测量相对化学发光强度定量检测甘氨酸. 在最佳试验条件下, 相对化学发光强度和甘氨酸的浓度在0.2～60 μmol/L范围内成良好的线性关系, 相关系数为r＝0.9972, 方法的检出限为0.07 μmol/L, 对1 μmol/L甘氨酸溶液进行11次平行测定, 相对标准偏差为3.3% (n＝11). 由于以甘氨酸为模板分子合成出来的分子印迹聚合物空腔比较小, 避免了非特异性吸附, 使它在识别丹磺酰氯标记的甘氨酸时特异性、响应速度和灵敏度都有所增强.     

Direct hapten coated immunoassay format for the detection of atrazine and 2,4-dichlorophenoxyacetic acid herbicides

A novel immunoassay format employing direct coating of small molecular hapten on microtiter plates is reported for the detection of atrazine and 2,4-dichlorophenoxyacetic (2,4-D). In this assay, the polystyrene surface of microtiter plates was first treated with an acid to generate -NO₂ groups on the surface. Acid treated plates were further treated with 3-aminoprpyltriethoxysilane (APTES) to functionalize the plate surface with amino groups for covalent linkage to small molecular hapten with carboxyl groups. The modified plates showed significantly high antibody binding in comparison to plates coated with hapten-carrier protein conjugates and presented excellent stability as a function of the buffer pH and reaction time. The developed assay employing direct hapten coated plates and using affinity purified atrazine and 2,4-D antibodies demonstrated very high sensitivity, IC₅₀ values for atrazine and 2,4-D equal to 0.8 ng mL⁻¹ and 7 ng mL⁻¹, respectively. The assay could detect atrazine and 2,4-D levels in standard water samples even at a very low concentration upto 0.02 and 0.7 ng mL⁻¹ respectively in the optimum working range between 0.01 and 1000 ng mL⁻¹ with good signal reproducibility (p values: 0.091 and 0.224 for atrazine and 2,4-D, respectively). The developed immunoassay format could be used as convenient quantitative tool for the sensitive screening of pesticides in samples.     

A sensitive immunoassay based on direct hapten coated format and biotin-streptavidin system for the detection of chloramphenicol

A novel enzyme liked immunosorbent assay (ELISA) was developed for the detection of chloramphenicol (CAP). In this assay, the small molecular hapten (Hap) was directly coated on the surface of microtiter plates and biotin-streptavidin system (BSAS) was employed to improve the sensitivity of immunoassay (BSAS-direct Hap coated ELISA). The surface of microtiter plates was treated with glutaraldehyde (GA) polymer network to introduce aldehyde group, which was used to cross-link with amino group of CAP. Compared with conventional ELISA (the plates were coated with Hap-carrier protein conjugates), the modified plates presented significantly high antibody and antigen (Ab-Ag) affinity and showed excellent stability. And then the biotinylated monoclonal antibody (mAb) and HRP-labeled streptavidin were employed in this assay for amplification of signals. The sensitivity of BSAS-direct Hap coated ELISA was increased by approximately 20-folds and the stability was also improved greatly compared to conventional ELISA. Its 50% inhibition concentration (IC₅₀) for CAP was 10.5 ng mL⁻¹ and the limit of detection (LOD) was 0.2 ng mL⁻¹ after optimization of reaction conditions. To our knowledge, this was one of the most sensitive immunoassay for CAP yet reported. In sample analysis, the results of CAP detected by this assay were in accordance with which obtained by conventional ELISA and high performance liquid chromatography (HPLC). Therefore, it is an attractive alternative compared to conventional immunoassays in routine supervision for residue detection in food and environment.     

Sensing of oxygen in microtiter plates: a novel tool for screening drugs against pathogenic yeasts

Most antibiotics were discovered via their inhibition of growth of target organisms. However, yeasts in particular have the capability to adapt metabolic pathways to the availability of nutrients e.g. yeasts can easily switch between respiratory and fermentative pathways in response to oxygen concentration, or can even use both simultaneously. Thus, we cultivated S. cerevisiae BY4741 and C. albicans 1386 in microtiter plates with integrated oxygen sensors to characterize the availability of oxygen for the organisms and to detect influences of fungicides on the oxygen consumption rates. The relevance of the respiratory pathway was indicated by the almost total consumption of oxygen during the first 1–3 h of the cultivation in the microtiter plates, when an increase in turbidity could hardly be seen. Moreover, the sensitivity of S. cerevisiae to inhibitors of the respiratory chain, such as myxothiazol, could be detected via a reduced oxygen consumption rate, whereas no inhibition of growth was observed. Thus, not only was the sensitivity of the test organism for the test compound detectable, but the affected pathway was also highlighted. Other compounds, such as pyrrolnitrin and ambruticin VS-3, inhibited growth of C. albicans 1386 and of S. cerevisiae (only pyrrolnitrin), which was additionally observed as reduced oxygen consumption rates. Thus, the determination of oxygen in microtiter plates via fluorescent dyes is a versatile supplement to standard growth inhibition tests.     

Chemiluminescent optical fiber immunosensor for the detection of anti-West Nile virus IgG

An ELISA-based optical fiber methodology developed for the detection of anti-West Nile virus IgG antibodies in serum was compared to standard colorimetric and chemiluminescent ELISA based on microtiter plates. Colorimetric ELISA was the least sensitive, especially at high titer dilutions. The fiber-optic immunosensor based on the same ELISA immunological rationale was the most sensitive technique.     

Analytical performance of luminescent immunoassays of different format for serum daidzein analysis

Two sensitive competitive-type solid-phase immunoassays for serum daidzein analysis have been developed and optimized. The first is a chemiluminescent enzyme immunoassay that uses black polystyrene microtiter wells in which daidzein-specific antibodies raised in rabbits are immobilized and a daidzein derivative is coupled to horseradish peroxidase (HRP) as a label. The HRP activity of the antibody-bound tracer is measured with an enhanced chemiluminescent system (luminol/H2O2/enhancer). The second immunoassay is based on the use of bovine serum albumin-daidzein derivative immobilized on microtiter plates and a secondary anti-rabbit IgG-Fc fragment conjugated with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Formation of the complex Eu3+-BCPDA enables time-resolved fluorescence-mode detection of the amount of antibody bound to the immobilized antigen. Both methods fulfilled all the requirements of accuracy and precision. The detection limit was the same for each method, 10 pg/well; this is better than that of other immunoassays. The specificity of the two methods was different, because of their competitive-type mechanisms. The performance of the chemiluminescence method is better, because the cross-reactivity of the main interfering compound (genistein) was 5%, compared with 25% for the time-resolved fluoroimmunoassay.     

Gold nanoparticles catalyzed chemiluminescence immunoassay for detection of herbicide 2,4-dichlorophenoxyacetic acid

We present a novel immunoassay format utilizing the catalytic properties of gold nanoparticles in the luminol-silver nitrate-gold nanoparticle based chemiluminescence (CL) system for the detection of widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Highly sensitive anti-2,4-D antibody was produced and conjugated with gold nanoparticles of various sizes. In the present assay format, employing a competitive inhibition approach, a well-characterized hapten-protein conjugate (2,4-D-BSA) was used to coat the microtiter plates. The analyte (2,4-D) was pre-incubated with anti-2,4-D antibody labeled with gold nanoparticles and added to each well of the microtiter plate. The gold label triggered the reaction between luminol and silver nitrate generating a luminescence signal at 425 nm. Under the optimized conditions, the CL based immunoassay showed the detection limit of 2,4-D in standard water samples around 3 ng mL(-1). The CL based immunoassay format, based on gold nanoparticles as a catalyst, could be used as a fast screening methodology (<30 min) for pesticide detection.     

Analytical performance of luminescent immunoassays of different format for serum daidzein analysis

Two sensitive competitive-type solid-phase immunoassays for serum daidzein analysis have been developed and optimized. The first is a chemiluminescent enzyme immunoassay that uses black polystyrene microtiter wells in which daidzein-specific antibodies raised in rabbits are immobilized and a daidzein derivative is coupled to horseradish peroxidase (HRP) as a label. The HRP activity of the antibody-bound tracer is measured with an enhanced chemiluminescent system (luminol/ H₂O₂/enhancer). The second immunoassay is based on the use of bovine serum albumin–daidzein derivative immobilized on microtiter plates and a secondary anti-rabbit IgG-Fc fragment conjugated with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Formation of the complex Eu³⁺-BCPDA enables time-resolved fluorescence-mode detection of the amount of antibody bound to the immobilized antigen. Both methods fulfilled all the requirements of accuracy and precision. The detection limit was the same for each method, 10 pg/ well; this is better than that of other immunoassays. The specificity of the two methods was different, because of their competitive-type mechanisms. The performance of the chemiluminescence method is better, because the cross-reactivity of the main interfering compound (genistein) was 5%, compared with 25% for the time-resolved fluoroimmunoassay.     

The radiation refinement of polystyrene surface applied in immunoanalysis

Polystyrene, material frequently used for ELISA microtiter plates, was modified by gamma irradiation in order to enhance the passive absorption binding capacity of immunoglobuline E. The dependences of this capacity increase on dose and irradiation conditions (atmosphere, presence of water) permitted the optimization this treatment of plates up to the technological scale. However the increase of passive absorption in the case of low-molecular weight synthetic peptides, such as anti-EBNA, was not sufficient and the reason for this remains to be clarified.

Radiation grafting made it possible to fix the hydrazide functional group onto the polysterene. It offers the possibility to of binding the proteins, via covalent linkage, with the carbonyl group formed by the oxidation of carbohydrates included in the protein molecule.     

Quantitative turbidity assay for lipolytic enzymes in microtiter plates

A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5–1 % tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1 % tributyrin emulsion in 2 % agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20–60 ng/well which is equivalent to 30 μU/well for T. lanuginosus lipase.

Covalent display of oligosaccharide arrays in microtiter plates

A covalent array for the display of complex oligosaccharides in microtiter plates has been developed. This strategy is conducive to the display of carbohydrates to proteins of interest such as lectins and antibodies, including the broadly neutralizing antibody 2G12 against HIV envelope oligomannose and can be cleaved from the surface for further characterization by mass spectrometry. The system was used to probe the multivalent interaction of 2G12 with an optimal epitope (Kd 0.1 muM).     

Parallel processing of microwave-assisted organic transformations

Microwave-assisted organic synthesis is an enabling technology that has been exploited for a variety of applications including medicinal chemistry/drug discovery projects where speed is often a critical factor. In this review, applications of microwave-assisted organic synthesis employing a parallel processing regime are summarized. Examples include parallel synthesis in domestic microwave ovens, the use of specialized multivessel rotors and microtiter plates in dedicated multimode microwave reactors, and applications of SPOT synthesis on cellulose matrices.     

Linked parallel synthesis and MTT bioassay screening of substituted chalcones

A 644-membered library of chalcones was prepared by parallel synthesis using the Claisen-Schmidt base-catalyzed aldol condensation of substituted acetophenones and benzaldehydes. The cytotoxicity of these chalcones was conveniently determined upon the crude products directly in 96-well microtiter test plates by the conventional MTT assay. This method revealed seven chalcones of IC(50) less than 1 microM of which 4'-hydroxy-2,4,6,3'-tetramethoxychalcone (5a) was the most active [IC(50) (K562), 30 nM]; it causes cell cycle arrest at the G(2)/M point and binds to tubulin at the colchicine binding site.