Heterogeneous Assay Research Articles

ChemInform Abstract: Development of Responsive Lanthanide‐Based Magnetic Resonance Imaging and Luminescent Probes for Biological Applications

AbstractReview: 93 refs.

Identification of a Novel Fibronectin-Aggrecan Complex in the Synovial Fluid of Knees with Painful Meniscal Injury

Molecular biomarkers associated with knee pain may be useful as diagnostic modalities, prognostic indicators, and surrogate end points for therapeutic trials. The present study describes a novel complex of fibronectin and aggrecan that is present in the affected knee of patients with pain and meniscal abnormality. The present prospective study included thirty patients with knee pain, mechanical symptoms, and magnetic resonance imaging findings that were positive for a meniscal tear who chose arthroscopic partial meniscectomy after unsuccessful nonoperative management. Synovial fluid was aspirated at the time of surgery and was assayed for the fibronectin-aggrecan complex with use of a heterogeneous enzyme-linked immunosorbent assay (ELISA). The results were compared with knee aspirates from ten asymptomatic volunteers with no pain who underwent magnetic resonance imaging of the knee. The mean optical density (and standard deviation) of the fibronectin-aggrecan complex was significantly greater in synovial fluid from knees undergoing arthroscopic surgery as compared with fluid from asymptomatic controls (13.29 ± 8.48 compared with 0.03 ± 0.09; p < 0.001). The mean age in the study group was significantly greater than in control group (46.0 ± 12.6 compared with 38.5 ± 6.0 years; p = 0.02), but controlling for age did not affect the results. Post hoc, an optical density cutoff value of 0.3 distinguished the study group from the control group with 100% accuracy. A novel fibronectin-aggrecan complex is present in the synovial fluid of painful knees with meniscal abnormality. The fibronectin-aggrecan complex may prove to be useful as a clinical biomarker or therapeutic target. Further research is warranted to correlate functional outcome after surgery with the fibronectin-aggrecan complex and other cartilage biomarkers.

Fluorescent quantification of amino groups on silica nanoparticle surfaces

Functionalization of the surfaces of silica particles is often the first step in their various applications. An improved heterogeneous Fmoc-Cl fluorescent assay using an aqueous solution was developed to detect the number of amino groups on solid-phase supports. The fluorescent Fmoc-Cl method is 50-fold more sensitive than the current UV assay using an organic solvent. This method, together with the homogeneous fluorescamine and OPA assays, is used to detect amino groups on the silica particle surface. The accuracy and effect factors of these methods were examined and the assays were optimized. The results showed that the amine groups on silica particles can produce stronger fluorescence than small amine molecules in solution, because the porous structure of the particle surface is a more hydrophobic environment. The number of active amino groups that can be conjugated with biomolecules is much less than the total number of amino groups on the silica particle. Compared with physical methods, chemical assays involving direct reaction with amino groups would furnish the closest result to the number of active amino groups on the particle surface.

Rapid detection of algal toxins by microfluidic immunoassay

Herein we report fabricating a microfluidic device to monitor harmful algal blooming (HAB). The heterogeneous immuno-enzyme assay was integrated into a self-designed microfluidic chip for rapid and automatic analysis of algal toxins. The device was made from polydimethylsiloxane (PDMS) and was assembled with a home-made control system. The performance of the system was demonstrated by the detection of microcystin, saxitoxin and cylindrospermopsin, the major cyanotoxins. In one single microfluidic chip, multiple samples were controlled and analysed in a parallel manner. Under the optimal conditions, the linear range and the limit of detection of microcystins were 0-5.0 ng mL(-1) and 0.02 ng mL(-1) respectively. The total analysis time was less than 25 min. The designed device was highly automatic, more efficient and economic compared to conventional techniques.

Homogeneous assays using aptamers

Aptamers are DNA or RNA oligonucleotides that can bind with high affinity and specificity to a wide range of targets such as proteins, metal ions or pathogenic microorganisms. Soluble aptamers and aptazymes have been used as sensing elements for developing homogeneous assays in a solution phase, the whole sensing process being carried out in a homogeneous solution. Contrary to most conventional heterogeneous assays that are time-consuming and labor-intensive, aptamer-based homogeneous assays are simple, easy-to-perform, rapid and do not require immobilization nor washing steps. To our knowledge, this review is the first entirely dedicated to aptamer-based homogeneous assays. Optical detection appears as the most developed technique. Colorimetry represents the simplest sensing mode that occupies a very important position among aptamer-based assays, involving gold nanoparticle aggregation (with unmodified or aptamer-modified gold NPs), the formation of HRP-mimicking DNAzyme with hemin, dye displacement or interactions with a cationic polymer. Fluorescence that is highly sensitive offers the most developed detection mode. Aptamers can be labeled or not, to give rise to turn-on or usually less sensitive turn-off fluorescent assays. Newly reported and thus less developed non-conventional magnetic resonance imaging (MRI) and electrochemistry also recently appeared in the literature, thrombin still remains the main detected target. Homogeneous assays based on aptazyme, an aptamer sequence connected to a known ribozyme motif, are also described in this review, involving optical detection, by colorimetry or fluorescence.

Spatially Addressable Chemoselective C-Terminal Ligation of an Intein Fusion Protein from a Complex Mixture to a Hydrazine-Terminated Surface

Protein immobilization on surfaces is useful in many areas of research, including biological characterization, antibody purification, and clinical diagnostics. A critical limitation in the development of protein microarrays and heterogeneous protein-based assays is the enormous amount of work and associated costs in the purification of proteins prior to their immobilization onto a surface. Methods to address this problem would simplify the development of interfacial diagnostics that use a protein as the recognition element. Herein, we describe an approach for the facile, site-specific immobilization of proteins on a surface without any preprocessing or sample purification steps that ligates an intein fusion protein at its C-terminus by reaction with a hydrazine group presented by a surface. Furthermore, we demonstrate that this methodology can directly immobilize a protein directly from cell lysate onto a protein-resistant surface. This methodology is also compatible with soft lithography and inkjet printing so that one or more proteins can be patterned on a surface without the need for purification.

Brain Disorders in Fetal Alcohol Syndrome

Intrauterine effect of alcohol on the development of cytomorphological structure of CNS in rats was studied by heterogeneous enzyme-linked immunosorbent assay. The level of transforming growth factor-β1 (TGF-β1) in animals during pregnancy was analyzed. Pronounced damaging effect of alcohol on brain cell in the progeny of alcoholized animals was demonstrated: loosening of nerve cells and degenerative changes in the form of pyknosis and chromatolysis in the cortex, hypothalamus, and cerebellum; subtotal decrease (sometimes complete absence) of neuroendocrine granules. The level of TGF-β1 was significantly increased in alcoholized pregnant females, which can attest to defects of the receptor apparatus of the target cells in both females and the progeny. Thus, the observed peculiarities of TGF-β1 expression are comparable to morphological changes in the brain and can be extrapolated to similar processes in humans (fetal alcohol syndrome).

Dry-Reagent Double-Monoclonal Assay for Cystatin C

Cystatin C is a low molecular weight cysteine proteinase inhibitor whose plasma or serum concentrations have been shown to be better correlated with glomerular filtration rate than serum creatinine concentrations. Routine assays for cystatin C are based on use of polyclonal antibodies and immunoturbidimetric and nephelometric designs. This study aimed to develop a double-monoclonal immunoassay for cystatin C. We tested functionality of 42 2-site antibody combinations involving 7 monoclonal antibodies with recombinant and plasma cystatin C. We developed a heterogeneous assay using 2 antibodies selected to give the best analytical performance. The assay used a dilution step and was based on a dry-reagent, all-in-one immunoassay concept with time-resolved fluorometry. The assay was performed on an automated immunoanalyzer in single wells that contained all the required assay components. We used heparin-derived plasma samples for methodological evaluation of the assay. From a relative epitope map involving 7 cystatin C-specific antibodies, we selected a pair of antibodies for a 2-site sandwich-type dry-reagent assay. Total assay time was 15 min, and 10 microL of a 100-fold diluted sample was used. The analytical detection limit (background + 3SD) and functional detection limit (CV 20%) were 0.01 mg/L and 0.02 mg/L, respectively. Within-run and total assay imprecision were <4.7% and <5.6% (at 0.84-3.2 mg/L), respectively, and plasma recoveries of added cystatin C were 94%-110%. Regression analysis with the Roche particle-enhanced immunoturbidimetric method yielded the following (SD): slope, 1.391 (0.029); y-intercept, -0.152 (0.045) mg/L; S(y logical or, bar belowx)=0.294 mg/L (n=131). The developed assay enables rapid and reliable measurement of cystatin C.

Rotationally Induced Hydrodynamics: Fundamentals and Applications to High-Speed Bioassays

Bioassays are indispensable tools in areas ranging from fundamental life science research to clinical practice. Improving assay speed and levels of detection will have a profound impact in all of these areas. We recently developed a rapid, sensitive format for immunosorbent assays that expedites antigen mass transport by rotating the capture substrate. This review outlines the theoretical foundation of rotationally induced hydrodynamics and its application in heterogeneous assays. We describe a general solution that solves the rates of immunoreactions on rotating capture substrates, taking into account both diffusion and the rate of reaction between antibody and antigen. The general solution applies to a wide range of rotation rates, including mass transport-limited to reaction rate-limited assays, and is validated experimentally. We discuss several applications that demonstrate how immunoassays can be tailored to increase speed as well as lower the limit of detection of viral particles, pathogens, toxins, and proteins.

Hapten synthesis and antibody production for the development of a melamine immunoassay

The incorporation of melamine into food products is banned but its misuse has been widely reported in both animal feeds and food. The development of a rapid screening immunoassay for monitoring of the substance is an urgent requirement. Two haptens of melamine were synthesized by introducing spacer arms of different lengths and structures on the triazine ring of the analyte molecular structure. 6-Aminocaproic acid and 3-mercaptopropionic acid were reacted with 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) to produce hapten 1 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid], respectively. The molecular structures of the two haptens were identified by 1H nuclear magnetic resonance spectrometry, mass spectrometry and infrared spectrometry. An immunogen was prepared by coupling hapten 1 to bovine serum albumin (BSA). Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to evaluate homogeneous and heterogeneous assay formats. The results showed that polyclonal antibodies with high titers were obtained, and the heterogeneous immunoassay format demonstrated a better performance with an IC 50 of 70.6 ng mL −1, a LOD of 2.6 ng mL −1 and a LOQ of 7.6 ng mL −1. Except for cyromazine, no obvious cross-reactivity to common compounds was found. The data showed that the hapten synthesis was successful and the resultant antisera could be used in an immunoassay for the rapid and sensitive detection of this banned chemical.

Homogeneous GTP Binding Assay Employing QRET Technology

Functional cell signaling assays have become important tools for measuring ligand-induced receptor activation in cell-based biomolecular screening. Guanosine-5'-triphosphate (GTP) is a generic signaling marker responsible for the first intracellular signaling event of the G-protein-coupled receptors (GPCRs). [(35)S]GTPgammaS binding assay is the classical well-established method for measuring agonist-induced G-protein activation requiring a separation of free and bound fractions prior to measurement. Here a novel, separation-free, time-resolved fluorescence GTP binding assay has been developed based on a non-fluorescence resonance energy transfer (FRET) single-label approach and quenching of a nonbound europium-labeled, nonhydrolyzable GTP analog (Eu-GTP). The quenching resonance energy transfer (QRET) method relies on the use of Eu-GTP, providing a time-resolved fluorescent detection as an alternative to the radiolabel [(35)S]GTPgammaS assay. Upon activation of recombinant human alpha(2A)-adrenoceptors (alpha(2A)-AR) expressed in Chinese hamster ovary cells, guanosine-5'-diphosphate is released from the alpha-subunit of Gi-proteins, enabling the subsequent binding of Eu-GTP. Activation of alpha(2A)-AR with 5 different alpha(2)-AR agonists was measured quantitatively using the developed QRET GTP assay and compared to [(35)S]GTPgammaS and heterogeneous Eu-GTP filtration assays. Equal potencies and efficacy rank orders were observed in all 3 assays but with a lower signal-to-background ratio and increased assay variation in the QRET assay compared to the Eu-GTP filtration and the nonhomogeneous [(35)S]GTPgammaS binding assays.

Peptide reporters of kinase activity in whole cell lysates

Kinase assays are used to screen for small-molecule inhibitors that may show promise as targeted pharmaceutical therapies. Using cell lysates instead of purified kinases provides a more accurate estimate of inhibitor sensitivity and selectivity in a biological setting. This review summarizes the range of homogeneous (solution-phase) and heterogeneous (solid-supported) formats available for using peptide substrates to monitor kinase activities in cell lysates. With a focus on heterogeneous kinase assays, the peptide substrate Abltide is used as a model to optimize presentation geometries and the modular arrangement of short sequences for kinase recognition. We present results from peptides immobilized on two- and three-dimensional surfaces such as hydrogels on 96-well plates and glass slides, and fluorescent Luminex beads. We discuss methods to increase assay sensitivity using chemifluorescent ELISAs, antibody-based recognition, and label-free mass spectrometry. Monitoring the activity of specific kinases in cell lysates presents challenges that can be overcome by manipulating peptide substrates to optimize assay conditions. In particular, signal-to-background ratios were improved by (1) adding long branched hydrophilic linkers between the substrate and the surface, (2) changing the orientation of peptides relative to the surface, and (3) including peptide ligands in cis or in trans to recruit kinases to the surface. By improving the accessibility of immobilized peptide substrates to kinases in solution, the apparent rate of phosphorylation increased and assays were more sensitive to changes in endogenous kinase activities. These strategies can be generalized to improve the reactivity of most peptide substrates used in heterogeneous kinase assays with cell lysates.

Development of Responsive Lanthanide-Based Magnetic Resonance Imaging and Luminescent Probes for Biological Applications

Lanthanide complexes have unique chemical characteristics compared with typical organic complexes, and have recently attracted much interest because of the expanding need for new bioanalytical sensors. For example, magnetic resonance imaging (MRI) permits noninvasive three-dimensional imaging inside opaque organisms, and gadolinium ion (Gd(3+)) complexes have become important tools as MRI contrast agents. However, most of them are nonspecific, and report solely on anatomy. Therefore, responsive MRI contrast agents, so-called "smart" MRI contrast agents whose ability to relax water protons is greatly enhanced by recognition of a particular biomolecule, have great potential for elucidating biological phenomena. On the other hand, lanthanide complexes such as europium (Eu(3+)) and terbium (Tb(3+)) complexes have excellent luminescence properties for biological applications, i.e., long luminescence lifetime of the order of milliseconds and a large Stoke's shift of >200 nm. Their long-lived luminescence is especially suitable for time-resolved measurements, because the interference from short-lived background fluorescence and scattered light rapidly decays to a negligible level after a pulse of excitation light is applied, and the emitted light can be collected after an appropriate delay time. These luminescent lanthanide complexes have already found commercial use as highly sensitive luminescent probes in heterogeneous and homogeneous assays. This paper reviews our research on the design and synthesis of responsive lanthanide-based MRI and luminescent probes for advanced bioimaging.

Feasibility Evaluation of 3 Automated Cellular Drug Screening Assays on a Robotic Workstation

This study presents the implementation and optimization of 3 cell-based assays on a TECAN Genesis workstation-the Caspase-Glo 3/7 and sulforhodamine B (SRB) screening assays and the mechanistic Caco-2 permeability protocol-and evaluates their feasibility for automation. During implementation, the dispensing speed to add drug solutions and fixative trichloroacetic acid and the aspiration speed to remove the supernatant immediately after fixation were optimized. Decontamination steps for cleaning the tips and pipetting tubing were also added. The automated Caspase-Glo 3/7 screen was successfully optimized with Caco-2 cells (Z' 0.7, signal-to-base ratio [S/B] 1.7) but not with DU-145 cells. In contrast, the automated SRB screen was successfully optimized with the DU-145 cells (Z' 0.8, S/B 2.4) but not with the Caco-2 cells (Z' -0.8, S/B 1.4). The automated bidirectional Caco-2 permeability experiments separated successfully low- and high-permeability compounds (Z' 0.8, S/B 84.2) and passive drug permeation from efflux-mediated transport (Z' 0.5, S/B 8.6). Of the assays, the homogeneous Caspase-Glo 3/7 assay benefits the most from automation, but also the heterogeneous SRB assay and Caco-2 permeability experiments gain advantages from automation.

Luminescent Polypyridinerhenium(I) Bis‐Biotin Complexes as Crosslinkers for Avidin

AbstractTwo novel luminescent polypyridinerhenium(I) bis‐biotin complexes [Re(NN)(CO)3(pyridine)](CF3SO3) {NN = 4,4′‐bis{[2‐(biotinamido)ethyl]aminocarbonyl}‐2,2′‐bipyridine, bpyC2B2 (1), 4,4′‐bis[(2‐{[6‐(biotinamido)hexanoyl]amino}ethyl)aminocarbonyl]‐2,2′‐bipyridine, bpyC2C6B2 (2)} and their biotin‐free counterpart [NN = 4,4′‐bis(n‐butylaminocarbonyl)‐2,2′‐bipyridine, bpyC4 (3)] have been synthesized and characterized. Upon irradiation, all the complexes exhibited triplet metal‐to‐ligand charge‐transfer (3MLCT) emission in fluid solutions at room temperature and alcohol glass at 77 K. The avidin‐binding properties of the bis‐biotin complexes 1 and 2 have been studied by 4′‐hydroxyazobenzene‐2‐carboxylic acid (HABA) assays, emission titrations, and dissociation assays. The potential use of the complexes as signal amplifiers for heterogeneous recognition assays has been demonstrated using avidin‐coated microspheres and one of the complexes. Additionally, the cytotoxicity of these rhenium(I) complexes towards the human cervix epithelioid carcinoma (HeLa) cell line has been evaluated by 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyltetrazolium bromide (MTT) assays. Furthermore, the cellular uptake of the complexes has been examined by laser‐scanning confocal microscopy. (© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2009)

Microfluidic, bead-based assay: Theory and experiments

Microbeads are frequently used as a solid support for biomolecules such as proteins and nucleic acids in heterogeneous microfluidic assays. However, relatively few studies investigate the binding kinetics on modified bead surfaces in a microfluidics context. In this study, a customized hot embossing technique is used to stamp microwells in a thin plastic substrate where streptavidin-coated agarose beads are selectively placed and subsequently immobilized within a conduit. Biotinylated quantum dots are used as a label to monitor target analyte binding to the bead's surface. Three-dimensional finite element simulations are carried out to model the binding kinetics on the bead's surface. The model accounts for surface exclusion effects resulting from a single quantum dot occluding multiple receptor sites. The theoretical predictions are compared and favorably agree with experimental observations. The theoretical simulations provide a useful tool to predict how varying parameters affect microbead reaction kinetics and sensor performance. This study enhances our understanding of bead-based microfluidic assays and provides a design tool for developers of point-of-care, lab-on-chip devices for medical diagnosis, food and water quality inspection, and environmental monitoring.

In Pursuit of Zero: Polymer Brushes that Resist the Adsorption of Proteins

AbstractProtein resistant or “non‐fouling” surfaces are of great interest for a variety of biomedical and biotechnology applications. This article briefly reviews the development of protein resistant surfaces, followed by recent research on a new methodology to fabricate non‐fouling surfaces by surface‐initiated polymerization. We show that polymer brushes synthesized by surface‐initiated polymerization that present short oligo(ethylene glycol) side chains are exceptionally resistant to protein adsorption and cell adhesion. The importance of the protein and cell resistance conferred by these polymer brushes is illustrated by their use as substrates for the fabrication of antibody microarrays that exhibit femtomolar limits of detection in complex fluids such as serum and blood with relaxed requirements for intermediate wash steps. This example highlights the important point that the reduction in background noise afforded by protein‐resistant surfaces can greatly simplify the development of ultrasensitive heterogeneous, surface‐based clinical and proteomic assays with increased sensitivity and utility.

A Complex-based Reconstruction of the Saccharomyces cerevisiae Interactome

Most cellular processes are performed by proteomic units that interact with each other. These units are often stoichiometrically stable complexes comprised of several proteins. To obtain a faithful view of the protein interactome we must view it in terms of these basic units (complexes and proteins) and the interactions between them. This study makes two contributions toward this goal. First, it provides a new algorithm for reconstruction of stable complexes from a variety of heterogeneous biological assays; our approach combines state-of-the-art machine learning methods with a novel hierarchical clustering algorithm that allows clusters to overlap. We demonstrate that our approach constructs over 40% more known complexes than other recent methods and that the complexes it produces are more biologically coherent even compared with the reference set. We provide experimental support for some of our novel predictions, identifying both a new complex involved in nutrient starvation and a new component of the eisosome complex. Second, we provide a high accuracy algorithm for the novel problem of predicting transient interactions involving complexes. We show that our complex level network, which we call ComplexNet, provides novel insights regarding the protein-protein interaction network. In particular, we reinterpret the finding that “hubs” in the network are enriched for being essential, showing instead that essential proteins tend to be clustered together in essential complexes and that these essential complexes tend to be large.

Confocal detection of planar homogeneous and heterogeneous immunosorbent assays

Optically sectioned detection of fluorescence immunoassays using a confocal microscope enables the creation of both homo- and heterogeneous planar format assays. We report a set assays requiring optically sectioned detection using a model system and analysis procedures for separating signals of a surface layer from an overlying solution. A model sandwich assay with human immunoglobulin G as the target antigen is created on a glass substrate. The prepared surfaces are exposed to antigen and a FITC-labeled secondary antibody. The resulting preparations are either read directly to provide a homogeneous assay or after wash steps, giving a heterogeneous assay. The simplicity of the object shapes arising from the planar format makes the decomposition of analyte signals from the thin film bound to the surface and overlayer straightforward. Measured response functions of the thin film and overlayer fit well to the Cauchy-Lorentz and cumulative Cauchy-Lorentz functions, respectively, enabling the film and overlayer to be separated. Under the conditions used, the detection limits for the homogeneous and heterogeneous forms of the assay are 2.2 and 5.5 ng/ml, respectively. Planar format, confocally read fluorescence assays enable wash-free detection of antigens and should be applicable to a wide range of assays involving surface-bound species.

Refined multivalent display of bacterial spore-binding peptides

A multiple antigen peptide display scaffold was used to create multivalent versions of a heptapeptide selected previously by phage display to bind to Bacillus subtilis spores. A simple flow cytometric assay was developed in which a biotinylated form of the peptide was first bound to fluorescent streptavidin, then the fluorescent streptavidin-peptide complex was bound to spores before introduction into the cytometer. This assay clearly demonstrated that the tetravalent scaffold enhanced the affinity for B. subtilis spores by greater than 1 and 2 orders of magnitude when compared to divalent and monovalent analogues, respectively. However, variations in the number and flexibility of spacer residues within the scaffold did not significantly affect the binding affinity of the tetravalent peptides. Similar to prior reports, these multivalent scaffolds are effective most likely because they mimic the multivalent display of the original peptide library on the phage coat. Moreover, the tetravalent peptides can be readily integrated into a variety of heterogeneous and homogeneous spore-detection assay formats.