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Immunochromatography and enzyme-linked immunosorbent assay (ELISA) represent selective and sensitive procedures based on solid-phases for separation/detection and quantification of anthropogenic pollutants in the aquatic environment. In contrast with batch-wise procedures, such as microplate-based platforms, automated methods reduce manual handling of reagents, thus increasing overall precision and decreasing time-to-result.
Microparticles have been shown to be an adequate support for carrying out immunoassays in meso and microfluidic systems. They offer a wide range of coupling sites for biomolecules such as antibodies, combined with specialised anti-fouling surfaces to prevent non-specific binding and high compressibility for optimum fluidics.
In this work we investigated the protein-coupling behaviour of two commercially available microsphere supports (Tentagel® polystyrene-PEG-COOH and PolyAn® PMMA beads with 3D antifouling surface) using DCC/EDC and NHS/S-NHS activation chemistry. The study of coupling conditions (pH, proportion of reagents and type of buffering system) was addressed. The success of the biomodification of the supports was demonstrated by using self-prepared fluorophore-protein conjugates (Fig. 1). Laser-scanning microscopy and flow cytometry were applied for further characterization of the functionalized particles. The applicability of the developed particles will be demonstrated through the design of suspension multiplex assays for the detection, quantification and preconcentration of bioactive substances such as caffeine and carbamazepine, using Lab-on-valve (LOV) platforms.
Biosensors, as defined by Pure and Applied Chemistry, are ‘chemical sensors in which the recognition System utilizes a biochemical mechanism. The biological recognition system translates information from the biochemical domain, usually an analyte concentration, into a chemical or physical output signal with a defined sensitivity’.(1) It is also appointed that chemical or biological sensors contain two basic components connected in series: a chemical or biomolecular recognition System (receptor) and a physicochemical transducer. According to this prerequisite, this overlook is confined to sensor devices that combine a biomolecular recognition element with an optical signal transducer. Homogeneous or intracellular assays using fluorescent molecular probes or nanoparticles are not considered, although they are frequently termed as molecular sensors or nanosensors in the literature.
Fluorescence-based biosensors are generalized as those devices that derive an analytical signal from a photoluminescent (either fluorescence or phosphorescence) emission process. Chemi- or bioluminescent detection systems are only briefly discussed in this review.
Biosensors are used for a wide variety of tasks, including detection of compounds of biomedical, environmental or defense interest; on-line monitoring for process control; quality control of foodstuffs; selective detection of compounds undergoing a chemical separation; and screening of drug compounds. Advantages of such devices include high selectivity, rapid response times, reusability, amenability to remote analysis, and immunity to electrical interferences. The selective nature of complexation between biomolecule and analyte and the small size of sensor devices can be combined with advanced detection techiques such as total internal reflection (TIR) spectroscopy. This results in an ability to measure analytes in complex matrices with unsurpassed sensitivity. Such samples may include highly scattering components such as milk or whole blood,(11) or relatively inaccessible locations such as groundwater wells, or even intracellular environments. The key limitation of such devices mainly centers on the poor stability of biological compounds, which can lead to a substantial drift in instrumental response over time. The so-called Cambridge Definition appoints another characteristic property of sensors. Therein, they are defined as ‘miniaturized devices which can deliver real-time and on-line information on the presence of specific compounds or ions in even complex samples’. Accordingly, a sensor is expected to respond reversibly and continuously. With the exception of some enzymatic sensors, these conditions are not fulfilled in case of most biosensors. Particularly, in devices where immunological reagents or DNA are used as recognition elements, they show a lack of reversibility and operate only as a ‘one-shot’ screen, without the potential for continuous, quantitative analysis. Nevertheless, the designations immunosensors or DNA sensors became accepted for such analytical or diagnostic tools.
Metal-organic framework (MOF) colloids hold great potential for bioanalytical and biomedical applications due to their unique features. These include responsive luminescent properties and exceptionally high loading capacities for small molecular drugs. However, currently the lack of a surface functionality independent method for biomolecule conjugation is strongly limiting the advancement of colloidal MOFs in bioanalytical or biomedical applications. Bioanalytical methods, especially for environmental analysis, would benefit dramatically when responsive luminescent properties of MOFs could be coupled with a specific antibody interaction. Targeted drug delivery in biomedical applications often requires specificity towards tissues of interest in addition to a high drug loading capacity. Thus, both envisioned applications require biomolecules and in particular antibodies to be conjugated to colloidal MOFs.
Here, we propose a robust and easy to handle method that is suitable for a wide range of MOF templates and that allows reliable conjugation of biomolecules.
Colloidal ZIF-8 MOFs with particle size between 300nm and 5µm were used as templates to adsorb polymeric multilayer through a Layer-by-Layer self-assembly process. Multilayer build up was confirmed by the change in zeta-potential of particles upon polyelectrolyte adsorption as well as dynamic light scattering experiments and SEM/TEM microscopy. Subsequent biomolecule conjugation to functional groups of the polymeric multilayer on the surface of colloidal MOFs was achieved by established carbodiimide/succinimide conjugation chemistry. Successful and stable conjugation was confirmed by employing fluorescent labeled biomolecules.
Subsequently, colloidal MOF-antibody (IgG) conjugates were utilized in a solid-phase immuno assay using antibodies against carbamazepine and lithocholic acid, two interesting markers in environmental analysis.
In brief, mouse anti-CBZ (Carbamazepine) and rabbit anti-lithocholic acid were separately and covalently immobilized on 2D polystyrene surfaces to form a small micro spot array. Two separate populations of colloidal MOF particles were conjugated with goat anti mouse IgG and goat anti rabbit IgG, respectively. The different fluorescent labels of the two MOF colloid populations allowed the determination of binding selectivity and strength of the colloidal MOF-antibody conjugates to the immobilized binding partners.
The ability to coat MOFs in solution independent of their surface charge with polymers and subsequently to conjugate biomolecules to their surface makes this method a powerful tool to foster bioanalytical, biomedical as well as other applications.
Fluorophore labeled proteins and antibodies, referred to also as targeted optical probes, present a promising strategy for a variety of applications from fundamental cell-based biological studies to in vivo diagnostics and image guided surgeries in humans. In this respect, design strategies for the preparation of such conjugates from different dyes including analyte-responsive fluorophores are presented as well as their analytical and spectroscopic characterization employing Absorption spectroscopy and steady state and time-resolved fluorometry. Special emphasis is dedicated to the influence of dye hydrophilicity and labeling density on the optical properties and binding behavior of these dye-bioconjugates including their performance in in vitro and in vivo bioimaging studies.
Nicht zuletzt bedingt durch den Pferdefleischskandal im Jahr 2013 wurden in Deutschland die Aktivitäten bei der Methodenstandardisierung im Bereich der Tierarten-Differenzierung in den vergangenen Jahren intensiviert. Wenn auch positive Befunde bei dem Nachweis von Bestandteilen aus Pferd in Lebensmitteln wie Lasagne derzeit so gut wie nicht mehr anzutreffen sind, so hat die Tierartendifferenzierung insgesamt beim Nachweis von Verfälschungen in Lebensmitteln einen hohen Stellenwert.
Diese Arbeit fasst daher den aktuellen Stand der Analytik in Deutschland mit Schwerpunkt bei der Standardisierung zusammen. Sie wurde erstellt durch die Arbeitsgruppe „Biochemische und molekularbiologische Analytik“ der Lebensmittelchemischen Gesellschaft mit Unterstützung von Experten der Arbeitsgruppe „Molekularbiologische Methoden zur Pflanzen- und Tierartendifferenzierung“ (§ 64 LFGB) sowie der ALTS-Arbeitsgruppe „Immunologie und Molekularbiologie“ (jeweils D).
Immunoassays are an important field of in vitro diagnostics, as they allow for a fast and highly sensitive detection of many biologically and diagnostically relevant analytes such as proteins, hormones, and pharmaceuticals. Fluorescence immunoassays (FIA), where the antibodies and/or antigens are labeled with luminescent reporters, can be easily read out directly by measuring the intensity, decay time, or polarization of the emitted light. Moreover, FIA enable the simultaneous detection of different analytes within a single sample (multiplexing) and are particularly suited for point-of-care (POC) diagnostics and high throughput screening (HTS). The application of luminescent nanoparticles as reporters in FIA could further improve assay sensitivity, as several 100 to 1000 luminophores can be incorporated or attached to such nanoscale carriers, thereby amplifying their absorption and/or emission signals simply by increasing the number of dye molecules.
Although dye-loaded polymeric and silica nanoparticles have been increasingly used as reporters in immunoassays, achievable signal amplification factors related to the use of particle reporters are still difficult to predict and quantify, which also hampers the comparability of different nanoscale reporters. To overcome this challenge, we performed a systematic comparison of spectroscopically and analytically well characterized particle labels in a homogeneous sandwich immunoassay format for the detection of the common inflammation biomarker C-reactive protein (CRP). Hereby, we studied the influence of particle parameters like size, surface chemistry, and dye loading concentration for different dye classes, i.e. organic dyes and metal ligand complexes, varying in their signal-relevant spectroscopic properties (molar absorption coefficients, photoluminescence quantum yields, Stokes shifts, and emission decay times), for different detection schemes (direct read-out vs. dye extraction). The emitters applied were chosen to be commercially available for a reasonable price, to absorb between 400 nm and 450 nm, and to emit in the visible region, as these parameters are accessible with most established microplate readers. Based upon our findings, we highlight the advantages and limitations of nanoscale reporters with respect to the choice of suitable particles, encoding dyes, and detection strategies, and compare the achievable sensitivities and dynamic ranges for our CRP model immunoassay.
Laser ablation with inductively coupled plasma is still more used in life science as biology and biomedicine and the utilization of metals and proteins determination simultaneously is also growing up. We have developed a new strategy of labeling of antibody (it can specific binds to proteins) by nanoparticles and quantum dots which is composed of thousands of atoms and thus increases the sensitivity enormously and of course decreases the Limit of detection, compare to lanthanoids labeling. The ability of successfully tagged antibodies bound to Antigen (protein) was proved by dot blot on membrane imaged by LA-ICP-MS.
Colloidal metalorganic frameworks as novel biofunctional nanoparticles for immunoassay applications
(2017)
Metal-organic framework (MOF) colloids have unique features that render them ideal signalling agents for realizing advanced immunoassay-based detection systems. MOFs are porous coordination polymers of metal nodes and organic linkers. The pore size of MOFs can be engineered and tailored to allow specific host (MOF) and guest (analyte) interactions. The particle sizes of the colloidal MOF can be tailored by employing methods from colloidal chemistry in wet synthesis. The adaption of established Layer-by-Layer polyelectrolyte coating protocols [1] allows equipping colloidal MOF particles with a nanometer thin polyelectrolyte membrane. This polyelectrolyte membrane serves as an interface for antibody binding. These biofunctional MOF nanoparticles have shown a strong immuno-binding that is sufficient for solid state immunoassays. Our current research addresses the design of luminescence encoded colloidal particle libraries by adjusting the ratios of e.g. Terbium (green) and Europium (red) metal nodes in mixed lanthanide based MOF-76. These mixed lanthanide MOF-76 particles are envisioned to allow multiplexed immuno-detection of endocrine disruptors such as bisphenol A.
In addition we investigate the detection of analytes that do not allow the production of antibodies due to their inherent properties. Such “difficult analytes” have a strong hydrophobicity or are very small or highly toxic molecules. One example is the common plasticizer dioctylphthalate that is also a potent endocrine disruptor. MOF colloids can address this issue by specific host (MOF) : guest (analyte) interactions that result in analyte-specific colour change or exciplex-based fluorescence emission. Our overall aim is to develop methodologies that allow parallel sensing of two endocrine disruptors (e.g. bisphenol A & phthalates) by simultaneous immuno-detection and MOF:analyte specific interactions.
Immunoassays are in the majority of formats heterogeneous assays with one of the commodities – antibody or antigen – immobilized on a solid substrate or sometimes to nanoparticles or beads. All these formats require a washing step in order to separate bound from non-bound species before generating the measurement signal.
Measuring fluorescence polarization is a method to distinguish between a fluorescent macromolecule and a low-molecular weight fluorophore. After irradiation of the sample with polarized light, a macromolecule like e.g. an antibody, will emit still highly polarized light (high polarization, resp. small difference in degree of polarization to the background). In a fluorescence polarization immunoassay, the fluorescence of an analyte surrogate (a fluorescence “tracer”) is followed. Depolarization is high and so the measured polarization is low. When antibody is added, the tracer is bound, depolarization decreases and the in polarization is high. Increasing amounts of analyte more and more impede the tracer being bound by the tracer and thus the decreases, the typical sigmoidal relationship is obtained (Fig. 1). We dispose of 5 polarization platforms reading in plate mode, strip mode and cuvette mode. One is able to register polarization changes with time and so allows for kinetic measurements another one is hand-held. We have been comparing FPIA formats before [1-4] and now a comprehensive view on sensitivities, the importance of fast vs. slow binding kinetics as well as the choice of format can be presented.