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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.
The lab-on-valve (LOV) is a mesofluidic platform that has been recently exploited for
the automation and miniaturization of bioanalytical assays, resorting namely to
molecular recognition schemes based on immunosensing. Due to its high versatility
for reagent accommodation, it is possible to establish immunoassays under several
formats (eg. direct competitive ELISA, sandwich ELISA or even label-free immunoaffinity
chromatography). For instance, the LOV has been used as a manifold for
UV-vis micro-Bead Injection Spectroscopy (μ-BIS), a technique that involves the
quantification of the target analyte by direct measurement on the surface of a solid
phase capable of retaining the target analyte by molecular recognition.
The μ-BIS-LOV strategy affords several analytical advantages, namely short time-toresult
intervals (3 to 15 min), low sample volume (1-20 μL), automated solution handling
and washing steps, downscaling of reagents’ consumption, low-cost analysis
and little generation of waste. Additionally, the solid support is renewed before each
determination, minimizing surface fouling, cross-contamination issues and functional
group deactivation. No sample clean-up steps are required because interferences
are separated from the target analyte upon quantification mediated by a molecular
recognition element attached to the micro-bead column. The portability of the LOV
device makes it compatible with point-of-care testing.
To our knowledge, this technique has been mainly employed for the evaluation and
optimization of bioaffinity processes, but its potential for clinical and environmental
analysis remains underexploited. Hence, in this communication, different immunosensing
strategies using the LOV platform will be addressed, namely the determination
of autoimmune IgG in human serum, and the assessment of drug (carbamazepine)
levels in wastewater samples.
Pollutants of low molecular weight, such as drug residues, are in the focus of water quality assessment: some of them, like carbamazepine are only partially degraded in wastewater treatment plants. Thus, these pollutants can serve as marker substances for elimination efficiencies. Monitoring water quality demands for selective, high-throughput and multi-target analytical methods. Immunoassays, such as ELISA, offer the possibility to be highly sensitive and selective due to the specific recognition by high affinity of target molecules to antibodies (Abs). Batch-wise processing in microtiter plates allows for the necessary high-throughput, however only a single analyte can be determined within one measurement.
To overcome these disadvantages, we developed a four-plex microbead-based flow cytometric assay, which is adaptable for the microtiter plate format. The modular and self-prepared bead support consists of polystyrene-core/silica-shell particles. While, the polystyrene core is used for encoding, by introducing different amounts of fluorescent dyes, the silica shell creates a solid support for the immunoassay: The target analytes, three drugs, carbamazepine, diclofenac and caffeine and the fecal marker isolithocholic acid are coupled covalently to the surface via NHS chemistry to amino groups on the surface.
For determination of the pollutants, a mixture of specific Abs is incubated with the samples, to bind competitively on the “anchor” molecules on the surface of the beads or the analyte in solution. Bound antibodies are then visualized via fluorescent dye-labelled secondary Abs. Flow-cytometry allows for decoding of the beads and signal read-out, without washing the system. In order to decrease non-specific binding, we investigated different types of surface modifications, finding, that a PEG-based surface is suitable to support our immunoassay format. For maximum sensitivity, a design-of-experiment approach was chosen for optimization of the assay parameters. The resulting immunoassay is appropriate to quantify the pollutants in the low μg/L-range.
Bisphenol A (BPA) is a common industrial chemical that is widely used as a monomer or additive in the production of polycarbonate plastics and other polymeric materials. The primary way of exposure to BPA for most people is assumed to be a direct con-tamination of food and water through use of containers, bottles, and other plastic stor-age. BPA has been identified as an important endocrine disrupting compound (EDC). It can interfere with hormonal activities like biosynthesis, metabolism and elimination of natural blood-borne hormones. Moreover, BPA can originate environmental problems. A spectrum of methods have been developed with the aim of determining BPA in water. Immunoassays appear to comply with sensitivity, rapidity, and low cost requirements for effective monitoring.
In this work a comparative analysis was conducted of the effect of temperature and the choice of buffers, as well as the substrate at various stages of an ELISA (enzyme-linked immunosorbent assay). The method has been optimized and has shown good repeatability and sensitivity. The results obtained from ELISA will be used as the basis for the development of a Lateral Flow ImmunoAssay (LFIA).
The assessment of water quality demands for multi-target compatible analytical methods. On the one hand, the influence of sewage on natural surface waters must be routinely monitored. On the other hand, estimation of removal efficiencies of pollutants, such as drug residues, is in the focus of industrial and public wastewater treatment. Immunoassays, such as ELISA, are often applied in this area, however, it is only possible to measure a single analyte in one measurement.
In contrast, array technology is capable for measuring multiple substances in parallel. Here we present a four-plex bead-based flow cytometric assay for measuring three drugs (carbamazepine, an antiepileptic drug, diclofenac, an antiphlogistic and caffeine, a psychoactive substance) and the bile acid isolithocholic acid, which is proposed to be used as a fecal marker.
In this suspension array core-shell particles, consisting of a polystyrene core and a silica shell, are used. They can be easily encoded with a fluorophore which is introduced to the core while the shell is functionalized with amino groups to which the analytes or their derivates are bound via NHS chemistry. They are the competitor binding sites for the antibodies.
For the determination of the pollutants, the beads are incubated with a mixture of the analyte-specific antibodies. The molecules on the surface of the beads and the free analyte in solution compete for the binding sites of the antibodies. Bead-bound antibodies can then be visualized via dye-labelled secondary antibodies. For read-out a flow cytometer is used to virtually separate the differently encoded beads. In order to obtain highly selective binding of the antibodies, we investigated different types of surface modifications to overcome unspecific binding, finding that a PEG-based surface is suitable to support our immunoassay format. The resulting multiplexing assay is appropriate to detect the marker substances in the low µg/L range.
Due to the demand of monitoring the water quality regarding emerging pollutants, such as drug residues, selective, high-throughput and multi-target analytical methods must be established. On the one hand, the influence of sewage on natural surface waters must be routinely monitored. On the other hand, estimation of removal efficiencies of pollutants, such as drug residues, is in the focus of industrial and public wastewater treatment. Immunoassays, such as ELISA, offer the possibility to be highly sensitive and selective due to the high target affine and specific recognition of antibodies to target molecules. Batch-wise processing in microtiter plates allow the necessary high-throughput, but only a single analyte can be determined within one measurement.
To overcome these disadvantages, we developed a four-plex micro-bead based flow cytometric assay, which is adaptable for the microtiter plate format. The modular and self-prepared bead support consists of polystyrene-core-silica-shell particles. While the polystyrene core is used for encoding, by introducing different amounts of fluorescent dyes, the silica-shell creates a solid-support for the immunoassay: The target analytes, three drugs, carbamazepine, diclofenac and caffeine and the f ecal marker isolithocholic acid are covalently coupled to amino-groups on the surface via NHS-chemistry. A competitive immunoassay is subsequently conducted in a simple mix-and-read procedure. Finally, we could use SAFIA to assess the influent of treated and untreated waste water on the Douro river estuary in Portugal. The results of the analysis are comparable to ELISA. However, measurements could be carried out in 25% the time of analysis.