Filtern
Dokumenttyp
- Zeitschriftenartikel (8)
- Vortrag (8)
- Posterpräsentation (8)
- Beitrag zu einem Tagungsband (1)
Schlagworte
- Immunoassay (12)
- Bead-based assay (8)
- Anthropogenic markers (5)
- Diclofenac (4)
- SAFIA (4)
- Wastewater (4)
- Flow cytometry (3)
- Automation (2)
- Bead-based-assay (2)
- Caffeine (2)
Organisationseinheit der BAM
Eingeladener Vortrag
- nein (8)
A bead suspension array for measuring emerging pollutants and anthropogenic markers in wastewater
(2016)
Background: The assessment of water quality demands for high-throughput and multi-target compatible analytical methods. On the one hand, the influence of sewage on natural surface waters must be routinely monitored - according to the European Water Framework Directive. 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 owing to their cost-efficiency and high-throughput capability. 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 anti-epileptic 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. The drugs can be detected from 1 to 4 µg/L and the bile acid can be detected up to 72 µg/L in wastewater, requiring a highly sensitive detection method.
Methods: We use core-shell particles as the spherical platform, consisting of a polystyrene core and a silica shell. Such beads can easily be encoded with an organic fluorophore in the core and functionalized with amino groups on the silica shell surface for creating binding sites for antibodies: Therefore, the marker substances are bound to the amino groups via NHS chemistry. The hybrid particles were synthesized in house. Polystyrene was chosen as core material because of its low density and high refractive index, both important requirements for the use in suspension arrays. Easy surface modifications using silane chemistry are accessible on the silica shell. For the determination of the pollutants, the beads are incubated with a mixture of the analyte-specific antibodies. The antibodies undergo a competitive reaction to bind the immobilized molecules on the surface of the beads or the free analyte in the solution, respectively. Bound antibodies can then be visualized via fluorophore-labelled secondary antibodies. For read-out a flow cytometer is used to virtually separate the different dye encoded beads and to measure the resulting Signal intensity of the immunoassay. A four-parametric function was used as calibration curve.
Results: In order to obtain highly selective binding of the antibodies, we investigated different types of silica shell surface modifications to overcome unspecific binding, finding that a PEG-modified surface is suitable to support our immunoassay format. Multiplexed small molecule detection can therefore be realized in a simple mix and read procedure without washing steps.
Conclusion: We presented a multiplexing suspension array for the detection of four marker substances in wastewater samples, based on an indirect immunoassay format with polystyrene core-silica shell microbeads. The resulting multiplexing assay is appropriate to detect the marker substances in the low µg/L range. Therefore, wastewater samples can be measured without further preconcentration.
A bead suspension array for measuring emerging pollutants and anthropogenic markers in wastewater.
(2016)
The assessment of water quality demands for high-throughput and multi-target compatible analytical methods. On the one hand, the influence of sewage on natural surface waters must be routinely monitored - according to the European Water Framework Directive. 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 owing to their cost-efficiency and high-throughput capability. 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 anti-epileptic 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 and functionalized with amino groups on the surface for creating binding sites for antibodies: Therefore, the marker substances are bound to the amino groups via NHS chemistry.
For the determination of the pollutants, the beads are incubated with a mixture of the analyte-specific antibodies. The antibodies undergo a competitive reaction to bind the immobilised molecules on the surface of the beads or the free analyte in the solution, respectively. Bound antibodies can then be visualized via fluorophore-labelled secondary antibodies. For read-out a flow cytometer is used to virtually separate the different encoded beads and to measure the resulting signal intensity of the immunoassay. 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.
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.
Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagentsmare expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via Nhydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve.
The development of an automated miniaturized analytical system that allows for the rapid monitoring of carbamazepine (CBZ) levels in serum and wastewater is proposed. Molecular recognition of CBZ was achieved through its selective interaction with microbeads carrying anti-CBZ antibodies. The proposed method combines the advantages of the micro-bead injection spectroscopy and of the flow-based platform lab-on-valve for implementation of automatic immunosorbent renewal, rendering a new recognition surface for each sample. The sequential (or simultaneous) perfusion of CBZ and the horseradish peroxidase-labelled CBZ through the microbeads is followed by real-time on-column Monitoring of substrate (3,30,5,50-tetramethylbenzidine) oxidation by colorimetry. The evaluation of the initial oxidation rate and also the absorbance value at a fixed time point provided a linear response versus the logarithm of the CBZ concentration. Under the selected assay conditions, a single analysis was completed after only 11 min, with a quantification range between 1.0 and 50 µg L⁻¹. Detection of CBZ levels in undiluted wastewater samples was feasible after a simple filtration step while good recoveries were attained for spiked certified human serum, analyzed without sample clean-up.
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.
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.
Electrochemical methods offer great promise in meeting the demand for user-friendly on-site devices for Monitoring important parameters. The food industry often runs own lab procedures, for example, for mycotoxin analysis, but it is a major goal to simplify analysis, linking analytical methods with smart technologies. Enzyme-linked immunosorbent assays, with photometric detection of 3,3’,5,5’-tetramethylbenzidine (TMB),form a good basis for sensitive detection. To provide a straightforward approach for the miniaturization of the detectionstep, we have studied the pitfalls of the electrochemical TMB detection. By cyclic voltammetry it was found that the TMB electrochemistry is strongly dependent on the pH and the electrode material. A stable electrode response to TMB could be achieved at pH 1 on gold electrodes. We created a smartphonebased, electrochemical, immunomagnetic assay for the detection of ochratoxin A in real samples, providing a solid basis forsensing of further analytes.
Monitoring water quality regarding emerging pollutants, such as drug residues, demands for selective, high-throughput and multi-target 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, offer the possibility to be highly sensitive and selective due to the high target affinity and specificity of target recognition by antibodies. Batch-wise processing in microtiter plates allows for the necessary high-throughput, but only a single analyte can be determined within one measurement.
To overcome these disadvantages, we developed a four-plex Suspension Array Fluorescence Immunoassay (SAFIA), which is adaptable for the microtiter plate format. The modular and self-prepared bead support consists of polystyrene-core silica-shell microparticles. 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 covalently coupled to amino groups on the surface via NHS chemistry. Specific recognition of these haptens is accomplished by further introduction of PEG moieties, suppressing non-specific binding. A competitive immunoassay is subsequently conducted in a simple mix-and-read procedure, eliminating the demand for laborious washing steps and decreasing time-to-result. An automated flow cytometer is used for simultaneous decoding and quantification.
After optimization, the SAFIA showed limits of detection for all analytes in the low µg L 1 range, meeting the sensitivity criteria for wastewater analysis. Then, applicability of the SAFIA was studied on real wastewater samples from three different wastewater treatment plants in Berlin. The results were in good comparability to LC-MS/MS indicating high matrix stability. Moreover, the accuracy of the assay exceeded that of the respective ELISA. Finally, we used SAFIA to assess the influent of treated and untreated wastewater on the Douro river estuary in Portugal. The obtained results of the analysis were comparable to ELISA. However, the measurements by SAFIA could be carried out in one quarter the time of analysis required for ELISA.
Acknowledgements: The authors acknowledge financial support from Deutscher Akademischer Austauschdienst and from Fundação das Universidades Portuguesas under the protocol CRUP-DAAD (Ações Integradas Luso-Alemãs nºE-20/16, DAAD Zuwendungsvertrag 57212899).