Filtern
Dokumenttyp
- Zeitschriftenartikel (3)
- Vortrag (2)
- Posterpräsentation (1)
Sprache
- Englisch (6)
Schlagworte
- Automation (4)
- Immunoassay (3)
- Lab-on-valve (2)
- Antikörper (1)
- Bead injection spectroscopy (1)
- Bead injections (1)
- Biomarkers (1)
- Biosensoren (1)
- Carbamazepine (1)
- ELISA (1)
Organisationseinheit der BAM
Eingeladener Vortrag
- nein (2)
The improvement of immunoanalytical methods for the determination of pharmaceuticals in wastewaters is a crucial yet challenging endeavor. In this work, the development of an automated miniaturized ELISA based on micro-Bead Injection Spectroscopy (μ-BIS) [1] for the determination of carbamazepine, a widely employed anti-epileptic drug and emergent pollutant [2], was pursued.
The experimental workflow comprised the offline functionalization of Sepharose beads with specific anti-CBZ antibodies via affinity immobilization using protein G, and 3 online steps inside the microfluidic analyzer lab-on-valve (LOV): I) packing of the bead column into the detection unit; II) sequential percolation of sample and a CBZ competitor- labeled with horseradish peroxidase (tracer) through the bead column; and III) on-column colorimetric detection employing the enzyme substrate 3,3’,5,5’-tetramethylbenzidine. After each analysis, the bead column was discarded, and the flow cell was washed before receiving new beads.
The elimination of manual washing steps is a novel feature compared to batch-wise ELISA, making the method less error-prone and therefore more robust. The replacement of the solid support prevents memory effects and cross-contamination between runs. The use of microparticles as solid support for the molecular recognition elements accounts for high area-to-volume ratios, and low molecular diffusion distances. For that reason, time-to-result was reduced from several hours to less than 10 min. The consumption of reagents was also very low. For instance, only ca. 200 μg of solid support and 900 ng of anti-CBZ antibody were required per determination. At last, the versatility of the LOV platform offers the possibility of adapting the assay to other relevant pharmaceuticals and anthropogenic markers in water.
Acknowledgements: Inês I. Ramos thanks FCT (Fundação para a Ciência e a Tecnologia) and POPH (Programa Operacional Potencial Humano) for her grant (SFRH/BD/97540/2013). This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC - Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013. Financial support from Deutscher Akademischer Austauschdienst and from Fundação das Universidades Portuguesas under the protocol CRUP-DAAD (Ações Integradas Luso-Alemãs nºE-20/16) is also acknowledged.
[1] Gutzman, Y.; Carrol, A. D. Analyst 2006, 131, 809.
[2] Murray, K. E.; Thomas, S. M.; Bodour, A. A. Environ. Pollut. 2010, 158, 3462.
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 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.
The presence of endocrine-disrupting compounds (EDCs) in water poses a significant threat to human and animal health, as recognized by regulatory agencies throughout the world. The Yeast Estrogen Screen (YES) assay is an excellent method to evaluate the presence of these compounds in water due to its simplicity and capacity to assess the bioaccessible forms/fractions of these compounds. In the presence of a compound with estrogenic activity, Saccharomyces cerevisiae cells, containing a lacZ reporter gene encoding the enzyme β-galactosidase, are induced, the enzyme is synthesised, and released to the extracellular medium. In this work, a YES-based approach encompassing the use of a lacZ reporter gene modified strain of S. cerevisiae, microcarriers as solid support, and a fluorescent substrate, fluorescein di-β-D-galactopyranoside, is proposed, allowing for the assessment of EDCs’ presence after only 2 h of incubation. The proposed method provided an EC50 of 0.17 ± 0.03 nM and an LLOQ of 0.03 nM, expressed as 17β-estradiol. The assessment of different EDCs provided EC50 values between 0.16 and 1.2 × 103 nM. After application to wastewaters, similar results were obtained for EDCs screening, much faster, compared to the conventional 45 h spectrophotometric procedure using a commercial kit, showing potential for onsite high-throughput screening of environmental contamination.
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.