Analytische Chemie
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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.
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 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.