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Pharmaceuticals, certain food ingredients, and mammalian endogenous metabolic products in wastewater are mostly of human origin. They are anthropogenic markers.
Proper knowledge of their levels in wastewater helps to track sources of pollutants in natural waters and allows for calculation of removal efficiencies in wastewater Treatment plants. Here, we describe the development and application of an indirect competitive, multiplexing suspension Array fluorescence immunoassay (SAFIA) for the detection of carbamazepine (CBZ), diclofenac (DCF), caffeine (CAF), and isolithocholic acid (ILA) in wastewater, covering those classes of anthropogenic markers. The assay consists of haptens covalently conjugated to fluorescence-encoded polystyrene core/silica shell microparticles to create a site for competitive binding of the antibodies (Abs). Bound Abs are then stained with fluorophore-labeled Abs. Encoding and signaling fluorescence of the particles are determined by an automated flow cytometer.
For compatibility of the immunoassay with the 96-well microtiter plate format, a stop reagent, containing formaldehyde, is used. This enables a wash-free procedure while decreasing time-to-result. Detection limits of 140 ± 40 ng/L for CBZ, 180 ± 110 ng/L for CAF, 4 ± 3 ng/L for DCF, and 310 ± 70 ng/L for ILA are achieved, which meet the sensitivity criteria of wastewater analysis. We demonstrate the applicability of SAFIA to real wastewater samples from three different wastewater Treatment plants, finding the results in good agreement with LC-MS/MS. Moreover, the accuracy in general exceeded that from classical ELISAs. We therefore propose SAFIA as a quick and reliable approach for wastewater analysis meeting the requirements for process analytical technology.
Early detection of cancer is essential for successful treatment and improvement in patient prognosis. Deregulation of post-translational modifications (PTMs) of proteins, especially phosphorylation, is present in many types of cancer. Therefore, the development of materials for the rapid sensing of low abundant phosphorylated peptides in biological samples can be of great therapeutic value. In this work, we have synthesised fluorescent molecularly imprinted polymers (fMIPs) for the detection of the phosphorylated tyrosine epitope of ZAP70, a cancer biomarker. The polymers were grafted as nanometer-thin shells from functionalised submicron-sized silica particles using a reversible addition-fragmentation chain-transfer (RAFT) polymerisation. Employing the combination of fluorescent urea and intrinsically cationic bis-imidazolium receptor cross-linkers, we have developed fluorescent sensory particles, showing an imprinting factor (IF) of 5.0. The imprinted polymer can successfully distinguish between phosphorylated and non-phosphorylated tripeptides, reaching lower micromolar sensitivity in organic solvents and specifically capture unprotected peptide complements in a neutral buffer. Additionally, we have shown the importance of assessing the influence of counterions present in the MIP system on the imprinting process and final material performance. The potential drawbacks of using epitopes with protective groups, which can co-imprint with targeted functionality, are also discussed.
Core-shell (CS) particles have been increasingly used for a wide range of applications due to their unique properties by merging individual characteristics of the core and the shell materials. The functionality of the designed particles is strongly influenced by their surface roughness. Quantitative evaluation of the roughness of CS microparticles is, however, a challenging task for Scanning Electron Microscopy (SEM).
The SEM images contain two-dimensional (2D) information providing contour roughness data only from the projection of the particle in the horizontal plane. This study presents a practical procedure to achieve more information by tilting the sample holder, hence allowing images of different areas of a single particle to be recorded at different orientations under the same view angle. From the analysis of these images, quasi three-dimensional (3D) information is obtained.
Three types of home-made particles were investigated: i) bare polystyrene (PS) particles, ii) PS particles decorated with a first magnetic iron oxide (Fe3O4) nanoparticle shell forming CS microbeads, iii) PS/Fe3O4 particles closed with a second silica (SiO2) shell forming core-shell-shell (CSS) microbeads. A series images of a single particle were taken with stepwise tilted sample holder up to 10° by an SEM with high-resolution and surface sensitive SE-InLens® mode.
A reliable analysis tool has been developed by a script in Python to analyze SEM images automatically and to evaluate profile roughness quantitatively, for individual core-shell microparticles. Image analysis consists of segmentation of the images, identifying contour and the centre of the particle, and extracting the root mean squared roughness value (RMS-RQ) of the contour profile from the particle projection within a few seconds.
The variation in roughness from batch-to-batch was determined with the purpose to set the method as a routine quality check procedure. Flow cytometry measurements provided complementary data. Measurement uncertainties associated to various particle orientations were also estimated.
Transparent dispersions of hydrophobic SrF2 :Eu3+ nanoparticles in cyclohexane with up to 20% europium were obtained by fluorolytic sol-gel synthesis followed by Phase transfer into cyclohexane through capping with sodium dodecylbenzenesulfonate (SDBS). The particles were characterized by TEM, XRD and DLS as spherical objects with a diameter between 6 and 11 nm in dry state. 1H-13CP MAS NMR experiments revealed the binding of the anionic sulfonate head group to the particle surface. The particles show bright red luminescence upon excitation of the aromatic capping agents, acting as antennas for an Energy transfer from the benzenesulfonate unit to the Eu3+ centers in the particles. This synthesis method overcomes the current obstacle of the fluorolytic sol-gel synthesis that transparent dispersions can be obtained directly only in hydrophilic solvents. To demonstrate the potential of such hydrophobized alkaline-earth fluoride particles, transparent luminescent organic-inorganic composites with 10% SrF2 :Eu3+ embedded into polyTEGDMA, polyBMA, poly-BDDMA and polyD3MA, respectively, were prepared, endowing the polymers with the luminescence features of the nanoparticles.
The use of macromolecules and materials immobilized in the detection zone of test strips for indicator capture and focusing in label-free lateral flow assays (LFAs) is described, with emphasis on its future use in low number multiplexing. Several materials such as polyelectrolytes, functionalized mesoporous silica micro- and nanoparticles, chemically modified cellulose or glass fibre (GF) membranes and molecularly imprinted polymer gels coated onto membranes were studied in model assays, before the most promising materials were combined with antibody-gated indicator delivering (gAID) sensor materials. Cellulose, nitrocellulose and GF membranes were used as supports and highly fluorescent dyes of different charge states as model indicators. Combination of the best performing capture materials with gAID systems made it possible to distinctly increase the sensitivity and reduce the measurement uncertainty in the LFA testing of pentaerythritol tetranitrate (PETN) in aqueous samples. In addition, dual-plexing of PETN and 2,4,6-trinitrotoluene (TNT) was realized on a single test strip containing two dedicated capture zones.
Bifunctional fluorescent molecular oxoanion probes based on the benzoxadiazole (BD) chromophore are described which integrate a thiourea binding motif and a polymerizable 2-aminoethyl methacrylate unit in the 4,7-positions of the BD core. Concerted charge transfer in this electron donor-acceptor-donor architecture endows the dyes with strongly Stokes shifted (up to >250 nm) absorption and fluorescence. Binding of electron-rich carboxylate guests at the thiourea receptor leads to further analyte-induced red-shifts of the emission, shifting the fluorescence maximum of the complexes to ≥700 nm. Association constants for acetate are ranging from 1–5×105 M−1 in acetonitrile. Integration of one of the fluorescent probes through its polymerizable moiety into molecularly imprinted polymers (MIPs) grafted from the surface of submicron silica cores yielded fluorescent MIP-coated particle probes for the selective detection of antibiotics containing aliphatic carboxylate groups such as enoxacin (ENOX) at micromolar concentrations in highly polar solvents like acetonitrile.
Gasoline adulteration is a frequent problem world-wide, because of the chance of quick, maximized profits. However, addition of cheaper ethanol or hydrocarbons like kerosene does not only result in economic damage but also poses problems for vehicles and the environment. To enable law enforcement forces, customers or enterprises to uncover such a fraudulent activity directly upon suspicion and without the need to organize for sampling and laboratory analysis, we developed a simple strip-based chemical test. Key to the favorable performance was the dedicated materials tailoring, which led to test strips that consisted of a cellulose support coated with silica, passivated with hexamethyldisilazane and functionalized covalently with a molecular probe. The probe fluoresces brightly across a broad solvent polarity range, enabling reliable quantitative measurements and data analysis with a conventional smartphone. The assays showed high reproducibility and accuracy, allowing not only for the detection of gasoline adulteration but also for the on-site monitoring of the quality of commercial E10 gasoline.
The use of gamma-hydroxybutyric acid (GHB) in drug-facilitated sexual assault has increased due to its availability and high solubility in aqueous solutions and alcoholic beverages, necessitating the development of rapid methods for GHB detection. In this respect, portable testing methods for use in the field, based on lateral flow assays (LFAs) and capable of detecting trace concentrations of target analytes, are particularly attractive and hold enormous potential for the detection of illicit drugs. Using this strategy, here we report a rapid, low cost, easy-to-handle strip-based LFA for GHB analysis employing a smartphone for fluorescence readout. At molecular signalling level, the ensemble is based on a Cu2+ complex with a tetradentate ligand and the fluorescent dye coumarin 343, which indicate GHB through an indicator displacement assay (IDA) in aqueous solution. When incorporated in a LFA-based strip test this system shows a detection limit as low as 0.03 μM for GHB in MES buffer solution and is able to detect GHB at concentrations of 0.1 μM in soft drinks and alcoholic beverages in only 1 min.
Die Kombination aus elektrogenerierter Chemilumineszenz (ECL) und Aptamer-gesteuerter Indikatorfreisetzung (gAID) aus magnetischen mesoporösen Silica-Nanopartikeln, die in mit Poly(ethylenglycol) und N-(3-Triethoxysilylpropyl) diethanolamin funktionalisiertes Glasfaserpapier eingebettet sind, ermöglichte die Entwicklung eines Schnelltests, mit dem sich Penicillin direkt in verdünnter Milch bis auf 50 +/- 9 ppt in <5 min nachweisen lässt. Die kovalente Bindung der Aptamer-Kappe an das Siliciumdioxid-Gerüst ermöglichte den Verschluss der Poren durch nicht-kovalente elektrostatische Wechselwirkungen mit Oberflächen-Aminogruppen. Die Bindung von Penicillin führte zu einer Faltung des Aptamers, wodurch der zuvor in das Material geladene ECL-Reporter Ru(bpy)32+ freigesetzt wurde und nach lateralem Fluss von einer Smartphone-Kamera nach elektrochemischer Anregung mit einer in einen 3D-gedruckten Halter eingesetzten Siebdruckelektrode detektiert wird. Der Ansatz ist einfach, generisch und bietet Vorteile hinsichtlich Empfindlichkeit, Messunsicherheit und Robustheit gegenüber konventioneller Fluoreszenz- oder elektrochemischer Detektion, insbesondere für Point-of-Need-Analysen anspruchsvoller Matrices und Analyten im Ultraspurenbereich.