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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.
Core-shell (CS) particles with a polymeric core and a silica shell play an important role in the materials and (bio)analytical sciences. Besides the establishment of reliable synthesis procedures, comprehensive particle characterization is essential for batch-to-batch reproducibility and objective performance assessment across architectures, protocols, and laboratories. Particle characterization with respect to size, size distribution, shell thickness and texture, surface area and roughness or materials composition is commonly conducted with different analytical methods, often on different samples. Our approach uses a dual-mode TSEM/SEM set-up with an EDX detector to obtain a complementary data set with sufficient statistical confidence of one and the same sample on a single instrument. Our protocol reveals information about size, size distribution and shell thickness of the various particles employed from overview images, while an increased field of view (FOV) and high-resolution EDX analysis yields detailed information on shell texture and elemental composition. An image analysis tool was developed to derive and quantify the profile roughness of CS particles from individual beads. Comparison with surface roughness data from AFM showed a similar trend in roughness across the series of particles. Reliable classification into smooth and rough is proposed and roughness changes within different particle batches were tracked systematically.
Multiplexed bead-based array formats play an increasingly important role in analytical laboratories. Due to the high surface-to-volume ratio, fast reaction kinetics and modular assay design, these sensor formats are applied in clinical diagnostic, drug development and classical biosensors with great success.
As the spherical platform, researchers utilize micron sized particles made from polymeric or silica material. Such beads are commercially available from vendors such as BD or Luminex. However, we have encountered several problematic issues which accompany these platforms: first, bigger sized beads, which are required for particle handling reasons, are difficult to prepare with high monodispersity - a key requirement for cytometric application. Second, plain beads, made from either polymer or silica, have each several disadvantages such as inferior scattering properties in case of silica or limited flexibility for coupling strategies in the case of latex beads.
In order to overcome this problem, we have developed a versatile core-shell (CS) platform which consists of a polymeric core with a structurally controlled silica shell. In our approach, the core building block can be easily prepared with high yields and high monodispersity in a dispersion polymerization from approximately 500 nm to 1.3 µm. Then, silica is coated in a classical sol-gel process to protect the core with a stable yet modifiable surface (see SEM image in Figure 1, platform). Here, we combine ideal scattering properties and easy preparation of the polymeric core with the chemical flexibility of a silica surface. Moreover, the additional shell domain adds density to the composite, which makes particle handling feasible also for nanometer sized beads.
In this contribution we present proof-of-principle results of competitive immunoassays with fluorescence detection using our CS beads – each performed in mix-and-read fashion without washing steps. All sizes are applicable in cytometric read-out which and can be used for size encoding (see Set 1 to 3 in Figure 1, size encoding). However, further multiplexing for a set of at least 20 parameters can be achieved by swelling hydrophobic dyes into the core. To the same time, precise tuning of the surface with mixed silane layers allowed us to improve the selectivity towards small molecules in competitive immunoassays significantly (see example in Figure 1, Application). We believe that our platform allows researchers to gain access to superior assay performance in combination with a low-threshold approach for the synthesis of the spherical platform.
The need for rapid and high-throughput screening in analytical laboratories has led to significant growth in interest in suspension array technologies (SATs), especially with regard to cytometric assays targeting a low to medium number of analytes. Such SAT or bead-based assays rely on spherical objects that constitute the analytical platform. Usually, functionalized polymer or silica (SiO2) microbeads are used which each have distinct advantages and drawbacks. In this paper, we present a straightforward synthetic route to highly monodisperse SiO2-coated polystyrene core−shell (CS) beads for SAT with controllable architectures from smooth to raspberry- and multilayer-like shells by varying the molecular weight of poly(vinylpyrrolidone) (PVP), which was used as the stabilizer of the cores. The combination of both organic polymer core and a structurally controlled inorganic SiO2 shell in one hybrid particle holds great promises for flexible next-generation design of the spherical platform. The particles were characterized by electron microscopy (SEM, T-SEM, and TEM), thermogravimetry, flow cytometry, and nitrogen adsorption/desorption, offering comprehensive information on the composition, size, structure, and surface area. All particles show ideal cytometric detection patterns and facile handling due to the hybrid structure. The beads are endowed with straightforward modification possibilities through the defined SiO2 shells. We successfully implemented the particles in fluorometric SAT model assays, illustrating the benefits of tailored surface area which is readily available for small-molecule anchoring. Very promising assay performance was shown for DNA hybridization assays with quantification limits down to 8 fmol.