Wash-Free Multiplexed Mix-and-Read Suspension Array Fluorescence Immunoassay for Anthropogenic Markers in Wastewater

Abstract

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.

A rapid magnetic bead-based immunoassay for sensitive determination of diclofenac

“…As reported before [28], diclofenac sodium salt (Sigma-Aldrich, Steinheim, Germany) was dissolved in dry N,Ndimethylformamide (DMF, Sigma-Aldrich) under argon atmosphere to a final concentration of 1/6 mol/L. Stock solutions of N-hydroxysuccinimide (NHS, Merck) and N,N'-dicyclohexylcarbodiimide (DCC, Sigma-Aldrich) of 1/2 mol/L each in DMF were prepared under argon.…”

“…This is why efforts have been made to transfer the immunochemical detection to faster and/or more mobile platforms. Faster immunoassays developed for DCF comprise an upconversion-linked immunosorbent assay (ULISA) [26], a fluorescence polarization immunoassay (FPIA) [27], and a suspension array fluorescence immunoassay (SAFIA) [28]. Steps forward to mobile and automated sensing of diclofenac have been made with several immunosensors for the detection of DCF reported in recent years [29][30][31][32][33][34][35].…”

On-Site Biolayer Interferometry-Based Biosensing of Carbamazepine in Whole Blood of Epileptic Patients

“…To achieve on-site TDM, a portable nano-plasmonic electrical field-enhanced resonating device and an immunochromatographic strip were built, but both failed to provide quantitative information on CBZ levels [ 16 , 17 ]. A suspension array fluorescence immunoassay that was developed to detect CBZ in wastewater has the potential to be applied for TDM of CBZ in clinical settings [ 18 ]. Surface plasmon resonance biosensors have long proven their capacity in detecting various small molecules [ 19 , 20 ].…”

Hydrogel Microparticles for Fluorescence Detection of miRNA in Mix-Read Bioassay

“…However, the majority require repeated separation and washing steps which is time-consuming, expensive, and subject to errors. For these reasons, in the last year, many no-wash or mix-read biosensors have been developed [1,2] and have led the way toward innovative and sensitive devices for Point of Care (POC) applications. Mix-read (or mix & read) bioassays are easily made by just mixing the sample solution with a signal generator probe (biosensor) and reading the detectable signal in solution or on solid surface.…”