Real-Time Immunosensor for Small-Molecule Monitoring in Industrial Food Processes.

Industrial food processes are monitored to ensure that food is being produced with good quality, yield, and productivity. For developing innovative real-time monitoring and control strategies, real-time sensors are needed that can continuously report chemical and biochemical data of the manufacturing process. Here, we describe a generalizable methodology to develop affinity-based biosensors for the continuous monitoring of small molecules in industrial food processes. Phage-display antibody fragments were developed for the measurement of small molecules, as exemplified with the measurement of glycoalkaloids (GAs) in potato fruit juice. The recombinant antibodies were selected for use in a competition-based biosensor with single-molecule resolution, called biosensing by particle motion, using assay architectures with free particles as well as tethered particles. The resulting sensor measures GAs in the micromolar range, is reversible, has a measurement response time below 5 min, and enables continuous monitoring of GAs in protein-rich solutions for more than 20 h with concentration measurement errors below 15%. The demonstrated biosensor gives the perspective to enable a variety of monitoring and control strategies based on continuous measurement of small molecules in industrial food processes.

Analysis of Neuron-Specific enolase isozymes in human serum using immunoaffinity purification and liquid chromatography-tandem mass spectrometry quantification.

Neuron-specific enolase (NSE) is a promising small-cell lung cancer (SCLC) biomarker composed of αγ and γγ isozyme dimers. As the conventional immunoassays are prone to interferences and cannot differentiate between the isozymes, we developed a multiplex immunoaffinity (IA) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of NSEα and NSEγ in human serum. A calibrator was prepared by performing cold denaturation of recombinantly expressed αα and γγ enolase dimers to induce a new dimer equilibrium that was determined to be approximately 1αγ:1γγ:1αα. Selective sample purification was achieved by performing IA extraction using an antibody specific towards NSEγ. The isolated αγ and γγ dimers were denatured and trypsin digested to allow quantification of the selected signature peptides and their corresponding isotopically labelled peptide internal standard. The obtained linear dynamic ranges were determined to be 1.5-56 ng/mL and 0.64-167 ng/mL for NSEα and NSEγ (R2 = 0.88 and 0.97 respectively). Validation of the assay showed acceptable accuracy and precision for NSEα and NSEγ. The method was successfully applied to patient serum in which both isozymes were detected. Compared to the conventional immunoassay, substantially lower total NSE concentrations were measured in IA LC-MS/MS. With this multiplex IA LC-MS/MS assay, the clinical value of quantifying the individual isozymes can be explored. In addition, together with the calibrator described here, it may be applied to standardize NSE immunoassays across different platforms.

A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays.

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.