Toward point-of-care diagnostics: Running enzymatic assays on a photonic waveguide-based sensor chip with a portable, benchtop measurement system.

We demonstrate a portable, compact system to perform absorption-based enzymatic assays at a visible wavelength of 639 nm on a photonic waveguide-based sensor chip, suitable for lab-on-a-chip applications. The photonic design and fabrication of the sensor are described, and a detailed overview of the portable measurement system is presented. In this publication, we use an integrated photonic waveguide-based absorbance sensor to run a full enzymatic assay. An assay to detect creatinine in plasma is simultaneously performed on both the photonic sensor on the portable setup and on a commercial microplate reader for a clinically relevant creatinine concentration range. We observed a high correlation between the measured waveguide propagation loss and the optical density measurement from the plate reader and measured a limit-of-detection of 4.5 µM creatinine in the sensor well, covering the relevant clinical range for creatinine detection.

Development and validation of assays for the quantification of ß-D-N4-hydroxycytidine in human plasma and ß-D-N4-hydroxycytidine-triphosphate in peripheral blood mononuclear cell lysates.

The novel antiviral prodrug molnupiravir is under evaluation for the treatment of SARS-CoV-2. Molnupiravir is converted to ß-D-N4-hydroxycytidine (NHC), which is the primary form found in systemic circulation. ß-D-N4-hydroxycytidine-triphosphate (NHCtp) is the bioactive anabolite produced intracellularly. Sensitive and accurate bioanalytical methods are required to characterize NHC and NHCtp pharmacokinetics in clinical trials. Human K2EDTA plasma or peripheral blood mononuclear cell (PBMC) lysates were spiked with NHC (plasma) or NHCtp (PBMC), respectively. Following the addition of isotopically-labeled internal standards and sample extraction via protein precipitation or lysate dilution, respectively, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Methods were validated in accordance with FDA Bioanalytical Method Validation recommendations. NHC can be quantified in plasma with a lower limit of quantification (LLOQ) of 1 ng/mL; the primary linearity of the assay is 1-5000 ng/mL. Assay precision and accuracy were ≤ 6.40% and ≤ ± 6.37%, respectively. NHC is unstable in whole blood and has limited stability in plasma at room temperature. The calibration range for NHCtp in PBMC lysates is 1-1500 pmol/sample, and the assay has an LLOQ of 1 pmol/sample. Assay precision and accuracy were ≤ 11.8% and ≤± 11.2%. Ion suppression was observed for both analytes; isotopically-labeled internal standards showed comparable ion suppression, resulting in negligible (<5%) relative matrix effects. Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the quantification of NHC in plasma and NHCtp in PBMC lysates. The described methods are appropriate for use in clinical trials.

Rapid Quantification of Plasma Creatinine Using a Novel Kinetic Enzymatic Assay.

BACKGROUND: Enzymatic assays are among the most common diagnostic tests performed in the clinical laboratory. Enzymatic substrate analysis is most commonly measured using endpoint methods; however, modulating the reaction kinetics allows fine control of the reaction rate, which can be adjusted based on specific monitoring technologies. METHODS: We developed and optimized an enzymatic method for measurement of creatinine in plasma, using commonly paired enzymes of creatininase (Crtnnase), creatinase (Crtase), sarcosine oxidase (SOX), ascorbate oxidase (AOX), and horseradish peroxidase (HRP). The novel aspect of the assay is that it is fast and uses SOX as the limiting enzyme. The assay performance was assessed with respect to precision, accuracy, and interferences. RESULTS: The intrarun %CV (n = 12) was approximately 5% for each concentration tested, with biases ranging from -3 to -9%. The interrun %CV (n = 39) ranged from 5 to 8%, with biases ranging from -2 to -6%. During the accuracy assessment (n = 127), only 4 samples did not meet the minimum acceptability criteria. Minimal interference was observed, except at low creatinine concentrations with elevated creatine. CONCLUSION: Our novel and versatile enzymatic assay to measure plasma creatinine using kinetic analysis with SOX as the limiting enzyme is rapid (<2 mins), sensitive, and specific and demonstrates excellent concordance with the laboratory standard. We anticipate this rapid kinetic assay to be compatible with emerging technologies in the field of portable diagnostic devices, such as the usage of silicon photonics to monitor biochemical reactions.