Rapid Quantification of Plasma Creatinine Using a Novel Kinetic Enzymatic Assay

Abstract

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.

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

“…The novelty of this report compared with our previous publication is running the real‐time enzymatic assay with clinically relevant concentration ranges on an integrated photonic waveguide‐based sensor with the presented portable setup. To demonstrate this, we perform a proof‐of‐principle experiment by running our previously reported creatinine enzymatic assay [14] on the colorimetric chip in plasma spiked with creatinine with concentrations ranging from 0 to 100 μM. All assay preparation steps and materials used to perform the assay can be found in the Supporting Information Section S3.…”

“…The waveguide-based sensor relies on optical absorption caused by the overlap of the waveguide's evanescent field with an analyte solution in close proximity to the waveguide. The creatinine assay that we developed and reported [14] is based on the reporter molecule methylene blue (MB) with an absorption peak at 664 nm. We used it for single-wavelength absorption measurements at a single wavelength line of 639 nm, which is compatible with available low-cost laser diodes.…”

“…Performing a rapid enzymatic assay to detect creatinine in plasma on the photonic colorimetric chip with a small, portable, benchtop tool is an important step toward the achievement of a lab-on-a-chip system for point-of-care diagnostics applications. Recently, we reported extensively on a creatinine assay development with MB used as reporter molecule for absorption measurements [14]. In a previous journal publication [16], we used a scientific microscope to demonstrate the functionality of the photonic waveguide chip by measuring the optical absorption caused by the exposure of the waveguide to a dilution series of MB in Tris (tris(hydroxymethyl)-aminomethane) buffer.…”