Single-point calibration and standard addition assays on calibrant-loaded paper-based analytical devices

This article describes a new, simplified approach for performing quantitative colorimetric assays on paper-based analytical devices that uses calibrant-loaded paper devices to perform external calibration and standard addition calibration using one calibration point. Calibrant-loaded devices consist of sensing areas pre-loaded with a colored product which is produced from the reaction of a standard solution of the analyte with the appropriate colorimetric reagents. When the sample is added into the calibrant-loaded sensing zone the analytical signal (i.e. color intensity) increases proportionally to the concentration of the analyte in the tested sample. The total measured signal corresponds to the sum of the concentration of the analyte in the sample and the standard solution pre-stored in the device and is used to calculate the concentration of the analyte in the sample based on the principles of linear calibration. The applicability of this approach was benchmarked in three colorimetric assays (i.e., for the determination of iron, nickel, and amino acids) that use different reaction chemistries. This work demonstrates a simplified calibration-free approach for assays performed on paper-based analytical devices that requires minimum experimental and computational effort, it can be used for either external or standard addition calibration and can be used to identify and eliminate the presence of interferences in a sample.