A method and optical references for compensating signal drift in a fluorescent microarray reader

[Display omitted] •A developed method addresses signal variations in a fluorescent microarray reader.•The method with no additional sensor employs two low-cost fluorescent references.•The temporal fluorescent reference is designed to produce gradient emissions.•Both temporal and spatial signal variations are mitigated using this method.•The detection repeatability improves significantly following the method. A point-of-need fluorescent microarray assay is a promising tool for on-site high-throughput and multiplex detection. However, its low-cost and portable read-out system, based on an image sensor, suffers from temporal and spatial signal variations caused by measurement drifts. This work presents a method to compensate for these variations without the need for an additional sensor. The method utilizes two inexpensive fluorescent references specifically designed to mitigate the variations while offering cost-saving benefits and broad portable applicability. This method relies on employing a representative signal instead of raw signals. Relevant systems with both variations were conducted to validate and demonstrate the method's effectiveness by varying light-source temperatures and analysis areas. Under investigation of different International Organization for Standardization (ISO) settings, shutter speeds, and light-source intensities, the method significantly reduced the influence of the variations, enhancing detection repeatability by up to a factor of 26-fold.