Uncovering the Formation of Color Gradients for Glucose Colorimetric Assays on Microfluidic Paper-Based Analytical Devices by Mass Spectrometry Imaging

Abstract

This study describes the use of mass spectrometry imaging with matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) to understand the color gradient generation commonly seen in microfluidic paper-based analytical devices (μPADs). The formation of color gradients significantly impacts assay sensitivity and reproducibility with μPADs but the mechanism for formation is poorly understood. The glucose enzymatic assay using potassium iodide (KI) as a chromogenic agent was selected to investigate the color gradient generated across a detection spot. Colorimetric measurements revealed that the relative standard deviation for the recorded pixel intensities ranged between 34 and 40%, compromising the analytical reliability. While a variety of hypotheses have been generated to explain this phenomenon, few studies have attempted to elucidate the mechanisms associated with its formation. Mass spectrometry imaging using MALDI and DESI was applied to understand the nonuniform color distribution on the detection zone. MALDI experiments were first explored to monitor the spatial distribution of the glucose oxidase and horseradish peroxidase mixture, before and after lateral flow assay with and without KI. MALDI(+)-TOF data revealed uniform enzyme distribution on the detection spots. On the other hand, after the complete assay DESI(-) measurements revealed a heterogeneous shape indicating the presence of iodide and triiodide ions at the zone edge. The reaction product (I) is transported by lateral flow toward the zone edge, generating the color gradient. Mass spectrometry imaging has been used for the first time to prove that color gradient forms as result of the mobility small molecules and not the enzyme distribution on μPAD surface.

Paper-based microfluidic devices based on 3D network polymer hydrogel for the determination of glucose in human whole blood

“…2 These characteristics make paper an ideal platform for point-of-care (POC) with the advantages of high sensitivity, faster analysis time, low-cost, low sample volume, environmental friendliness, exibility and portability. 2 With the development of mPADs, several detection techniques, such as colorimetric change, 3,4 electrochemical properties, 3 light absorption and emission, 5 mass spectrum, 6 and surface-enhanced Raman spectroscopy, 7,8 have been applied to the fabrication of microuidic channels on paper substrates. 9 Among these methods, the advantages of the luminescencebased detection technique integrated with a paper-based device includes low cost, fast detection times, no need for signicant technicians, and high sensitivity.…”

Paper Stacks for Uniform Rehydration of Dried Reagents in Paper Microfluidic Devices

“…Of further note is the uniformity of pink color generated over the 1 cm 2 area of the paper sensor when used with a surface distributor. Colorimetric signals generated in traditional µPADs are associated with steep gradients with standard deviations in pixel intensities ranging from 34 to 40% 15 , which makes it difficult to interpret the signal 7 . Recently, it was shown that these gradients arise as a result of wicking fluid pushing small colorimetric molecules to the edge of the detection zones 15 .…”

“…Colorimetric signals generated in traditional µPADs are associated with steep gradients with standard deviations in pixel intensities ranging from 34 to 40% 15 , which makes it difficult to interpret the signal 7 . Recently, it was shown that these gradients arise as a result of wicking fluid pushing small colorimetric molecules to the edge of the detection zones 15 . No obvious steep chemical gradients were observed in our glucose sensors utilizing surface distributors, despite using relatively large fluid volumes (57 µl) and sensor surface areas (1 cm 2 ).…”

Fully inkjet-printed glucose assay fabricated on highly porous pigment coating

“…Furthermore, multiple stabilisers, such as trehalose (Martinez et al 2007;Gabriel et al 2016), polyvinyl alcohol (Olkkonen et al 2010;Soni and Jha 2015), gelatin (Cha et al 2012;Zhu et al 2014;Liu et al 2019) and dithiothreito (DTT) (Soni and Jha 2017), have been used to ensure long-term stability of the assays. The glucose enzymes have been often spotted onto a substrate (Martinez et al 2007;Santana-Jiménez et al 2018;Dungchai et al 2010;Klasner et al 2010;Gabriel et al 2017;Olkkonen et al 2010;de Freitas et al 2018) though other deposition methods, such as screen printing (Määttänen et al 2011;Lamas-Ardisana et al 2018) and inkjet printing (Abe et al 2010;Wilson et al 2015) take the µPAD a step closer to mass production.…”