Detection of Biomarkers of Periodontal Disease in Human Saliva Using Stabilized, Vertical Flow Immunoassays

Abstract

We report methods for stabilizing cellulose-based immunoassays and using this platform to analyze human saliva. Stabilization treatments of immunoassays for matrix metalloproteinases (MMP)-8 and -9, biomarkers of periodontal disease, were conducted and compared, revealing that anti-MMP-8 and -9 capture antibodies could be stabilized with the addition of a 5% trehalose solution to the test zones, followed by drying in a vacuum oven. After stabilization, the paper devices retained equivalent binding activity to that of freshly prepared tests for 14 days-a time frame that enables US-based clinical testing of this diagnostic assay. A saliva pretreatment method was developed to remove viscous elements without reducing the concentration or binding activity of dissolved proteins. Immunoassays were stored in ziplock bags containing desiccant, and used to detect nanomolar concentrations of MMP-9 in human saliva across the relevant clinical concentration range. These methods and findings facilitate rapid, affordable validation studies of this and other biomarkers that are found in saliva using vertical flow immunoassays.

Development of Paper-Based Analytical Devices for Minimizing the Viscosity Effect in Human Saliva

“…In this case, glass fiber was used to remove proteinaceous substances and cellular debris from saliva and a flow regulator was used to regulate the flow in strip 35 . A vertical flow immunoassay was also developed for the detection of matrix metalloproteinase-8 and -9 where the viscous mucin was removed by freezing, thawing, and centrifuging the sample before testing on PADs 39 . While this method was effective at reducing the viscosity issue, it would be difficult to implement this sample pretreatment step in the field.…”

Label-Free Detection of Salivary Pepsin Using Gold Nanoparticle/Polypyrrole Nanocoral Modified Screen-Printed Electrode

“…However, appropriate sample preparation procedures, including dilution, centrifugation, filtration, precipitation and extraction, can help eliminate or minimize matrix effects [ 35 ]. For example, freezing/centrifugation treatment was very effective to minimize the clogging effect of highly viscous mucins in saliva [ 36 ]. Therefore, saliva samples in this study went through 1.5 times dilution with citric acid, freezing and centrifugation to reduce matrix effects.…”

Exponential Amplification Using Photoredox Autocatalysis

“…Advances in point-of-care diagnostic tests have enabled rapid detection of high concentrations of biomarkers at low cost, 1,2 but these tests have limited utility for clinical decision-making due to lower sensitivity than other laboratory methods such as the polymerase chain reaction (PCR). [3][4][5] On the other hand, PCR can detect low-abundance target nucleic acids 6 or antigens (via immuno-PCR) 7 by amplifying specific nucleic acids exponentially, but this method often takes several hours, and is not easily translated to resource-limited settings due to reliance on laboratory instruments (thermal cyclers and analyzers), poor stability of enzyme-based reagents, and requirement of trained personnel. [8][9][10] To address these issues, efforts have been made to develop isothermal nucleic acid amplification strategies, 9,11 long-term storage methods for reagents, 12 and automated formats.…”

“…Inspired by the activation of non-fluorescent probes such as 1 and 2 via photo-induced oxidation (Scheme 1A), [29][30][31][32][33][34] we designed eosin Y (EY)-based photoredox autocatalysis where doubly reduced and protonated eosin Y (EYH2) is converted into EY by triplet EY ( 3 EY * ) or other oxidizing species produced during the photoredox catalysis of EY (Scheme 1B). EY was chosen as the photocatalyst for the autocatalytic reaction because (1) EY-conjugated affinity reagents can be readily prepared, [35][36][37] (2) EY has a high triplet quantum yield (0.6 -0.7) 38,39 and a long triplet lifetime (1.85 ms) 40 in water, (3) EYH2 cannot absorb visible light, 41,42 (4) both EY and EYH2 are water-soluble due to ionizable groups, and (5) EYH2 is potentially stable against oxidation during storage. 41,43 Our initial hypothesis was that the additional EY generated in previous photoredox cycles can activate EYH2 in the next cycle, so it was expected that the amount of EY would increase exponentially (Scheme 2).…”