Development of a Microfluidic Paper-Based Analytical Device for Myeloperoxidase Detection in Periodontitis

Objectives: To develop a microfluidic paper-based analytical device (μPAD) that identifies myeloperoxidase (MPO) levels in the saliva of healthy patients and those with periodontal disease. Materials and Methods: A platform similar to a 96-well plate was printed on Watman^® chromatography paper to run the experimental analysis with unstimulated saliva samples were collected from two groups of patients: those with periodontal health (H, n = 15) and established periodontitis (PD, n = 15). Then, three types of chromophore substrates were pipetted into the wells of the prototype: (1) Guaiacol; (2) Guaiacol, 4,4 '-diaminodifenilsulfon (DAB) and hydrogen peroxide in Tris-HCl buffer; and (3) 3,3',5,5'-Tetramethylbenzidine (TMB), followed by saliva samples. The reaction images were analyzed by numbering according to the intensity scale. Results: The comparative results of the reactions using μPAD demonstrated that both the H and PD groups were compatible with each other without differences among the chromophore substrates (p > 0.05). However, the protocol with TMB showed a faster reaction and better color difference when comparing 15.62 ng/mL and 7.81 ng/mL of MPO in the plate embedded with Guaiacol; 1000 ng/mL and 62.5 ng/mL on the Guaiacol and DAB plate; and 62.5 ng/mL of TMB. The average detectable concentrations of MPO in saliva using TMB were H = 21.2 ± 10.4 ng/mL and PD = 28.9 ± 12.8 ng/mL (p = 0.08). Conclusions: The developed microfluidic paper-based analytical device has been tested for identifying the myeloperoxidase saliva levels of healthy patients and those with periodontal disease. This rapid test demonstrated its possible applicability mainly when associated with the TMB chromophore, but further studies are required with different biomarkers to explore this promising diagnostic platform.