Fluorescence determination of soluble pyrophosphate levels in synovial fluid as a marker of pseudogout using middle point of quantification concept and molecular sensor

Pseudogout is a type of joint inflammations caused by deposition of calcium pyrophosphate (CaPPi) crystals in the affected joint. As Ca²⁺ is abundant in the synovial fluid (SF), high levels of soluble PPi in the SF could be one of the key factors that contribute to CaPPi formation in the joint and may serve as a biomarker for pseudogout. Here, we developed and applied an artificial molecular sensor to selective fluorescent detection of soluble PPi in SF of the arthritis patients. The sensor employed xanthene as a fluorophore and the Dpa/Zn(II) as two specific binding sites for PPi. When titrated with serially diluted aqueous PPi solutions, the sensor displayed high sensitivity and exhibited the detection limit of 0.01 µM. The effect of salt concentration was normalized via the concept of Middle Point of Quantification (MPOQ) firstly proposed in this study. The performance of this sensor was also further validated by testing with SF samples extracted from eight clinical patients. The results revealed that six patients had the PPi levels in the range of 60 and 200 µM, indicating moderate likelihood of having pseudogout. Hence, our new method for determining the soluble PPi levels in SF shows promise as a robust, sensitive, and accurate diagnostic tool for the pseudogout.