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The authors have developed a surface-enhanced Raman spectroscopic (SERS) method for the determination of pyrophosphate ion (PPi). It is based on the competitive coordination of Cu(II) between cysteine (Cys) and PPi using silver nanoparticles (AgNPs) modified with Cys and Rhodamine 6G (R6G). Cys was attached to the surface of the AgNPs via Ag−S bond, and the amino acid unit in Cys bind to Cu(II) to form a chelate complex. This results in the aggregation of AgNPs and a strong SERS signal for the probe R6G. However, in the presence of PPi, the aggregated AgNPs are solubilized because of the stronger affinity between PPi and Cu(II). This leads to a decrease of the SERS signal and forms the basis for the quantitation of PPi. The amount of AgNPs, the concentration of Cu(II), and the mixing time were optimized. The method displays a linear response in the 0.1 to 80 μM PPi concentration range, and the limit of detection is as low as 20 nM. The method was applied to the determination of PPi in spiked serum samples and urine samples, with recoveries between 95.2 to 100.5% and relative standard deviations of <4.4%. [ABSTRACT FROM AUTHOR]