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Molecular Beacon-Based Fluorescent Assay for Specific Detection of Oversulfated Chondroitin Sulfate Contaminants in Heparin without Enzyme Treatment

Lee, Chih-Yi, Tseng, Wei-Lung
Analytical chemistry 2015 v.87 no.10 pp. 5031-5035
adenosine, calcium, chondroitin sulfate, electrostatic interactions, enzymatic treatment, fluorescence, fluorescent dyes, heparin, ions, neutralization
Oversulfated chondroitin sulfate (OSCS) is a harmful contaminant in the pharmaceutical heparin. The development of a rapid, convenient, sensitive, and selective method is required for routine analysis of OSCS in pharmaceutical heparin. Here we report a simple, rapid, sensitive, and enzyme-free method for detecting OSCS in heparin based on the competitive binding between OSCS and the adenosine-repeated molecular beacon (MB) stem to coralyne in the presence of Ca²⁺ ions. The MB (A₈–MB–A₈) contains a 22-mer loop, a stem of a pair of 8-mer adenosine (A) bases, a fluorophore unit at the 5′-end, and a quencher at the 3′-end. The presence of coralyne promotes these A–A mismatches to form a hairpin-shaped MB. However, this kind of MB is incapable of differentiating between heparin and OSCS because they both exhibit strong electrostatic attraction with coralyne. This study found that while Ca²⁺ ions can efficiently suppress the negative charges of heparin, they do not neutralize the negative charge of OSCS. Thus, in the presence of Ca²⁺ ions, OSCS can remove coralyne from the MB stem, initiating fluorescence of the MB. Under optimal conditions (10 nM A₈–MB–A₈, 800 nM coralyne, and 0.5 mM Ca²⁺ ions), the proposed system can detect 0.01% w/w OSCS in heparin in under 5 min without enzyme treatment. This study also validates the practicality of the proposed system to determine 0.01% w/w OSCS in the pharmaceutical heparin.