Molecular Beacon-Based Fluorescent Assay for Specific Detection of Oversulfated Chondroitin Sulfate Contaminants in Heparin without Enzyme Treatment.
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 Ca(2+) 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 Ca(2+) ions can efficiently suppress the negative charges of heparin, they do not neutralize the negative charge of OSCS. Thus, in the presence of Ca(2+) 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 Ca(2+) 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.
†Department of Chemistry, National Sun Yat-sen University, 70, Lien-hai Road, Kaohsiung 80424, Taiwan.
Chih-Yi
CY
Lee
†Department of Chemistry, National Sun Yat-sen University, 70, Lien-hai Road, Kaohsiung 80424, Taiwan.|‡School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, 100, Shih-Chuan 1st Road, Kaohsiung 80708, Taiwan.
Wei-Lung
WL
Tseng
D001483:Base Sequence; D001600:Berberine Alkaloids / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000737:chemistry; D002809:Chondroitin Sulfates / Q000032:analysis* / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D013997:Time Factors
D001600:Berberine Alkaloids; D015345:Oligonucleotide Probes; C000666:coralyne; D006493:Heparin; D002809:Chondroitin Sulfates; D002118:Calcium