Abstract
This study presents two sensitive fluorescent assays for sensing heparin on the basis of the electrostatic interaction between heparin and Naja naja atra cardiotoxin 3 (CTX3). Owing to CTX3-induced folded structure of an adenosine-based molecular beacon (MB) or a DNA aptamer against CTX3, a reduction in the fluorescent signal of the aptamer or MB 5′-end labeled with carboxyfluorescein (FAM) and 3′-end labeled with 4-([4-(dimethylamino)phenyl]azo)-benzoic acid (DABCYL) was observed upon the addition of CTX3. The presence of heparin and formation of the CTX3–heparin complex caused CTX3 detachment from the MB or aptamer, and restoration of FAM fluorescence of the 5′-FAM-and-3′-DABCYL-labeled MB and aptamer was subsequently noted. Moreover, the detection of heparin with these CTX3-aptamer and CTX3-MB sensors showed high sensitivity and selectivity toward heparin over chondroitin sulfate and hyaluronic acid regardless of the presence of plasma. The limit of detection for heparin in plasma was determined to be 16 ng/mL and 15 ng/mL, respectively, at a signal-to-noise ratio of 3. This study validates the practical utility of the CTX3-aptamer and CTX3-MB systems for determining the concentration of heparin in a biological matrix.
Highlights
Heparin, which consists of a trisulfated repeating disaccharide unit, is widely used as an anticoagulant or antithrombotic agent during clinical procedures such as cardiovascular surgery.An overdose of heparin can cause adverse effects such as hemorrhages and thrombocytopenia [1,2].The proper therapeutic concentration range of heparin in plasma has been found to be 17–67 μM during cardiovascular surgery and 1.7–10 μM in the postoperative period [3]
The amino groups of cardiotoxin 3 (CTX3) may interact with negatively charged sulfateand and
Heparin possesses three sulfate groups and one carboxylate per repeat unit. These results indicate that the CTX3-aptamer and CTX3-molecular beacon (MB) sensors have higher selectivity for heparin
Summary
Heparin, which consists of a trisulfated repeating disaccharide unit, is widely used as an anticoagulant or antithrombotic agent during clinical procedures such as cardiovascular surgery.An overdose of heparin can cause adverse effects such as hemorrhages and thrombocytopenia [1,2].The proper therapeutic concentration range of heparin in plasma has been found to be 17–67 μM during cardiovascular surgery and 1.7–10 μM in the postoperative period [3]. Measurements of activated clotting time or activated partial thromboplastin time are the traditional procedures for heparin detection [2,4]. These methods are not sufficiently sensitive or accurate for detection of heparin because of their lack of specificity and potential interference from other factors [5]. A number of methods for the quantitative detection of heparin has been developed including a fluorescent method [6], a colorimetric assay [7,8], and an electrochemical method [9] These methods offer reasonable sensitivity and specificity, but some are complicated and time-consuming. Many reported methods are based on complicated nano-organic hybrid systems
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