Rapid and sensitive detection of Nampt (PBEF/visfatin) in human serum using an ssDNA aptamer-based capacitive biosensor.
A single-stranded DNA (ssDNA) aptamer was successfully developed to specifically bind to nicotinamide phosphoribosyl transferase (Nampt) through systematic evolution of ligands by exponential enrichment (SELEX) and successfully implemented in a gold-interdigitated (GID) capacitor-based biosensor. Surface plasmon resonance (SPR) analysis of the aptamer revealed high specificity and affinity (K(d)=72.52 nM). Changes in surface capacitance/charge distribution or dielectric properties in the response of the GID capacitor surface covalently coupled to the aptamers in response to changes in applied AC frequency were measured as a sensing signal based on a specific interaction between the aptamers and Nampt. The limit of detection for Nampt was 1 ng/ml with a dynamic serum detection range of up to 50 ng/ml; this range includes the clinical requirement for both normal Nampt level, which is 15.8 ng/ml, and Nampt level in type 2 diabetes mellitus (T2DM) patients, which is 31.9 ng/ml. Additionally, the binding kinetics of aptamer-Nampt interactions on the capacitor surface showed that strong binding occurred with increasing frequency (range, 700 MHz-1 GHz) and that the dissociation constant of the aptamer under the applied frequency was improved 120-240 times (K(d)=0.3-0.6 nM) independent on frequency. This assay system is an alternative approach for clinical detection of Nampt with improved specificity and affinity.
College of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-Gu, Seoul 136-713, South Korea.
Jee-Woong
JW
Park
Sreenivasa Saravan
SS
Kallempudi
Javed H
JH
Niazi
Yasar
Y
Gurbuz
Byung-Soo
BS
Youn
Man Bock
MB
Gu
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D015374:Biosensing Techniques / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D004277:DNA, Single-Stranded / Q000737:chemistry*; D003924:Diabetes Mellitus, Type 2 / Q000097:blood / Q000201:enzymology; D017097:Electric Impedance; D004867:Equipment Design; D006801:Humans; D057230:Limit of Detection; D008969:Molecular Sequence Data; D054409:Nicotinamide Phosphoribosyltransferase / Q000097:blood* / Q000302:isolation & purification; D052156:SELEX Aptamer Technique; D020349:Surface Plasmon Resonance
D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D054409:Nicotinamide Phosphoribosyltransferase