Rapid and simple G-quadruplex DNA aptasensor with guanine chemiluminescence detection.
Cost-effective and sensitive aptasensor with guanine chemiluminescence detection capable of simply quantifying thrombin in human serum was developed using thrombin aptamer (TBA), one of the G-quadruplex DNA aptamers, without expensive nanoparticles and complicated procedures. Guanines of G-quadruplex TBA-conjugated carboxyfluorescein (6-FAM) bound with thrombin do not react with 3,4,5-trimethoxylphenylglyoxal (TMPG) in the presence of tetra-n-propylammonium hydroxide (TPA), whereas guanines of free TBA- and TBA-conjugated 6-FAM immobilized on the surface of graphene oxide rapidly react with TMPG to emit light. Thus, guanine chemiluminescence in 5% human serum with thrombin was lower than that without thrombin when TBA-conjugated 6-FAM was added in two samples and incubated for 20 min. In other words, the brightness of guanine chemiluminescence was quenched due to the formation of G-quadruplex TBA-conjugated 6-FAM bound with thrombin in a sample. High-energy intermediate, capable of emitting dim light by itself, formed from the reaction between guanines of TBA and TMPG in the presence of TPA, transfers energy to 6-FAM to emit bright light based on the principle of chemiluminescence energy transfer (CRET). G-quadruplex TBA aptasensor devised using the rapid interaction between TBA-conjugated 6-FAM and thrombin quantified trace levels of thrombin without complicated procedures. The limit of detection (LOD = background + 3 × standard deviation) of G-quadruplex TBA aptasensor with good linear calibration curve, accuracy, precision, and recovery was as low as 12.3 nM in 5% human serum. Using the technology reported in this research, we expect that various types of G-quadruplex DNA aptasensors capable of specifically sensing a target molecule such as ATP, HIV, ochratoxin, potassium ions, and thrombin can be developed.
Luminescent MD, LLC, 20140 Scholar Dr., Hagerstown, MD 21742, United States.
Sandy
S
Cho
Lucienne
L
Park
Richard
R
Chong
Young Teck
YT
Kim
Ji Hoon
JH
Lee
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D004735:Energy Transfer; D054856:G-Quadruplexes; D006108:Graphite; D006147:Guanine / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D057230:Limit of Detection; D049449:Luminescence
D052157:Aptamers, Nucleotide; C081676:thrombin aptamer; D006147:Guanine; D006108:Graphite
3,4,5-trimethoxylphenylglyoxal hydrate (TMPG); Chemiluminescence resonance energy transfer (CRET); G-quaduplex DNA Aptasensor; Guanine chemiluminescence; Magnetic Fe(3)O(4) graphene oxide; Thrombin