Rapid and simple G-quadruplex DNA aptasensor with guanine chemiluminescence detection

Abstract

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.

Rapid aptasensor capable of simply diagnosing prostate cancer

“…6-FAM labeled with PSA aptamers was selected based on the research results we previously reported (Cho et al, 2014). PSA was purchased from Sigma-Aldrich (Milwaukee, WI, USA).…”

“…The procedures for synthesizing DNA and RNA aptamers are more cost-effective, faster, simpler, and stable than those of antibodies using small animals such as mouse, rat, and sheep. As the additional advantage of aptamer, an aptamer conjugated with fluorescent dye can capture a specific biomarker in a sample as well as detect the concentration of biomarker for the diagnosis of diseases (Chen et al, 2012;Cho et al, 2014;Choi and Lee, 2013;Choi et al, 2010;He et al, 2012;Leung et al, 2011;Ma et al, 2013).…”

“…Yamasuji reported that fluorescein-5-isothiocyanate (FITC) conjugated with G-rich single strand DNA (ssDNA) can emit bright chemiluminescence based on the principle of chemiluminescence resonance energy transfer (CRET) (Yamasuji et al, 2011). Recently, we observed that G-rich ssDNA aptamer-conjugated 6-carboxyfluorescein (6-FAM), instead of FITC, capable of binding thrombin in human serum can emit bright light in guanine chemimiluminescence system (Cho et al, 2014). However, G-rich ssDNA aptamer-conjugated 6-FAM (Gquadruplex-conjugated 6-FAM) transformed in the presence of thrombin cannot emit light.…”

“…However, G-rich ssDNA aptamer-conjugated 6-FAM (Gquadruplex-conjugated 6-FAM) transformed in the presence of thrombin cannot emit light. This is because that primary and secondary amines of guanine immobilized due to strong hydrogen bonding between guanines to form G-quadruplex cannot react with TMPG in aqueous solution (Cho et al, 2014).…”

“…Our recent report (Cho et al, 2014) implies that guanine used to capture a biomarker in a sample cannot react with TMPG to emit bright light. If the hypothesis is correct, relative CL intensity of G-rich ssDNA aptamer emitted in the presence of a biomarker will be lower than that in the absence of a biomarker.…”