Jun Ki Ahn

A novel electrochemical method to detect theophylline utilizing silver ions captured within abasic site-incorporated duplex DNA

We herein describe a novel and label-free electrochemical system to detect theophylline. The system was constructed by immobilizing duplex DNA containing an abasic site opposite cytosine on the gold electrode surface. In the absence of theophylline in a sample, silver ions freely bind to the empty abasic site in the duplex DNA leading to the highly elevated electrochemical signal by the redox reaction of silver ions. On the other hand, when theophylline is present, it binds to the abasic site by pseudo base pairing with the opposite cytosine nucleobase, which consequently prevents silver ions from binding to the abasic site. As a result, redox reaction of silver ions would be greatly reduced resulting in the accordingly decreased electrochemical signal. By employing this electrochemical strategy, theophylline was reliably detected at a concentration as low as 3.2 μM with the high selectivity over structurally similar substances such as caffeine and theobromine. Finally, the diagnostic capability of this method was also successfully verified by reliably detecting theophylline present in a real human serum sample with an excellent recovery ratio within 100±6%.

A new s-adenosylhomocysteine hydrolase-linked method for adenosine detection based on DNA-templated fluorescent Cu/Ag nanoclusters

We herein describe a novel fluorescent method for the rapid and selective detection of adenosine by utilizing DNA-templated Cu/Ag nanoclusters (NCs) and employing s-adenosylhomocysteine hydrolase (SAHH). SAHH is allowed to promote hydrolysis reaction of s-adenosylhomocysteine (SAH) and consequently produces homocysteine, which would quench the fluorescence signal from DNA-templated Cu/Ag nanoclusters employed as a signaling probe in this study. On the other hand, adenosine significantly inhibits the hydrolysis reaction and prevent the formation of homocysteine. Consequently, highly enhanced fluorescence signal from DNA-Cu/Ag NCs is retained, which could be used to identify the presence of adenosine. By employing this design principle, adenosine was sensitively detected down to 19nM with high specificity over other adenosine analogs such as AMP, ADP, ATP, cAMP, guanosine, cytidine, and urine. Finally, the diagnostic capability of this method was successfully verified by reliably detecting adenosine present in a real human serum sample.

Abasic Site-Assisted Inhibition of Nicking Endonuclease Activity for the Sensitive Determination of Uracil DNA Glycosylase

We herein describe A novel strategy to accurately determine uracil DNA glycosylase (UDG) activity is described based on the finding that nicking endonuclease-assisted cleavage reaction can be regulated by the presence of abasic site. This strategy utilizes DNA probes rationally designed to contain uracil base at the cleavage site for nicking endonuclease, which is coupled to the isothermal nicking endonuclease amplification reaction (NEAR) method. In the absence of UDG, intact DNA probes generate a large number of double-stranded (ds) DNA products through the NEAR, but the presence of UDG that converts uracil base into abasic site suppresses nicking endonuclease activity and the subsequent NEAR. As a result, dsDNA products are not produced, which is simply monitored by the dsDNA specific fluorescence dye, SYBR green I. By employing this strategy, we sensitively determined the UDG activity down to 0.003 U mL-1 with high specificity over other base excision enzymes. In addition, the diagnostic capability of this method was successfully verified by reliably assaying UDG present in a human serum sample.

Rapid and label-free strategy for the sensitive detection of Hg2+ based on target-triggered exponential strand displacement amplification

We herein describe a rapid and label-free strategy for the sensitive detection of Hg2+ based on target-triggered exponential strand displacement amplification (eSDA). The developed strategy utilizes specially designed DNA probes that form mismatched thymine–thymine (T–T) base pairs at their 3′-ends. In the absence of Hg2+, the mismatched T–T base pairs prohibit the DNA polymerase-mediated extension and the subsequent eSDA does not occur. On the other hand, the presence of Hg2+ that mediates the formation of stable T–Hg2+–T base pairs, enables the extension of DNA probes by DNA polymerase. As a result, a large number of duplexes are produced through the eSDA, which is simply monitored in real-time by the gradual fluorescence enhancement from SYBR green I. By employing this strategy, we detected Hg2+ down to 2.95 pM within 30 min, and the practical applicability of this method was successfully demonstrated by detecting Hg2+ in tap water.

An impedimetric determination of alkaline phosphatase activity based on the oxidation reaction mediated by Cu2+ bound to poly-thymine DNA

We herein describe a novel impedimetric method to determine alkaline phosphatase (ALP) activity based on the Cu2+-mediated oxidation of ascorbic acid on a specific DNA probe-modified electrode. In this method, pyrophosphate (PPi) capable of complexing with Cu2+ is employed as a substrate of the ALP enzyme. In the presence of ALP, PPi is hydrolyzed to phosphate (Pi), which is not able to entrap Cu2+. The free Cu2+ are specifically bound to a poly-thymine DNA probe immobilized on the electrode surface and reduced to form copper nanoparticles by a concomitant oxidation of ascorbic acid. As a result, the oxidation products of ascorbic acid are accumulated on the electrode surface, which consequently increase electron transfer resistance (Ret) by interrupting the electron transfer on the electrode. On the other hand, in the absence of ALP, PPi remains intact to effectively capture Cu2+, consequently preventing the oxidation of ascorbic acid and the subsequent increase of Ret. Based on this design principle, the change in Ret, which is proportional to ALP activity, was measured by electrochemical impedance spectroscopy (EIS) and ALP activities were successfully determined down to 6.5 pM (7.2 U L−1) with excellent selectivity.