A novel fluorescence aptasensor for 8-hydroxy-2'-deoxyguanosine based on the conformational switching of K(+)-stabilized G-quadruplex.

A sensitive, lable-free and low cost fluorescence aptasensor was developed for detecting 8-hydroxy-2'-deoxyguanosine (8-OHdG) by using 8-OHdG aptamer (Apt) as a recognition probe and N-methyl mesoporphyrin IX (NMM) as a reporter. The method is based on the conformational switching of a K(+)-stabilized G-quadruplex to a 8-OHdG-stabilized one. NMM can selectively bind to K(+)-stabilized G-quadruplex instead of 8-OHdG-stabilized one. The addition of 8-OHdG in the solution of Apt - K(+) ions leads to a sharp change in fluorescence intensity, which showed a good linear response toward 8-OHdG concentration ranging from 3.96 nM to 211 nM with a detection limit of 1.19 nM. The relative standard deviation and the recovery were 1.23-3.26% (n=11) and 94.8-106.7%, respectively. The proposed aptasensor consists of only an aptamer probe and a specific dye NMM, avoiding the complex and expensive labeling procedure. Thus it is much cheaper and more applicable.

An enzyme-free strategy for ultrasensitive detection of adenosine using a multipurpose aptamer probe and malachite green.

We report on an enzyme-free and label-free strategy for the ultrasensitive determination of adenosine. A novel multipurpose adenosine aptamer (MAAP) is designed, which serves as an effective target recognition probe and a capture probe for malachite green. In the presence of adenosine, the conformation of the MAAP is converted from a hairpin structure to a G-quadruplex. Upon addition of malachite green into this solution, a noticeable enhancement of resonance light scattering was observed. The signal response is directly proportional to the concentration of adenosine ranging from 75 pM to 2.2 nM with a detection limit of 23 pM, which was 100-10,000 folds lower than those obtained by previous reported methods. Moreover, this strategy has been applied successfully for detecting adenosine in human urine and blood samples, further proving its reliability. The mechanism of adenosine inducing MAAP to form a G-quadruplex was demonstrated by a series of control experiments. Such a MAAP probe can also be used to other strategies such as fluorescence or spectrophotometric ones. We suppose that this strategy can be expanded to develop a universal analytical platform for various target molecules in the biomedical field and clinical diagnosis.

A colorimetric aptasensor for the highly sensitive detection of 8-hydroxy-2'-deoxyguanosine based on G-quadruplex-hemin DNAzyme.

A highly sensitive, low-cost colorimetric aptasensor was developed for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in human urine. The method is based on a conformational switching of the 8-OHdG aptamer to form a G-quadruplex structure in the presence of 8-OHdG. The resulting G-quadruplex assembles into a peroxidase-like DNAzyme with hemin, which effectively catalyzes the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS(2-)) by H2O2 to ABTS(+), resulting in an increase in the absorption signal at 416nm along with a color change of the solution. The response signals linearly correlated with the concentration of 8-OHdG, ranging from 466pM to 247nM with a detection limit of 141pM. The relative standard deviation and the recovery were 1.97-3.47% (n=11) and 98.8-100.2%, respectively. The proposed method avoids the label and derivatization steps in common methods and allows direct analysis of the samples by the naked eye without costly instruments, which is reliable, inexpensive, and sensitive.

An ultrasensitive label-free assay of 8-hydroxy-2'-deoxyguanosine based on the conformational switching of aptamer.

We developed a highly sensitive label-free assay of 8-hydroxy-2'-deoxyguanosine (8-OHdG) using 8-OHdG-aptamer (Apt) as the recognition element. The Apt was adsorbed onto the surface of gold nanoparticles (AuNPs), which prevents them from aggregating under high-salt conditions. Upon addition of 8-OHdG, the conformation of the Apt changes to form a G-quadruplex structure, which leads to the aggregation of the AuNPs along with the increase of the resonance light scattering intensity. The mechanism of 8-OHdG that induces Apt to form G-quadruplexes structure was studied by circular dichroism. The response signals linearly correlated with the concentration of 8-OHdG ranging from 90.8pM to 14.1nM with a detection limit of 27.3pM, which is much lower than that obtained by other methods. This method does not need any label steps and sophisticated equipment. The application for detection of 8-OHdG in real samples further demonstrated its reliability. This strategy would be helpful for developing a universal analytical method by simply replacing corresponding aptamers for various target molecules.

Fluorescence quenching determination of metallothioneins using 8-hydroxyquinoline-5-sulphonic acid-Cd(II) chelate.

A novel method for the determination of metallothioneins (MTs) in urine was developed by fluorescence quenching strategy. The response signals linearly correlated with the concentration of MTs in the ranges of 3.12×10(-8)-1.23×10(-6) mol L(-1), and the limit of detection (LOD) was 9.36×10(-9) mol L(-1). The proposed method avoids the label and derivatization steps in common methods, and is reliable, inexpensive and sensitive. Furthermore, the interaction of MTs and 8-hydroxyquinoline-5-sulphonic acid (HQS)-Cd(II) chelate was investigated, and a static quenching mode was proposed to be primarily responsible for the fluorescence quenching event. It could provide a promising potential for the detection of the biomacromolecules which have no native fluorescence, and be benefit to extend the application of fluorescence strategy.