Si-Han Chen

A novel strategy for dual-channel detection of metallothioneins and mercury based on the conformational switching of functional chimera aptamer

A novel strategy for dual-channel detection of metallothioneins (MTs) and Hg(2+) has been proposed. In the absence of Hg(2+), the functional chimera aptamer (FCA) designed can form an intact G-quadruplex with flexibility, which was demonstrated to have peroxidase-like activities upon hemin binding. In the presence of Hg(2+), the formation of T-Hg(2+)-T complex results in the conformational switching of FCA, which lost the peroxidase-like activities and cannot catalyze the oxidation of ABTS by H2O2. Upon addition of MTs in this solution, MTs could interact with Hg(2+) to form a MTs-Hg(2+) complex, leading to the recovery of the G-quadruplex DNAzyme. The color and absorbance of the sensing system were also changed accordingly. In the optimizing condition, ΔA was directly proportional to the concentration ranging from 8.84 nM to 1.0 μM for Hg(2+), and 7.82 nM to 0.462 μM for MTs with the detection limits of 2.65 nM and 2.34 nM, respectively. The proposed dual-channel method avoids the label steps in common methods, and allows direct analysis of the samples without costly instruments, and is reliable, inexpensive and sensitive.

Ultrasensitive detection of uranyl by graphene oxide-based background reduction and RCDzyme-based enzyme strand recycling signal amplification

We proposed a novel strategy which combines graphene oxide-based background reduction with RCDzyme-based enzyme strand recycling amplification for ultrahigh sensitive detection of uranyl. The RCDzyme is designed to contain a guanine (G)-rich sequence that replaces the partial sequence in an uranyl-specific DNAzyme. This multifunctional probe can act as the target recognition element, DNAzyme and the primer of signal amplification. The presence of UO2(2+) can induce the cleavage of the substrate strands in RCDzyme. Then, each released enzyme strand can hybridize with another substrate strands to trigger many cycles of the cleavage by binding uranyl, leading to the formation of more G-quadruplexes by split guanine-rich oligonucleotide fragments. The resulting G-quadruplexes could bind to N-methyl-mesoporphyrin IX (NMM), causing an amplified detection signal for the target uranyl. Next, graphene oxide-based background reduction strategy was further employed for adsorbing free ssDNA and NMM, thereby providing a proximalis zero-background signal. The combination of RCDzyme signal amplification and proximalis zero-background signal remarkably improves the sensitivity of this method, achieving a dynamic range of two orders of magnitude and giving a detection limit down to 86 pM, which is much lower than those of related literature reports. These achievements might be helpful in the design of highly sensitive analytical platform for wide applications in environmental and biomedical fields.

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.

Dual-channel detection of metallothioneins and mercury based on a mercury-mediated aptamer beacon using thymidine–mercury–thymidine complex as a quencher

A novel dual-channel strategy for the detection of metallothioneins (MTs) and Hg(2+) has been developed based on a mercury-mediated aptamer beacon (MAB) using thymidine-mercury-thymidine complex as a quencher for the first time. In the presence of Hg(2+), the T-rich oligonucleotide with a 6-carboxyfluorescein (TRO-FAM) can form an aptamer beacon via the formation of T-Hg(2+)-T base pairs, which results in a fluorescence quenching of the sensing system owing to the fluorescence resonance energy transfer (FRET) from the fluorophore of FAM to the terminated T-Hg(2+)-T base pair. The addition of MTs into this solution leads to the disruption of the T-Hg(2+)-T complex, resulting in an increase of the fluorescent signal of the system. In the optimizing condition, ΔF was directly proportional to the concentrations ranging from 5.63 nM to 0.275 μM for MTs, and 14.2 nM to 0.30 μM for Hg(2+) with the detection limits of 1.69 nM and 4.28 nM, respectively. The proposed dual-channel method avoids the label steps of a quencher in common molecular beacon strategies, without tedious procedure or the requirement of sophisticated equipment, and is rapid, inexpensive and sensitive.

Exonuclease III-assisted substrate fragment recycling amplification strategy for ultrasensitive detection of uranyl by a multipurpose DNAzyme

We report on a novel and highly sensitive strategy for UO22+ detection. The designed single-labeled hairpin signal probe (SSP) was employed as a reporter, and a multipurpose RNA-cleaving DNAzyme as a target recognition element, catalytic DNAzyme and the primer of substrate fragment recycling amplification. The presence of UO22+ results in the substrate strand in DNAzyme being cleaved at the RNA site, releasing short substrate fragments. The fragment that is complementary partially with SSP could open the hairpin of SSP to form a double-stranded DNA with recessed 3′-termini, which could be selectively digested by exonuclease III. After the double-stranded DNA is fully consumed, the substrate fragment is released. This one could hybridize with another SSP to initiate the next round of digestion by exonuclease III. The recycling of substrate fragments can be repeated multiple times, resulting in the accumulation of the more 6-carboxyfluorescein in the solution along with an amplified fluorescence signal. By this strategy, we achieved hitherto an unreported UO22+ detection limit of 2.4 pM. Therefore, this novel exonuclease III-amplified strategy has great potential for highly sensitive and selective quantification of UO22+ in biomedical research and environmental science fields.

Ultrasensitive detection of Ag(I) based on the conformational switching of a multifunctional aptamer probe induced by silver(I)

We for the first time confirmed that the low concentrations of Ag(I) could induce a silver specific aptamer probe (SAP) from a random coil sequence form to G-quadruplex structure. Thereby, a novel highly sensitive fluorescence strategy for silver(I) assay was established. The designed multifunctional SAP could act as a recognition element for Ag(I) and a signal reporter. The use of such a SAP can ultrasensitively and selectively detect Ag(I), giving a detection limit down to 0.64nM. This is much lower than those reported by related literatures. This strategy has been applied successfully for the detection of Ag(I) in real samples, further proving its reliability. Taken together, the designed SAP is not only a useful recognition and signal probe for silver, but also gives a platform to study the interaction of monovalent cations with DNA.

A label-free ultrasensitive and selective strategy for Pb(II) assay by a multifunctional DNA probe-mediated rolling-circle amplified synthesis of the G-quadruplexes

We report a label-free ultrasensitive and selective strategy for Pb(II) assay by a multifunctional DNA probe (MDP)-mediated rolling-circle amplified synthesis of the G-quadruplexes. The MDP acted as a target recognition probe, a catalytic DNAzyme and a primer of rolling-circle amplification (RCA). The presence of Pb(II) can induce the conformational switching of the MDP, resulting in the cleavage of the S-DNA in the MDP to release an E-DNA. The released E-DNA then initiated a RCA reaction with a reasonably devised padlock DNA template to produce an accumulated amount of repeated sequences. The RCA product in the present work was designed as a G-rich sequence, which could fold into thousands of G-quadruplex units. The G-quadruplex formed by RCA can specifically bind to NMM to result in an amplified fluorescence signal. Through these cascade amplifications, Pb(II) ions can be detected at as low as 94.29 pM, which is much lower than those reported in related literature. We expect that this amplification strategy might be helpful in the design of a highly sensitive analytical platform for wide application in environmental and biomedical fields.