Methylene Blue Tag Research Articles

Aptamer/Protein Proximity Binding-Triggered Molecular Machine for Amplified Electrochemical Sensing of Thrombin.

The development of convenient and sensitive methods without involving any enzymes or complex nanomaterials for the monitoring of proteins is of great significance in disease diagnostics. In this work, we describe the validation of a new aptamer/protein proximity binding-triggered molecular machinery amplification strategy for sensitive electrochemical assay of thrombin in complex serum samples. The sensing interface is prepared by self-assembly of three-stranded DNA complexes on the gold electrode. The association of two distinct functional aptamers with different sites of thrombin triggers proximity binding-induced displacement of one of the short single-stranded DNAs (ssDNAs) from the surface-immobilized three-stranded DNA complexes, exposing a prelocked toehold domain to hybridize with a methylene blue (MB)-tagged fuel ssDNA strand (MB-DNA). Subsequent toehold-mediated strand displacement by the MB-DNA leads to the release and recycling of the aptamer/protein complexes and the function of the molecular machine. Eventually, a large number of MB-DNA strands are captured by the sensor surface, generating drastically amplified electrochemical responses from the MB tags for sensitive detection of thrombin. Our signal amplified sensor is completely enzyme-free and shows a dynamic range from 5 pM to 1 nM with a detection limit of 1.7 pM. Such sensor also has a high specificity for thrombin assay in serum samples. By changing the affinity probe pairs, the developed sensor can be readily expanded as a more general platform for sensitive detection of different types of proteins.

Homogeneous Electrochemical Method for Ochratoxin A Determination Based on Target Triggered Aptamer Hairpin Switch and Exonuclease III-Assisted Recycling Amplification

A simple, fast, sensitive, and homogeneous electrochemical method has been developed for ochratoxin A (OTA) determination, which combines the advantage of the high selectivity of aptamer to OTA and high efficiency of exonuclease (Exo) III-assisted recycling amplification mechanism. The metastable hairpin probe (HP) was designed into three parts: regions I is the aptamer of OTA as the target recognition; regions II is poly[dA] sequence as extended DNA (E-DNA); regions III as the target DNA (T-DNA) is complementary to a part of the aptamer to stabilize the hairpin-sharp form of the HP in the absence of OTA. The DNA probe with a methylene blue tag at its 3′ terminus (MB-DNA) was designed to be complementary with T-DNA. Introduction of OTA into the assay leads to the formation change of HP from hairpin shape to open form, thus facilitating the hybridization between T-DNA and MB-DNA. The electrochemical signal is amplified through continuous Exo III cleavage. Under the optimal conditions, the differential pulse voltammetric (DPV) response had a linear relationship with the logarithm of OTA concentration in the range of 0.001~0.5 ng mL−1. In addition, the developed method has been successfully applied to detect OTA in wheat standard quality control sample. This homogeneous electrochemical sensor may have a potential prospect in detecting other molecules or proteins, which possesses the corresponding aptamer, through easily designing the hairpin probe.

Ultrasensitive Electrochemical Detection of Nucleic Acids Based on the Dual-Signaling Electrochemical Ratiometric Method and Exonuclease III-Assisted Target Recycling Amplification Strategy.

Because of the intrinsic importance of nucleic acids as biotargets, the simple and sensitive detection of nucleic acids is very essential for biological studies and medical diagnostics. In this work, a novel, simple, and selective electrochemical DNA biosensor for the sensitive detection of target DNA (T-DNA) has been developed based on the dual-signaling electrochemical ratiometric method and exonuclease III (Exo III)-assisted target recycling amplification strategy. The assay strategy includes both "signal-on" and "signal-off" elements. The stem-loop (hairpin) DNA capture probe (HP), which was labeled by thiolated methylene blue (MB) at the 3'-protruding termini and ferrocene (Fc) in the middle of the loop, first self-assembled on the gold electrode surface via a Au-S bond. In the presence of T-DNA, the T-DNA hybridized with HP, which triggered the Exo III cleavage process and accompanied the release of T-DNA. As a result, the MB tags were away from and the Fc tags close to the gold electrode surface, leading to the decrease of the oxidation peak current of MB (I(MB)) and the increase of that of Fc (I(Fc)). The value of ΔI(Fc)/|ΔI(MB)| (ΔI(Fc) and ΔI(MB) are the change values of the oxidation peak currents of Fc and MB, respectively) is linear with the concentration of T-DNA from 0.01 pM to 0.8 pM. The detection limit (4.16 fM) of the developed method is much lower than that of the most reported electrochemical DNA biosensors. This strategy provides a simple and sensitive approach for the detection of T-DNA and has promising applications in bioanalysis, disease diagnostics, and clinical biomedicine.

A ratiometric electrochemical biosensor for sensitive detection of Hg2+ based on thymine–Hg2+–thymine structure

In this paper, a simple, selective and reusable electrochemical biosensor for the sensitive detection of mercury ions (Hg2+) has been developed based on thymine (T)-rich stem–loop (hairpin) DNA probe and a dual-signaling electrochemical ratiometric strategy. The assay strategy includes both “signal-on” and “signal-off” elements. The thiolated methylene blue (MB)-modified T-rich hairpin DNA capture probe (MB-P) firstly self-assembled on the gold electrode surface via Au–S bond. In the presence of Hg2+, the ferrocene (Fc)-labeled T-rich DNA probe (Fc-P) hybridized with MB-P via the Hg2+-mediated coordination of T–Hg2+–T base pairs. As a result, the hairpin MB-P was opened, the MB tags were away from the gold electrode surface and the Fc tags closed to the gold electrode surface. These conformation changes led to the decrease of the oxidation peak current of MB (IMB), accompanied with the increase of that of Fc (IFc). The logarithmic value of IFc/IMB is linear with the logarithm of Hg2+ concentration in the range from 0.5nM to 5000nM, and the detection limit of 0.08nM is much lower than 10nM (the US Environmental Protection Agency (EPA) limit of Hg2+ in drinking water). What is more, the developed DNA-based electrochemical biosensor could be regenerated by adding cysteine and Mg2+. This strategy provides a simple and rapid approach for the detection of Hg2+, and has promising application in the detection of Hg2+ in real environmental samples.

A ratiometric electrochemical aptasensor for sensitive detection of protein based on aptamer–target–aptamer sandwich structure

A novel ratiometric electrochemical aptasensor for sensitive and selective analysis of thrombin has been developed based on aptamer–target–aptamer sandwich structure. In the absence of thrombin, the methylene blue (MB) tagged thrombin aptamer (MB-TBA) was introduced on the electrode through the hybridization with the immobilized DNA sequences, thereby observing an oxidation signal of MB due to the flexible, single-stranded element of 5′ end of MB-TBA. After the addition of thrombin, the specific thrombin–aptamer interaction drove the conformational change of MB-TBA (from the unfolded conformation to the G-quadruplex), resulting in the MB tag far from the electrode surface. The second thrombin aptamer with ferrocene (Fc) tagged on both chain ends (Fc-TBA-Fc) interacted with thrombin to form another G-quadruplex conformation. Such sandwich structure and the G-quadruplex conformation acted cooperatively to allow the two Fc tags to be close to the electrode surface. These conformational changes resulted in a decrease of the oxidation peak current of MB (IMB) and an increase of that of Fc (IFc), and the logarithmic value of IFc/IMB was linear with the logarithm of thrombin concentration. The developed aptasensor shows good response toward the target with a wide linear range from 1nM to 600nM and a low detection limit of 170pM.

Aptamer-based homogeneous protein detection using cucurbit[7]uril functionalized electrode

A new strategy for homogeneous protein detection is developed based on a cucurbit[7]uril (CB[7]) functionalized electrode. The analytical procedure consists of the binding of target protein to its aptamer in the test solution, followed by an exonuclease-catalyzed digestion of methylene blue (MB) tag labeled DNA oligonucleotides. Since CB[7] molecules immobilized on the electrode may efficiently capture the released MB-labeled nucleotides, the MB tags are concentrated to the electrode surface and subsequently yield highly sensitive electrochemical signal, which is related to the concentration of the target protein. The method combines the host–guest properties of CB[7] with the immobilization-free homogeneous assay, providing a powerful tool for protein detection. Taking the detection of osteopontin as an example, the proposed method can have a linear response to the target protein in a range from 50 to 500ngmL−1 with a detection limit of 10.7ngmL−1. It can also show high specificity and good reproducibility, and can be used directly for the assay of osteopontin in serum samples.

A Reagentless Signal-On Architecture for Electronic, Aptamer-Based Sensors via Target-Induced Strand Displacement

Thrombin binding stabilizes the alternative G-quadruplex conformation of the aptamer, liberating the methylene blue (MB)-tagged oligonucleotide to produce a flexible, single-stranded DNA element. This allows the MB tag to collide with the gold electrode surface, producing a readily detectable Faradaic current at thrombin concentrations as low as approximately 3 nM.