Jiaqing Xie

Highly sensitive electrochemical detection of cocaine on graphene/AuNP modified electrode via catalytic redox-recycling amplification.

We demonstrated a new strategy for highly sensitive electrochemical detection of cocaine by using two engineered aptamers in connection to redox-recycling signal amplification. The graphene/AuNP nanocomposites were electrochemically deposited on a screen printed carbon electrode to enhance the electron transfers. The cocaine primary binding aptamers were self-assembled on the electrode surface through sulfur-Au interactions. The presence of the target cocaine and the biotin-modified secondary binding aptamers leads to the formation of sandwich complexes on the electrode surface. The streptavidin-conjugated alkaline phosphatases (ALPs) were used as labels to generate quantitative signals. The addition of the ALP substrate and the co-reactant NADH results in the formation of a redox cycle between the enzymatic product and the electrochemically oxidized species and the signal is thus significantly amplified. Because of the effective modification of the sensing surface and signal amplification, low nanomolar (1 nM) detection limit for cocaine is achieved. The proposed aptamer-based sandwich sensing approach for amplified detection of cocaine thus opens new opportunities for highly sensitive determination of other small molecules.

Label-free and amplified aptasensor for thrombin detection based on background reduction and direct electron transfer of hemin.

In this work, we describe the development of a sensitive and label-free aptasensor for thrombin detection based on background noise reduction by exonuclease I (Exo I) and signal amplification by direct electron transfer (DET) of hemin. The thrombin binding aptamers (TBAs) are self-assembled on a sensing electrode. In the absence of the target thrombin, the TBAs are digested by Exo I, which avoids the association of hemin and significantly minimizes the background current noise. The presence of thrombin stabilizes the folded TBA G-quadruplex and prevents it from degrading by Exo I. The G-quadruplex bound hemin thus generates amplified signal output. In our sensing approach, the introduction of Exo I significantly enhances the signal to noise ratio of the sensor response and achieves sensitive detection of thrombin. Our new method is also coupled with good selectivity against other non-target proteins and thus holds great potential for the development of robust aptasensors for the detection of different types of targets.

Electrochemiluminescence recovery-based aptasensor for sensitive Ochratoxin A detection via exonuclease-catalyzed target recycling amplification

Based on the recovery of the quantum dot (QD) electrochemiluminescence (ECL) and exonuclease-catalyzed target recycling amplification, the development of a highly sensitive aptasensor for Ochratoxin A (OTA) detection is described. The duplex DNA probes containing the biotin-modified aptamer are immobilized on a CdTe QD composite film-coated electrode. The presence of the OTA target leads to effective removal of the biotin-aptamers from the electrode surface via exonuclease-catalyzed recycling and reuse of OTA, which prevents the attachment of streptavidin-alkaline phosphatase (STV-ALP) through biotin-STV interaction. The electron transfer (ET) from the excited state CdTe QD ([CdTe](⁎)) to the electro-oxidized species of the enzymatic product of ALP during the potential scan is thus inhibited and the QD ECL emission is restored for quantitative OTA detection. Due to the exonuclease-catalyzed target recycling amplification, the inhibition effect of ET is significantly enhanced to achieve sensitive detection of OTA down to 0.64 pg mL(-1). The proposed method is selective for OTA and can be used to monitor OTA in real red wine samples. Our developed ECL recovery-based aptasensor thus offers great potential for the development of new ECL sensing platforms for various target analytes.

Amplified terminal protection assay of small molecule/protein interactions via a highly characteristic solid-state Ag/AgCl process.

In this work, we describe a new sensitive strategy for electrochemical detection of protein via small molecule/protein interactions. This assay is based on a terminal protection mechanism that small molecule-linked single-stranded DNA (ssDNA) is protected against hydrolysis by exonuclease I when the target protein is captured by the corresponding small molecule recognition element. Positively charged gold nanoparticles (AuNPs) are attached to the termini-protected and negatively charged ssDNA through electrostatic interactions. Subsequent AuNP-catalyzed silver enhancement followed by a highly characteristic and sensitive solid-state Ag/AgCl process is introduced to the sensing platform to amplify the signal output. By combining the amplification ability resulting from the silver deposition on the surface-captured AuNPs with the inherent high sensitivity of the electrochemical solid-state Ag/AgCl process, our method expands its range to the detection of macromolecules that bind to specific small molecules and enables low picomolar detection of protein. As a model of biotin/streptavidin interaction, a detection limit of 10 pM for streptavidin is readily achieved with desirable sensitivity and specificity, which indicates that the terminal protection assay coupled with the electrochemical solid-state Ag/AgCl process can offer a promising platform for the determination of various of types of proteins or small molecule-protein interactions.

Metallo-Toehold-Activated Catalytic Hairpin Assembly Formation of Three-Way DNAzyme Junctions for Amplified Fluorescent Detection of Hg2+

Because of their irreversible toxicological impacts on the environment and human body, the development of reliable and sensitive Hg2+ detection methods with high selectivity is of great significance. On the basis of the substantial signal amplification by metallo-toehold-triggered, catalytic hairpin assembly (CHA) formation of three-way DNAzyme junctions, we have constructed a highly selective and sensitive fluorescent sensing system for the determination of Hg2+ in different environmental water samples. The presence of the target Hg2+ ions can lead to the generation of T-Hg2+-T base mismatched metallo-toeholds, which trigger the catalytic assembly of three split-DNAzyme containing hairpins to form many Mg2+-dependent DNAzyme junction structures upon binding to the fluorescently quenched substrate sequences. The Mg2+ ions then cyclically cleave the fluorescently quenched substrate sequences of the Mg2+-dependent DNAzymes to generate drastically enhanced fluorescent signals for sensitively detecting Hg2+ at the low 4.5 pM level. The developed sensing method offers high selectivity toward the target Hg2+ over other possible competing metal ions due to the specific T-Hg2+-T bridge structure chemistry in the metallo-toehold domain, and reliable detection of spiked Hg2+ in environmentally relevant water samples with this method is also verified. Considering the nucleic acid nature of the trigger and assembly sequences, the developed approach thus holds great potentials for designing new enzyme-free signal amplification strategies to achieve highly sensitive determination of different DNA and RNA targets.

Coupling of background reduction with rolling circle amplification for highly sensitive protein detection via terminal protection of small molecule-linked DNA.

In this work, by coupling background current reduction with rolling circle amplification (RCA), we describe the development of an ultrasensitive electrochemical sensing method for protein detection based on a small molecule-linked DNA terminal protection strategy. Our detection platform employs a typical streptavidin (STV)-biotin interaction system. Biotin-linked single-stranded DNA (SH-ssDNA-biotin) is self-assembled on a gold electrode to capture the target protein, STV. The binding of STV with the biotin small molecule recognition element protects the SH-ssDNA-biotin against hydrolysis by exonuclease I (Exo I), while the unbound SH-ssDNA-biotin is effectively hydrolyzed and removed from the electrode surface. The bound STV further interacts with long, RCA-amplified biotin DNAs to facilitate the adsorption of numerous electroactive reporters, hexaammineruthenium(III) chloride (RuHex) via electrostatic interactions, which results in significantly amplified signals for the quantitative determination of STV. Moreover, the removal of the unbound SH-ssDNA-biotin probes from the sensing electrode obviates the accumulation of RuHex and leads to a highly minimized background current. The simultaneous RCA signal amplification and background current reduction is expected to significantly enhance the signal-to-noise ratio and to achieve ultrahigh sensitivity. The results reveal that the developed strategy provides a low detection limit of 0.4 pM with high selectivity.

Terminal protection of small molecule-linked ssDNA for label-free and sensitive fluorescent detection of folate receptor.

In this work, based on terminal protection of folate-linked ssDNA (FA-ssDNA) and the SYBR Gold fluorescent dye, we describe the development of a label-free fluorescent strategy for the detection of folate receptors (FRs). The binding between the target FR and the FA moiety of the FA-ssDNA protects the FR bound FA-ssDNA from digesting by Exo I. The binding of SYBR Gold to the terminal protected, un-digested FA-ssDNA leads to enhanced fluorescent emission for the monitoring of FR with a detection limit of 30 pM. Besides, the developed method also shows high selectivity toward FR against other control proteins. Moreover, our approach avoids the labeling of the probes with fluorescent tags and achieves label-free detection of FR. With these advantages, the proposed method thus holds promising potential for the development of simple and convenient strategies for the detection of other proteins by using different small molecule receptor/protein ligand pairs.

Preparation of a new metallomicelle catalyst for the hydrolysis of bis(4-nitrophenyl) phosphate

A new metallomicellar system containing cerium(III), a macrocylic polyamine ligand, and the nonionic surfactant Brij35(polyoxyethylene(23) lauryl ether) was prepared and used as a catalyst in the hydrolysis of bis(4-nitrophenyl) phosphate (BNPP). Catalytic rate of the BNPP hydrolysis was measured kinetically using the UV-VIS spectrophotometric method. The results indicate that the metallomicellar system has relatively high stability and excellent catalytic function in the BNPP hydrolysis; also, the reaction rate of the BNPP catalytic hydrolysis increased by a factor of ca. 1 × 1010 compared to the BNPP spontaneous hydrolysis due to the catalytic effect of the active species and the local concentration effect of the micelles in the metallomicellar system. Experimental results also showed that the mono-hydroxy complex containing the macrocyclic polyamine ligand and cerium(III) is the real active species in the BNPP catalytic hydrolysis, and that the micelles provide a useful catalytic environment for the reaction. On basis of the research results, the reaction mechanism of BNPP catalytic hydrolysis has been proposed.

Development of the aza-crown ether metal complexes as artificial hydrolase

Hydrolases play a crucial role in the biochemical process, which can catalyze the hydrolysis of various compounds like carboxylic esters, phosphoesters, amides, nucleic acids, peptides, and so on. The design of artificial hydrolases has attracted extensive attention due to their scientific significance and potential applications in the field of gene medicine and molecular biology. Numerous macrocyclic metal complexes have been used as artificial hydrolase in the catalytic hydrolysis of the organic substrate. Aza-crown ether for this comment is a special class of the macrocyclic ligand containing both the nitrogen atoms and oxygen atoms in the ring. The studies showed that the aza-crown complexes exhibited high activity of hydrolytic enzyme. However, the aza-crown ether metal complex as artificial hydrolase is still very limited because of its difficulty in synthesis. This review summarizes the development of the aza-crown ether metal complexes as the artificial hydrolase, including the synthesis and catalysis of the transition metal complexes and lanthanide metal complexes of aza-crown ethers. The purpose of this review is to highlight: (1) the relationship between the structure and hydrolytic activity of synthetic hydrolase; (2) the synergistic effect of metal sites and ligands in the course of organic compound hydrolysis; and (3) the design strategies of the aza-crown ethers as hydrolase.

Cascaded signal amplification via target-triggered formation of aptazyme for sensitive electrochemical detection of ATP

The construction of reliable sensors for adenosine triphosphate (ATP) detection gains increasing interest because of its important roles in various enzymatic activities and biological processes. Based on a cascaded, significant signal amplification approach by the integration of the aptazymes and catalytic hairpin assembly (CHA), we have developed a sensitive electrochemical sensor for the detection of ATP. The target ATP leads to the conformational change of the aptazyme sequences and their association with the hairpin substrates to form active aptazymes, in which the hairpin substrates are cyclically cleaved by the metal ion cofactors in buffer to release the enzymatic sequences that can also bind the hairpin substrates to generate active DNAzymes. The catalytic cleavage of the hairpin substrates in the aptazymes/DNAzymes thus results in the generation of a large number of intermediate sequences. Subsequently, these intermediate sequences trigger catalytic capture of many methylene blue-tagged signal sequences on the electrode surface through CHA, producing significantly amplified current response for sensitive detection of ATP at 0.6nM. Besides, the developed sensor can discriminate ATP from analogous interference molecules and be applied to human serum samples, making the sensor a useful addition to the arena for sensitive detection of small molecules.