Qinpeng Shen

A simple "clickable" biosensor for colorimetric detection of copper(II) ions based on unmodified gold nanoparticles.

A novel colorimetric copper(II) biosensor has been developed based on the high specificity of alkyne-azide click reaction to the catalysis of copper ions and unmodified gold nanoparticles (AuNPs) as the signal reporter. The clickable DNA probe consists of two parts: an azide group-modified double-stranded DNA (dsDNA) hybrid with an elongated tail and a short alkyne-modified single-stranded DNA (ssDNA). Because of low melting temperature of the short ssDNA, these two parts are separated in the absence of Cu(2+). Copper ion-induced azide-alkyne click ligation caused a structural change of probe from the separated form to entire dsDNA form. This structural change of probe can be monitored by the unmodified AuNPs via mediating their aggregation with a red-to-blue colorimetric read-out because of the differential ability of ssDNA and dsDNA to protect AuNPs against salt-induced aggregation. Under the optimum conditions, this biosensor can sensitively and specifically detect Cu(2+) with a low detection limit of 250 nM and a linear range of 0.5-10 μM. The method is simple and economic without dual-labeling DNA and AuNPs modification. It is also highly selective for Cu(2+) in the presence of high concentrations of other environmentally relevant metal ions because of the great specificity of the copper-caused alkyne-azide click reaction, which potentially meets the requirement of the detection in real samples.

A sensitive, label free electrochemical aptasensor for ATP detection

A sensitive, label free electrochemical aptasensor for small molecular detection has been developed in this work based on gold nanoparticles (AuNPs) amplification. This aptasensor was fabricated as a tertiary hybrid DNA-AuNPs system, which involved the anchored DNA (ADNA) immobilized on gold electrode, reporter DNA (RDNA) tethered with AuNPs and target-responsive DNA (TRDNA) linking ADNA and RDNA. Electrochemical signal is derived from chronocoulometric interrogation of [Ru(NH(3))(6)](3+) (RuHex) that quantitatively binds to surface-confined DNA via electrostatic interaction. Using adenosine triphosphate (ATP) as a model analyte and ATP-binding aptamer as a model molecular reorganization element, the introduction of ATP triggers the structure switching of the TRDNA to form aptamer-ATP complex, which results in the dissociation of the RDNA capped AuNPs (RDNA-AuNPs) and release of abundant RuHex molecules trapped by RDNA-AuNPs. The incorporation of AuNPs in this strategy significantly enhances the sensitivity because of the amplification of electrochemical signal by the RDNA-AuNPs/RuHex system. Under optimized conditions, a wide linear dynamic range of 4 orders of magnitude (1 nM-10 microM) was reached with the minimum detectable concentration at sub-nanomolar level (0.2 nM). Those results demonstrate that our nanoparticles-based amplification strategy is feasible for ATP assay and presents a potential universal method for other small molecular aptasensors.

A DNA-based electrochemical strategy for label-free monitoring the activity and inhibition of protein kinase

A novel label-free electrochemical strategy for monitoring the activity and inhibition of protein kinase is developed, based on the linkage between the phosphorylated peptide and DNA functionalized Au nanoparticles (DNA-AuNPs) by Zr(4+) and the chronocoulometric response of [Ru(NH(3))(6)](3+) absorbed on the DNA-AuNPs.

A fluorometric assay for acetylcholinesterase activity and inhibitor detection based on DNA-templated copper/silver nanoclusters

A novel label-free, rapid, cost-effective, and highly sensitive fluorometric sensor has been constructed for the detection of acetylcholinesterase (AChE) activity and its inhibitor based on the fluorescence quenching of DNA-templated copper/silver nanoclusters (DNA-Cu/AgNCs). In this assay, AChE catalyzes the hydrolysis of acetylthiocholine (ATCh) to form thiocholine which induces fluorescence quenching of DNA-Cu/AgNCs. The AChE activity could be detected as low as 0.05mU/mL and with a linear range from 0.05 to 2.0mU/mL. This assay offers a very convenient mix and detect approach for AChE activity. On the other hand, tacrine and organophosphorus pesticides (OPPs) were employed to inhibit the hydrolysis of ATCh, which could eliminate the fluorescence quenching of DNA-Cu/AgNCs. The IC50 of tacrine and methamidophos were estimated to be 16.9nM and 0.075mg/L, respectively. This method was also used to detect spiked OPPs in agricultural products successfully. The present work may expand the use of DNA-Cu/AgNCs to the field of enzyme sensors.

Intra-molecular G-quadruplex structure generated by DNA-templated click chemistry: "turn-on" fluorescent probe for copper ions.

A novel homogenous fluorescent sensor for signal-on detection of Cu(2+) has been developed based on intra-molecular G-quadruplex formed by DNA-templated click reaction and crystal violet (CV) as label-free signal reporter. The clickable DNA probe consists of two G-rich strands (A and B) bearing azide and alkyne group, respectively, and a template strand (C) locating two proximate reactants by pairing with A and B. The sequences of A and B are derived from asymmetric split of the G-quadruplex sequence (TTAGGG)4. In the presence of Cu(2+), the whole G-quadruplex sequence A-B is generated by chemical ligation of A and B via copper ion-catalyzed alkyne-azide cycloaddition, then released from template by toehold strand displacement, and consequently forming a stable intra-molecular G-quadruplex, which binds with CV to generate a strong fluorescent signal. Oppositely, weak fluorescence was obtained without Cu(2+) because of unstable intermolecular G-quadruplex formed by A and B and lack of lateral loop connection. Therefore, the Cu(2+) can be sensitively and specifically detected by the fluorescence of the CV-stained G-quadruplex with a low detection limit of 65nM and a linear range of 0.1-3µM. This method rationally integrated the DNA-templated synthesis and G-quadruplex structure-switch, presenting a simple and promising approach for biosensor development.

A TdT-mediated cascade signal amplification strategy based on dendritic DNA matrix for label-free multifunctional electrochemical biosensing

We describe a novel label-free amplified multifunctional strategy of dendritic electrochemical DNA sensor based on terminal deoxynucleotidyl transferase (TdT). We have found that the sequence composition of TdT-yielded DNA is largely dependent on the constitution of substrate deoxynucleotides (dNTPs) pool. After rational design of dNTPs pool and controllable TdT polymerization, dendritic protocol has been developed involving two-type amplification strategies; one is the formation of trunk and branch of the dendritic electrochemical sensor by TdT amplification; the other is the introduction of nucleic acid functionalized Au nanoparticles (DNA-AuNPs) for multiple branching. The results indicate that the G-rich ssDNA, which is synthesized under the condition of 40% deoxyadenosine triphosphate (dATP) and 60% deoxyguanosine triphosphate (dGTP), can be induced to form a long signal strand to G-quadruples (G4) in the presence of Pb(2+). The electrochemical sensing platform is employed for sequence-specific DNA detection and the detection limit is as low as 1 fM. Our multifunctional strategy is further extended to Pb(2+) detection and thrombin aptasensor. This proposed sensor displays excellent sensitivity and selectivity, and is applied for detection in complicated samples successfully.

The Ag+–G interaction inhibits the electrocatalytic oxidation of guanine – A novel mechanism for Ag+ detection

The heavy metal ions-nucleobases interaction is an important research topic in environmental and biochemical analysis. The presence of the silver ion (Ag(+)) may influence the formation of oxidation intermediate and the electrocatalytic oxidation activity of guanine (G), since Ag(+) can interact with guanine at the binding sites which are involved in the electrocatalytic oxidation reaction of guanine. According to this principle, a new electrochemical sensor for indirectly detecting Ag(+) based on the interaction of Ag(+) with isolated guanine base using differential pulse voltammetry (DPV) was constructed. Among the heavy metal ions examined, only Ag(+) showed the strongest inhibitory effect on the electrocatalytic oxidation of guanine at the multi-walled carbon nanotubes modified glassy carbon electrode (CNTs/GC). And the quantitative study of Ag(+) based on Ag(+)-G sensing system gave a linear range from 100 nM to 2.5 μM with a detection limit of 30 nM. In addition, this modified electrode had very good reproducibility and stability. The developed electrochemical method is an ideal tool for Ag(+) detection with some merits including remarkable simplicity, low-cost, and no requirement for probe preparation.

Fluorescent detection of copper(II) based on DNA-templated click chemistry and graphene oxide.

A novel DNA-templated click chemistry strategy for homogenous fluorescent detection of Cu(2+) has been developed based on click ligation-dependent DNA structure switch and the selective quenching ability of graphene oxide (GO) nanosheet. The clickable duplex probe consists of two DNA strands with alkyne and azide group, respectively, and Cu(+)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction can chemically ligate these two strands. Toehold sequence displacement was consequently exploited to achieve DNA structure transformation bearing fluorescent tag FAM. Cu(2+)-induced chemical ligation caused the probe transfer to hybrid structure with single stranded DNA (ssDNA) tail, while only duplex structure was obtained without Cu(2+). This structural difference can be probed by GO-based fluorescence detection due to the preferential binding of GO to ssDNA. Under the optimum conditions, this sensor can sensitively and specifically detect Cu(2+) with a low detection limit of 58 nM and a linear range of 0.1-10 μM. This new strategy is highly sensitive and selective for Cu(2+) detection because of the great specificity of click chemistry and super-quenching ability of GO. Moreover, with the aid of high efficient DNA templated synthesis, the detection process requires only about half an hour which is much quicker than previous click-chemistry-based Cu(2+) sensors.

Nitrogen doped graphene quantum dots based long-persistent chemiluminescence system for ascorbic acid imaging

High photo-intensity and sluggish flight attenuation are important to highly sensitive chemluminescence imaging. Herein, we present a copper ion catalyzed long-persistent chemiluminescent imaging system of nitrogen-doped graphene quantum dots (NGQDs) for ascorbic acid detection in fruit. NGQDs as luminescent probe are fabricated, emitting out chemluminescence with the direct oxidation by H2O2. In addition, Cu2+ ion enlarges over two order magnitudes of NGQDs CL intensity (214 times) due to its catalyzed Fenton-like reaction for H2O2 decomposition, and displaying unique specificity against other metal ions. As a result, the twinkling luminescence of NGQDs is boosted and changes to hold persistent with small decay in the presence of copper ion exhibiting potential for CL imaging. As an imaging model, a visual sensor based on Cu2+/NGQDs/H2O2 is developed for AA quantitative monitoring with a limit of detection (LOD) of 0.5μM (S/N=3) and applied in real AA detection in fruit. The CL imaging method demonstrated with high stability and proper sensitivity would provide a convenient and visual tool for AA determination, displaying promising candidates for imaging sensing.

Layer‐by‐Layer Assembly of Polyelectrolyte and Nanoparticles, Monitored by Capillary Electrophoresis

Layer-by-layer (LBL) assembly is a versatile nanofabrication technique, and investigation of its kinetics is essential for understanding the assembly mechanism and optimizing the assembly procedure. In this work, the LBL assembly of polyelectrolyte and nanoparticles were monitored in situ by capillary electrophoresis (CE) for the first time. The assembly of poly(diallyldimethylammonium chloride) (PDDA), and gold nanoparticles (AuNPs) on capillary walls causes surface-charge neutralization and resaturation, and thus yields synchronous changes in the electroosmotic flow (EOF). The EOF data show that formation of multilayers follows first-order adsorption kinetics. On the basis of the fit results, influencing factors, including number of layers, concentration of materials, flow rate, and size of AuNPs, were investigated. The stability and robustness of the assembled coatings were also characterized by CE. It was found that degradation of PDDA layers follows first-order chemical kinetics, while desorption of AuNPs takes place in a disorderly manner. The substrate strongly affects assembly of the underlying layer, while this effect is rapidly screened with increasing number of layers. Furthermore, we demonstrate that the EOF measuring step does not disturb LBL assembly, and the proposed method is reliable and rugged. This work not only studies in detail the LBL adsorption/desorption process of polyelectrolyte and nanoparticles, but also offers an alternative tool for monitoring multilayer buildup. It may also reveal the potential of CE in fields other than analytical separation.