Bingying Jiang

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.

Quantum dot layer-by-layer assemblies as signal amplification labels for ultrasensitive electronic detection of uropathogens.

The preparation and use of a new class of signal amplification label, quantum dot (QD) layer-by-layer (LBL) assembled polystyrene microsphere composite, for amplified ultrasensitive electronic detection of uropathogen-specific DNA sequences is described. The target DNA is sandwiched between the capture probes immobilized on the magnetic beads and the signaling probes conjugated to the QD LBL assembled polystyrene beads. Because of the dramatic signal amplification by the numerous QDs involved in each single DNA binding event, subfemtomolar level detection of uropathogen-specific DNA sequences is achieved, which makes our strategy among the most sensitive electronic approach for nucleic acid-based monitoring of pathogens. Our signal amplified detection scheme could be readily expanded to monitor other important biomolecules (e.g., proteins, peptides, amino acids, cells, etc.) in ultralow levels and thus holds great potential for early diagnosis of disease biomarkers.

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.

DNAzyme-based highly sensitive electronic detection of lead via quantum dot-assembled amplification labels.

An electronic DNAzyme sensor for highly sensitive detection of Pb(2+) is demonstrated by coupling the significant signal enhancement of the layer-by-layer (LBL) assembled quantum dots (QDs) with Pb(2+) specific DNAzymes. The presence of Pb(2+) cleaves the DNAzymes and releases the biotin-modified fragments, which further hybridize with the complementary strands immobilized on the gold substrate. The streptavidin-coated, QD LBL assembled nanocomposites were captured on the gold substrate through biotin-streptavidin interactions. Subsequent electrochemical signals of the captured QDs upon acid dissolution provide quantitative information on the concentrations of Pb(2+) with a dynamic range from 1 to 1000 nM. Due to the dramatic signal amplification by the numerous QDs, subnanomolar level (0.6 nM) of Pb(2+) can be detected. The proposed sensor also shows good selectivity against other divalent metal ions and thus holds great potential for the construction of general DNAzyme-based sensing platform for the monitoring of other heavy metal ions.

Aptamer pseudoknot-functionalized electronic sensor for reagentless and single-step detection of immunoglobulin E in human serum.

The development of electronic sensors with minimized usage of reagents and washing steps in the sensing protocols will significantly facilitate the detection of biomolecules. In this work, by using a new pseudoknot design of the aptamer probes, the construction of an electronic sensor for reagentless and single-step detection of immunoglobulin E (IgE) in human serum is described. The pseudoknot aptamer probes are self-assembled on the disposable electrode surface. The association of IgE with the aptamer probes leads to conformational changes of the pseudoknot aptamer structures and brings the redox-tags in close proximity to the electrode, resulting in amplified current response for monitoring IgE. The effects of the pseudoknot structure and the immobilization concentration of the aptamer probes on the sensor performance are evaluated. Under optimal conditions, the detection limit for IgE is estimated to be 60 pM. The sensor is also selective and can be employed to detect IgE in human serum samples. The developed sensor can achieve reagentless, washing-free and low-cost (with the disposable electrode) electrochemical detection of proteins, making this device a convenient sensing platform for the monitoring of different biomarkers when coupled with the appropriate aptamer probes.

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.

Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences.

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.