Novel Electrochemical Biosensors Based On Self-assembly And Target-recycling For Signal Amplification

Rapid, simple and sensitive determination of biomolecules has become increasingly important in clinical diagnosis, food analysis and bioterrorism/environmental monitoring over the past few years. Electrochemical biosensor has gained increasing interest due to its inherent advantages such as simplicity, sensitivity and low cost in cooperation with the comprehensive applications in different fields. Various signal amplification methods have been reported to achieve high sensitivity for biomolecules determination. Therefore, aiming to simplify the operation steps, this research mainly focuses on the self-assembly (including the assembly of monomolecular layer on the electrode surface, assembly of nanomaterials and DNA self-assembly) and target (DNA, biological small molecules) recycling technologies as the amplification methods to creatively develop electrochemical biosensors. The corresponding principle and performance of each work have been explored carefully. The research contents are mainly as follows:1. Aptamer-based highly sensitive electrochemiluminescent detection of thrombin via nanoparticle layer-by-layer assembled amplification labels.The preparation and use of a new class of signal amplification label, the CdTe quantum dot (QD) nanoparticle layer-by-layer (LBL) assembled polystyrene microbead composite, for amplified ultrasensitive electrochemiluminescent detection of thrombin is described. The presence of the thrombin leads to the association of thrombin with the primary binding aptamers immobilized on the electrode surface and the secondary signaling aptamers conjugated to the QD LBL assembled polystyrene beads. Due to the dramatic signal amplification of the numerous CdTe QDs involved in each aptamers/thombin binding event, subfemtomolar level detection of thrombin is achieved, which makes our strategy among the most sensitive approach for aptamer-based thrombin monitoring. Our new signal amplified detection scheme could be readily expended 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.2. Target recycling amplification for sensitive and label-free impedimetric genosensing based on hairpin DNA and graphene/Au nanocomposites.A convenient signal amplification strategy for sensitive and label-free impedimetric detection of specific DNA sequences is described. The strategy is based on enzymatic target recycling and graphene/AuNP nanocomposites as well as the hairpin DNA probes. The target DNAs hybridize with the hairpin DNA probes self-assembled on a graphene/AuNP modified screen printed carbon electrode. The enzyme, exonulease Ⅲ (Exo Ⅲ), selectively cleaves the open and hybridized hairpin DNA probes to release the target DNAs. These released target DNAs again hybridize with the remaining hairpin DNA probes and trigger another cleavage cycle in the presence of Exo Ⅲ. As a result, trace amount of target DNA leads to the removal of a large number of hairpin DNA probes from the electrode surface, leading to a substantial decrease in electron transfer resistance monitored by electrochemical impedance spectroscopy (EIS). Due to the effective catalytic target recycling signal amplification, low femtomolar (10 fM) level of target DNA sequences could be detected in a simple and label-free fashion. Our sensing protocol thus could be readily expanded for amplified, label-free EIS detection of a wide range of DNA sequences at trace levels.3. In Situ Hybridization Chain Reaction Amplification for Universal and Highly Sensitive Electrochemiluminescent Detection of DNA.In this work, we describe a new universal and highly sensitive strategy for electrochemiluminescent (ECL) detection of sequence specific DNA at the femtomolar level via in situ hybridization chain reaction (HCR) signal amplification. The DNA capture probes are self-assembled on a gold electrode. The presence of the target DNA and two hairpin helper DNAs leads to the formation of extended dsDNA polymers through HCR on the electrode surface. The in situ, HCR-generated dsDNA polymers cause the intercalation of numerous ECL indicators (Ru(phen)32+) into the dsDNA grooves, resulting in significantly amplified ECL signal output. The proposed strategy combines the amplification power of the DNA HCR and the inherent high sensitivity of the ECL technique and enables low femtomolar detection of sequence specific DNA. The developed strategy also shows high selectivity against single-base mismatch sequences, which makes our new universal and highly sensitive HCR-based method a useful addition to the amplified DNA detection arena.4. A new hybrid signal amplification strategy for ultrasensitive electrochemical detection of DNA based on enzyme-assisted target recycling and DNA supersandwich assemblies.Herein, we describe a highly sensitive strategy for electrochemical detection of sequence-specific DNA by coupling N.BstNB I (a nicking endonuclease)-assisted target recycling amplification with DNA supersandwich assembly signal enhancement. The target DNA hybridizes with the hairpin probes immobilized on the surfaces of the magnetic beads to form partial double-stranded structures and specific recognition sites for N.BstNB I. The N.BstNB I enzyme selectively cleaves the hybridized DNA probes and releases the target DNA, which further hybridizes with other hairpin probes to initiate the target recycling cycles and produces massive intermediate DNA fragments. Subsequently, the numerous intermediate DNA fragments act as bridges to assemble multiple helper DNA probes to form DNA supersandwich structures on the electrode surfaces. The resulting DNA supersandwiches bind with numerous redox-active probes. [Ru(NH3)6]3+, within the vicinity of the sensing surface and lead to dramatically amplified analytical signals for DNA quantitation. The integration of the N.BstNB I-assisted target recycling with the supersandwich assemblies thus provides a powerful amplification route for ultrasensitive electrochemical detection of DNA down to the sub-femtomolar level (0.36 fM). Our proposed sensing strategy not only offers high sensitivity and good selectivity but also is easy to operate as it avoids extra chemical labeling steps and takes the advantages of magnetic beads (easy separation and concentration), which facilitates the DNA assay processes and makes this protocol hold great potentials for early diagnosis of gene-related diseases.5. Binding-induced autonomous disassembly of aptamer-DNAzyme supersandwich nanostructures for sensitive electrochemiluminescence turn-on detection of OTA.A new The DNA-based self-assembly nanostructure has been one of the most interesting research area in the field of nanoscience, and the application of the DNA self-assembled nanostructures in biosensing is still in the early stage. In this work, based on the target-induced autonomous disassembly of the aptamer-DNAzyme supersandwich nanostructures, we demonstrated a highly sensitive strategy for electrochemiluminescent (ECL) detection of Ochratoxin A (OTA). The aptamer-DNAzyme supersandwich nanostructures, which exhibited significant ECL quenching effect toward the O2/S2O82- system, were self-assembled on the gold electrode surface. The presence of the target OTA and the exonuclease (RecJf) resulted in autonomous disassembly of the nanostructures and cyclic reuse of OTA, leading to efficient recovery of the ECL emission and highly sensitive detection of OTA. Our developed method also showed high selectivity against other interference molecules and can be applied for the detection of OTA in real red wine samples, which offers the proposed method opportunities for designing new DNA-based nanostructures for biosensing applications.