Fenglei Gao

Ultrasensitive electrochemical detection of DNA based on Zn2+ assistant DNA recycling followed with hybridization chain reaction dual amplification

A new strategy to combine Zn(2+) assistant DNA recycling followed with hybridization chain reaction dual amplification was designed for highly sensitive electrochemical detection of target DNA. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of the target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme hybridized with the MB and catalyzed its cleavage in the presence of Zn(2+) cofactor and resulting in a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles triggering the cleavage of MB, thus forming numerous MB fragments. The MB fragments triggered the HCR and formed a long double-helix DNA structure. Because both H1 and H2 were labeled by biotin, a lot of SA-ALP was captured on the electrode surface, thus catalyzing a silver deposition process for electrochemical stripping analysis. This novel cascade signal amplification strategy can detect target DNA down to the attomolar level with a dynamic range spanning 6 orders of magnitude. This highly sensitive and specific assay has a great potential to become a promising DNA quantification method in biomedical research and clinical diagnosis.

A sensitive sandwich-type electrochemical aptasensor for thrombin detection based on platinum nanoparticles decorated carbon nanocages as signal labels

In this work, a novel and sensitive sandwich-type electrochemical aptasensor has been developed for thrombin detection based on platinum nanoparticles (Pt NPs) decorated carbon nanocages (CNCs) as signal tags. The morphological and compositional of the Pt NPs/CNCs were examined using transmission electron microscopy, X-ray diffraction, and Raman spectroscopy. The results showed that the Pt NPs with about 3-5nm in diameter were well dispersed on the surface of CNCs. The thiolated aptamer was firstly immobilized on the gold electrode to capture the thrombin molecules, and then aptamer functionalized Pt NPs/CNCs nanocomposites were used to fabricate a sandwich sensing platform. Then, the high-content Pt NPs on carbon nanocages acting as hydrogen peroxide-mimicking enzyme catalyzed the reduction of H2O2, resulting in significant electrochemical signal amplification. Differential pulse voltammetry is employed to detect thrombin with different concentrations. Under optimized conditions, the approach provided a good linear response range from 0.05 pM to 20nM with a low detection limit of 10fM. This Pt NPs/CNCs-based aptasensor shows good precision, acceptable stability and reproducibility, which provided a promising strategy for electrochemical aptamer-based detection of other biomolecules.

Proximity hybridization triggered hemin/G-quadruplex formation for construction a label-free and signal-on electrochemical DNA sensor

We describe a novel label-free and signal-on electrochemical DNA sensing platform via proximity hybridization triggered hemin/G-quadruplex formation based on the direct electron transfer of hemin. The thiolated modified G-DNA1 was first immobilized onto the Au electrode surface. In the presence of target DNA, Y-junction-structure ternary complex can be formed to trigger the proximity assembly of G-DNA1, hemin, and G-DNA2, which leads to the formation of hemin/G-quadruplex for generation an amplified electrochemical signal by differential pulse voltammetry. The observed signal gain was sufficient to achieve a demonstrated detection limit of 54 fM, with a wide linear dynamic range from 10-13 to 10-9 M and discriminated mismatched DNA from perfect matched target DNA with a high selectivity. Benefiting from the one step proximity dependent hemin/G-quadruplex formation, the assay method is extremely simple and can be carried out within 40min. The advantages of free of any label conjugation step, and versatility make it a promising candidate for point-of-care testing and commercial application.

Highly efficient electrochemical sensing platform for sensitive detection DNA methylation, and methyltransferase activity based on Ag NPs decorated carbon nanocubes

In this paper, we reported a sensitive and selective electrochemical method for quantify DNA methylation, analyzing DNA MTase activity and screening of MTase inhibitor based on silver nanoparticles (Ag NPs) decorated carbon nanocubes (CNCs) as signal tag. The Ag NPs/CNCs was prepared by in situ growth of nanosilver on carboxylated CNCs and used as a tracing tag to label antibody. The sensor was prepared by immobilizing the double DNA helix structure on the surface of gold electrode. When DNA MTase was introduced, the probe was methylated. Successively, anti-5-methylcytosine antibody labeled Ag NPs/CNCs was specifically conjugated on the CpG methylation site. The electrochemical stripping signal of the Ag NPs was used to monitor the activity of MTase. The electrochemical signal has a linear relationship with M.SssI activities ranging from 0.05 to 120U/mL with a detection limit of 0.03U/mL. In addition, we also demonstrated the method could be used for rapid evaluation and screening of the inhibitors of MTase. The newly designed strategy avoid the requirement of deoxygenation for electrochemical assay, and thus provide a promising potential in clinical application.

Enzyme-free amplification for sensitive electrochemical detection of DNA via target-catalyzed hairpin assembly assisted current change.

An isothermal, enzyme-free and sensitive method for electrochemical detection of DNA is proposed based on target catalyzed hairpin assembly and for signal amplification. Molecular beacon 1 (MB1) contains a ferrocene (Fc) tag, which was immobilized on the gold electrode as recognition probe to hybridize with target DNA. Then, molecular beacon 2 hybridized with the opened MB1, allowing the target to be displaced. The displaced target again triggered the next round of strand exchange reaction resulting in many Fc far away from the GE to achieve signal amplification for sensitive DNA detection. The current signal amplification strategy is relatively simple and inexpensive owing to avoid the use of any kind of enzyme or sophisticated equipment. It can achieve a sensitivity of 42 fM with a wide linear dynamic range from 10(-13) to 10(-9)M and discriminate mismatched DNA from perfect matched target DNA with a high selectivity. The proposed method showed excellent specificity, high sensitivity and low detection limit, and could be applied in analysis of real samples.

A novel label-free aptasensor based on target-induced structure switching of aptamer-functionalized mesoporous silica nanoparticles

In this study, a sensitive protocol for the high-performance liquid chromatography (HPLC) detection of adenosine triphosphate (ATP) based on mesoporous silica nanoparticles (MSN) functionalized with an aptamer as a cap has been designed. Aminopyrine (AP) is sealed in the inner pores of MSN with single-stranded DNA. In the presence of ATP, an ATP aptamer combines with ATP and escapes from a pore and thus opens the DNA biogate to release AP. The released signal tags can be easily read out by HPLC. The designed protocol provides sensitive HPLC detection of ATP down to 0.41 nM with a linear range of four orders of magnitude (from 1.0 nM to 10 μM) and has high selectivity toward its target ATP, which can be attributed to the large loading capacity and highly ordered pore structure of mesoporous silica nanoparticles, as well as the selective binding of the aptamer with ATP. This proof of concept might promote the application of ATP-responsive devices and also provide ideas for designing various target-responsive systems using other aptamers as caps.

Assistant deoxyribonucleic acid recycling with Zn2+ and molecular beacon for electrochemical detection of deoxyribonucleic acid via target-triggered assembly of mutated DNAzyme

A novel enzyme-free amplification strategy was designed for sensitive electrochemical detection of deoxyribonucleic acid (DNA) based on Zn(2+) assistant DNA recycling via target-triggered assembly of mutated DNAzyme. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme first hybridized and then cleaved the MB in the presence of cofactor Zn(2+). After cleavage, the MB was cleaved into two pieces and the ferrocene (Fc) labeled piece dissociated from the gold electrode, thus obviously decreasing the Fc signal and forming a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles to trigger the cleavage of many MB substrates. Therefore, the peak current of Fc dramatically decreased to approximately zero. The strategy showed a detection limit at 35 fM levels, which was about 2 orders of magnitude lower than that of the conventional hybridization without Zn(2+)-based amplification. The Zn(2+) assistant DNA recycling offers a versatile platform for DNA detection in a cost-effective manner, and has a promising application in clinical diagnosis.

Proximity hybridization triggered strand displacement and DNAzyme assisted strand recycling for ATP fluorescence detection in vitro and imaging in living cells

We developed a novel strategy for ATP detection in vitro and imaging in living cells based on integrating proximity hybridization-induced strand displacement and metal ion-dependent DNAzyme recycling amplification. Four DNA oligonucleotides were used in the sensing system including two aptamer probes, enzymatic sequences and FAM-linked substrate strands. Upon the addition of ATP, the proximity binding of two aptamers to ATP led to the release of the enzymatic sequences, which hybridized with the FAM-linked substrate strand on the graphene oxide (GO) surface to form the ion-dependent DNAzyme. Subsequent catalytic cleavage of the DNAzyme by the corresponding metal ions results in recycling of the enzymatic sequences and cyclic cleavage of the substrate strand, liberating many short FAM-linked oligonuleotide fragments separated from the GO surface, which results in fluorescence enhancement due to the weak affinity of the short FAM-linked oligonuleotide fragment to GO. The amount of produced short FAM-linked oligonuleotide fragments is positively related to the concentration of ATP. This means that one target binding could result in cleaving multiplex fluorophore labelled substrate strands, which provided effective signal amplification. The vivo studies suggested that the nanoprobe was efficiently delivered into living cells and worked for specific, high-contrast imaging of target ATP. More importantly, this target-responsive nanoscissor model is an important approach for intracellular amplified detection and imaging of various analytes by selecting appropriate affinity ligands.

Nitrogen doped carbon nanocage modulated turn-on fluorescent probes for ATP detection in vitro and imaging in living cells

In this work, nitrogen-doped carbon nanocage (N-CNC) modulated turn-on fluorescent probes were developed for selective detection of adenosine monophosphate (ATP), and their application for imaging of ATP in living cells was demonstrated. The as-synthesized N-CNC nanoprobes exhibited minimal background fluorescence due to the interaction between ssDNA and the N-CNCs. The aptamer was adsorbed onto the surface of N-CNCs forming an aptamer/N-CNC nanocomplex, and the fluorescence was quenched by the N-CNCs through Forster resonance energy transfer. In the in vitro assay, the introduction of ATP led to the dissociation of the aptamer from the N-CNCs’ surface. The limit of detection was as low as 5 μM in the range of 5 μM to 2 mM, and the nanocomplex was highly selective toward ATP. The in vivo studies suggested that the N-CNC nanoprobes showed excellent biocompatibility and successfully worked for specific, high-contrast imaging of ATP in living cells. Noncovalent binding between N-CNCs and oligonucleotides along with excellent solubility and biocompatibility have shown N-CNCs to be an efficient carrier and provide excellent protection for cellular delivery of genes. They may be an excellent candidate for many biological applications, such as gene and drug delivery, intracellular imaging, and in vivo molecular analysis.