Chang Feng

A dual-colorimetric signal strategy for DNA detection based on graphene and DNAzyme

In this work, by employing graphene together with a peroxidase-mimic DNAzyme, we have developed a novel dual-colorimetric strategy for DNA detection. In this strategy, a bi-functional probe DNA with both the sequence to have peroxidase activity and the sequence to be complementary to the target DNA is designed. Through π–π stacking, the probe DNA can interact with graphene; however, when the target DNA is present, the graphene-probe DNA interaction will be interrupted, resulting in the peroxidase activity being transferred from the precipitated graphene to the supernatant under centrifugation. Consequently, colorimetric signals can be obtained due to the catalytic reactions by the formed peroxidase-mimic DNAzyme. By observing the changes of the color depth of either the precipitate or the supernate, we are able to detect the target DNA very easily and sensitively with the naked eye. The dual colorimetric signals (signal-off for the precipitate and signal-on for the supernate) can also be integrated through mathematical operations, which may greatly improve the performance of the sensing platform.

Fabrication of magneto-controlled moveable architecture to develop reusable electrochemical biosensors

Electrochemical biosensors have been studied intensively for several decades. Numerous sensing concepts and related interface architectures have been developed. However, all such architectures suffer a trade-off: simple architectures favour usability, whereas complex architectures favour better performance. To overcome this problem, we propose a novel concept by introducing a magneto-controlled moveable architecture (MCMA) instead of the conventional surface-fixed architecture. As a model, human breast cancer cells were used in this study. The results showed that a detection range from 100 to 1 × 106 cells could be achieved. Moreover, the whole detection cycle, including the measurement and the regeneration, could be completed in only 2 min. Thus, usability and excellent performance can be achieved in a single biosensor.

Detection of microRNA: A Point-of-Care Testing Method Based on a pH-Responsive and Highly Efficient Isothermal Amplification

Laborious and costly detection of miRNAs has brought challenges to its practical applications, especially for home health care, rigorous military medicine, and the third world. In this work, we present a pH-responsive miRNA amplification method, which allows the detection of miRNA just using a pH test paper. The operation is easy and no other costly instrument is involved, making the method very friendly. In our strategy, a highly efficient isothermal amplification of miRNA is achieved using an improved netlike rolling circle amplification (NRCA) technique. Large amounts of H+ can be produced as a byproduct during the amplification to induce significant changes of pH, which can be monitored directly using a pH test paper or pH-sensitive indicators. The degree of color changes depends on the amount of miRNA, making it possible for quantitative analysis. As an example, the method is successfully applied to quantify a miRNA (miR-21) in cancer cells. The results agree well with that from the prevalent qRT-PCR analysis. It is the first time that a paper-based point-of-care testing (POCT) is developed for the detection of miRNAs, which might promote the popularization of miRNAs working as biomarkers for diagnostic purposes.

Fabrication of nanozyme@DNA hydrogel and its application in biomedical analysis

Nanozymes have received great attention owing to the advantages of easy preparation and low cost. Unlike natural enzymes that readily adapt to physiological environments, artificial nanozymes are apt to passivate in complex clinical samples (e.g., serum), which may damage the catalytic capability and consequently limit the application in biomedical analysis. To conquer this problem, in this study, we fabricated novel nanozyme@DNA hydrogel architecture by incorporating nanozymes into a pure DNA hydrogel. Gold nanoparticles (AuNPs) were adopted as a model nanozyme. Results indicate that AuNPs incorporated in the DNA hydrogel retain their catalytic capability in serum as they are protected by the hydrogel, whereas AuNPs alone totally lose the catalytic capability in serum. The detection of hydrogen peroxide and glucose in serum based on the catalysis of the AuNPs@DNA hydrogel was achieved. The detection limit of each reaches 1.7 and 38 μM, respectively, which is equal to the value obtained using natural enzymes. Besides the mechanisms, some other advantages, such as recyclability and availability, have also been explored. This nanozyme@DNA hydrogel architecture may have a great potential for the utilization of nanozymes as well as the application of nanozymes for biomedical analysis in complex physiological samples.

From Interface to Solution: Integrating Immunoassay with Netlike Rolling Circle Amplification for Ultrasensitive Detection of Tumor Biomarker.

Both the 3D solution and the 2D interface play important roles in bioanalysis. For the former, reactions can be carried out adequately; while for the latter, interfering substance can be eliminated simply through wash. It is a challenge to integrate the advantages of solution-based assays and the interface-based assays. Here, we report an immuno-NRCA (netlike rolling circle amplification) strategy, which integrates immunoassay with NRCA for the ultrasensitive detection of tumor biomarker, by taking the assay of a tumour marker as an example. In this strategy, immunoreactions occur on interface, while the target-induced signal amplification can be completed totally in solution. As a result, this system has the merits of both solution- and interface-based assays. The whole procedure of this novel strategy is similar to the conventional ELISA, inheriting the usability. But in comparison with ELISA, the performance is greatly improved. The detection limit can be lowered to 5.5 fg/L, making it possible to detect the target tumour marker in one drop of blood. Also, in comparison with established immuno-PCR method, which integrates immunoassay with the commonly used nucleic acid amplification approach, this system has no requirement for thermal cycler owing to the isothermal amplification, and it has the ability to retain the immunoreactivities. So, the new immunoassay method proposed in this study may have more feasible applications in the future.

A one-pot strategy for the detection of proteins based on sterically and allosterically tunable hybridization chain reaction

In this work, we report a facile one-pot strategy for protein detection based on sterically and allosterically tunable hybridization chain reaction (HCR). In our strategy, DNA hairpins H1 and H2 are dual-labeled with pyrene moieties through a six-carbon-atom spacer at each end; and a single-stranded DNA primer is designed to contain two small molecules near each end. In the absence of target protein, the primer can trigger HCR events between alternating H1 and H2 hairpins to form a nicked double-helix. As a result, the pyrene excimers are formed to emit at approximately 485nm. On the contrary, upon binding of the specific target protein onto the primer through the protein-small molecule interaction, the HCR will be inhibited due to the steric and allosteric effect. The changes of the fluorescent signals of pyrene excimers are in response to the concentration of target protein, so that the detection of protein can be realized. We have demonstrated the feasibility of this strategy by using streptavidin (SA) and folate receptor (FR) as model targets. Results show that both of them can be well detected with a detection limit of 1.07nM and 2.7nM, respectively. The developed method for protein assay is flexible, so we infer that the one-pot strategy holds great potential for the detection of other proteins.

Electrochemical detection of Nanog in cell extracts via target-induced resolution of an electrode-bound DNA pseudoknot

Nanog is among the most important indicators of cell pluripotency and self-renew, so detection of Nanog is critical for tumor assessment and monitoring of clinical prognosis. In this work, a novel method for Nanog detection is proposed by using electrochemical technique based on target-induced conformational change of an electrode-bound DNA pseudoknot. In the absence of Nanog, the rigid structure of the pseudoknot will minimize the connection between the redox tag and the electrode, thus reducing the obtained faradaic current. Nevertheless, the Nanog binding may liberate the flexible single-stranded element that transforms the DNA pesudokont into DNA hairpin structure due to steric hindrance effect, thus making the electrochemical tag close to the electrode surface. Consequently, electron transfer can be enhanced and very well electrochemical response can be observed. By using the proposed method, Nanog can be determined in a linear range from 2nM to 25nM with a detection limit of 163 pM. Furthermore, the proposed method can be directly used to assay Nanog not only in purified samples but also in complex media (cell extracts), which shows potential applications in Nanog functional studies as well as clinical diagnosis in the future.

Assembly of Self-Cleaning Electrode Surface for the Development of Refreshable Biosensors

Passivation of electrode surface and tedious reconstruction of biosensing architectures have long plagued researchers for the development of electrochemical biosensors. Here, we report a novel self-cleaning electrode by modifying the commonly used working electrode with superhydrophobic and conductive nanocomposite. Owing to the superhydrophobicity and the chemical stability, the electrode avoids passivation result from both adsorption of molecules and oxidation in air. The high conductivity and the high effective area also allow the achievement of enhanced electrochemical signals. On the basis of comprehensive studies on this novel electrode, we have applied it in the fabrication of refreshable electrochemical biosensors for both electro-active and electro-inactive targets. For both cases, detection of the targets can be well performed, and the self-cleaning electrode can be refreshed by simply washing and applied for successive measurements in a long period.

Electrochemical Analysis of Enzyme Based on the Self-Assembly of Lipid Bilayer on an Electrode Surface Mediated by Hydrazone Chemistry

In this work, a new strategy for electrochemical analysis of enzyme has been proposed based on a self-assembled lipid bilayer on an electrode surface mediated by hydrazone chemistry. Taking aldolase as an example, the enzyme can catalyze the formation of products containing carbonyl groups. These groups can react with hydrazine groups of the functional lipid derivative, resulting in the self-assembly of a lipid bilayer on a guanidinium modified electrode surface. The lipid bilayer will then prevent the movement of hydrophilic electrochemical probes. Consequently, the catalytic reaction of the enzyme may result in the change of the obtained electrochemical peak current. Experimental results reveal that aldolase activity can be analyzed over a widely linear detection range from 5 mU/L to 100 U/L with a low detection limit of 1 mU/L. Meanwhile, the method can exhibit good precision and reproducibility and it can be applied for real sample analysis. What is more, because the lipid bilayer is the universal basis for cell-membrane structure, while hydrazone chemistry is popular in nature, this work may also provide a new insight for the development of electrochemical analysis and electrochemical biosensors.

Nicking Enzyme-Assisted Branched-Chain RCA Reaction for Cascade DNA Amplification

Nicking enzymes are a special family of restriction endonucleases, which recognize a specific sequence along double-stranded DNA, but cut only one DNA strand, thus creating a DNA nick in the corresponding strand. The nick can be employed in the rolling circle amplification (RCA) reaction, which has attracted substantial interest lately due to its high sensitivity, excellent specificity, and detection versatility. We have recently developed a new nonlinear RCA format by introducing a nicking enzyme into the hyperbranched RCA system. This innovative DNA amplification technique, named as netlike RCA (NRCA) and described in this chapter, resulted in cubic amplification of a HIV-1 DNA target sequence and strongly discriminated it from its single-base mutants.