Bing Bo

A simple and general approach to assay protease activity with electrochemical technique

Proteases are involved in a large number of serious disease processes, while the assay of proteolytic activity can be used for clinical diagnostics. In this paper we report a simple electrochemical method to assay protease activity. This method makes use of an unlabeled peptide that comprises the specific substrate domain of a protease, which can be easily operated and generalized for assay of various kinds of proteases. Specifically, the peptide is immobilized onto a gold electrode surface via the chemical adsorption of the C-terminal cysteine residue, forming a positively charged interface derived from the N-terminal cationic residue. Therefore, the positive electrochemical probes [Ru(NH3)5Cl](2+) cannot get across to the electrode to generate signal. Nevertheless, the proteolytic digestion of the peptide will decrease the number of positive charges on the electrode surface and weaken the blocking effect against the positive electrochemical species, resulting in an increased electrochemical signal. Under optimized conditions, the activity of the model protease, trypsin, can be assayed with a detection limit of 0.026 U/mL. The method may also allow the determination of trypsin activity in serum samples. Moreover, since this approach can be used for the assay of other proteases by simply changing the substrate domain of the peptide, it may have great potential in biomedical applications in the future.

An electrochemical biosensor for clenbuterol detection and pharmacokinetics investigation.

Clenbuterol is a member of β2 adrenergic agonists, which is widely used not only as a food additive for livestocks, but also a kind of stimulant for athletes; however, the abuse of clenbuterol may pose a significant negative impact on human health. Since it is highly required to develop fast, sensitive and cost-effective method to determine clenbuterol level in the suspected urine or blood, we herein have fabricated an electrochemical biosensor for the determination of clenbuterol. Measurement of the species with the proposed biosensor can also have the advantages of simplicity, high sensitivity and selectivity. Moreover, the sensor can be directly used for clenbuterol determination in rat urine. We have further studied the pharmacokinetics of clenbuterol by using this proposed electrochemical biosensor, so a new tool to investigate pharmacokinetic is developed in this work.

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.

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.

GOLD NANOPARTICLES-BASED BIOSENSORS FOR BIOMEDICAL APPLICATION

Gold nanoparticles are the most extensively studied nanomaterials for biomedical application due to their unique properties, such as rapid and simple synthesis, large surface area, strong adsorption ability and facile conjugation to various biomolecules. The remarkable photophysical properties of gold nanoparticles have provided plenty of opportunities for the preparation of gold nanoparticles-based optical biosensors, while the excellent biocompatibility, conductivity, catalytic properties and large surface-to-volume ratio have facilitated the application of gold nanoparticles in the construction of electrochemical biosensors. In this review, we mainly detail the gold nanoparticles-based optical and electrochemical biosensors for biomedical application in the recent two years, which have exhibited greatly enhanced analytical performances in the detection of DNA, proteins and some important small molecules.

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.