Yongqian Shu

Enhanced Charge Transfer by Gold Nanoparticle at DNA Modified Electrode and Its Application to Label-Free DNA Detection

Rational utilization of nanomaterials to construct electrochemical nucleic acid sensors has attracted large attention in recent years. In this work, we systematically interrogate the interaction between gold nanoparticles (GNPs) and single-strand DNA (ssDNA) immobilized on an electrode surface and then take advantage of the ultrahigh charge-transfer efficiency of GNPs to develop a novel DNA sensing method. Specifically, ssDNA modified gold electrode can adsorb GNPs because of the interaction between gold and nitrogen-containing bases; thus, the negative electrochemical species [Fe(CN)6](3-/4-) may transfer electrons to electrode through adsorbed GNPs. In the presence of target DNA, the formed double-strand DNA (dsDNA) cannot capture GNPs onto the electrode surface and the dsDNA may result in a large charge-transfer resistance owing to the negatively charged phosphate backbones of DNA. So a simple but sensitive method for the detection of target DNA can be developed by using GNPs without any requirement of modification. Experimental results demonstrate that the electrochemical method we have proposed in this work can detect as low as 1 pM breast cancer gene BRCA1 in a 10 μL sample volume without any signal amplification process or the involvement of other synthesized complex, which may provide an alternative for cancer DNA detection. This method may also be generalized for detecting a spectrum of targets using functional DNA (aptamer, metal-specific oligonucleotide, or DNAzyme) in the future.

Double recognition of oligonucleotide and protein in the detection of DNA methylation with surface plasmon resonance biosensors

DNA methylation plays an essential role in maintenance of cellular function. A growing number of human diseases have been found to be associated with aberrant DNA methylation, especially cancer. However, current technologies used in DNA methylation detection are complicated and time consuming. A promotor of the Adenomatous polyposis coli (APC) gene, a well-studied tumor suppressor gene, was used as the detection target DNA sequence. The double recognition mechanism was realized with oligonucleotide probe hybridization and specific protein binding. First, complementary target DNA was captured by the probe immobilized onto a surface plasmon resonance (SPR) sensor chip. Then, the recombinant methyl-CpG binding domain (MBD) protein was passed over the surface to recognize and bind to methylated CpG sites. Binding resulted in an increase in the refractive index, and a detectable optical signal was generated. Five picomoles of methylated APC promotor DNA could be easily detected with this method. The entire detection could be completed within 1h. This work represents the first SPR based biosensor technology, which achieves simple and specific DNA methylation detection and avoids complicated bisulfite treatment and methylation-sensitive restriction digestion. It will improve our ability to detect DNA methylation specifically and rapidly, and promote our understanding of the role of DNA methylation in gene regulation and diseases.

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.

Binding-regulated click ligation for selective detection of proteins

Herein, a binding-regulated click ligation (BRCL) strategy for endowing selective detection of proteins is developed with the incorporation of small-molecule ligand and clickable DNA probes. The fundamental principle underlying the strategy is the regulating capability of specific protein-ligand binding against the ligation between clickable DNA probes, which could efficiently combine the detection of particular protein with enormous DNA-based sensing technologies. In this work, the feasibly of the BRCL strategy is first verified through agarose gel electrophoresis and electrochemical impedance spectroscopy measurements, and then confirmed by transferring it to a nanomaterial-assisted fluorescence assay. Significantly, the BRCL strategy-based assay is able to respond to target protein with desirable selectivity, attributing to the specific recognition between small-molecule ligand and its target. Further experiments validate the general applicability of the sensing method by tailoring the ligand toward different proteins (i.e., avidin and folate receptor), and demonstrate its usability in complex biological samples. To our knowledge, this work pioneers the practice of click chemistry in probing specific small-molecule ligand-protein binding, and therefore may pave a new way for selective detection of proteins.

One-Step Modification of Electrode Surface for Ultrasensitive and Highly Selective Detection of Nucleic Acids with Practical Applications

Electrochemistry-based nucleic acid sensors have long been plagued by the limited accessibility of target molecules to the capture probes immobilized on heterogeneous surfaces, which largely hinders their practical application. In this work, we find that dual-thiolated hairpin DNA immobilized on an electrode surface as the capture probe cannot only efficiently bind with target molecule as well as the signal probe but also process impressive protein-repelling ability, which allows us to directly detect as few as attomolar targets (∼300 copies in 100 μL sample) with single-base discrimination ability. Meanwhile, the preparation of functional electrode surface becomes simple (one step), fast (30 min), and homogeneous (just one probe modified surface without small molecules coassembled). These advantages are attributed to the unique probe design, where the stem of the capture probe can act as rigid scaffold to keep it upright, and the loop of the capture probe may provide an enclosed platform for target and signal probe binding. More importantly, through tuning the distance between enzyme and the electrode surface (from 8.5 to 13.6 nm), we find that the performance of the sensor can be favorably controlled. Furthermore, taking advantage of this new binding model, different complex samples including polymerase chain reaction (PCR) product, mRNA, and micro RNA can be conveniently analyzed, which may hold great potential for real application.

Rapid detection of acute myocardial infarction-related miRNA based on a Capture-interCalation-electroCatalysis (3C) strategy.

Acute myocardial infarction (AMI) is one of the most urgent and serious diseases that may cause cardiac death in a few hours. Rapid diagnosis of AMI is the pre-requisite for timely interventions. Recently, several specific circulating miRNAs have been proven to have high correlation with AMI. To adopt miRNA as a biomarker may improve the diagnostic accuracy. However, it is a pity that the current available methods for the detection of miRNA usually require a few hours, which is too long for the diagnosis of AMI. In this paper, by adopting a capture DNA, an electrochemical active intercalator and an unimmobilized enzyme, we develop a Capture-interCalation-electroCatalysis (3C) strategy for the rapid detection of AMI-related miRNA. The whole detection process can be completed in 35 min, which is much shorter than most current methods and is acceptable for the diagnosis of AMI. This strategy also shows favorable sensitivity and selectivity, thus provides an alternative for the detection of miRNA. Most importantly, this effort may promote miRNA to work as an effective biomarker in the diagnosis of AMI.