Lanying Li

A carbon nanotube-based high-sensitivity electrochemical immunosensor for rapid and portable detection of clenbuterol

Carbon nanotubes have shown their unique advantages of mechanical, chemical and electronic properties in bioanalysis. We herein report a new method to efficiently and reproducibly prepare multi-walled carbon nanotubes (MWNTs)-protein sensing layers for electrochemical immunosensors. This method employs centrifugation to prepare a conjugate of MWNTs and goat anti mouse-immunoglobulin G (IgG) (secondary antibody). The conjugates were then deposited on screen-printed electrodes to form a nanostructured layer (MWNT-I layer). CLB monoclonal antibody was assembled through its binding to the secondary antibody. The MWNT-I layer-based electrodes were used for rapid and sensitive amperometric immunosensing detection of clenbuterol (CLB) in swine urine samples. Horseradish peroxidase-coupled CLB (CLB-HRP) competed with free CLB in the samples to bind the monoclonal antibody. It has shown significantly higher sensitivity and better reproducibility than the chemical conjugation method. This MWNT-based immunosensor is highly sensitive, leading to a limit of detection of 0.1 ng/mL within a rapid assay time of 16 min. Its sensitivity is at least 1 order of magnitude higher than that of a normal immunosensor (without MWNTs). The sensing device is portable with disposable screen-printed electrode, satisfactorily meeting the requirements for field detection of food security-related species.

Target-Responsive, DNA Nanostructure-Based E-DNA Sensor for microRNA Analysis

Because of the short size and low abundance of microRNAs, it is challenging to develop fast, inexpensive, and simple biosensors to detect them. In this work, we have demonstrated a new generation (the third generation) of E-DNA sensor for the sensitive and specific detection of microRNAs. Our third generation of E-DNA sensor can sensitively detect microRNA target (microRNA-141) as low as 1 fM. The excellent specificity has been demonstrated by its differential ability to the highly similar microRNA analogues. In our design, the use of DNA tetrahedron ensures the stem-loop structure in well controlled density with improved reactivity. The regulation of the thermodynamic stability of the stem-loop structure decreases the background signal and increases the specificity as well. The enzymes attached bring the electrocatalytic signal to amplify the detection. The combination of these effects improves the sensitivity of the E-DNA sensor and makes it suitable to the microRNA detection. Finally, our third generation of E-DNA sensor is generalizable to the detection of other micro RNA targets (for example, microRNA-21).

DNA nanostructure-based ultrasensitive electrochemical microRNA biosensor

MicroRNAs (miRNAs) are key regulators of a wide range of cellular processes, and have been identified as promising cancer biomarkers due to their stable presence in serum. As an surface-based electrochemical biosensors which offer great opportunities for low-cost, point-of-care tests (POCTs) of disease-associated miRNAs. Nevertheless, the sensitivity of miRNA sensors is often limited by mass transport and the surface crowding effect at the water-electrode interface. Here, we present a protocol as well as guidelines for ultrasensitive detection of miRNA with DNA nanostructure-based electrochemical miRNA biosensor. By employing the three-dimensional DNA nanostructure-based interfacial engineering approach, we can directly detect as few as attomolar (<1000 copies) miRNAs with high single-base discrimination ability. Since this ultrasensitive electrochemical miRNA sensor (EMRS) is highly reproducible and essentially free of prior target labeling and PCR amplification, it can conveniently and reliably analyze miRNA expression levels in clinical samples from esophageal squamous cell carcinoma (ESCC) patients.

Development of mercury (II) ion biosensors based on mercury-specific oligonucleotide probes

Mercury (II) ion (Hg(2+)) contamination can be accumulated along the food chain and cause serious threat to the public health. Plenty of research effort thus has been devoted to the development of fast, sensitive and selective biosensors for monitoring Hg(2+). Thymine was demonstrated to specifically combine with Hg(2+) and form a thymine-Hg(2+)-thymine (T-Hg(2+)-T) structure, with binding constant even higher than T-A Watson-Crick pair in DNA duplex. Recently, various novel Hg(2+) biosensors have been developed based on T-rich Mercury-Specific Oligonucleotide (MSO) probes, and exhibited advanced selectivity and excellent sensitivity for Hg(2+) detection. In this review, we explained recent development of MSO-based Hg(2+) biosensors mainly in 3 groups: fluorescent biosensors, colorimetric biosensors and electrochemical biosensors.

Single-nucleotide polymorphism genotyping using a novel multiplexed electrochemical biosensor with nonfouling surface

A novel electrochemical DNA biosensor for single-nucleotide polymorphism (SNP) analysis was developed. In this work, an oligonucleotide-incorporated nonfouling surface (ONS) was constructed to resist nonspecific absorption. The biosensor was developed using a 16-electrode array for high-throughput SNP analysis. The proposed strategy was primarily based on specific oligonucleotide ligation. Fully matched target DNA templated the ligation between a capture probe assembled on gold electrodes and a tandem signal probe with a biotin moiety that could capture avidin-horseradish peroxidase and sequentially generate a catalysed amperometric signal. A pre-core mutation in the hepatitis B virus (HBV) genome at G1896A and two adjacent polymorphisms in the human CYP2C19 genome at C680T and G681A were analysed. Polymerase chain reaction (PCR) products were used as real-life samples and analysed. Our results showed that 10% of a single-mismatched mutant gene was clearly distinguished with a current signal 16 times higher than that of the blank sample, demonstrating the selectivity and practicability of the multiplexed electrochemical DNA biosensor.

Detection of Single-Nucleotide Polymorphism on uidA Gene of Escherichia coli by a Multiplexed Electrochemical DNA Biosensor with Oligonucleotide-Incorporated Nonfouling Surface

We report here a practical application of a multiplexed electrochemical DNA sensor for highly specific single-nucleotide polymorphism (SNP) detection. In this work, a 16-electrode array was applied with an oligonucleotide-incorporated nonfouling surfaces (ONS) on each electrode for the resistance of unspecific absorption. The fully matched target DNA templated the ligation between the capture probe assembled on gold electrodes and the tandem signal probe with a biotin moiety, which could be transduced to peroxidase-based catalyzed amperometric signals. A mutant site (T93G) in uidA gene of E. coli was analyzed in PCR amplicons. 10% percentage of single mismatched mutant gene was detected, which clearly proved the selectivity of the multiplexed electrochemical DNA biosensor when practically applied.

A Sensitive and Label-Free Pb(II) Fluorescence Sensor Based on a DNAzyme Controlled G-Quadruplex/Thioflavin T Conformation

Pb(II) can cause serious damaging effects to human health, and thus, the study of Pb2+ detection methods to sensitively and selectively monitor Pb(II) pollution has significant importance. In this work, we have developed a label-free fluorescence sensing strategy based on a Pb(II) DNAzyme cleavage and the ThT/G-quadruplex complex. In the presence of Pb(II), a G-rich tail was cut and released from the substrate strand, which then would form a G-quadruplex structure by combination with ThT dye. The fluorescence signal increase was then measured for sensitive Pb(II) quantification with a limit of detection of 0.06 nM. Our sensor also demonstrated high selectivity against six different metal ions, which is very important for the analysis of complex samples.