Zhiyang Li

Selection of HBsAg-Specific DNA Aptamers Based on Carboxylated Magnetic Nanoparticles and Their Application in the Rapid and Simple Detection of Hepatitis B Virus Infection

Aptamers are short single-stranded DNA or RNA oligonucleotides and can be selected from synthetic combinatorial libraries in vitro. They have a high binding affinity and specificity for their targets. Agarose gels, nitrocellulose membranes, and adsorptive microplates are often used as carriers to immobilize targets in the SELEX (systematic evolution of ligands by exponential enrichment) process, but the subsequent separation step is tedious and time-consuming. Therefore, we used magnetic nanoparticles (MNPs) as carriers to immobilize the target, hepatitis B surface antigen (HBsAg), which is convenient for fast magnetic separation. In this study, we first selected DNA aptamers against HBsAg by immobilizing HBsAg on the surface of carboxylated MNPs. The ssDNA library of each selection round was prepared by asymmetric PCR amplification for the next selection round. To obtain aptamer sequences, the final selected products were purified by gel electrophoresis, then cloned, and sequenced. DNA aptamers that specifically bind to HBsAg were successfully obtained after 13 selection rounds. The selected aptamers were used to construct a chemiluminescence aptasensor based on magnetic separation and immunoassay to detect HBsAg from pure protein or actual serum samples. There was a linear relationship between HBsAg concentration and chemiluminescent intensity in the range of 1-200 ng/mL. The aptasensor worked well even in the presence of interfering substances and was highly specific in the detection of HBsAg in serum samples, with a detection limit 0.1 ng/mL lower than the 0.5 ng/mL limit of an ELISA in use at the hospital. This aptasensor can contribute to better detection of hepatitis B virus infection.

Highly Sensitive and Rapid Detection of Pseudomonas aeruginosa Based on Magnetic Enrichment and Magnetic Separation

A method for highly sensitive and rapid detection of Pseudomonas aeruginosa, based on magnetic enrichment and magnetic separation, is described in this paper. The magnetic nanoparticles (MNPs) were applied to adsorb genome DNA after the sample was lysed. The DNA binding MNPs were directly subjected to polymerase chain reaction (PCR) to amplify gyrB specific sequence of Pseudomonas aeruginosa. The biotin labeled PCR products were detected by chemiluminescence when they were successively incubated with the probes-modified MNPs and alkaline phosphatase (ALP) labeled streptavidin (SA). Agarose gel electrophoresis analyses approved the method of in situ PCR to be highly reliable. The factors which could affect the chemiluminiscence were studied in detail. The results showed that the MNPs of 400 nm in diameter are beneficial to the detection. The sequence length and the binding site of the probe with a target sequence have obvious effects on the detection. The optimal concentration of the probes, hybridization temperature and hybridization time were 10 μM, 60 ºC and 60 mins, respectively. The method of in situ PCR based on MNPs can greatly improve the utilization rate of the DNA template ultimately enhancing the detection sensitivity. Experiment results proved that the primer and probe had high specificity, and Pseudomonas aeruginosa was successfully detected with detection limits as low as 10 cfu/mL by this method, while the detection of a single Pseudomonas aeruginosa can also be achieved.

Chemiluminescent labels released from long spacer arm-functionalized magnetic particles: a novel strategy for ultrasensitive and highly selective detection of pathogen infections.

Previously, the unique advantages provided by chemiluminescence (CL) and magnetic particles (MPs) have resulted in the development of many useful nucleic acid detection methods. CL is highly sensitive, but when applied to MPs, its intensity is limited by the inner filter-like effect arising from excess dark MPs. Herein, we describe a modified strategy whereby CL labels are released from MPs to eliminate this negative effect. This approach relies on (1) the magnetic capture of target molecules on long spacer arm-functionalized magnetic particles (LSA-MPs), (2) the conjugation of streptavidin-alkaline phosphatase (SA-AP) to biotinylated amplicons of target pathogens, (3) the release of CL labels (specifically, AP tags), and (4) the detection of the released labels. CL labels were released from LSA-MPs through LSA ultrasonication or DNA enzymolysis, which proved to be the superior method. In contrast to conventional MPs, LSA-MPs exhibited significantly improved CL detection, because of the introduction of LSA, which was made of water-soluble carboxymethylated β-1,3-glucan. Detection of hepatitis B virus with this technique revealed a low detection limit of 50 fM, high selectivity, and excellent reproducibility. Thus, this approach may hold great potential for early stage clinical diagnosis of infectious diseases.

Simultaneous extraction of DNA and RNA from Escherichia coli BL 21 based on silica-coated magnetic nanoparticles

The extraction of nucleic acid is recognized as one of the most essential steps in molecular biology for initiating other downstream applications such as sequencing, amplification, hybridization, and cloning. Many commercial kits and methods are currently available that allow the extraction of only one type of nucleic acids-DNA or RNA. However, in parallel clinical detection of several diseases, a method for simultaneous extraction of both DNA and RNA from the same source is needed in such cases. In this study, a method for simultaneous extraction of DNA and RNA from bacteria based on magnetic nanoparticles (MNPs) was described. Lysis buffers were prepared to help the nucleic acid released and adsorbed to MNPs. Then, two washing buffers were used to remove the contamination of proteins and carbohydrates. The nucleic acids were finally eluted by Deoxyribonuclease (DNase) and Ribonucleases (RNase) free water. Different factors which might affect the purification of the nucleic acid were investigated, and the quantity and quality parameters of the nucleic acid were also recorded. The DNA and RNA extracted from bacteria were then respectively subjected to polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) to further confirm its quality. The results indicated that our method can be successfully used to simultaneously extract DNA and RNA from bacteria.

Mass spectrometry-assisted gel-based proteomics in cancer biomarker discovery: approaches and application

There is a critical need for the discovery of novel biomarkers for early detection and targeted therapy of cancer, a major cause of deaths worldwide. In this respect, proteomic technologies, such as mass spectrometry (MS), enable the identification of pathologically significant proteins in various types of samples. MS is capable of high-throughput profiling of complex biological samples including blood, tissues, urine, milk, and cells. MS-assisted proteomics has contributed to the development of cancer biomarkers that may form the foundation for new clinical tests. It can also aid in elucidating the molecular mechanisms underlying cancer. In this review, we discuss MS principles and instrumentation as well as approaches in MS-based proteomics, which have been employed in the development of potential biomarkers. Furthermore, the challenges in validation of MS biomarkers for their use in clinical practice are also reviewed.

Ultrasensitive quantitation of MicroRNAs via magnetic beads-based chemiluminesent assay

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level, and their aberrant expression occurs during the development of malignant diseases. Recently, miRNAs have been proposed as potential prognostic and predictive biomarkers for early diagnosis. However, a major obstacle in rapid miRNA analysis from real samples is the lack of ultrasensitive and quantitative techniques. In this regard, the use of chemiluminescence (CL) system offers a highly sensitive strategy for detecting miRNAs. In this article, an ultrasensitive approach has been established for the quantification of miRNAs, using magnetic beads (MBs) and alkaline phosphatase (AP)-based CL system. This technique depends on sandwich hybridization among MBs-labeled capture probes, target miRNAs and biotin-labeled reporter probes, conjugation of streptavidin-alkaline phosphatase (SA-AP) to biotin-labeled reporter probes, and CL detection of AP-linked targets. Detection of miR-21 with this technique demonstrated a high selectivity and an ultralow limit of detection (LOD) of 60 fM with an extraordinarily wide range of six orders of magnitudes. The quantitation could be achieved by direct detecting target miRNA in serum samples within a total time of 1.5 h and did not require reverse transcription and polymerase chain reaction (PCR) amplification. Therefore, this developed method shows great potential for early cancer diagnosis based on miRNAs as biomarkers.

A Portable Multi-Channel Turbidity System for Rapid Detection of Pathogens by Loop-Mediated Isothermal Amplification

This study aimed at developing a portable multi-channel turbidity system (21 cm in length, 15.5 cm in width and 11.5 cm in depth) by real-time loop-mediated isothermal amplification (LAMP) method for rapid detection of pathogens. The developed system herein includes temperature control unit, photoelectric detection unit, turbidity calibration unit, power management unit, human machine unit, communication unit and ARM-based microcontroller. The coefficient of variation for eight channels is less than 0.25% in noise analysis. Legionella bacteria (LEG) and H7 subtype virus (H7) were successively detected by the designed and developed system within 60 minutes. Moreover, its specificity for LEG is satisfactory and its sensitivity for H7 is 10 copies/mL. Besides, this system for point-of-care diagnosis allows a rapid, small size, low cost, and automatic detection with the characteristics of high-efficiency, excellent stability and high uniformity.

Biological Application of Digital Microfluidics Technology

Digital microfluidics technology offers a platform for developing diagnostic applications with the advantages of portability, sample and reagent volume reduction, faster analysis, increased automation, low power consumption, compatibility with mass manufacturing and high throughput. In addition to diagnostics, digital microfluidics is finding use in nucleic acid analysis, peptide and protein analysis, cell analysis, drug analysis and delivery and immunization analysis. In this review, we describe these applications, their implementation, and associated design issues. As other review in the digital microfluidics technology, there have been and will be unexpected developments as DMF matures, but we predict that the future is bright for this promising technology at the last section.