Hongna Liu

An integrated and sensitive detection platform for biosensing application based on Fe@Au magnetic nanoparticles as bead array carries

A sensitive and selective biosensor platform suited for SNP type using Fe@Au magnetic nanoparticles (GMNPs) to fabricate bead array is described. This new platform integrates the rapid binding kinetics of magnetic nanoparticles carriers, the multiplexing and encoding capabilities of chips, and tagged array. As a DNA sensor, the biotinylated single-stranded DNA was obtained by asymmetry PCR amplification, and then captured by GMNPs modified with streptavidin to form GMNP-ssDNA complexes without further purification. The complexes were immobilized on the slide to fabricate bead array through magnetic field. The bead array was hybridized with the corresponding allele-specific tag probes for each locus, and a pair of given universal detectors were applied to these markers analysis. Using bead array, all samples can be analyzed in one hybridization chamber which lowers the cost of the assay. Using universal tags, only a pair of universal dual-color probes labeled fluorophores was used for multiplex genotyping. Without the need of laborious and time-consuming elution, the experiment process was simple, reproducible and easy to handle. Two SNPs loci from 12 individual samples were discriminated using this platform and the results demonstrated that the expected scores and good discrimination were obtained between the two alleles from the two SNP loci. In summary, the integrated sensitive platform is adaptable and versatile, while offering a high-throughput capability needed for genome research and clinical applications.

A Novel SNPs Detection Method Based on Gold Magnetic Nanoparticles Array and Single Base Extension

To fulfill the increasing need for large-scale genetic research, a high-throughput and automated SNPs genotyping method based on gold magnetic nanoparticles (GMNPs) array and dual-color single base extension has been designed. After amplification of DNA templates, biotinylated extension primers were captured by streptavidin coated gold magnetic nanoparticle (SA-GMNPs). Next a solid-phase, dual-color single base extension (SBE) reaction with the specific biotinylated primer was performed directly on the surface of the GMNPs. Finally, a “bead array” was fabricated by spotting GMNPs with fluorophore on a clean glass slide, and the genotype of each sample was discriminated by scanning the “bead array”. MTHFR gene C677T polymorphism of 320 individual samples were interrogated using this method, the signal/noise ratio for homozygous samples were over 12.33, while the signal/noise ratio for heterozygous samples was near 1. Compared with other dual-color hybridization based genotyping methods, the method described here gives a higher signal/noise ratio and SNP loci can be identified with a high level of confidence. This assay has the advantage of eliminating the need for background subtraction and direct analysis of the fluorescence values of the GMNPs to determine their genotypes without the necessary procedures for purification and complex reduction of PCR products. The application of this strategy to large-scale SNP studies simplifies the process, and reduces the labor required to produce highly sensitive results while improving the potential for automation.

A novel automated assay with dual-color hybridization for single-nucleotide polymorphisms genotyping on gold magnetic nanoparticle array

A high-throughput and cost-effective single-nucleotide polymorphism (SNP) genotyping method based on a gold magnetic nanoparticle (GMNP) array with dual-color hybridization has been designed. Biotinylated single-strand polymerase chain reaction (PCR) products containing the SNP locus were captured by the GMNPs that were coated with streptavidin. The GMNP array was fabricated by immobilizing single-stranded DNA (ssDNA)-GMNP complexes onto a glass slide using a magnetic field, and SNPs were identified with dual-color fluorescence hybridization. Three different SNP loci from 24 samples were genotyped successfully using this platform. This procedure allows the user to directly analyze the bead fluorescence to determine the SNP genotype, and it eliminates the need for background subtraction for signal determination. This method also bypasses tedious PCR purification and concentration procedures, and it facilitates large-scale SNP studies by using a method that is highly sensitive, simple, labor-saving, and potentially automatable.

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.

Copy Number Variation Analysis by Ligation-Dependent PCR Based on Magnetic Nanoparticles and Chemiluminescence

A novel system for copy number variation (CNV) analysis was developed in the present study using a combination of magnetic separation and chemiluminescence (CL) detection technique. The amino-modified probes were firstly immobilized onto carboxylated magnetic nanoparticles (MNPs) and then hybridized with biotin-dUTP products, followed by amplification with ligation-dependent polymerase chain reaction (PCR). After streptavidin-modified alkaline phosphatase (STV-AP) bonding and magnetic separation, the CL signals were then detected. Results showed that the quantification of PCR products could be reflected by CL signal values. Under optimum conditions, the CL system was characterized for quantitative analysis and the CL intensity exhibited a linear correlation with logarithm of the target concentration. To validate the methodology, copy numbers of six genes from the human genome were detected. To compare the detection accuracy, multiplex ligation-dependent probe amplification (MLPA) and MNPs-CL detection were performed. Overall, there were two discrepancies by MLPA analysis, while only one by MNPs-CL detection. This research demonstrated that the novel MNPs-CL system is a useful analytical tool which shows simple, sensitive, and specific characters which are suitable for CNV analysis. Moreover, this system should be improved further and its application in the genome variation detection of various diseases is currently under further investigation.

High-Throughput SNP Detection Based on PCR Amplification on Magnetic Nanoparticles Using Dual-Color Hybridization

A microarray-based method for detecting single nucleotide polymorphisms (SNPs) using solid-phase polymerase chain reaction (PCR) on magnetic nanoparticles (MNPs) was developed. In this method one primer with a biotin label is captured by streptavidin-coated MNPs (SA-MNPs), and the PCR products are directly amplified on the surface of SA-MNPs in a 96-well plate. The samples are further probed by hybridization with a pair of dual-color probes to determine SNP. The genotype of each sample can be simultaneously identified by scanning the microarray printed with the denatured fluorescent probes. All the reactions can be performed in the same reaction volume without the necessity of purification of intermediates. This approach represents a novel, simple, and labor-saving method for SNP genotyping and can be applied in automation system(s) to achieve high-throughput SNP detection.

Fabrication of Porous Pseudo-Carbon Paste Electrode as a Novel High-Sensitive Electrochemical Biosensor

Abstract Porous pseudo-carbon paste electrode (PPCPE) as a novel high-sensitive electrochemical biosensor was fabricated by mixing polymethyl methacrylate (PMMA) microspheres for use as the template, graphite powders for the filler, and pyrrole as the precursor of the polymer which acted as the paste. After the polymerization of pyrrole catalyzed by Fe3+, the PMMA microspheres were removed to form PPCPE. The pore size was determined by SEM observations, with diameters ranging from 2 to 5 µm. The anodic stripping voltammetry response of DNA indicated that the adsorption of oligonucleotide on PPCPE was enhanced. The PPCPE was easy to preserve and had a good reusability in comparison with that of a pure carbon paste electrode (CPE) and a carbon nanotube-modified carbon paste electrode (CNTPE). The detection limits of guanine and adenine were 20 nM and 8 nM, respectively.

One-Step Synthesis of DNA Templated Water-Soluble Au–Ag Bimetallic Nanoclusters for Ratiometric Fluorescence Detection of DNA

DNA strands have been used as templates to form different sized silver nanoclusters. Although DNA templated silver nanoclusters (NCs) probes which show outstanding fluorescence properties have been widely applied in chemical sensing and cellular imaging, synthesis of DNA template Au-Ag bimetallic nanoclusters remains a challenge. A facile one-step synthesis method was thus developed in this study to form Au-Ag NCs using C4-ATAT-C4 as template. C4-ATAT-C4 acted as a stabilizer for preventing aggregation of the Au-Ag NCs. The obtained Au-Ag NCs stabilized by C4-ATAT-C4 showed bright fluorescence and high stability. A series of experiments showed that temperature, citrate-citric acid buffer, sequence and concentration of DNA played an important role in the synthesis of fluorescent Au-Ag NCs. In addition, a ratiometric fluorescence probe was designed through combining the nucleotide sequence (C4-ATAT-C4) and hybridization sequence in the presence of the double-strand-chelating dye Super SYBR Green (SG). The combination of prepared Au-Ag NCs, Super SG, and perfectly matched DNA emitted fluorescence at 500 and 590 nm, respectively, when the composite was excited at 290 nm. A gastric cancer gene was also selectively detected by our developed ratiometric fluorescence probe.

Aptamer-Based Electrochemical Biosensor for Mercury Ions Detection Using AuNPs-Modified Glass Carbon Electrode

A mercury ion aptamer electrochemical biosensor based on a Thymine-Hg2+-Thymine (T-Hg2+-T) structure has been constructed and successfully used to detect mercury ions in tap water samples. The aptamer electrochemical biosensor was assembled using a mercury ion aptamer-functionalized AuNPs-modified glass carbon electrode (aptamer/(AuNPs/CS)₂/GCE) capable of specifically detecting mercury ions through a T-Hg2+-T structure. The experimental results indicated the optimum electrochemical performance of the prepared aptamer biosensor when the (AuNPs/CS)₂/GCE surface was modified with 1.0 μM aptamer and incubated with Hg2+ for 60 min. Moreover, the aptamer biosensor exhibits a good linear relationship between the logarithm of the Hg2+ concentration and the DPV peak current in the range from 0.01 to 500 nM following the linearization equation Ip (μA) = 2.59902 + 0.2097logC (R² = 0.9994) with a limit of detection as low as 0.005 nM. Therefore, the constructed aptamer biosensor provides a simple and sensitive approach for Hg2+ detection in aqueous solution with promising application for trace Hg2+ detection in real samples.

Applications of Magnetic Nanoparticle-Based High-Throughput Single-Nucleotide Polymorphism Genotyping Platforms in Gastric Cancer Research

To realize SNP genotyping of gastric cancer can direct reasonable choice of therapeutic drugs of gastric cancer. This chapter summarizes magnetic nanoparticles-based high-throughput single-nucleotide polymorphism genotyping platforms that were developed and successfully used for SNP genotyping of clinical gastric cancer specimens, and have great potential in genotyping and directing choice of therapeutic drugs of gastric cancer.