Axial assembly of AuNR for tumor theranostics via Zn2+-GSH chelation induced degradation of AuNR@ZIF-8 heterostructures.

Tumor microenvironment responsive photothermal ablation is a noninvasive and accurately targeted approach for cancer therapy. Herein, an intracellular directional assembly strategy for enhanced photothermal therapy (PTT) was realized by using ZIF-8 encapsulated Au nanorod (AuNR) heterostructure as the precursor of photothermal convertible material. The ZIF-8 shell selectively degraded in tumor cells upon the chelation between GSH and Zn2+, while the as-formed Zn(SG) connected the released AuNR in end-to-end fashion. The coating of ZIF-8 shell significantly improves the stability and targeting of AuNR, and the released Zn2+ shielded the GSH binding site on the lateral side of AuNR, increased the plasmonic coupling efficiency of AuNR assembly geometer. This design enabled atomic-economical, efficient and low-side effect targeted photothermal therapy through the effective integration of heterostructures.

Progress in the Mechanism of the Effect of Fe3O4 Nanomaterials on Ferroptosis in Tumor Cells.

Ferroptosis is a new form of iron-dependent programmed cell death discovered in recent years, which is caused by the accumulation of lipid peroxidation (LPO) and reactive oxygen species (ROS). Recent studies have shown that cellular ferroptosis is closely related to tumor progression, and the induction of ferroptosis is a new means to inhibit tumor growth. Biocompatible Fe3O4 nanoparticles (Fe3O4-NPs), rich in Fe2+ and Fe3+, act as a supplier of iron ions, which not only promote ROS production but also participate in iron metabolism, thus affecting cellular ferroptosis. In addition, Fe3O4-NPs combine with other techniques such as photodynamic therapy (PDT); heat stress and sonodynamic therapy (SDT) can further induce cellular ferroptosis effects, which then enhance the antitumor effects. In this paper, we present the research progress and the mechanism of Fe3O4-NPs to induce ferroptosis in tumor cells from the perspective of related genes and chemotherapeutic drugs, as well as PDT, heat stress, and SDT techniques.

Biologic Mechanisms of Macrophage Phenotypes Responding to Infection and the Novel Therapies to Moderate Inflammation.

Pro-inflammatory and anti-inflammatory types are the main phenotypes of the macrophage, which are commonly notified as M1 and M2, respectively. The alteration of macrophage phenotypes and the progression of inflammation are intimately associated; both phenotypes usually coexist throughout the whole inflammation stage, involving the transduction of intracellular signals and the secretion of extracellular cytokines. This paper aims to address the interaction of macrophages and surrounding cells and tissues with inflammation-related diseases and clarify the crosstalk of signal pathways relevant to the phenotypic metamorphosis of macrophages. On these bases, some novel therapeutic methods are proposed for regulating inflammation through monitoring the transition of macrophage phenotypes so as to prevent the negative effects of antibiotic drugs utilized in the long term in the clinic. This information will be quite beneficial for the diagnosis and treatment of inflammation-related diseases like pneumonia and other disorders involving macrophages.

Integration and Quantitative Visualization of 3,3',5,5'-Tetramethylbenzidine-Probed Enzyme-Linked Immunosorbent Assay-like Signals in a Photothermal Bar-Chart Microfluidic Chip for Multiplexed Immunosensing.

The photothermal effect shows significant promise for various biomedical applications but is rarely exploited for microfluidic lab-on-a-chip bioassays. Herein, a photothermal bar-chart microfluidic immunosensing chip, with the integration of the conventional 3,3',5,5'-tetramethylbenzidine (TMB)-probed enzyme-linked immunosorbent assay (ELISA)-like system, was developed based on exploiting the photothermal pumping technique for visual bar-chart microfluidic immunosensing. Both the sandwich ELISA-like system and the photothermal pumping protocol were integrated into a single photothermal bar-chart chip. On-chip immunocaptured iron oxide nanoparticles catalyzed the oxidation of the chromogenic substrate, TMB, to produce a sensitive photothermal and chromogenic dual-functional probe, oxidized TMB. As the result of heat generation and the subsequent production of elevating vapor pressure in the sealed microfluidic environment, the on-chip near-infrared laser-driven photothermal effect of the probe served as a dose-dependent pumping force to drive the multiplexed quantitative display of the immunosensing signals as visual dye bar charts. Prostate-specific antigen as a model analyte was tested at a limit of detection of 1.9 ng·mL-1, lower than the clinical diagnostic threshold of prostate cancer. This work presents a new perspective for microfluidic integration and multiplexed quantitative bar-chart visualization of the conventional TMB-probed ELISA signals possibly by means of an affordable handheld laser pointer in a lab-on-a-chip format.

Development of Poloxamer Hydrogels Containing Antibacterial Guanidine-Based Polymers for Healing of Full-Thickness Skin Wound.

A series of hydrogels containing guanidine-based polymers using a poloxamer as the matrix were prepared to provide novel wound dressings with antibacterial and repairing-promotion properties for skin wounds. Herein, we developed a series of antibacterial hydrogels, the cationic guanidine-based polymer polyhexamethylene guanidine hydrochloride (PHMG) with poloxamer aqueous solution (12%, w/w) simplified as PHMGP, chitosan (CS)-cross-linked PHMG (referred to as PHMC) with poloxamer aqueous solution simplified as PHMCP, and hyaluronic acid (HA)-modified PHMG (referred to as PHMH) with poloxamer aqueous solution simplified as PHMHP, for enhancing full-thickness skin wound healing. The characterizations, antimicrobial activity, cytotoxicity, and in vivo full-thickness wound-healing capability of these hydrogels were analyzed and evaluated. The results show that though PHMGP possesses great bactericide properties, its cytotoxicity is too strong to support skin regeneration. However, after modified with CS or HA, PHMCP and PHMHP showed good biocompatibility and antimicrobial properties against Gram-positive and Gram-negative bacteria that are commonly present in injured skin. Both PHMCP and PHMHP hydrogels exhibited upgraded wound-healing efficiency in full-thickness skin defects, characterized by a shorter wound closure time, faster re-regeneration, and the earlier formation of skin appendages, compared with those of control or pure poloxamer treatments. Their biological mechanism was detected. Both PHMCP and PHMHP can regulate the related biofactors during the skin repair process such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), transforming growth factor beta-1(TGF-ß1), alpha-smooth muscle actin (α-SMA), and vascular endothelial growth factor, to promote wound healing with less serious scarring. In short, hydrogels with excellent capabilities to inhibit microorganism infection and promote wound healing were developed, which will shed light on designing and producing wound dressings with promising applications in future.

Differentiation of bMSCs on Biocompatible, Biodegradable, and Biomimetic Scaffolds for Largely Defected Tissue Repair.

Biocompatible, biodegradable, and biomimetic scaffolds in combination with stem cells are of great importance for tissue engineering, especially the repairing and regeneration of defected organs. As a case in point, esophageal diseases have become serious clinical problems because of the poor self-repairing ability of the organ. It is crucial to prepare artificial replacements with biological function for serious lesions of the esophagus. However, the pH value, mechanical strength, thickness, and other physical conditions are very different in different organs or different parts of the same organ, which pose high difficulty for successful tissue engineering. In this work, bone marrow mesenchymal stem cells (bMSCs) were isolated from rabbits and transfected with green fluorescent protein (GFP) to follow their capabilities of growth, stemness, and differentiation in ex vivo culture. These bMSCs were seeded on biocompatible, biodegradable, and biomimetic scaffolds to detect the tissue regenerative capability of the esophagus with multilayer hierarchical structure. According to the esophageal bilayer muscle architecture, we designed discontinuous and continuous microchannel patterned scaffolds with medical level polyurethane (PU) as the matrix to guide the inner-circular and outer-longitudinal muscle growth. The gap on the discontinuous walls not only helped cells to communicate with each other but also assisted cells to infiltrate through the gap and grew into the inner circular muscle. The graft of silk fibroin on the scaffold surface using the aminolysis and glutaraldehyde cross-linking method enhanced the substrate's hydrophilicity and biocompatibility. Mucosa-submucosa tissue of rabbit's esophagus was decellularized to obtain the extracellular matrix (ECM) and implanted in situ after recellularizing with bMSCs to repair the partially defected rabbits' esophagus. On the basis of both in vitro and in vivo results, we concluded that esophagus regeneration was promoted by the differentiation of bMSCs on the biocompatible, biodegradable, and biomimetic scaffolds, starting from tissue "niches", to repair the largely defected esophagus, which paves the way for tissue engineering and defected organ treatments.

In-Situ Mutation Detection by Magnetic Beads-Probe Based on Single Base Extension and Its Application in Genotyping of Hepatitis B Virus Pre-C Region 1896nt Locus Single Nucleotide Polymorphisms.

Hepatitis B virus (HBV) is closely related to occurrence and development of viral hepatitis. A mutation of 1896nt locus in its pre-C region can promote replication of HBV DNA and improve stability of pre-genome RNA structure, and can even help HBV evade immune clearance. In this study, magnetic beads-probe (MBs@probe) method, combined with single base extension (SBE) technology, was developed for in-situ mutation detection of HBV pre-C region 1896nt locus. Before successfully completing the genotyping of 165 HBV samples, the crucial reaction conditions were first optimized, such as SBE temperature, MBs size and amount, and probe concentration on the surface of MBs. Experimental results showed that these conditions had significant effects on MBs@probe in-situ mutation detection. Comprehensive considerations, such as 58 °C of SBE temperature, high fluorescence intensity and signal-to-noise ratios (SNRs) were obtained when MBs@probe complex was made by 100 µg of 300 nm-MBs and 3.0 µM of probes in the system. Finally, 1896nt locus mutation in pre-C region of 165 HBV samples was successfully genotyped, among which 71 HBV samples were wild types and the remaining 94 samples were mutant types. Meanwhile, 14 randomly chosen samples were taken to further analyze fluorescence intensity and SNRs respectively, and sequencing results for the first two samples were consistent with results from the MBs@probe in-situ mutation detection method. Compared with two-color fluorescence hybridization (TCFH) genotyping technology, this method generally improves the SNRs to more than 10 (which is more than 2-fold), has higher reliability and is more suitable to detect SNPs for known sites.

Identification of candidate genes for the diagnosis and treatment of cholangiocarcinoma using a bioinformatics approach.

Cholangiocarcinoma (CCA) is a biliary epithelial tumor with poor prognosis. As the key genes and signaling pathways underlying the disease have not been fully elucidated, the aim of the present study was to improve the understanding of the molecular mechanisms associated with CCA. The microarray datasets GSE26566 and GSE89749 were downloaded from the Gene Expression Omnibus and differentially expressed genes (DEGs) between CCA and normal bile duct samples were identified. Gene and pathway enrichment analyses were performed, and a protein-protein interaction network was constructed and analyzed. A total of 159 DEGs and 10 hub genes were identified. The functions and pathways of the DEGs were mainly enriched in 'heparin binding', 'serine-type endopeptidase activity', 'calcium ion binding', 'pancreatic secretion', 'fat digestion and absorption' and 'protein digestion and absorption'. Survival analysis revealed that the upregulated expression of carboxypeptidase B1 and Kruppel like factor 4 was significantly associated with lower overall survival rate. In summary, the present study identified DEGs and hub genes associated with CCA, which may serve as potential diagnostic and therapeutic targets for the disease.

A Highly Sensitive Strategy for Low-Abundance Hepatitis B Virus Detection via One-Step Nested Polymerase Chain Reaction, Chemiluminescence Technology and Magnetic Separation.

Hepatitis B virus (HBV) has been always threatening to health. It is significant to establish an effective strategy for HBV carriers and hepatitis B patients diagnosis at early stage of infection in clinic. Herein, we combined nested PCR, chemiluminescence assay and magnetic separation technology to establish a highly sensitive and stable strategy for low-abundance HBV-DNA detection. Six types of monodisperse Fe3O4 magnetic beads (MBs) were fabricated and coated with SiO2 layer (MBs@SiO2). The effect of MBs@SiO2 size on HBV-DNA extraction and chemiluminescence assay was explored, and results displayed that the MBs@SiO2-F particles with largest size were much more suitable for HBV-DNA extraction than others, and MBs@SiO2-C with the size range between 330 nm-450 nm performed best in chemiluminescence assay. As one of key factors, the concentration (or density) of the probe fixed on MBs@SiO2 surface was optimized for chemiluminescence assay, exhibiting the best assay result when reaction system contained 2.0 µM of probe. Finally, under the best reaction conditions, low-abundance HBV serum specimens were sensitively and stably detected, and the result of HBV specimen with 34 copy/mL loads showed significant difference with negative and blank (p < 0.001).

Biological properties of a bionic scaffold for esophageal tissue engineering research.

Polyurethane is a good matrix material with wide application prospects in tissue engineering because of its adjustable and mechanical properties. A novel biodegradable crosslinked poly(ester urethane) (CPU) with flexible poly(caprolactone) (PCL) and hydrophilic poly(ethylene glycol) (PEG) components has been synthesized using a ferric iron catalyst in our laboratory. In the present study, to promote the interaction between the CPU material and cells, the material was superficially modified by silk fibroin (SF) grafting using an aminolysis and glutaraldehyde crosslinking method to achieve a biocompatible material, CPU-SF. Considering the esophageal-specific architecture, three types of scaffolds were fabricated. S1 was a CPU-SF channel (200 µm in diameter and 30 µm in depth with 30 µm of wall thickness) to support muscle regeneration; S2 was the decellularized matrix of the esophageal mucosa/submucosa obtained by enzyme treatment; and S3 was a combination of S1 and S2, aiming to promote esophageal regeneration with histological structure and function. The biological properties and functions of the materials and scaffolds were investigated by qualitative and quantitative analyses using scanning electron microscopy, immunofluorescence staining, cell adhesion and proliferation measurements, and western blotting technology. The results showed that esophageal smooth muscle cells (SMCs) and epithelial cells (ECs) were very well supported by the scaffolds. In particular, SMCs exhibited guided directional growth and ECs infiltrated the acellular mucosa with retained biological functions when co-cultured on the composite scaffold S3. These findings suggest that the composite bionic scaffold will be a good alternative for esophageal replacement.

An Aptamer-Based Probe for Molecular Subtyping of Breast Cancer.

Molecular subtyping of breast cancer is of considerable interest owing to its potential for personalized therapy and prognosis. However, current methodologies cannot be used for precise subtyping, thereby posing a challenge in clinical practice. The aim of the present study is to develop a cell-specific single-stranded DNA (ssDNA) aptamer-based fluorescence probe for molecular subtyping of breast cancer. Methods: Cell-SELEX method was utilized to select DNA aptamers. Flow cytometry and confocal microscopy were used to study the specificity, binding affinity, temperature effect on the binding ability and target type analysis of the aptamers. In vitro and in vivo fluorescence imaging were used to distinguish the molecular subtypes of breast cancer cells, tissue sections and tumor-bearing mice. Results: Six SK-BR-3 breast cancer cell-specific ssDNA aptamers were evolved after successive in vitro selection over 21 rounds by Cell-SELEX. The Kd values of the selected aptamers were all in the low-nanomolar range, among which aptamer sk6 showed the lowest Kd of 0.61 ± 0.14 nM. Then, a truncated aptamer-based probe, sk6Ea, with only 53 nt and high specificity and binding affinity to the target cells was obtained. This aptamer-based probe was able to 1) differentiate SK-BR-3, MDA-MB-231, and MCF-7 breast cancer cells, as well as distinguish breast cancer cells from MCF-10A normal human mammary epithelial cells; 2) distinguish HER2-enriched breast cancer tissues from Luminal A, Luminal B, triple-negative breast cancer tissues, and adjacent normal breast tissues (ANBTs) in vitro; and 3) distinguish xenografts of SK-BR-3 tumor-bearing mice from those of MDA-MB-231 and MCF-7 tumor-bearing mice within 30 min in vivo. Conclusion: The results suggest that the aptamer-based probe is a powerful tool for fast and highly sensitive subtyping of breast cancer both in vitro and in vivo and is also very promising for the identification, diagnosis, and targeted therapy of breast cancer molecular subtypes.

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.

Injectable hydrogels for cartilage and bone tissue engineering.

Tissue engineering has become a promising strategy for repairing damaged cartilage and bone tissue. Among the scaffolds for tissue-engineering applications, injectable hydrogels have demonstrated great potential for use as three-dimensional cell culture scaffolds in cartilage and bone tissue engineering, owing to their high water content, similarity to the natural extracellular matrix (ECM), porous framework for cell transplantation and proliferation, minimal invasive properties, and ability to match irregular defects. In this review, we describe the selection of appropriate biomaterials and fabrication methods to prepare novel injectable hydrogels for cartilage and bone tissue engineering. In addition, the biology of cartilage and the bony ECM is also summarized. Finally, future perspectives for injectable hydrogels in cartilage and bone tissue engineering are discussed.

Integration of Nucleic Acid Extraction Protocol with Automated Extractor for Multiplex Viral Detection.

The isolation of nucleic acids (NA) is the preliminary step to carry out genetic studies and DNA biosensor development. The presence of inhibitors in the purified NA interferes with the downstream application. These salts and other organic contaminations particularly challenge the analytical sensitivity of DNA biosensors. The detailed study was carried out to optimize the factors which might affect viral nucleic acid purification. The results suggested that 6 M guanidinium hydrochloride salt concentration was critical for NA isolation. The inverse relation has been found in the pH of the lysis buffer and quality and quantity of NA. The NA yield was relatively stable at pH 4­5. It has been observed that the use of carrier RNA was indispensable for viral genome isolation. The addition of ethanol to lysate in 1:1 ratio greatly improved NA recovery. The elution efficiency of DNase and RNase free water, 1× TE buffer and 1× PCR buffer was compared. The carrier RNA was best eluted in DNase and RNase free water and 1× TE buffer. It was further demonstrated that this method can be automatized for high throughput detection. A simple experiment was conducted to optimize the different parameters of an automated NA extractor to simultaneously extract HBV DNA and HCV RNA. The purified NA was successfully amplified in PCR and RT-PCR to verify the reliability of the established protocol. Thus a semi-automated system for the simultaneous detection of multiple viruses has been demonstrated.

Genetic Variation of BCL2 (rs2279115), NEIL2 (rs804270), LTA (rs909253), PSCA (rs2294008) and PLCE1 (rs3765524, rs10509670) Genes and Their Correlation to Gastric Cancer Risk Based on Universal Tagged Arrays and Fe3O4 Magnetic Nanoparticles.

With the help of Fe3O4 nagnetic nanoparticles as a solid carrier and an excellent tool for separation, six SNP loci (rs2279115 of BCL2 gene, rs804270 of NEIL2 gene, rs909253 of LTA gene, rs2294008 of PSCA gene, rs3765524 and rs10509670 of PLCE1 gene) were selected to evaluate their relation to gastric cancer risk. Using two kinds of functionalized magnetic nanoparticles and universal tagged arrays, the whole operation procedure including genome DNA extraction and SNP genotyping was performed. All genotypes and allele frequencies were calculated in the cases and controls respectively to analyze their association with gastric cancer risk. Totally 200 pathological samples and 134 normal control subjects were collected. The results demonstrated that four SNP loci (rs2279115, rs804270, rs909253 and rs3765524) showed a potential association with gastric cancer risk, and the other two (rs2294008, rs10509670) possessed no difference/association among cases and controls.

Applications of Magnetic Nanoparticles in Targeted Drug Delivery System.

Magnetic nanoparticles (MNPs) are a special kind of nanomaterials and widely used in biomedical technology applications. Currently they are popularly customized for disease detection and treatment, particularly as drug carriers in drug targeted delivery systems, as a therapeutic in hyperthermia (treating tumors with heat), and as contrast agents in magnetic resonance imaging (MRI). Due to their biocompatibility and superparamagnetic properties, MNPs as next generation drug carriers have great attraction. Although the potential benefits of MNPs are considerable, any potential toxicity associated with these MNPs should be identified distinctly. The drug loading capability and the biomedical properties of MNPs generated by different surface coatings are the most sensitive parameters in toxicity. A lot of organic and inorganic materials are utilized as coating materials for surface functionalization and reducing toxicity of MNPs. pH or temperature sensitivity materials are widely used to manage drug loading and targeted release. In addition, MNPs can be controlled and directed to the desired pathological region by using external magnetic files (EMF). The realization of targeted drug delivery has decreased the dosage and improved the efficiency of drugs, which results in reduced side effects to normal tissues. This review discussed the possible organ toxicities of MNPs and their current advances as a drug delivery vehicle.

Application of functional microsphere in human hepatitis B virus surface antigen detection.

A novel and simple emulsifier-free emulsion polymerization technique was developed for preparation of mono-dispersed amino functionalized polymer microspheres with well defined diameters (about 400 nm). Various characterization methods demonstrated that the obtained amino microspheres had a uniform size and good dispersity which were confirmed by scanning electron microscope (SEM). Zeta potential and Fourier transform infrared spectrometer (FT-IR) demonstrated that amino groups have been successfully introduced to the microsphere surface. These functionalized microspheres have been shown to be efficient and controllable carriers capable of immobilizing and enriching monoclonal antibodies. Moreover, a newest chemiluminescent enzyme-linked immunoassay (ELISA) approach has been developed for human Hepatitis B virus surface antigen (HBsAg) detection. HBsAg was sandwiched between goat anti-HBsAg polyclonal antibody and mouse anti-HBsAg antibody. Alkaline phosphatase (ALP) conjugated horse anti-mouse immunnogloblin was used to bond with monoclonal antibody. Finally, chemiluminesent (CL) signals were recorded after adding 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD) which was used as a chemiluminescent substrate reagent of ALP. This novel chemiluminescent ELISA assay was proved to be of excellent specificity and high sensitivity when using ALP and AMPPD luminescence systems for specific HBsAg detection.

Magnetic beads-based chemiluminescent assay for ultrasensitive detection of pseudorabies virus.

A rapid, ultrasensitive and economical Pseudorabies virus (PRV) detection system based on magnetic beads (MBs) and chemiluminescence was developed in this paper. The carboxyl functionalized MBs (MBs-COOH) were covalently coupled with aminated DNA probes for capturing PRV biotinylated amplicon, the product of polymerase chain reaction (PCR). Agarose gel electrophoresis analysis approved the reliability of biotinylated amplicon. The MBs composites were incubated with alkaline phosphatase labeled streptavidin (ALP-SA) and chemiluminescene was determined by subsequently adding 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD). The optimal conditions of the PRV detection method were 10 microM for probe concentration, 50 degrees C for hybridization temperature and 30 min for hybridization time. The limit of detection (LOD) was as low as 100 amol/5 pM of amplicon which proved that this approach for PRV detection was ultrasensitive.

Detection of Pseudomonas aeruginosa based on magnetic enrichment and nested PCR.

Pseudomonas aeruginosa is a common opportunistic pathogen in clinics. The species-specific ecfX gene of Pseudomonas aeruginosa has high specificity. In this experiment, we are intended to develop a new method for the detection of Pseudomonas aeruginosa based on magnetic enrichment and nested PCR, and the specific ecfX gene of Pseudomonas aeruginosa was used as the detection object. The genomic DNA of Pseudomonas aeruginosa was extracted using amino-modified magnetic nanoparticles (MNPs). The ecfX gene was amplified by nested PCR and the product of PCR was detected by agarose gel electrophoresis. The results showed that the optimal annealing temperature was 64 degrees C and 62 degrees C respectively in the first and the second rounds of PCR. The lowest concentration of Pseudomonas aeruginosa that could be detected was 10 cfu/mL. The method provides a reliable, timely and accurate technology for early detection of Pseudomonas aeruginosa. Furthermore, the method can shorten the procedure and time from DNA extraction to detection, which made automation more convenient.

An automatic high-throughput single nucleotide polymorphism genotyping approach based on universal tagged arrays and magnetic nanoparticles.

Recent developments in highly parallel genome-wide studies are transforming the association of human health and diseases. In these studies, multiple SNP loci from large amount of samples need to be investigated to obtain a result with a high degree of confidence. Herein, we describe a novel, cost-effective and automated method for high-throughput single nucleotide polymorphisms (SNPs) genotyping based on universal tagged array and magnetic separation. By using two kinds of functionalized magnetic nanoparticles, the whole operation procedure including genome DNA extraction and SNP genotyping can be automatically performed by JANUS automated workstation (Perkin Elmer Inc.). Four different SNPs loci from 80 samples were scored using only one pair of universal dual-color probes, the phase of numerous SNPs can be automated assessed simultaneously. The results demonstrated that the expected scores and good discrimination were obtained between the two alleles from these four SNP loci. Due to adequately taking the advantages of high parallel read-out and intrinsically scalable properties of microarray, and the automated magnetic separation handling technology is highly adaptable fro multiplexing sample preparation and automated SNP analysis, also avoid the complex procedure including purification and concentration, the new strategy is high-throughput, simple, flexible, cost-effective, and will be very suitable for large-scale genotyping.