Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration.

CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications. However, it is still infeasible because homologous recombination (HR) is inefficient, especially for non-dividing cells. To overcome the challenge, we report that a homology-independent targeted integration (HITI) strategy is used for permanent integration of high-specificity-activity Factor IX variant (F9 Padua, R338L) at the albumin (Alb) locus in a novel hemophilia B (HB) rat model.

A rapid nucleic acid concentration measurement system with large field of view for a droplet digital PCR microfluidic chip.

Droplet digital polymerase chain reaction (ddPCR) is an effective technique, with unparalleled sensitivity, for the absolute quantification of target nucleic acids. However, current commercial ddPCR devices for detecting the gene chip are time consuming due to complex image stitching. To address this issue, we propose a universal concentration determination system and realize one-time gene chip imaging with high resolution.

A Self-Priming Microfluidic Chip with Cushion Chambers for Easy Digital PCR.

A polydimethylsiloxane (PDMS)-based self-priming microfluidic chip with cushion chambers is presented in this study for robust and easy-operation digital polymerase chain reaction (dPCR). The chip has only one inlet and can partition samples autonomously through negative pressure, provided by a de-gassed PDMS layer with a multi-level vertical branching microchannel design. Meanwhile, cushion chambers make the chip capable of very robust use for sample partitioning.

A Double-Deck Self-Digitization Microfluidic Chip for Digital PCR.

In this work, a double-deck microfluidic chip was presented for digital PCR application. This chip consists of two reverse-placed micro-patterned polydimethylsiloxane (PDMS) layers between the top and bottom glass substrates. Each micropatterned PDMS layer contains more than 20,000 cylindrical micro-chambers to hold the partitioned droplets.

Fast and robust sample self-digitization for digital PCR.

We present a facile sample partitioning method to enable rapid and low-cost digital PCR (dPCR) assays. By subdividing a high percentage of the sample volume into a large number of equal volume compartments with a self-digitization (SD) chip, this method can achieve a low-waste and high-order sample discretization in a matter of minutes. The SD chip contains a set of parallel microfluidic channels used for sample delivery, and each channel is connected with two rows of cylindrical wells to hold the discretized sample.

A self-digitization chip integrated with hydration layer for low-cost and robust digital PCR.

We present a self-discretization and zero-water-loss microfluidic digital PCR (dPCR) device to enable low-cost and robust quantitative nucleic acid assays. In this device, a thin void is integrated beneath the reaction chamber array. By utilizing the permeability of polydimethylsiloxane (PDMS) film, the integrated void serves a dual function: vacuum "accumulator" and hydration "reservoir".

Highly sensitive detection of multiple tumor markers for lung cancer using gold nanoparticle probes and microarrays.

Assay of multiple serum tumor markers such as carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen (CYFRA21-1), and neuron specific enolase (NSE), is important for the early diagnosis of lung cancer. Dickkopf-1 (DKK1), a novel serological and histochemical biomarker, was recently reported to be preferentially expressed in lung cancer. Four target proteins were sandwiched by capture antibodies attached to microarrays and detection antibodies carried on modified gold nanoparticles.

Analysis of circulating tumor cells from lung cancer patients with multiple biomarkers using high-performance size-based microfluidic chip.

Circulating tumor cells (CTCs) have attracted pretty much attention from scientists because of their important relationship with the process of metastasis. Here, we developed a size-based microfluidic chip containing triangular pillar array and filter channel array for detecting single CTCs and CTC clusters independent of tumor-specific markers. The cell populations in chip were characterized by immune-fluorescent staining combining an epithelial marker and a mesenchymal marker.

Androgen-induced miR-27A acted as a tumor suppressor by targeting MAP2K4 and mediated prostate cancer progression.

Prostate cancer (PCa) is the most commonly diagnosed and secondly leading cause of cancer death among males. But the precise mechanism of prostate cancer progression, including microRNAs (miRNAs) functioning in it, is still needs further study. We found miR-27a to be down-regulated in prostate cancer, and we investigated the mechanism and role of miRNA-27a in prostate cancer.

Multiplexed detection of lung cancer biomarkers based on quantum dots and microbeads.

We have developed a multiplexed fluoroimmunoassay of three lung cancer biomarkers based on multicolor quantum dots (QDs) as detection elements and micro-magnetic beads as immune carriers. QDs have the ability to simplify multiplexed analysis. In our method, the fluorescent signals derived from three cross-talk-free QD conjugated probes with emission maxima at 525, 585 and 625nm could be analyzed to determine the concentrations of the target proteins.

Bead-based microarray immunoassay for lung cancer biomarkers using quantum dots as labels.

In this study, we developed a multiplex immunoassay system that combines the suspension and planar microarray formats within a single layer of polydimethylsiloxane (PDMS) using soft lithography technology. The suspension format was based on the target proteins forming a sandwich structure between the magnetic beads and the quantum dot (QD) probes through specific antibody-antigen interactions. The planar microarray format was produced by fabricating an array of micro-wells in PDMS.

Absolute quantification of lung cancer related microRNA by droplet digital PCR.

Digital polymerase chain reaction (digital PCR) enables the absolute quantification of nucleic acids through the counting of single molecules, thus eliminating the need for standard curves or endogenous controls. In this study, we developed a droplet digital PCR (ddPCR) system based on an oil saturated PDMS (OSP) microfluidic chip platform for quantification of lung cancer related microRNA (miRNA). The OSP chip was made with PDMS and was oil saturated to constrain oil swallow and maintain the stability of droplets.

A microfluidic droplet digital PCR for simultaneous detection of pathogenic Escherichia coli O157 and Listeria monocytogenes.

Sensitive and rapid identification of pathogenic bacterial is extremely important due to the serious threat of pathogens to human health. In this study, we demonstrate the simultaneous and sensitive detection of pathogenic Escherichia coli O157 and Listeria monocytogenes using a novel duplex droplet digital PCR (ddPCR) platform. The ddPCR platform, which uses a mineral oil-saturated polydimethylsiloxane (OSP) chip to overcome the problem of droplet evaporation, integrates the functions of droplet generation, on-chip amplification and end-point fluorescence readout.

Early Detection of Lung Cancer in Serum by a Panel of MicroRNA Biomarkers.

Introduction: The objective of the study was to develop a panel of microRNAs (miRNAs) as highly sensitive and specific biomarkers for lung cancer early detection.

Materials And Methods: The study contained 2 phases: first, preliminary marker selection based on previous reports on the serum of 24 early stage lung cancer patients and 24 healthy control subjects by TaqMan probe-based real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR); and second, validation of miRNA markers on 94 early stage lung cancer, 48 stage III to IV lung cancer, and 111 healthy control serum samples.

Results: A total of 3 miRNAs (miR-125a-5p, miR-25, and miR-126) were selected for further analysis in this study.

Multi-nanomaterial electrochemical biosensor based on label-free graphene for detecting cancer biomarkers.

Developing a rapid, accurate and sensitive electrochemical biosensor for detecting cancer biomarkers is important for early detection and diagnosis. This work reports an electrochemical biosensor based on a graphene (GR) platform which is made by CVD, combined with magnetic beads (MBs) and enzyme-labeled antibody-gold nanoparticle bioconjugate. MBs coated with capture antibodies (Ab1) were attached to GR sheets by an external magnetic field, to avoid reducing the conductivity of graphene.

High-performance size-based microdevice for the detection of circulating tumor cells from peripheral blood in rectal cancer patients.

Since individualized therapy becomes more and more important in the treatment of rectal cancer, an accurate and effective approach should be established in the clinical settings to help physicians to make their decisions. Circulating tumor cells (CTCs), originated from either primary or metastatic cancer, could provide important information for diagnosis and monitoring of cancer. However, the implication and development of CTCs are limited due to the extreme rarity of these tumor cells.

Highly sensitive enumeration of circulating tumor cells in lung cancer patients using a size-based filtration microfluidic chip.

Circulating tumor cells (CTCs) in the peripheral blood could serve as a surrogate marker for the diagnosis of cancer metastasis and for therapeutic evaluation. However, the separation and characterization of CTCs is technically challenging owing to the extremely low number of CTCs present. Here we developed a size-based and high-throughput microfluidic chip, which exploits filtration microchannels to isolate the relatively larger CTCs from the rest of the blood constituents.

Integrated biochip for label-free and real-time detection of DNA amplification by contactless impedance measurements based on interdigitated electrodes.

Here, we introduce an integrated biochip which offers accurate thermal control and sensitive electrochemical detection of DNA amplification in real-time. The biochip includes a 10-μl microchamber, a temperature sensor, a heater, and a contactless impedance biosensor. A pair of interdigitated electrodes is employed as the impedance biosensor and the products of the amplification are determined directly through tracing the impedance change, without using any labels, redox indicators, or probes.

Real-time monitoring of strand-displacement DNA amplification by a contactless electrochemical microsystem using interdigitated electrodes.

This paper reports the design and implementation of a contactless conductivity detection system which combines a thermal control cell, a data processing system and an electrochemical (EC) cell for label-free isothermal nucleic acid amplification and real-time monitoring. The EC cell consists of a microchamber and interdigitated electrodes as the contactless conductivity biosensor with a cover slip as insulation. In our work, contactless EC measurements, the effects of trehalose on amplification, and chip surface treatment are investigated.

[Membrane transfer-based colorimetric DNA detection using enzyme modified gold nanoparticles].

We report here a novel membrane transfer-based DNA detection method, in which alkaline phosphatase labeled gold nanoparticle (AuNP) probes were used as a means to amplify the detection signal. In this method, the capture probe P1, complimentary to the 3' end of target DNA, was immobilized on the chip. The multi-component AuNP probes were prepared by co-coating AuNPs with the detecting probe P2, complimentary to the 5' end of target DNA, and two biotin-labeled signal probes (T10 and T40) with different lengths.

Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori.

Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H.

Nano-ELISA for highly sensitive protein detection.

Highly sensitive protein detection method based on nanoparticles and enzyme-linked immunosorbent assays (ELISAs), named Nano-ELISA, was introduced. In this method, the micro-magnetic beads were modified with monoclonal antibody of the target protein p53. Gold nanoparticles (AuNPs) were modified with another monoclonal detector antibody and Horseradish peroxidase (HRP, for signal amplification).

[The detection of plasmid pCAMBIA1301 in transgenic rice by arrayed primer extension].

Biochip technology which had emerged from the fusion of biotechnology and micro/nanofabrication technology at the end of 1980s has been widely used in life science, medicine, clinical diagnosis, drug development, agriculture, environmental protection and strategies. DNA microarray (also call gene chip, DNA chip), one kind of biochips, is small chip containing many oligonucleotide probes. It can hybridize with labelled sample, making it possible to detect large numbers of oligonucleotides at one time.

[Detection for single nucleotide polymorphisms].

As the third generation of genetic markers SNPs (single nucleotide polymorphisms) has been used extensively in gene mapping,disease-correlativity analysis ,population genetics and drug research. Here methods for detection are reviewed. Most SNP genotyping are a combination of method for interrogating SNPs and analysis technique.