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Find video protocols related to scientific articles indexed in Pubmed.
Design and test of a flat-top magnetic field system driven by capacitor banks.
Rev Sci Instrum
PUBLISHED: 05-03-2014
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An innovative method for generating a flat-top pulsed magnetic field by means of capacitor banks is developed at the Wuhan National High Magnetic Field Center (WHMFC). The system consists of two capacitor banks as they are normally used to generate a pulsed field. The two discharge circuits (the magnet circuit and the auxiliary circuit) are coupled by a pulse transformer such that the electromotive force (EMF) induced via the transformer in the magnet circuit containing the magnet coil is opposed to the EMF of the capacitor bank. At a certain point before the current pulse in the coil reaches its peak, the auxiliary circuit is triggered. With optimized parameters for charging voltage and trigger delay, the current in the magnet circuit can be approximately kept constant to obtain a flat-top. A prototype was developed at the WHMFC; the magnet circuit was energized by seven 1 MJ (3.2 mF/25?kV) capacitor modules and the auxiliary circuit by four 1 MJ modules. Fields up to 41 T with 6?ms flat-top have been obtained with a conventional user magnet used at the WHMFC.
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Simultaneous determination of bosentan and glimepiride in human plasma by ultra performance liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study.
J Pharm Biomed Anal
PUBLISHED: 03-03-2014
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A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine bosentan (BOS) and glimepiride (GPD) in human plasma simultaneously. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 552.0?202.1 and m/z 491.2?125.9 were used to quantify BOS and GPD, respectively. This assay method has been fully validated in terms of selectivity, linearity, recovery and matrix effect, accuracy, precision and stability. The linearity of this method was found to be within the concentration range of 5-1000 ng/mL for BOS, and 2.5-500 ng/mL for GPD in human plasma. Only 1.5 min was needed for an analytical run. This assay was used to support a clinical study where multiple oral doses were administered to healthy Chinese subjects to investigate the pharmacokinetics of BOS and GPD.
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An equipment-free polydimethylsiloxane microfluidic spotter for fabrication of microarrays.
Biomicrofluidics
PUBLISHED: 03-01-2014
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This paper presents a low-cost, power-free, and easy-to-use spotter system for fabrication of microarrays. The spotter system uses embedded dispensing microchannels combined with a polydimethylsiloxane (PDMS) membrane containing regular arrays of well-defined thru-holes to produce precise, uniform DNA or protein microarrays for disease diagnosis or drug screening. Powered by pre-evacuation of its PDMS substrate, the spotter system does not require any additional components or external equipment for its operation, which can potentially allow low-cost, high-quality microarray fabrication by minimally trained individuals. Polyvinylpyrrolidone was used to modify the PDMS surface to prevent protein adsorption by the microchannels. Experimental results indicate that the PDMS spotter shows excellent printing performance for immobilizing proteins. The measured coefficient of variation (CV) of the diameter of 48 spots was 2.63% and that of the intensity within one array was 2.87%. Concentration gradient experiments revealed the superiority of the immobilization density of the PDMS spotter over the conventional pin-printing method. Overall, this low-cost, power-free, and easy-to-use spotting system provides an attractive new method to fabricate microarrays.
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?-Opioid receptor activation reduces ?-synuclein overexpression and oligomer formation induced by MPP(+) and/or hypoxia.
Exp. Neurol.
PUBLISHED: 02-14-2014
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Hypoxic/ischemic brain injury is a potential cause of Parkinson's disease (PD) with ?-synuclein playing a critical role in the pathophysiology. Since ?-opioid receptor (DOR) is neuroprotective against hypoxic/ischemic insults, we sought to determine if DOR regulates ?-synuclein under hypoxia and/or MPP(+) stress. We found that in HEK293 cells 1) MPP(+) in normoxia enhanced ?-synuclein expression and the formation of ?-synuclein oligomers thereby causing cytotoxic injury; 2) hypoxia at 1% O2 for 48h or at 0.5% O2 for 24h also induced ?-synuclein overexpression and its oligomer formation with cell injury; 3) however, hypoxia at 1% O2 for 24h, though increasing ?-synuclein expression, did not cause ?-synuclein oligomer formation and cell injury; 4) UFP-512 mediated DOR activation markedly attenuated the hypoxic cell injury and ?-synuclein overexpression, which was largely attenuated by DOR antagonism with naltrindole or siRNA "knock-down" of the DOR; and 5) DOR activation enhanced CREB phosphorylation and prevented the collapse of mitochondrial membrane potential (??m). These findings suggest that DOR activation attenuates MPP(+) or severe hypoxia induced ?-synuclein expression/aggregation via a CREB pathway.
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Activation of multiple signaling pathways during the differentiation of mesenchymal stem cells cultured in a silicon nanowire microenvironment.
Nanomedicine
PUBLISHED: 01-31-2014
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Mesenchymal stem cells (MSC) offer an optimal source for bone tissue engineering due to their capability of undergoing multilineage differentiation, where the mechanical properties of the microenvironment of MSCs are vital for osteochondral formation. However, the mechanisms of how mechanical and microenvironmental cues control osteogenesis and chondrogenesis are yet to be elucidated. In this study, we investigated the effects of vertically aligned silicon nanowire (SiNW) array on the differentiation of MSCs and the associated molecular mechanisms involved in osteogenesis and chandrogenesis. The results showed that the microenvironment of SiNW array activated a number of mechanosensitive pathways (including Integrin, TGF-?/BMP, Akt, MAPK, Insulin, and Wnt pathways) in MSCs, which converged to stimulate the osteogenesis and chondrogenesis via the Ras-Raf-MEK-ERK cascade.
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Immobilization of biomolecules on cysteamine-modified polyaniline film for highly sensitive biosensing.
Talanta
PUBLISHED: 01-29-2014
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We present a new cysteamine (CS)-modified polyaniline (PANI) film for highly efficient immobilization of biomolecules in biosensing technology. This electrochemical deposited PANI film treated with CS and glutaraldehyde could be employed as an excellent substrate for biomolecules immobilization. The parameters of PANI growth were optimized to obtain suitable surface morphology of films for biomolecules combination with the help of electron and atomic force microscopy. Cyclic voltammetry (CV) was utilized to illustrate the different electrochemical activities of each modified electrode. Due to the existence of sulfydryl group and amino group in CS, surface modification with CS was proven to reduce oxidized units on PANI film remarkably, as evidenced by both ATR-FTIR and Raman spectroscopy characterizations. Furthermore, bovine serum albumin (BSA) was used as the model protein to investigate the immobilization efficiency of biomolecules on the PANI film, comparative study using quartz crystal microbalance (QCM) showed that BSA immobilized on CS-modified PANI could be increased by at least 20% than that without CS-modified PANI in BSA solution with the concentration of 0.1-1mg/mL. The CS-modified PANI film would be significant for the immobilization and detection of biomolecules and especially promising in the application of immunosensor for ultrasensitive detection.
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Multi-nanomaterial electrochemical biosensor based on label-free graphene for detecting cancer biomarkers.
Biosens Bioelectron
PUBLISHED: 01-28-2014
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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. Sensitivity was also enhanced by modifying the gold nanoparticles (AuNPs) with horseradish peroxidase (HRP) and the detection antibody (Ab2), to form the conjugate Ab2-AuNPs-HRP. Electron transport between the electrode and analyte target was accelerated by the multi-nanomaterial, and the limit of detection (LOD) for carcinoembryonic antigen (CEA) reached 5 ng mL(-1). The multi-nanomaterial electrode GR/MBs-Ab1/CEA/Ab2-AuNPs-HRP can be used to detect biomolecules such as CEA. The EC biosensor is sensitive and specific, and has potential in the detection of disease markers.
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The advantage of circulating tumor cells over serum carcinoembryonic antigen for predicting treatment responses in rectal cancer.
Future Oncol
PUBLISHED: 10-11-2013
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The objective of this study was to investigate the clinical significance of circulating tumor cells (CTCs) on the evaluation and prediction of treatment responses in rectal cancer patients compared with serum carcinoembryonic antigen (CEA).
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Signal-to-noise ratio enhancement of silicon nanowires biosensor with rolling circle amplification.
Nano Lett.
PUBLISHED: 08-15-2013
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Herein, we describe a novel approach for rapid, label-free and specific DNA detection by applying rolling circle amplification (RCA) based on silicon nanowire field-effect transistor (SiNW-FET) for the first time. Highly responsive SiNWs were fabricated with a complementary metal oxide semiconductor (CMOS) compatible anisotropic self-stop etching technique which eliminated the need for hybrid method. The probe DNA was immobilized on the surface of SiNW, followed by sandwich hybridization with the perfectly matched target DNA and RCA primer that acted as a primer to hybridize the RCA template. The RCA reaction created a long single-stranded DNA (ssDNA) product and thus enhanced the electronic responses of SiNW significantly. The signal-to-noise ratio (SNR) as a figure-of-merit was analyzed to estimate the signal enhancement and possible detection limit. The nanosensor showed highly sensitive concentration-dependent conductance change in response to specific target DNA sequences. Because of the binding of an abundance of repeated sequences of RCA products, the SNR of >20 for 1 fM DNA detection was achieved, implying a detection floor of 50 aM. This RCA-based SiNW biosensor also discriminated perfectly matched target DNA from one-base mismatched DNA with high selectivity due to the substantially reduced nonspecific binding onto the SiNW surface through RCA. The combination of SiNW FET sensor with RCA will increase diagnostic capacity and the ability of laboratories to detect unexpected viruses, making it a potential tool for early diagnosis of gene-related diseases.
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Single layer linear array of microbeads for multiplexed analysis of DNA and proteins.
Biosens Bioelectron
PUBLISHED: 07-29-2013
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In this study, a microfluidic platform was developed to generate single layer, linear array of microbeads for multiplexed high-throughput analysis of biomolecules. The microfluidic device is comprised of eight microbead-trapping units, where microbeads were immobilized in a linear array format by the exertion of a negative pressure in the control channel connected to each sieving microstructure. Multiplexed assays were achieved by using a mixture of different spectrally-encoded microbeads functionalized with specific probes, followed by on-chip reaction and detection. The microfluidic-based microbeads array platform was employed for multiplexed analysis of DNA and proteins, as demonstrated by the simultaneous discrimination of four HPV genotypes and the parallel detection of six different proteins. Compared with the off-chip protocols, the on-chip analysis exhibited better reaction efficiency, higher sensitivity and wider linear detection range. Visual inspection and identification of functionalized microbeads were facilitated by the single layer arrangement of microbeads so that accurate data acquisition can be performed during the detection process.
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A colorimetric method for H1N1 DNA detection using rolling circle amplification.
Analyst
PUBLISHED: 05-08-2013
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A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay has been successfully developed as a method for the effective detection of H1N1 DNA. Specific oligonucleotide and reporter primer probes were designed together with a circular template, and the oligonucleotide probes were attached to the surfaces of magnetic beads (MBs) to form functional MB-DNA conjugates as capture probes for the target H1N1 DNA molecules. Together with the addition of DNA targets and reporter primer probes to the MB-DNA conjugates, sandwiched hybrids were formed. The initiation of RCA amplification using the circular template in the presence of phi29 polymerase allowed for the amplification of a large number of repeat sequences of the single-stranded (ss)-DNA product. This RCA product accumulated gold nanoparticles (AuNPs), resulting in a colorimetric change that could be viewed by the naked eye or detected using UV-vis spectroscopy. According to this method, H1N1 DNA could be detected at the 1 pmol L(-1) level. This platform exhibited design convenience, simplicity, and cost-effectiveness, and could be used to provide a new diagnostic assay for H1N1, and other infectious diseases.
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Integrated biochip for label-free and real-time detection of DNA amplification by contactless impedance measurements based on interdigitated electrodes.
Biosens Bioelectron
PUBLISHED: 01-05-2013
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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-?l 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 amplification and rolling circle amplification was successfully performed on the biochip and a detection limit of 1 nM was achieved. Amplification starting at an initial concentration of 10 nM could be discriminated from that starting at 1 nM started concentration as well as from the negative control. Since an insulation layer covers the electrodes, the electrodes are spared from erosion, hydrolysis and bubble formation on the surface, thus, ensuring a long lifetime and a high reusability of the sensor. In comparison to bench-top apparatus, our chip shows good efficiency, sensitivity, accuracy, and versatility. Our system requires only simple equipments and simple skills, and can easily be miniaturized into a micro-scale system. The system will then be suitable for a handheld portable device, which can be applied in remote areas. It covers merits such as low cost, low-power consumption, rapid response, real-time monitoring, label-free detection, and high-throughput detection.
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Photopatterning of self-assembled poly (ethylene) glycol monolayer for neuronal network fabrication.
J. Neurosci. Methods
PUBLISHED: 01-03-2013
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The ability to culture individual neurons and direct their connections on functional interfaces provides a platform for investigating information processing in neuronal networks. Numerous methods have been used to design ordered neuronal networks on microelectrode arrays (MEAs) for neuronal electrical activities recording. However, so far, no method has been implemented, which simultaneously provides high-resolution neuronal patterns and low-impedance microelectrode. To achieve this goal, we employed a chemical vapor-deposited, non-fouling poly (ethylene) glycol (PEG) self-assembled monolayer to provide a cell repellant background on the MEAs. Photolithography, together with plasma etching of the PEG monolayer, was used to fabricate different patterns on MEAs. No electrode performance degradation was observed after the whole process. Dissociated cortical neurons were cultured on the modified MEAs, and the patterns were maintained for more than 3 weeks. Spontaneous and evoked neuronal activities were recorded. All of the results demonstrate this surface engineering strategy allows successful patterning of neurons on MEAs, and is useful for future studies of information processing in defined neuronal networks on a chip.
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High-performance size-based microdevice for the detection of circulating tumor cells from peripheral blood in rectal cancer patients.
PLoS ONE
PUBLISHED: 01-01-2013
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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. In this study we fabricated a simple and high-performance microfluidic device, which exploited numerous filtered microchannels in it to enrich the large-sized target tumor cells from whole blood. A very high CTC capture efficiency (average recovery rate: 94%) was obtained in this device at the optimum flow rate of 0.5 mL/h and channel height of 5 µm. Additionally, we used this device for detecting CTCs in 60 patients with rectal cancer. The CTC counts of rectal cancer patients were significantly higher than those in healthy subjects. Furthermore, the CTC counts detected by this device were significantly higher than those by EpCAM bead-based method for rectal cancer patients with various stage. Especially, for localized rectal cancer patients, the positive rates of samples with more than 3 CTCs per 5 mL blood by use of microdevice vs. EpCAM-based ones were 100% vs. 47%, respectively. Thus, this device provides a new and effective tool for accurate identification and measurement of CTCs in patients with rectal cancer, and has broad potential in clinical practice.
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DNAzyme-based fluorescent microarray for highly selective and sensitive detection of lead(II).
Analyst
PUBLISHED: 10-28-2011
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A facile microarray-based fluorescent sensor for the detection of lead (II) was developed based on the catalytic cleavages of the substrates by a DNAzyme upon its binding to Pb(2+). The release of the fluorophore labelled substrates resulted in the decrease of fluorescence intensity. The sensor had a quantifiable detection range from 1 nM to 1 ?M and a selectivity of >20 fold for Pb(2+) over other metal ions.
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Simultaneous detection of high-sensitivity cardiac troponin I and myoglobin by modified sandwich lateral flow immunoassay: proof of principle.
Clin. Chem.
PUBLISHED: 10-13-2011
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Although numerous lateral flow immunoassays (LFIAs) have been developed and widely used, inadequate analytical sensitivity and the lack of multiple protein detection applications have limited their clinical utility. We developed a new LFIA device for the simultaneous detection of high-sensitivity cardiac troponin I (hs-cTnI) and myoglobin (Myo).
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Sensitive label-free oligonucleotide-based microfluidic detection of mercury (II) ion by using exonuclease I.
Biosens Bioelectron
PUBLISHED: 07-04-2011
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Mercury is a highly toxic metal that can cause significant harm to humans and aquatic ecosystems. This paper describes a novel approach for mercury (Hg(2+)) ion detection by using label-free oligonucleotide probes and Escherichia coli exonuclease I (Exo I) in a microfluidic electrophoretic separated platform. Two single-stranded DNAs (ssDNA) TT-21 and TT-44 with 7 Thymine-Thymine mispairs are employed to capture mercury ions. Due to the coordination structure of T-Hg(2+)-T, these ssDNAs are folded into hairpin-like double-stranded DNAs (dsDNA) which are more difficult to be digested by Exo I, as confirmed by polyacrylamide gel electrophoresis (PAGE) analysis. A series of microfluidic capillary electrophoretic separation studies are carried out to investigate the effect of Exo I and mercury ion concentrations on the detected fluorescence intensity. This method has demonstrated a high sensitivity of mercury ion detection with the limit of detection around 15 nM or 3 ppb. An excellent selectivity of the probe for mercury ions over five interference ions Fe(3+), Cd(2+), Pb(2+), Cu(2+) and Ca(2+) is also revealed. This method could potentially be used for mercury ion detection with high sensitivity and reliability.
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A simplified poly(dimethylsiloxane) capillary electrophoresis microchip integrated with a low-noise contactless conductivity detector.
Electrophoresis
PUBLISHED: 02-22-2011
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A contactless conductivity detector integrated into a poly(dimethylsiloxane) microchip for electrophoresis is presented. It adopted the simplest configuration of electrodes commonly used in this detection mode for capillary electrophoresis microchips. Although the chip is based on a simple and effective design, it is able to obtain low detection levels due to the low noise of the detection circuit. A circuit based on a lock-in amplifier was designed on printed circuit boards to read out the signal. The property of the detection cell was studied by applying excitation signals of different frequencies and different amplitudes. It was found that the best detection limit could be achieved with a frequency of 50?kHz and amplitude of 20?V. The performance of the detector was demonstrated by successfully separating and detecting several inorganic ions and also a mixture of heavy metal ions. An average detection limit of 0.4??M was obtained for inorganic cations. This value is significantly improved compared to similar microchip-based detectors. The presented detector could be promising for mass production due to its properties, such as simple construction, high degree of integration, high performance and low cost.
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Label-free optical detection of single-base mismatches by the combination of nuclease and gold nanoparticles.
Biosens Bioelectron
PUBLISHED: 01-21-2011
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We report here an optical approach that enables highly selective and colorimetric single-base mismatch detection without the need of target modification, precise temperature control or stringent washes. The method is based on the finding that nucleoside monophosphates (dNMPs), which are digested elements of DNA, can better stabilize unmodified gold nanoparticles (AuNPs) than single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with the same base-composition and concentration. The method combines the exceptional mismatch discrimination capability of the structure-selective nucleases with the attractive optical property of AuNPs. Taking S1 nuclease as one example, the perfectly matched 16-base synthetic DNA target was distinctively differentiated from those with single-base mutation located at any position of the 16-base synthetic target. Single-base mutations present in targets with varied length up to 80-base, located either in the middle or near to the end of the targets, were all effectively detected. In order to prove that the method can be potentially used for real clinic samples, the single-base mismatch detections with two HBV genomic DNA samples were conducted. To further prove the generality of this method and potentially overcome the limitation on the detectable lengths of the targets of the S1 nuclease-based method, we also demonstrated the use of a duplex-specific nuclease (DSN) for color reversed single-base mismatch detection. The main limitation of the demonstrated methods is that it is limited to detect mutations in purified ssDNA targets. However, the method coupled with various convenient ssDNA generation and purification techniques, has the potential to be used for the future development of detector-free testing kits in single nucleotide polymorphism screenings for disease diagnostics and treatments.
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[Membrane transfer-based colorimetric DNA detection using enzyme modified gold nanoparticles].
Sheng Wu Gong Cheng Xue Bao
PUBLISHED: 11-25-2010
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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. In the presence of target DNA, DNA hybridization led to the attachment of AuNPs on the chip surface where specific DNA sequences were located in a "sandwich" format. Alkaline phosphatase was then introduced to the surface via biotine-streptavidin interaction. By using BCIP/NBT alkaline phosphatase color development kit, a colorimetric DNA detection was achieved through membrane transfer. The signal on the membrane was then detected by the naked eye or an ordinary optical scanner. The method provided a detection of limit of 1 pmol/L for synthesized target DNA and 0.23 pmol/L for PCR products of Mycobacterium tuberculosis 16S rDNA when the ratio of probes used was 9:1:1 (T10:T40:P2). The method described here has many desirable advantages including high sensitivity, simple operation, and no need of sophisticated equipment. The method can be potentially used for reliable biosensings.
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A compact disk-like centrifugal microfluidic system for high-throughput nanoliter-scale protein crystallization screening.
Anal. Chem.
PUBLISHED: 05-13-2010
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A centrifuge-based microfluidic system has been developed that enables automated high-throughput and low-volume protein crystallizations. In this system, protein solution was automatically and accurately metered and dispensed into nanoliter-sized multiple reaction chambers, and it was mixed with various types of precipitants using a combination of capillary effect and centrifugal force. It has the advantages of simple fabrication, easy operation, and extremely low waste. To demonstrate the feasibility of this system, we constructed a chip containing 24 units and used it to perform lysozyme and cyan fluorescent protein (CyPet) crystallization trials. The results demonstrate that high-quality crystals can be grown and harvested from such a nanoliter-volume microfluidic system. Compared to other microfluidic technologies for protein crystallization, this microfluidic system allows zero waste, simple structure and convenient operation, which suggests that our microfluidic disk can be applied not only to protein crystallization, but also to the miniaturization of various biochemical reactions requiring precise nanoscale control.
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Novel colorimetric enzyme immunoassay for the detection of carcinoembryonic antigen.
Talanta
PUBLISHED: 03-08-2010
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We developed a highly sensitive colorimetric enzyme immunoassay for the detection of carcinoembryonic antigen (CEA). This method employed gold nanoparticles (AuNPs) as carriers of anti-CEA antibody labeled with biotin, which served as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP) binding. Using this strategy, about 12 HRP molecules were coated onto each AuNP. Through "sandwich-type" immunoreaction, the AuNP-anti-CEA-HRP complex was brought into the proximity of magnetic microparticle. As a result, HRP molecules confined at the surface of the "sandwich" immunocomplexes catalyzed the enzyme substrate and generated an optical signal. The spectrophotometric measurement confirmed effective signal amplification. The signals were linearly dependent on CEA concentrations from 0.05 to 50ngmL(-1) in a logarithmic plot, with a detection limit of 48pgmL(-1). Intra- and inter-assay coefficients of variation were <8.5%. The CEA concentrations of serum specimens assayed by the developed immunoassay showed consistent results in comparison with those obtained by a conventional enzyme-linked immunosorbent assay. The developed method thus proved its potential use in clinical immunoassay of CEA.
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QDs-DNA nanosensor for the detection of hepatitis B virus DNA and the single-base mutants.
Biosens Bioelectron
PUBLISHED: 01-08-2010
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We report here a quantum dots-DNA (QDs-DNA) nanosensor based on fluorescence resonance energy transfer (FRET) for the detection of the target DNA and single mismatch in hepatitis B virus (HBV) gene. The proposed one-pot DNA detection method is simple, rapid and efficient due to the elimination of the washing and separation steps. In this study, the water-soluble CdSe/ZnS QDs were prepared by replacing the trioctylphosphine oxide (TOPO) on the surface of QDs with 3-mercaptopropionic acid (MPA). Subsequently, oligonucleotides were attached to the QDs surface to form functional QDs-DNA conjugates. Along with the addition of DNA targets and Cy5-modified signal DNAs into the QDs-DNA conjugates, sandwiched hybrids were formed. The resulting assembly brings the Cy5 fluorophore, the acceptor, and the QDs, the donor, into proximity, leading to fluorescence emission from the acceptor by means of FRET on illumination of the donor. In order to efficiently detect single-base mutants in HBV gene, oligonucleotide ligation assay was employed. If there existed a single-base mismatch, which could be recognized by the ligase, the detection probe was not ligated and no Cy5 emission was produced due to the lack of FRET. The feasibility of the proposed method was also demonstrated in the detection of synthetic 30-mer oliginucleotide targets derived from the HBV with a sensitivity of 4.0nM by using a multilabel counter. The method enables a simple and efficient detection that could be potentially used for high throughput and multiplex detections of target DNA and the mutants.
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Highly sensitive protein detection using enzyme-labeled gold nanoparticle probes.
Analyst
PUBLISHED: 12-10-2009
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A highly sensitive protein detection method based on a novel enzyme-labeled gold nanoparticle (AuNP) probe has been developed. In this method, we firstly prepared the enzyme-labeled AuNP probe by coating AuNP with antibody, single-stranded DNA (ssDNA), and horseradish peroxidase (HRP). Magnetic microparticle (MMP) functionalized with another antibody was used as capture probe. Then, target protein was sandwiched by the enzyme-labeled AuNP probe and the capture probe through immunoreaction. The target immunoreaction event could be sensitively transduced via the enzymatically amplified optical signal. By using this strategy, carcinoembryonic antigen (CEA), as a model protein, was detected with high sensitivity and good specificity. The detection limit for this approach was 12 ng L(-1), which was approximately 130-fold more sensitive than the conventional enzyme-linked immunosorbent assay (ELISA). The practical application of the proposed immunoassay was carried out for determination of CEA in serum samples. The demonstrated capability of the proposed method shows potentially applications for early diagnoses of diseases.
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[Patterning different cells based on microfluidics and self-assembled monolayers].
Sheng Wu Gong Cheng Xue Bao
PUBLISHED: 11-27-2009
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The ability to pattern multiple cells through precise surface engineering of cell culture substrates has promoted the development of cellular bioassays, such as differentiation, interaction and molecular signaling pathways. There are several well developed ways to pattern cells. This report describes a method for patterning multiple types of cells based on microfluidics and self-assembled monolayers. We developed two types of micro-dam structures by soft-lithography to locate cells precisely and modified the substrate by a kind of self-assembled monolayer with property of electrochemical desorption to confine cells in specific areas. Finally we could pattern an array of two different types of cells closely and precisely. Cells were confined in specific areas but still shared the same microenvironment, so they could interact through soluble molecules. The substrate was transparent and open, so we could easily apply several instruments for research. With these merits, this cell chip is appropriate for investigating the interaction between different types of cells.
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Integrated microfluidic chip for endothelial cells culture and analysis exposed to a pulsatile and oscillatory shear stress.
Lab Chip
PUBLISHED: 08-18-2009
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For a comprehensive understanding of cells or tissues, it is important to enable multiple studies under the controllable microenvironment of a chip. In this report, we present an integrated microfluidic cell culture platform in which endothelial cells (ECs) are under static conditions or exposed to a pulsatile and oscillatory shear stress. Through the integration of a microgap, self-contained flow loop, pneumatic pumps, and valves, the novel microfluidic chip achieved multiple functions: pulsatile and oscillatory fluid circulation, cell trapping, cell culture, the formation of ECs barrier, and adding shear stress on cells. After being introduced into the chip by gravity, the ECs arranged along the microgap with the help of hydrodynamic forces and grew in the microchannel for more than 7 days. The cells proliferated and migrated to form a barrier at the microgap to mimic the vessel wall, which separated the microenvironment into two compartments, microchannel and microchamber. An optimized pneumatic micropump was embedded to actuate flow circulation in a self-contained loop that induced a pulsatile and oscillatory shear stress at physiological levels on the ECs in the microchannel. All the analyses were performed under either static or dynamic conditions. The performance of the barrier was evaluated by the diffusion and distribution behaviors of fluorescently labeled albumin. The permeability of the barrier was comparable to that in traditional in vitro assays. The concentration gradients of the tracer formed in the microchamber can potentially be used to study cell polarization, migration and communications in the future. Additionally, the morphology and cytoskeleton of the ECs response to the pulsatile and oscillatory shear stress were analyzed. The microfluidic chip provided a multifunctional platform to enable comprehensive studies of blood vessels at the cell or tissue level.
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[Microfluidic cell culture array chip for drug screening assays].
Sheng Wu Gong Cheng Xue Bao
PUBLISHED: 08-13-2009
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We developed a novel microfluidic cell chip, which enabled drug delivery, fluid control and cell co-culture. The device consisted of an array of 6x6 cell culture chambers, a drug gradient generator and fluidic control valves. Micro-dam structures of the chambers were able to trap cells while loading and drug gradient network generated drug gradient of 6 different concentrations. Also we applied hydraulic valves to control the microfluid and simulate the microenvironment of cells. We had investigated the viability of co-culturing cells in the chip and the ability for drug screening. This microfluidic cell chip has the potential in cell-based research of high throughput drug screening.
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Clinical evaluation of a colorimetric oligonucleotide chip for genotyping hepatitis C virus.
Clin. Biochem.
PUBLISHED: 06-04-2009
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Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. It is associated with the development of end-stage liver disease and hepatocellular carcinoma. Studies have shown that determination of hepatitis C virus (HCV) genotypes is clinically important for prediction of the clinical course and the outcome of antiviral therapy. The aim of this study was to evaluate a colorimetric oligonucleotide chip, which can be used for the rapid and economical detection of the genotypes/subtypes of hepatitis C virus.
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Application of poly(dimethylsiloxane)/glass microchip for fast electrophoretic separation of serum small, dense low-density lipoprotein.
J Chromatogr A
PUBLISHED: 03-21-2009
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Due to the mounting evidence of altered low-density lipoprotein (LDL) size in several disease states, there has been an increasing interest in developing new analytical methods for small, dense low-density lipoprotein (sdLDL) for diagnosis. The present report demonstrates that sdLDL analysis can be performed in a poly(dimethylsiloxane) (PDMS/glass) microchannel. n-Dodecyl beta-D-maltoside (DDM) was utilized to alter channel surface to make it become hydrophilic and nonionic, thus reducing the interaction between the protein and the surface. Moreover, hydroxypropylcellulose (HPC) was added into the running buffer to suppress the adsorption of analytes and also to serve as a sieving matrix. Under optimal conditions, two baseline separations of lipoproteins including high-density lipoprotein (HDL), sdLDL, and lLDL were achieved with different selectivity. LDL particles shown on the electropherogram were also identified by several procedures. This method affords high separation speed and high reproducibility. The intraassay and interassay RSDs of lipoprotein migration times were in the range of 2.01-2.45%. The variation of serum sdLDL of a patient between prior treatment and post-treatment was assessed by this method. This system has the potential for rapid and sensitive detection of different LDL forms, and thus will be applicable to clinical diagnosis.
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Detection of known base substitution mutations in human mitochondrial DNA of MERRF and MELAS by biochip technology.
Biosens Bioelectron
PUBLISHED: 01-22-2009
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We developed a DNA biochip specialized for detection of known base substitution mutations in mitochondrial DNA causing mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) and myoclonic epilepsy associated with ragged-red fibers (MERRF). A set of probes sharing a given allele-specific sequence with a single base substitution near the middle of the sequence was covalently immobilized. Cy5-labeled DNA targets were amplified from sample DNAs containing 31 potential MELAS and/or MERRF mutations by a multiplex PCR method. Detection parameters for the DNA biochip-based assay were accordingly optimized. Seven clinically confirmed patients with MELAS, 5 patients with MERRF, 1 suspected MERRF case and 25 healthy controls were tested using the DNA biochip. For discriminating of homoplasmic and heteroplasmic point mutations in mtDNA, a diagnostic factor based on the ratio between the hybridization signals from the reference and test targets with each probe was used. The results showed that all the cases with MELAS had a causal heteroplasmic A3243G tRNA(Leu(UUR)) mutation. In the MERRF patients, four cases were found to be a homoplasmic A8344G tRNA(Lys) mutation and one case was a heteroplasmic T8356C tRNA(Lys) mutation. None of the healthy controls carried the potential mutations. The results of the DNA biochip were completely consistent with those by DNA sequencing. Thus, the DNA biochip would potentially become a valuable tool in clinical specific screening of the mtDNA point mutations associated with MELAS and/or MERRF syndrome.
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Evaluation of a modified lateral flow immunoassay for detection of high-sensitivity cardiac troponin I and myoglobin.
Biosens Bioelectron
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We prospectively evaluated the use of lateral flow immunoassay (LFIA) test modified with nanoparticles for combined detection of high-sensitivity cardiac troponin I (hs-cTnI) and myoglobin with the aim of excluding acute myocardial infarction (AMI). Specimens from 173 patients with symptoms suggestive of AMI were collected to measure hs-cTnI and myoglobin using an electrochemiluminescence immunoassay (ECLI) and the LFIA test modified with nanoparticles, and a comparison was performed between the modified method and a commercial LFIA test for detection of the two proteins. The accuracy of the modified LFIA test was also evaluated. Consistent agreement was observed in the quantitative comparison of 173 clinical samples using the modified LFIA and ECLI, and the modified method was more sensitive than the commercial LFIA test. The accuracy of the modified LFIA was <12% for both hs-cTnI and myoglobin. Thus, the new approach has great potential to improve LFIAs test, demonstrating its usefulness for simple screening applications and for sensitivity and quantitative immunoassays for diagnosis ofAMI.
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Highly sensitive and selective chip-based fluorescent sensor for mercuric ion: development and comparison of turn-on and turn-off systems.
Anal. Chem.
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Miniaturization is currently an important trend in environmental and food monitoring because it holds great promise for on-site monitoring and detection. We report here two ready-to-use chip-based fluorescent sensors, compatible with microarray technology for reagentless, one-step, fast, highly sensitive and selective detection of the mercuric ion (Hg(2+)) in the turn-on and turn-off operation modes. Both operation modes are based on the highly selective T-Hg(2+)-T coordination between two neighboring polythymine (T) strands at a high probe density and its induced displacement of the complementary polyadenine strand labeled with either fluorophore or quencher, which enables the turn-off and turn-on detection of Hg(2+), respectively. The turn-off sensor is slightly more sensitive than the turn-on sensor, and their detection limits are 3.6 and 8.6 nM, respectively, which are both lower than the U.S. Environmental Protection Agency limit of [Hg(2+)] for drinkable water (10 nM, 2 ppb). Compared to the turn-off sensor with the dynamic Hg(2+) detection range from 3.6 nM to 10 ?M (R(2) = 0.99), the turn-on sensor has a broader dynamic Hg(2+) detection range, from 8.6 nM to 100 ?M (R(2) = 0.996). Both sensors exhibited superior selectivity over other reported sensors using thymine-rich probes for Hg(2+) detection over other common metal ions. In addition, the practical application of the chip-based sensors was demonstrated by detecting spiked Hg(2+) in drinking water and fresh milk. The sensor has great potential for on-site practical applications due to its operational convenience, simplicity, speed, and portability.
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Real-time monitoring of strand-displacement DNA amplification by a contactless electrochemical microsystem using interdigitated electrodes.
Lab Chip
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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. With the superior performance of the biosensor, the device can detect the amount of pure DNA at concentrations less than 0.1 pg ?l(-1). The EC cell, integrated with a heater and a temperature sensor, has successfully implemented nicking-based strand-displacement amplification at an initial concentration of 2.5 ?M and the yields are monitored directly (dismissing the use of probes or labels) on-line. This contactless detector carries important advantages: high anti-interference capability, long detector life, high reusability and low cost. In addition, the small size, low power consumption and portability of the detection cell give the system the potential to be highly integrated for use in field service and point of care applications.
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A "place n play" modular pump for portable microfluidic applications.
Biomicrofluidics
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This paper presents an easy-to-use, power-free, and modular pump for portable microfluidic applications. The pump module is a degassed particle desorption polydimethylsiloxane (PDMS) slab with an integrated mesh-shaped chamber, which can be attached on the outlet port of microfluidic device to absorb the air in the microfluidic system and then to create a negative pressure for driving fluid. Different from the existing monolithic degassed PDMS pumps that are generally restricted to limited pumping capacity and are only compatible with PDMS-based microfluidic devices, this pump can offer various possible configures of pumping power by varying the geometries of the pump or by combining different pump modules and can also be employed in any material microfluidic devices. The key advantage of this pump is that its operation only requires the user to place the degassed PDMS slab on the outlet ports of microfluidic devices. To help design pumps with a suitable pumping performance, the effect of pump module geometry on its pumping capacity is also investigated. The results indicate that the performance of the degassed PDMS pump is strongly dependent on the surface area of the pump chamber, the exposure area and the volume of the PDMS pump slab. In addition, the initial volume of air in the closed microfluidic system and the cross-linking degree of PDMS also affect the performance of the degassed PDMS pump. Finally, we demonstrated the utility of this modular pumping method by applying it to a glass-based microfluidic device and a PDMS-based protein crystallization microfluidic device.
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Determination of heavy metal ions by microchip capillary electrophoresis coupled with contactless conductivity detection.
Electrophoresis
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An integrated detection circuitry based on a lock-in amplifier was designed for contactless conductivity determination of heavy metals. Combined with a simple-structure electrophoresis microchip, the detection system is successfully utilized for the separation and determination of various heavy metals. The influences of the running buffer and detection conditions on the response of the detector have been investigated. Six millimole 2-morpholinoethanesulfonic acid + histidine were selected as buffer for its stable baseline and high sensitivity. The best signals were recorded with a frequency of 38 kHz and 20 V(pp). The results showed that Mn(2+), Cd(2+), Co(2+), and Cu(2+) can be successfully separated and detected within 100 s by our system. The detection limits for five heavy metals (Mn(2+), Pb(2+), Cd(2+), Co(2+), and Cu(2+)) were determined to range from about 0.7 to 5.4 ?M. This microchip system performs a crucial step toward the realization of a simple, inexpensive, and portable analytical device for metal analysis.
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Cytotoxicity of cadmium-containing quantum dots based on a study using a microfluidic chip.
Nanotechnology
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There is a lack of reliable nanotoxicity assays available for monitoring and quantifying multiple cellular events in cultured cells. In this study, we used a microfluidic chip to systematically investigate the cytotoxicity of three kinds of well-characterized cadmium-containing quantum dots (QDs) with the same core but different shell structures, including CdTe core QDs, CdTe/CdS core–shell QDs, and CdTe/CdS/ZnS core-shell-shell QDs, in HEK293 cells. Using the microfluidic chip combined with fluorescence microscopy, multiple QD-induced cellular events including cell morphology, viability, proliferation, and QD uptake were simultaneously analysed. The three kinds of QDs showed significantly different cytotoxicities. The CdTe QDs, which are highly toxic to HEK293 cells, resulted in remarkable cellular and nuclear morphological changes, a dose-dependent decrease in cell viability, and strong inhibition of cell proliferation; the CdTe/CdS QDs were moderately toxic but did not significantly affect the proliferation of HEK293 cells; while the CdTe/CdS/ZnS QDs had no detectable influence on cytotoxicity with respect to cell morphology, viability, and proliferation. Our data indicated that QD cytotoxicity was closely related to their surface structures and specific physicochemical properties. This study also demonstrated that the microfluidic chip could serve as a powerful tool to systematically evaluate the cytotoxicity of nanoparticles in multiple cellular events.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.