Preparation of uniform magnetic recoverable catalyst microspheres with hierarchically mesoporous structure by using porous polymer microsphere template.

Merging nanoparticles with different functions into a single microsphere can exhibit profound impact on various applications. However, retaining the unique properties of each component after integration has proven to be a significant challenge. Our previous research demonstrated a facile method to incorporate magnetic nanoparticles into porous silica microspheres. Here, we report the fabrication of porous silica microspheres embedded with magnetic and gold nanoparticles as magnetic recoverable catalysts. The as-prepared multifunctional composite microspheres exhibit excellent magnetic and catalytic properties and a well-defined structure such as uniform size, high surface area, and large pore volume. As a result, the very little composite microspheres show high performance in catalytic reduction of 4-nitrophenol, special convenient magnetic separability, long life, and good reusability. The unique nanostructure makes the microspheres a novel stable and highly efficient catalyst system for various catalytic industry processes.

Mechanism of mercury detection based on interaction of single-strand DNA and hybridized DNA with gold nanoparticles.

Mechanisms of interaction of single-strand DNA and hybridized DNA on gold nanoparticles in the presence of Hg(2+) was studied in this work. Recently the detection of Hg(2+) using unmodified gold nanoparticles (AuNPs) combined with DNA is becoming a promising technique with the advantages of simplicity, cost-effectiveness and high sensitivity. However, few studies focused on the interaction of ssDNA and hybridized DNA on AuNPs to date. In the present work, we compared the interactions of different DNA probes on AuNPs using both absorption and fluorescence detection. It was found that there were only small partial dsDNA dissociated from the surface of AuNPs after hybridization in the presence of Hg(2+). Moreover, we found that the aggregated AuNPs/DNA system tended to be dispersed again with increasing Hg(2+) concentration up to 250µM. Based on these results, the mechanisms of mercury detection based on interaction between DNA-conjugated gold nanoparticles were investigated. Positively charged dsDNA could bind to the surface of AuNPs and dominate the electrostatic interactions and consequently aggregation of the AuNPs/DNA system.

Nucleic acids detection using cationic fluorescent polymer based on one-dimensional microfluidic beads array.

An assay for rapid and direct detection of DNA/mRNA using cationic fluorescent polymer based on one-dimensional microfluidic beads array (1-D chip) has been developed. The cationic water-soluble polythiophene derivatives can easily transduce hybridization events into measurable optical signal due to the conformational changes of the conjugated backbone, when mixed with single-stranded or double-stranded oligonucleotides. In this paper, the polymer was introduced into 1-D chip for fluorescence detection of nucleic acids, and demonstrated its capability on rapid detection of p53 complementary DNA (cDNA) with different concentration. Using this system, we have evaluated the mRNA expression changes of three tumor-associated genes (p53, c-myc and cyclin-d1) in human nasopharyngeal carcinoma CNE2 cell lines before and after 5-flouorouracil (5-FU) stimuli. These results were validated by the conventional reverse transcriptase-PCR. The most important advantage of this assay is not needed target or report labeling prior to hybridization, which makes the experiment process easy to handle and low-cost for multi-target measurement.

Telomerase catalyzed fluorescent probes for sensitive protein profiling based on one-dimensional microfluidic beads array.

A nucleic acid-based signal amplified method for multiple proteins detection based on one-dimensional beads array using telomerase catalyzed fluorescent probes has been developed in this paper. The biotin labeled fluorescent probes were synthesized by telomerase in homogeneous solution. Approximately 360-480 fluorescein molecules were inserted in each probe. The limit of detection for p53 protein is 1.1 pM (S/N=3) and a 3 orders of linear dynamic range is obtained. The sensitivity is nearly two orders higher than the two-site "sandwich" immunoassay using the same platform. Using this method, cellular p53 protein contents of as few as 85 CNE2 cells per mul sample can be determined specifically. The expression changes of p53, c-myc and beta-actin in CNE2 cells were further examined as a function of anti-cancer drug treatment, and the results are consistent with our previous reports. Compared with immuno-polymerase chain reaction and immuno-rolling circle amplification, this method is simple, fast, cheap and suitable for multi-protein analysis. The multiplexed proteins profiling of cellular samples should facilitate the new opportunities to the fundamental research of tumor development and progression, especially to the low abundant tumor-associated proteins analysis.

On-chip oligonucleotide ligation assay using one-dimensional microfluidic beads array for the detection of low-abundant DNA point mutations.

The detection of low-abundant DNA point mutations is very important for the early prediction of cancer, diagnostics of disease and clinical prognosis. In this paper, an on-chip oligonucleotide ligation approach that arrayed a series of functionalized beads in a single microfluidic channel was described for detection of low-abundant point mutations in p53 gene. This gene carried the point mutation with high diagnostic value for assessment of tumor progression and resectional borders. This work extended our prior efforts using one-dimensional (1-D) microfluidic beads array for protein and nucleic acid molecular profiling, and displayed high discrimination sensitivity to mutations detection due to the enhanced mass transport capability caused by microfluidic addressing format of beads array. As a demonstration, it was found that the on-chip beads ligation held high mutation discrimination sensitivity in 1 pM quantities at a SNR (signal-to-noise ratio) >2 using synthesized DNA oligonucleotides in accordance with target fragment. The RT-PCR products of tumor cell line A549, CNE2 and SKBr-3 were further examined to distinguish the point mutation at codon 175 of p53 gene. This approach was capable of detecting a point mutation in a p53 oncogene at a level of 1 mutant in 1000 wild-type sequences using PCR products without the need of LDR amplification. Additionally, this on-chip beads ligation approach also displayed other microfluidic-based advantages of simple handling (one sample injection per test), little reagent quantities, and low potential of contaminations.

Detection of single-base mutations using 1-D microfluidic beads array.

The application of a 1-D microfluidic beads array that is composed of individually addressable functionalized SiO2 beads has been demonstrated for detection of single-base mutations based on "sandwich" hybridization assay without additional sample labeling and PCR amplification. We concentrated on detection of mutations in the human p53 tumor suppressor gene with more than 50% mutation frequency in the known human cancers. Using a microinjection system, functionalized beads could be selectively and linearly arrayed in a single microfluidic channel comprising many periodic chambers. This 1-D microfluidic beads array was sufficiently sensitive to identify single-nucleotide mutations in 40 pM quantities of DNA targets and could discriminate the mutated alleles in an excess of nonmutated alleles at a level of one mutant in 100 wild-type sequences. The surface of beads was regenerated and rehybridized up to six times without obvious loss of signal. The entire reaction process was done at room temperature within minutes, and only 2-10 microL sample solution was needed to complete the whole detection process. The p53 genotypes of A549, CNE2, and SKBr-3 cell lines were also correctly evaluated by using mRNA extracts as target without need for sample labeling and amplification. Thus, this platform enabled rapid and exact discrimination of gene mutations with the advantages of reusability, simple handling of liquid, low cost, and little reagent consumption.

A novel sandwich assay with molecular beacon as report probe for nucleic acids detection on one-dimensional microfluidic beads array.

A novel sandwich assay with molecular beacons as report probes has been developed and integrated into one-dimensional microfluidic beads array (1-D chip) to pursue a label-free and elution-free detection of DNA/mRNA targets. In contrast with the immobilized molecular beacons, this sandwich assay can offer lower fluorescence background and correspondingly higher sensitivity. Furthermore, this sandwich assay on 1-D chip operating in conjunction with molecular beacon technique allows multiple targets detection without the need of laborious and time-consuming elution, which makes the experiment process simple, easy to handle, and reproducible results. In the experiment, the synthesized DNA targets with different concentrations were detected with a detection limit of approximately 0.05 nM. Moreover, the mRNA expression changes in A549 cells before and after anticancer drug 5-flouorouracil treatments were detected and the results were validated by the conventional RT-PCR method.

One-dimensional microfluidic beads array for multiple mRNAs expression detection.

A one-dimensional microfluidic beads array for in vitro rapid measurement of multiple mRNAs expression is presented in this paper. Gene specific capture DNA-functional beads were deposited along a microchannel to form an addressable beads array. We demonstrated that the one-dimensional beads array could perform simultaneous multiple nucleic acid targets detection and a DNA detection limit of 0.02 nM was obtained. Using this array, transcripts expression of three tumor-associated genes, including p53, H-ras, and NME1, both in CNE2 nasopharyngeal carcinoma cell lines and in normal human nasopharyngeal epithelial cells were evaluated. The responses of these three genes expression in CNE2 cells to 5-flouorouracil (5-Fu) stimuli were also assessed. Validation of these results was performed using reverse transcriptase-PCR. The presented methodology combines high throughput of microarrays and low sample consumption, convenient liquid handling of microfluidics. It enables rapid and facile determination of multi-gene expression and holds great potential in early cancer diagnostics and molecular biology.

Quantitative intracellular molecular profiling using a one-dimensional flow system.

We report on the development of one-dimensional microfluidic bead arrays for rapid and quantitative molecular profiling of human cancer cells. This new bioanalytical platform integrates the rapid binding kinetics of suspension bead carriers, the multiplexing and encoding capabilities of gene/protein chips, and the liquid handling advantages of microfluidic devices. Using antibody-conjugated beads in a two-site "sandwich" format, we demonstrate that the proteomic contents of as few as 56 human lung epithelial cancer cells can be determined with high sensitivity and specificity. The results indicate that each cell contains approximately 6 x 10(5) copies of the tumor suppressor protein P53. We have further examined the expression changes of P53, c-Myc, and beta-Actin as a function of anticancer drug treatment and have validated these changes by using Western blotting. This ability to quantitatively analyze normal and diseased cells raises new possibilities in studying cancer heterogeneity and circulating tumor cells.

Separation and determination synchronously by multichannel mode-filtered light capillary electrochromatography.

Mode-filtered light capillary electrophoresis (CE) was developed for capillary electrochromatography (CEC) by applying annular columns of different size (250, 320, and 530 micro m) and a bovine serum albumin (BSA)-modified chiral stationary phase. BSA was encapsulated on the pretreated surface of an optic fiber and the inner wall of a capillary using sol-gel technique with a resultant larger active stationary phase area. The coating was examined by atomic force microscopy (AFM) and Fourier transform infrared (FT-IR). The separation of DL-tryptophan (10 mM) was investigated under different voltage/current conditions.

Mixed C18 and C1 modification on an optical fiber for chromatographic sensing.

An optical fiber-chromatographic sensor, aiming at simultaneous and selective response to multiple components following a chromatographic separation, is described. We report an improved approach for immobilization of octadecyl (C(18)) and methyl (C(1)) moieties as stationary phase on an optical fiber suitable as a sensing phase for organic solutes. By this approach, the stability and lifetime of the sensing layer as well as the detectability and retention behavior of the chromatographic sensor could be improved. Infrared spectroscopy was employed to confirm the presence of C(18) and C(1) moieties on the modified surface of the optical fiber. The chromatographic sensor was applied, with good sensitivity and chemical selectivity, to the simultaneous separation and detection of bromobenzene and toluene, using water as the mobile phase.