Efficient detection of Escherichia coli O157:H7 using a reusable microfluidic chip embedded with antimicrobial peptide-labeled beads.

The ability of antimicrobial peptides (AMPs) for effective binding to multiple target microbes has drawn lots of attention as an alternative to antibodies for detecting whole bacteria. We investigated pathogenic Escherichia coli (E. coli) detection by applying a microfluidic based biosensing device embedded with AMP-labeled beads. According to a new channel design, our device is reusable by the repeated operation of detection and regeneration modes, and the binding rate is more enhanced due to even distribution of the bacterial suspension inside the chamber by implementing influx side channels. We observed higher binding affinity of pathogenic E. coli O157:H7 for AMP-labeled beads than nonpathogenic E. coli DH5α, and the fluorescence intensity of pathogenic E. coli was about 3.4 times higher than the nonpathogenic one. The flow rate of bacterial suspension should be applied above a certain level for stronger binding and rapid detection by attaining a saturation level of detection within a short time of less than 20 min. A possible improvement in the limit of detection in the level of 10 cells per mL for E. coli O157:H7 implies that the AMP-labeled beads have high potential for the sensitive detection of pathogenic E. coli at an appropriate flow rate.

Rapid detection of specific genes from human genomic DNA using the microbead-quantum dot complexes in microfluidic chip.

In the clinic, it is important to prepare for single stranded DNA from genomic DNA to detect the target gene. In this study, we have investigated the detection of single stranded DNA (ssDNA) obtained by ultrasonication of human genomic DNA via fluorescence quenching on microfluidic chip with pillars at the channel to entrap the microbead-QD complexes (MQCs). The QDs with carboxyl group bind to microbeads with amine group by EDC/NHS coupling reaction. The thiolated probe DNA conjugates strongly with the metal ions on the surface of QDs. The MQCs were packed into a chamber on the channel blocked by pillars. ssDNA and TOTO-3 (intercalating dye) were introduced into the microchannel. After hybridization of probe DNA and target DNA, fluorescence quenching was observed at the surface of the MQDs by FRET between QD and TOTO-3. This experiment shows the possibility of rapid detection of genomic DNA from clinical samples via microbead-QD complexs on microfluidic chip.

The preparation of core-shell magnetic silica nanospheres for enhancing magnetism and fluorescence intensity.

Recently, magnetic and luminescent composite silica with structure of micro- and nanospheres containing both magnetic (Fe3O4) nanoparticles (MPs) and quantum dots (QDs) has attracted great interests. In this study, we have prepared core-shell structure of silica spheres in which magnets are incorporated into silica core and QDs into a mesoporous silica shell by using C18-TMS (octade-cyltrimethoxysilane). MPs were synthesized by a co-precipitation method from ferrous and ferric solutions with a molecular ratio of 2:3. Monodisperse magnetic silica cores have been prepared via sol-gel reaction of TEOS (tetraethoxysilane) and water using base catalyst. The size of magnetic silica nanospheres was confirmed by dynamic laser light scattering system (DLS) and scanning electoron microscope (SEM). The pore volume and surface area were calculated by using BET after calcination. The core-shell structure plays an important role in providing more domains for MPs in silica Core and QDs in silica shell. QDs were incorporated into the mesoporous shell by hydrophobic interactions. Magnetic characterization was performed using a superconducting quantum interference device (SQUID). The optical properties of the particles were characterized with UV/Vis spectrometer, PL spectrometer, and fluorescence microscope.

The detection of p53 gene via fluorescence quenching of quantum dot in microfluidic chip.

Recently, quantum dot (QD) has been used widely in the field of bio assay including cell imaging, biomarker, and fluorescence resonance energy transfer (FRET) sensor. The DNA assay without labeling process has several advantages including low cost, short time, and simplicity. Microbeads of agarose, glass, and polystyrene have been used as a solid support in microfluidic devices to trace molecules. The main advantages of microfluidics include high throughput, short analysis time, small sample volume, and high sensitivity. PDMS based microfluidic chips were prepared for the detection of p53 gene by using QD-DNA conjugate. The microfluidic chip has a weir in the channel to trap microbeads to which QD-DNA probes bind. Carboxylated CdSe/ZnS QDs (wavelength of emission: 605 nm) could bind to microbeads of polystyrene/divinyl benzene via EDC/NHS crosslinking reaction. The target gene and DNA intercalating dye (TOTO-3) were loaded into the micro-channel. Fluorescence quenching from QDs by intercalating dye was observed after hybridization of DNA at the weir in the channel of microfluidic chip. The fluorescence quenching from QDs by TOTO-3 was dependent on the concentration of target gene. This experiment shows the possibility of rapid detection of DNA via bead-QD complex on microfluidic chip.

Identification of p53 gene by using CdSe/ZnS conjugation and hybridization.

Recently Quantum Dots (QDs) have been of great interest due to their unique optical properties such as size-dependent, symmetric, narrow, and stable emissions, allowing for prolonged observation and multiplexing. We have prepared oligonucleotide conjugated to QD as a probe to detect p53 tumor suppressor gene related to hereditary cancer. QDs with carboxyl functional group have been conjugated to thiol-modified oligo nucleotides, which have been used as a hybridization probe for p53 gene. Target gene was added and hybridized with the QD bound probe. The conjugation of QD and thiolated oligonucleotide was stained by gel electrophoresis using Etrium Bromide (EtBr) as intercalating dye. Fluorescence resonance energy transfer (FRET) has been observed between QD and intercalating dye (Propidium Iodide) after hybridization of target and probe. FRET efficiency was increased with the increase of dye and DNA concentration. This shows the possibility of specific detection of low concentration of unlabeled complimentary DNA via quantum dots.

The specific hybridization of p53 gene on bead-quantum dot complex in microfluidic chip.

Recently, nanobiosensors using nanoparticles, such as gold, silver, and quantum dots, have been studied extensively. Among them, fluorescence resonance energy transfer (FRET)-based DNA sensor is prominent device, especially for the medical diagnosis and biomolecular investigations. FRET is a phenomenon of the emitted energy transfer from one fluorescent dye to another dye through a convoluted wavelength for the excitation. PDMS-based microfluidic chips with pillar structure were prepared for the detection of exon 7 of p53 gene by using QD-DNA probe attached to polystyrene micro beads. The specific hybridization was investigated with 4 different target oligonucleotides. Fluorescence quenching was observed only from the target oligonucleotide for exon 7 with proper sequence for the hybridization. The fluorescence intensity from QDs decreased rapidly due to hybridization and FRET between QDs and intercalating dyes.

Realisation and advanced engineering of true optical rugate filters based on nanoporous anodic alumina by sinusoidal pulse anodisation.

This study is the first realisation of true optical rugate filters (RFs) based on nanoporous anodic alumina (NAA) by sinusoidal waves. An innovative and rationally designed sinusoidal pulse anodisation (SPA) approach in galvanostatic mode is used with the aim of engineering the effective medium of NAA in a sinusoidal fashion. A precise control over the different anodisation parameters (i.e. anodisation period, anodisation amplitude, anodisation offset, number of pulses, anodisation temperature and pore widening time) makes it possible to engineer the characteristic reflection peaks and interferometric colours of NAA-RFs, which can be finely tuned across the UV-visible-NIR spectrum. The effect of the aforementioned anodisation parameters on the photonic properties of NAA-RFs (i.e. characteristic reflection peaks and interferometric colours) is systematically assessed in order to establish for the first time a comprehensive rationale towards NAA-RFs with fully controllable photonic properties. The experimental results are correlated with a theoretical model (Looyenga-Landau-Lifshitz - LLL), demonstrating that the effective medium of these photonic nanostructures can be precisely described by the effective medium approximation. NAA-RFs are also demonstrated as chemically selective photonic platforms combined with reflectometric interference spectroscopy (RIfS). The resulting optical sensing system is used to assess the reversible binding affinity between a model drug (i.e. indomethacin) and human serum albumin (HSA) in real-time. Our results demonstrate that this system can be used to determine the overall pharmacokinetic profile of drugs, which is a critical aspect to be considered for the implementation of efficient medical therapies.

Identification of potential biomarkers for diazinon exposure to Japanese Medaka (Oryzias latipes) using annealing control primers.

A new differential display-polymerase chain reaction (PCR) method based on annealing control primers was used to screen and identify potential biomarkers from differentially expressed genes (DEGs) in medaka exposed to sub-lethal concentration of diazinon (100 ppb). Among the differentially expressed genes identified, the majority were in functional categories of protein biosynthesis, transport and metabolism according to the gene ontology classification. The differential expression of ribosomal protein genes was quantified by real time PCR. The genes encoding ribosomal proteins including L3 and S17 were selected as potential biomarkers for diazinon exposure in medaka fish.