Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB.

Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R² = 0.8131).

Experimental investigation of magnetically actuated separation using tangential microfluidic channels and magnetic nanoparticles.

A novel continuous switching/separation scheme of magnetic nanoparticles (MNPs) in a sub-microlitre fluid volume surrounded by neodymium permanent magnet is studied in this work using tangential microfluidic channels. Polydimethylsiloxane tangential microchannels are fabricated using a novel micromoulding technique that can be done without a clean room and at much lower cost and time. Negligible switching of MNPs is seen in the absence of magnetic field, whereas 90% of switching is observed in the presence of magnetic field. The flow rate of MNPs solution had dramatic impact on separation performance. An optimum value of the flow rate is found that resulted in providing effective MNP separation at much faster rate. Separation performance is also investigated for a mixture containing non-magnetic polystyrene particles and MNPs. It is found that MNPs preferentially moved from lower microchannel to upper microchannel resulting in efficient separation. The proof-of-concept experiments performed in this work demonstrates that microfluidic bioseparation can be efficiently achieved using functionalised MNPs, together with tangential microchannels, appropriate magnetic field strength and optimum flow rates. This work verifies that a simple low-cost magnetic switching scheme can be potentially of great utility for the separation and detection of biomolecules in microfluidic lab-on-a-chip systems.

Magnetite nanoparticles doped photoresist derived carbon as a suitable substratum for nerve cell culture.

A method which alters the substrate's physical and electrochemical properties by doping photoresist derived carbon with magnetite nanoparticles has been developed to enhance the existing substrate's ability to foster cell growth. Cyclic voltammetry, scanning electron microscopy and atomic force microscopy are used to evaluate the characters of the prepared film. And then, the magnetite nanoparticles doped carbon film is used as substrate for the growth of nerve cell. Here, rat pheochromocytoma cells are used for culture to test substrate-cell interactions. The results showed an increase in cell concentration and average neurite length with the increase of nanoparticle concentration on the surface. Importantly, the nerve cells can be grown on the magnetite nanoparticles doped carbon even in the absence of nerve growth factor. This finding will potentially provide a new material for nerve regeneration.

Fe3O4 nanoparticles-enhanced SPR sensing for ultrasensitive sandwich bio-assay.

Magnetic nanoparticles (MNPs) have been receiving increasing attention because of its great potentials in bioseparation. However, the separation products are difficult to be detected by general method due to their extremely small size. Here, we demonstrate that MNPs can greatly enhance the signal of surface plasmon resonance spectroscopy (SPR). Features of MNPs-aptamer conjugates as a powerful amplification reagent for ultrasensitive immunoassay are reported in this work for the first time. In order to evaluate the sensing ability of MNPs-aptamer conjugates as an amplification reagent, a sandwich SPR sensor is constructed by using thrombin as model analyte. Thrombin, captured by immobilized anti-thrombin aptamer on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 0.017 nM after MNPs-aptamer conjugates is used as amplification reagent. At the same time, the excellent selectivity of the present biosensor is also confirmed by using three kinds of proteins (BSA, human IgM and human IgE) as controls. These results confirm that MNPs is a powerful sandwich element and an excellent amplification reagent for SPR based sandwich immunoassay and SPR has a great potential for the detection of MNPs-based bioseparation products.

Magnetic nanoparticle enhanced surface plasmon resonance sensing and its application for the ultrasensitive detection of magnetic nanoparticle-enriched small molecules.

Magnetic nanoparticles (MNPs) have been frequently used in bioseparation, but their applicability in bioassays is limited due to their extremely small size so that sensitive detection is difficult to achieve using a general technique. Here, we present an amplification technique using MNPs for an enhanced surface plasmon resonance (SPR) bioassay. The amplification effect of carboxyl group modified Fe(3)O(4) MNPs of two sizes on SPR spectroscopy is first demonstrated by assembling MNPs on amino group modified SPR gold substrate. To further evaluate the feasibility of the use of Fe(3)O(4) MNPs in enhancing a SPR bioassay, a novel SPR sensor based on an indirect competitive inhibition assay (ICIA) is developed for detecting adenosine by employing Fe(3)O(4) MNP-antiadenosine aptamer conjugates as the amplification reagent. The results confirm that Fe(3)O(4) MNPs can be used as a powerful amplification agent to provide a sensitive approach to detect adenosine by SPR within the range of 10-10,000 nM, which is much superior to the detection result obtained by a general SPR sensor. Importantly, the present detection methodology could be easily extended to detect other biomolecules of interest by changing the corresponding aptamer in Fe(3)O(4) MNP-aptamer conjugates. This novel technique not only explores the possibility of the use of SPR spectroscopy in a highly sensitive detection of an MNP-based separation product but also offers a new direction in the use of Fe(3)O(4) MNPs as an amplification agent to design high performance SPR biosensors.

Aptamer-Au NPs conjugates-accumulated methylene blue for the sensitive electrochemical immunoassay of protein.

In this paper, we combine the advantages of aptamer, nanomaterial and antibody to design an electrochemical sandwich immunoassay for the ultrasensitive detection of human immunoglobulin E (IgE) by using methylene blue (MB) as electrochemical indicator. The sandwich structure is fabricated by using goat anti-human IgE as capturing probe. Aptamer-Au nanoparticles (NPs) conjugates are used both as a sandwich amplification element as well as an accumulation reagent of MB. Once the aptamer-Au NPs conjugates specifically bind to electrode surface, MB molecules are accumulated on its surface by the specific interaction of MB with G base of aptamer-Au NPs conjugates. Therefore, with the increase of human IgE concentration, more aptamer-Au-NPs conjugates are bound, and thus, more MB molecules are accumulated. A good linear relationship is obtained for the detection of human IgE over a range of 1-10,000 ng/ml with a lowest detection limit of 0.52 ng/ml. In addition, by using BSA, human IgA and human IgM as contrast, the excellent specificity of this sensing system for the detection of human IgE is also demonstrated.

Aptamer-Au NPs conjugates-enhanced SPR sensing for the ultrasensitive sandwich immunoassay.

The goal of this work is to explore the amplification effect of aptamer-gold nanoparticles (Au NPs) conjugates for ultrasensitive detection of large biomolecules by surface plasmon resonance (SPR). A novel sandwich immunoassay is designed to demonstrate the amplification effect of aptamer-Au NPs conjugates by using human immunoglobulin E (IgE) as model analyte. Human IgE, captured by immobilized goat anti-human IgE on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 1 ng/ml after anti-human IgE aptamer-Au NPs conjugates is used as amplification reagent. Meanwhile, the non-specific adsorption of aptamer-Au NPs conjugates on goat anti-human IgE is confirmed by SPR spectroscopy and then it is minimized by treating aptamer-Au NPs conjugates with 6-mercaptohexan-1-ol (MCH). These results confirm that aptamer-Au NPs conjugates is a powerful sandwich element and an excellent amplification reagent for SPR-based sandwich immunoassay.

Au NPs-aptamer conjugates as a powerful competitive reagent for ultrasensitive detection of small molecules by surface plasmon resonance spectroscopy.

Features of Au NPs-aptamer conjugates as a powerful competitive reagent to substitute antibody in enhancing surface plasmon resonance spectroscopy (SPR) signal for the detection of small molecule are explored for the first time. In order to evaluate the sensing ability of Au NPs-aptamer conjugates as a competitive reagent, a novel SPR sensor based on indirect competitive inhibition assay (ICIA) for the detection of adenosine is constructed by employing the competitive reaction between antiadenosine aptamer with adenosine and antiadenosine aptamer with its partial complementary ss-DNA. The partial complementary ss-DNA of antiadenosine aptamer is firstly immobilized on SPR gold film as sensing surface. When the Au NPs-antiadenosine aptamer conjugates solution is added to SPR cell in the absence of adenosine, Au NPs-antiadenosine aptamer conjugates is adsorbed to SPR sensor by the DNA hybridization reaction, and results in a large change of SPR signal. However, the change of SPR signal is decreased when the mixing solution of adenosine with Au NPs-antiadenosine aptamer conjugates is added. This is because adenosine reacts with antiadenosine aptamer in Au NPs-antiadenosine aptamer conjugates and changes its structure from ss-DNA to tertiary structure, which cannot hybridize with its partial complementary ss-DNA immobilized on SPR gold surface. Based on this principle, a SPR sensor for indirect detection of adenosine can be developed. The experimental results confirm that the SPR sensor possesses a good sensitivity and a high selectivity for adenosine, which indirectly confirms that Au NPs-aptamer conjugates is a powerful competitive reagent. More significantly, it can be used to develop other SPR sensors based on ICIA to detect different targets by changing the corresponding type of aptamer in Au NPs-aptamer conjugates.