Disposable on-chip microfluidic system for buccal cell lysis, DNA purification, and polymerase chain reaction.

This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off-chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.

A novel microfluidic biosensor based on an electrical detection system for alpha-fetoprotein.

Conventional immunoassays are labor intensive, expensive and time consuming and require large pieces of equipment for detection. Therefore, we have developed and characterized a novel immunoassay methodology comprised of microbeads and microbiochips. In this method, microbeads are used to filter and immobilize antibodies and an immuno-gold silver staining (IGSS) method is then used to amplify electrical signals that correspond to the bound antibodies. The chip used for this system is composed of an inexpensive and biocompatible polydimethylsiloxane (PDMS) layer over a Pyrex glass substrate that contains a platinum (Pt) microelectrode, which is used to detect the electrical signal in this system, the microelectrode is fabricated on the substrate and a microchannel and pillar-type microfilter is formed in the PDMS layer. A sandwich immunoassay approach was applied to detect alpha-fetoprotein (AFP), a cancer biomarker, using this system. The results of this study showed that the time required for a complete assay was reduced by 1h and a detection limit as low as 1 ng/mL was attained when this system used, which indicates that similar bead-based electrical detection systems could be used for the diagnosis of many forms of cancer.

Design, Fabrication, and Testing of a Microfluidic Device for Thermotaxis and Chemotaxis Assays of Sperm.

Infertile couples needing assisted reproduction are increasing, so a fundamental understanding of motile sperm migration is required. This paper presents an advanced microfluidic device for sperm motion analysis utilizing chemotaxis and thermotaxis simultaneously for the first time. The proposed device is a transparent polydimethylsiloxane- and glass-based microfluidic chip system providing a low-cost, useful, and disposable platform for sperm analysis. The concentration gradient of the chemoattractant (acetylcholine) and the temperature difference are formed along the microchannel. The temperature gradient is generated and controlled by a microheater and microsensor. Thermotactic and chemotactic responses of mouse sperm were examined using the proposed device. Experimental results show that motile mouse sperm are attracted more sensitively under integrated conditions of chemotaxis and thermotaxis rather than individual conditions of chemotaxis and thermotaxis. This sperm analysis device is expected to be a useful tool for the study of mammalian sperm migration and the improvement of artificial insemination techniques.

Fluorogenic nanoreactor assembly with boosted sensing kinetics for timely imaging of cellular hydrogen peroxide.

The precise detection of endogenous H2O2 has been considered to be a useful tool for understanding cell physiology. Here, we have developed a nanoreactor co-incorporated with a H2O2-responsive fluorogenic molecule and a catalytic additive. The fast sensing kinetics allows us to visualize a subcellular response in real-time.

Tuning solid-state fluorescence to the near-infrared: a combinatorial approach to discovering molecular nanoprobes for biomedical imaging.

Dyes showing solid-state fluorescence (SSF) are intriguing molecules that can emit bright fluorescence in the condensed phase. Because they do not suffer from self-quenching of fluorescence, nanoscopic dense integration of those molecules produces particulate nanoprobes whose emission intensity can be boosted by raising the intraparticle dye density. In spite of the potential promise for imaging applications demanding intense emission signals, their excitation and emission spectra are generally limited to the visible region where biological tissues have less transparency. Therefore, the SSF-based nanoprobes have rarely been applied to noninvasive in vivo imaging. Here we report a combinatorial chemistry approach to attain a high level of tissue transparency of SSF by tuning its excitation and emission wavelengths to the truly near-infrared (NIR) region. We built a dipolar arylvinyl (ArV) scaffold-based chemical library where the optical bandgap could be narrowed to the NIR above 700 nm by combinatorial modulation of the π-electron push-pull strengths. The ArV-aggregated nanoparticles (FArV NPs) with a colloidal size less than 20 nm were formulated using a polymeric surfactant (Pluronic F-127) and applied to bioimaging in cells and in vivo. We demonstrate that some of FArV NPs have truly NIR excitation and emission of SSF, capable of noninvasive in vivo imaging (efficient lymph node mapping and early diagnosis of tumor) in mouse models by virtue of bright solid-state NIR fluorescence and high signal-to-background contrast (S/B ≈ 8) as well as facile circulation in the living body.

Separation of progressive motile sperm from mouse semen using on-chip chemotaxis.

We present a novel method for the separation of progressive motile sperm from non-progressive motile and immotile sperm. This separation was accomplished by inducing chemotaxis along a longitudinal chemical gradient in a microchip composed of a biocompatible polydimethysiloxane layer and a glass substrate. In a preliminary experiment using fluorescent rhodamine B as a marker, we verified that a chemical gradient was generated by diffusion within the microchannel. We used acetylcholine as a chemoattractant to evaluate the chemotactic response of sperm. We tested the response to a 1/2 to 1/64 dilution series of acetylcholine. The results of a mouse sperm chemotaxis assay showed that progressive motile sperm swam predominantly toward the outlet at an optimal chemical gradient of 0.625 (mg/ml)/mm of acetylcholine. This device provides a convenient, disposable, and high-throughput platform that could function as a progressive motile sperm sorter for potential use in intracytoplasmic sperm injection.

Potential application of antibody-mimicking peptides identified by phage display in immuno-magnetic separation of an antigen.

Phage display was performed against human IgG (hIgG) through five rounds of 'biopanning'. Each round consisted of: (1) incubating a library of phage-displayed 12-mer peptides sequences on hIgG-coated magnetic beads, (2) washing the unbound phages, and (3) eluting the bound phages. The eluted phages were either amplified to enrich the pool of positive clones or subjected to the next round without amplification. Through ELISA, four clones (F9, D1, G5, and A10) showing specific binding affinity to hIgG were identified. Among these, F9 had the highest affinity (K(d)=6.2 nM), only one order of magnitude lower than the native anti-hIgG antibody (0.66 nM). Following the DNA sequences of the selected clones, four 12-mer peptides were chemically synthesized. Among them, D1 peptide showed the highest binding affinity to hIgG via SPR biosensor measurements. This peptide was conjugated to biofunctionalized magnetic beads, and its immuno-binding ability was compared with that of the native antibody immobilized to magnetic beads. The mol-to-mol binding efficacy of the peptide-coated magnetic beads was approximately 1000-fold lower than that of the antibody-coated magnetic beads. Our results suggest a feasibility of using antibody-mimicking peptides identified by phage display technique for immuno-magnetic separation of an antigen.

Microchip-based multiplex electro-immunosensing system for the detection of cancer biomarkers.

Microfluidic-based microchips have become the focus of research interest for immunoassays and biomarker diagnostics. This is due to their aptitude for high-throughput processing, small sample volume, and short analysis times. In this paper, we describe the development of a microchip-based multiplex electro-immunosensing system for simultaneous detection of cancer biomarkers using gold nanoparticles and silver enhancer. Our microchip is composed of biocompatible poly(PDMS) and glass substrates. To fix the antibody-immobilized microbeads, we used pillar-type microfilters within a reaction chamber. An immunogold silver staining (IGSS) method was used to amplify the electrical signal that corresponded to the immune complex. To demonstrate this approach, we simultaneously assayed three cancer biomarkers, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate-specific antigen (PSA) on the microchip. The electrical signal generated from the result of the immunoreaction was measured and monitored by a PC-based system. The overall assay time was reduced from 3-8 h to about 55 min when compared to conventional immunoassays. The working range of the proposed microchip was from 10(-3) to 10(-1) microg/mL of the target antigen.