微流控芯片细胞实验室

以作者所在课题组近年开展的研究工作为基础,阐述了微流控芯片细胞实验室的平台特征,并从细胞个体、群体和多细胞生命体研究等三个方面概述微流控芯片细胞实验室的应用对象特征,显示其在生物医学领域的应用前景。     

Rapid and inexpensive method for the simple fabrication of PDMS‐based electrochemical sensors for detection in microfluidic devices

The fabrication of PDMS microfluidic structures through soft lithography is widely reported. While this well‐established method gives high precision microstructures and has been successfully used for many researchers, it often requires sophisticated instrumentation and expensive materials such as clean room facilities and photoresists. Thus, we present here a simple protocol that allows the rapid molding of simple linear microchannels in PDMS substrates aiming microfluidics‐based applications. It might serve as an alternative to researchers that do not have access to sophisticated facilities such as clean rooms. The method developed here consists on the use of pencil graphite leads as template for the molding of PDMS channels. It yields structures that can be used for several applications, such as housing support for electrochemical sensors or channels for flow devices. Here, the microdevices produced through this protocol were employed for the accommodation of carbon black paste, which was utilized for the first time as amperometric sensor in microchip electrophoresis. This platform was successfully used for the separation and detection of model analytes. Ascorbic acid and iodide were separated within 45 s with peak resolution of 1.2 and sensitivities of 198 and 492 pA/μM, respectively. The background noise was ca. 84 pA. The analytical usefulness of the system developed was successfully tested through the quantification of iodide in commercial pharmaceutical formulations. It demonstrates good efficiency of the microfabrication protocol developed and enables its use for the easy and rapid prototyping of PDMS structures over a low fabrication cost.     

Single-molecule studies on DNA and RNA.

DNA and RNA are the most individual molecules known. Therefore, single-molecule experiments with these nucleic acids are particularly useful. This review reports on recent experiments with single DNA and RNA molecules. First, techniques for their preparation and handling are summarised including the use of AFM nanotips and optical or magnetic tweezers. As important detection techniques, conventional and near-field microscopy as well as fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are touched on briefly. The use of single-molecule techniques currently includes force measurements in stretched nucleic acids and in their complexes with binding partners, particularly proteins, and the analysis of DNA by restriction mapping, fragment sizing and single-molecule hybridisation. Also, the reactions of RNA polymerases and enzymes involved in DNA replication and repair are dealt with in some detail, followed by a discussion of the transport of individual nucleic acid molecules during the readout and use of genetic information and during the infection of cells by viruses. The final sections show how the enormous addressability in nucleic acid molecules can be exploited to construct a single-molecule field-effect transistor and a walking single-molecule robot, and how individual DNA molecules can be used to assemble a single-molecule DNA computer.     

Brownian dynamics simulations of electrophoretic DNA separations in a sparse ordered post array

We use Brownian dynamics simulations to analyze the electrophoretic separation of λ-DNA (48.5 kbp) and T4-DNA (169 kbp) in a hexagonal array of 1 μm diameter posts with a 3 μm center-to-center distance. The simulation method takes advantage of an efficient interpolation algorithm for the non-uniform electric field to reach an ensemble size (100 molecules) and simulation length scale (1 mm) that produces meaningful results for the average electrophoretic mobility and effective diffusion (dispersion) coefficient of these macromolecules as they move through the array. While the simulated electrophoretic mobility for λ-DNA is close to the experimental data, the simulation underestimates the magnitude of the corresponding dispersion coefficient. The simulations predict baseline resolution in a 15 mm device after 7 min using an electric field around 30 V/cm, with the resolution increasing exponentially as the electric field further decreases. The mobility and dispersivity data point out two essential phenomena that have been overlooked in previous models of DNA electrophoresis in post arrays: the relaxation time between collisions and simultaneous collisions with multiple posts.     

Sequencing Single DNA Molecules in Real Time

One's enough : The direct observation of a DNA‐polymerase‐based “sequencing engine” using single‐molecule detection recently allowed single‐molecule sequencing by synthesis in real time. Nucleotides with a fluorescent marker at the 5′‐phosphate unit and zero‐mode waveguides are crucial components of this approach, which at last promises low‐cost genome‐scale sequencing.

     

Simultaneous AFM manipulation and fluorescence imaging of single DNA strands.

We report combined atomic force and far-field fluorescence microscopic experiments which allow the simultaneous atomic force manipulation and optical observation of individual dye-labeled DNA molecules. A detailed understanding of the binding properties of DNA to different transparent surfaces is prerequisite for these investigations. Atomic force spectroscopy and fluorescence microscopy of single DNA strands yielded detailed insight into two different types of DNA binding onto transparent polylysine-coated and silanized glass surfaces. We subsequently demonstrate how the different binding can be exploited to perform two types of nanomanipulation experiments: On polylysine, strong electrostatic interactions over the whole length of the DNA strand enable the writing of micrometer-sized patterns. By contrast, the strong pointwise attachment of DNA to silanized surfaces allows horizontal stretching of single DNA strands to lengths exceeding 1.6 times the contour length of the DNA strand. With this new approach it is possible to directly observe the rupture of the strongly bonded DNA strand.     

Selection of phage-displayed antibodies with high affinity and specificity by electrophoresis in microfluidic devices

A method development aimed for high-throughput and automated antibody screening holds great potential for areas ranging from fundamental molecular interactions to the discovery of novel disease markers, therapeutic targets, and monoclonal antibody engineering. Surface display techniques enable efficient manipulation of large molecular libraries in small volumes. Specifically, phage display appeared as a powerful technology for selecting peptides and proteins with enhanced, target-specific binding affinities. Here, we present a phage-selection microfluidic device wherein electrophoresis was performed under two orthogonal electric fields through an agarose gel functionalized with the respective antigen. This microdevice was capable of screening and sorting in a single round high-affinity phage-displayed antibodies against virus glycoproteins, including human immunodeficiency virus-1 glycoprotein 120 or the Ebola virus glycoprotein (EBOV-GP). Phages were differentially and laterally swept depending on their antigen affinity; the high-affinity phages were recovered at channels proximal to the application site, whereas low-affinity phages migrated distal after electrophoresis. These experiments proved that the microfluidic device specifically designed for phage-selection is rapid, sensitive, and effective. Therefore, this is an efficient and cost-effective method that allowed highly controlled assay conditions for isolating and sorting high-affinity ligands displayed in phages.     

Loading Dynamics of a Sliding DNA Clamp

Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E. coli β clamp in real time by using single‐molecule FRET (smFRET). Three successive FRET states were retained for 0.3 s, 0.7 s, and 9 min: Hydrolysis of the first ATP molecule by the γ clamp loader resulted in closure of the clamp in 0.3 s, and after 0.7 s in the closed conformation, the clamp was released to diffuse on the dsDNA for at least 9 min. An additional single‐molecule polarization study revealed that the interfacial domain of the clamp rotated in plane by approximately 8° during clamp closure. The single‐molecule polarization and FRET studies thus revealed the real‐time dynamics of the ATP‐hydrolysis‐dependent 3D conformational change of the β clamp during loading at a ss/dsDNA junction.     

Single carbon nanotube optical spectroscopy.

This Minireview discusses novel insights into the electronic structure of carbon nanotubes obtained using single-molecule fluorescence spectroscopy. Fluorescence spectra from single nanotubes are well described by a single, Lorentzian lineshape. Nanotubes with identical structures fluoresce with different energies due to local electronic perturbations. Carbon nanotube fluorescence unexpectedly does not-show any intensity or spectral fluctuations at 300 K The lack of intensity blinking or bleaching demonstrates that carbon nanotubes have the potential to provide a stable, single-molecule infrared photon source, allowing for the exciting possibility of applications in quantum optics and biophotonics.