DNA analysis by single molecule stretching in nanofluidic biochips (original) (raw)
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Nature communications, 2017
Wafer-scale fabrication of complex nanofluidic systems with integrated electronics is essential to realizing ubiquitous, compact, reliable, high-sensitivity and low-cost biomolecular sensors. Here we report a scalable fabrication strategy capable of producing nanofluidic chips with complex designs and down to single-digit nanometre dimensions over 200 mm wafer scale. Compatible with semiconductor industry standard complementary metal-oxide semiconductor logic circuit fabrication processes, this strategy extracts a patterned sacrificial silicon layer through hundreds of millions of nanoscale vent holes on each chip by gas-phase Xenon difluoride etching. Using single-molecule fluorescence imaging, we demonstrate these sacrificial nanofluidic chips can function to controllably and completely stretch lambda DNA in a two-dimensional nanofluidic network comprising channels and pillars. The flexible nanofluidic structure design, wafer-scale fabrication, single-digit nanometre channels, rel...
Nanoimprint lithography for the fabrication of DNA electrophoresis chips
Microelectronic Engineering, 2002
We describe the fabrication of microfluidic devices for bio-molecule separation using an array of well-defined nanostructures. Two types of pattern replication of the same device configuration are considered, based on different material processing. In the first approach we use a tri-layer nanoimprint lithography process to pattern a silicon dioxide substrate, on top of which we stick a transparent elastomer cover plate. The second approach relies on direct imprinting of thermoplastic polymer pellets to form two bulk plastic plates later assembled together by thermal bonding. As a result, novel microfluidic devices combining deep and wide channels and a shallower nanostructure array are obtained. The fabricated devices have been characterized by epifluorescence microscopy, using a fluorescein solution to track fluid penetration inside the high density nanostructured region. These realisations not only demonstrate that nanofluidic devices are achievable, but also that they can be manufactured for mass production via nanoimprint-based techniques.
Nanofluidic system for the studies of single DNA molecules
ELECTROPHORESIS, 2008
Here, we describe a simple and low-cost lithographic technique to fabricate size-controllable nanopillar arrays inside the microfluidic channels for the studies of single DNA molecules. In this approach, nanosphere lithography has been employed to grow a single layer of well-ordered close-packed colloidal crystals inside the microfluidic channels. The size of the polymeric colloidal nanoparticles could be trimmed by oxygen plasma treatment. These size-trimmed colloidal nanoparticles were then used as the etching mask in a deep etching process. As a result, well-ordered size-controllable nanopillar arrays could be fabricated inside the microfluidic channels. The gap distance between the nanopillars could be tuned between 20 and 80 nm allowing the formation of nanofluidic system where the behavior of a single l-phage DNA molecule has been investigated. It was found that the lphage DNA molecule could be fully stretched in the nanofluidic system formed by nanopillars with 50 nm gap distance at a field of 50 V/cm.
Micro- and nanofluidics for DNA analysis
Analytical and Bioanalytical Chemistry, 2004
Miniaturization to the micrometer and nanometer scale opens up the possibility to probe biology on a length scale where fundamental biological processes take place, such as the epigenetic and genetic control of single cells. To study single cells the necessary devices need to be integrated on a single chip; and, to access the relevant length scales, the devices need to be designed with feature sizes of a few nanometers up to several micrometers. We will give a few examples from the literature and from our own research in the field of miniaturized chip-based devices for DNA analysis, including dielectrophoresis for purification of DNA, artificial gel structures for rapid DNA separation, and nanofluidic channels for direct visualization of single DNA molecules. The length scales of the fundamental building blocks of biology overlap with the length scales realizable using micro-and nanofabrication technology from the microelectronics industry. The persistence length (L p ) of DNA is given for standard physiological conditions. The gene size corresponds to the typical size of 1 kbp excluding any non-coding regions (introns). The chromosome size range corresponds to metaphase condensed chromosomes. The radius of gyration is indicated for DNA molecules ranging in size from 100 kbp to 100 Mbp C.
Amplified stretch of bottlebrush-coated DNA in nanofluidic channels
Nucleic acids research, 2013
The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy. An enhanced stretch along the channel is observed with increased binding of the cationic block of the polypeptide to DNA. A maximum stretch of 85% of the contour length can be achieved inside a channel with a cross-sectional diameter of 200 nm and at a 2-fold excess of polypeptide with respect to DNA charge. With site-specific fluorescence labelling, it is demonstrated that this maximum stretch is sufficient to map large-scale genomic organization. Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA. The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on s...
An easy method is introduced allowing fast polydimethylsiloxane (PDMS) replication of nanofluidic lab-on-chip devices using accurately fabricated molds featuring cross-sections down to 60 nm. A high quality master is obtained through proton beam writing and UV lithography. This master can be used more than 200 times to replicate nanofluidic devices capable of handling single DNA molecules. This method allows to fabricate nanofluidic devices through simple PDMS casting. The extensions of YOYO-1 stained bacteriophage T4 and kÀDNA inside these nanochannels have been investigated using fluorescence microscopy and follow the scaling prediction of a large, locally coiled polymer chain confined in nanochannels.
Single-molecule denaturation mapping of DNA in nanofluidic channels
Proceedings of the National Academy of Sciences of the United States of America, 2010
Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO®-1labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence. Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells.
On-Chip Stretching, Sorting, and Electro-Optical Nanopore Sensing of Ultralong Human Genomic DNA
ACS Nano
Solid-state nanopore sensing of ultralong genomic DNA molecules has remained challenging, as the DNA must be controllably delivered by its leading end for efficient entry into the nanopore. Herein, we introduce a nanopore sensor device designed for electro-optical detection and sorting of ultralong (300+ kilobase pair) genomic DNA. The fluidic device, fabricated in-silicon and anodically bonded to glass, uses pressure-induced flow and an embedded pillar array for controllable DNA stretching and delivery. Extremely low concentrations (50 fM) and sample volumes (∼1 μL) of DNA can be processed. The low height profile of the device permits high numerical aperture, high magnification imaging of DNA molecules, which remain in focus over extended distances. We demonstrate selective DNA sorting based on sequence-specific nick translation labeling and imaging at high camera frame rates. Nanopores are fabricated directly in the assembled device by laser etching. We show that uncoiling and stretching of the ultralong DNA molecules permits efficient nanopore capture and threading, which is simultaneously and synchronously imaged and electrically measured. Furthermore, our technique provides key insights into the translocation behavior of ultralong DNA and promotes the development of all-in-one micro/nanofluidic platforms for nanopore sensing of biomolecules.
Automation of a single-DNA molecule stretching device
Review of Scientific Instruments, 2015
We automate the manipulation of genomic-length DNA in a nanofluidic device based on real-time analysis of fluorescence images. In our protocol, individual molecules are picked from a microchannel and stretched with pN forces using pressure driven flows. The millimeter-long DNA fragments free flowing in micro-and nanofluidics emit low fluorescence and change shape, thus challenging the image analysis for machine vision. We demonstrate a set of image processing steps that increase the intrinsically low signal-to-noise ratio associated with single-molecule fluorescence microscopy. Furthermore, we demonstrate how to estimate the length of molecules by continuous real-time image stitching and how to increase the e↵ective resolution of a pressure controller by pulse width modulation. The sequence of image-processing steps addresses the challenges of genomic-length DNA visualization; however, they should also be general to other applications of fluorescence-based microfluidics.
DNA detection with a polymeric nanochannel device
Lab on a Chip, 2011
We present the development and the electrical characterization of a polymeric nanochannel device. Standard microfabrication coupled to Focused Ion Beam (FIB) nanofabrication is used to fabricate a silicon master, which can be then replicated in a polymeric material by soft lithography. Such an elastomeric nanochannel device is used to study DNA translocation events during electrophoresis experiments. Our results demonstrate that an easy and low cost fabrication technique allows creation of a low noise device for single molecule analysis.