Article

Resistive Pulse Sensing Device with Embedded Nanochannel (nanochannel-RPS) for Label-free Biomolecule and Bionanoparticle Analysis

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Abstract

This paper reports an IC-compatible method for fabricating a PDMS-based resistive pulse sensing (RPS) device with embedded nanochannel (nanochannel-RPS) for label-free analysis of biomolecules and bionanoparticles, such as plasmid DNAs and exosomes. Here, a multilayer lithography process was proposed to fabricate the PDMS mold for the microfluidic device comprising a bridging nanochannel, as the sensing gate. RPS was performance by placing the sensing and excitation electrodes symmetrically upstream and downstream the sensing gate. In order to reduce the noise level, a reference electrode was designed and placed beside the excitation electrode. To demonstrate the feasibility of the proposed nanochannel-RPS device and sensing system, polystyrene micro- and nanoparticles with diameters of 1 μm and 300 nm were tested by the proposed device with signal-to-noise ratios (SNR) ranging 9.1-30.5 and 2.2-5.9, respectively. Furthermore, nanochannel with the height of 300 nm was applied for 4 kb plasmid DNA detection, implying the potential of the proposed method for label-free quantification of nanoscale biomolecules. Moreover, HeLa cell exosomes, known as a well-studied subtype of extracellular vesicles, were measured and analyzed by their size distribution. The result of the resistive pulse amplitude was well corresponding to the that of nanoparticle tracking analysis (NTA). The proposed nanochannel-RPS device and the sensing strategy are not only capable for label-free analysis for nanoscale biomolecules and bionanoparticle, but also cost-effective for large-scale manufacturing.

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... In principle, scaling down the dimensions of the microfluidic channel to submicrometer levels could be a solution for improving their ma nipulation ability. For example, nanofluidic devices (28,29), such as nanoscale deterministic lateral displacement (30), have been used to detect and manipulate sub100nm bioparticles. However, the en hanced interactions between the nanoparticles in such limited space induce uncontrolled agglomeration, adhesion, and even damage to the bioparticles. ...
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It has recently been recognized that solid-state nanopores in single-atomic-layer graphene membranes can be used to electronically detect and characterize single long charged polymer molecules. We have now fabricated nanopores in single-layer graphene that are closely matched to the diameter of a double-stranded DNA molecule. Ionic current signals during electrophoretically driven translocation of DNA through these nanopores were experimentally explored and theoretically modeled. Our experiments show that these nanopores have unusually high sensitivity (0.65 nA/Å) to extremely small changes in the translocating molecule's outer diameter. Such atomically short graphene nanopores can also resolve nanoscale-spaced molecular structures along the length of a polymer, but do so with greatest sensitivity only when the pore and molecule diameters are closely matched. Modeling confirms that our most closely matched pores have an inherent resolution of ≤0.6 nm along the length of the molecule.
Article
The first electronic measurement of DNA translocation through ultrathin BN nanopores is demonstrated. BN nanopores show much higher detection sensitivity compared with SiN nanopores. BN has as high a spatial resolution as graphene. The ultrathin BN nanopores provide substantial opportunities in realizing high-spatial-sensitivity nanopore electrical devices for various applications.
Article
Ion current through a single-walled carbon nanotube (SWCNT) was monitored at the same time as fluorescence was recorded from charged dye molecules translocating through the SWCNT. Fluorescence bursts generally follow ion-current peaks with a delay time consistent with diffusion from the end of the SWCNT to the fluorescence collection point. The fluorescence amplitude distribution of the bursts is consistent with single molecule signals. Thus each peak in the ion current flowing through the SWCNT is associated with the translocation of a single molecule. Ion current peaks (as opposed to blockades) were produced by both positively (Rhodamine 6G) and negatively (Alexa 546) charged molecules, showing that the charge filtering responsible for the current bursts is caused by the molecules themselves.
Article
This paper reports a lab-on-a-chip device that can automatically detect and sort particles based on their size differences with a high resolution. The PDMS-glass microfluidic chip is made by soft lithography technique. A differential resistive pulse sensor (RPS) is employed to electrically detect the sizes of the particles in electroosmotic flow generated by applying DC voltages across channels. The detected PRS signals, whose amplitudes are proportional to particles' sizes, will automatically trigger the sorting process that is controlled by applying a voltage pulse (36V) whenever a target particle is detected. This method was applied to automatically detect and sort polystyrene particles and micro algae in aqueous solutions. Sorting 5μm polymer particle from a mixture of 4μm and 5μm polystyrene particles in aqueous solution, i.e., 1μm sorting resolution, was demonstrated. The device described in this paper is simple, automatic and label-free with high sorting resolution. It has wide applications in sample pretreatment and target particles detection.
Article
We have used micromolding techniques to embed a nanoscale pore in PDMS. This novel design allows rapid and reproducible fabrication of pores, is extremely flexible, and can be modified both structurally and chemically for a variety of single-molecule detection applications. We demonstrate the capabilities of the device to sense electronically single DNA molecules. Because ion-channel proteins are so finely tuned to respond to a single molecule, they have served as a model for developing nanopore devices for biomolecular sensing. 1 Two strategies for engineered nanoporesstransmembrane protein pores suspended in lipid bilayers 2-5 and molecular-scaled holes in silicon nitride 6 shave achieved success in detecting single biological molecules; however, further development of these strategies is impeded by several technological barriers, including difficulties in creating an effective pore or array of pores and stabilizing it over a period of time. Here we report on a fundamentally different artificial nanopore that can be fabricated with great ease and control using micromolding techniques, is capable of sensing single molecules of unlabeled λ-phage DNA, and provides op-portunities for diverse single-molecule detection applications. Figure 1 shows one of our devices: a pore of length 3 µm and diameter 200 nm connecting two 5-µm-deep reservoirs. Well-established lithographic techniques are used to create a negative master of the pore and reservoirs, which is subsequently cast into a poly(dimethylsiloxane) (PDMS) slab. The master is created in two steps: first, electron-beam lithography is used to pattern a 200-nm-wide, 200-nm-thick polystyrene line on a silicon substrate, creating the negative of the pore. Next, photolithography is used to pattern an SU-8 photoresist on the substrate to form the negatives of the reservoirs. Both resists (polystyrene and SU-8) are excep-tionally durable once cross linked, allowing us to reuse each master indefinitely. Following standard micromolding tech-niques, 7 we pour PDMS (Sylgard 184) over the master and cure it at 80 °C for at least 24 h. The PDMS slab is then removed from the master and sealed to a glass substrate that has previously defined platinum electrodes. The device is now complete and can be wet with the solution to be studied. Molecules in the solution are driven through the pore either electrophoretically or by applying pressure to one of the reservoirs. When in the pore, the molecules partially block the flow of current, leading to transient increases in the pore's electrical resistance. Molecular sensing is accomplished by performing a four-point measurement of the electrical current through the pore using the platinum electrodes. The current is low-pass filtered below 0.3 ms in rise time and is sampled at 1 kHz using a voltmeter. To demonstrate the sensing capabilities of our nanopore, we have measured solutions of 2.5 µg/mL λ-phage DNA in a 0.1 M KCl, 2 mM Tris (pH 8.4) buffer. Typical traces of measured current are shown in Figure 2. The striking downward peaks, of height 10-30 pA and width 2-10 ms, correspond to individual molecules of DNA passing through the pore. In contrast, such peaks are absent when measuring only buffer. We further note that peaks are present only when using pores with diameters of 300 nm or less.
Article
A method for detecting and counting pollen particles based on Coulter counting principle is presented. This approach also provides information on the size and surface charges of the micro particles, allowing for preliminary differentiation of pollens from other micro particles. Three samples are studied: polymethyl methacrylate particles, tree pollens from Juniperus Scopulorum and grass pollens from Secale Cerale. The samples, suspended in diluted KCl aqueous solutions in an electrochemical cell, were allowed to pass through a microchannel and the conductance of the microchannel was sampled with a Gamry® Potentiostat. The changes in the conductance due to the passing of the micro particles was thus recorded and analyzed. The experimental results showed that tree pollens and grass pollens display distinctive behaviors. The phenomena may be attributed to the differences in the surface characteristics of the pollens and is potentially useful for counting and differentiating different micro particles.
Article
We report fabrication and characterization of nanochannel devices with two nanopores in series for resistive-pulse sensing of hepatitis B virus (HBV) capsids. The nanochannel and two pores are patterned by electron beam lithography between two microchannels and etched by reactive ion etching. The two nanopores are 50-nm wide, 50-nm deep, and 40-nm long and are spaced 2.0-μm apart. The nanochannel that brackets the two pores is 20× wider (1 μm) to reduce the electrical resistance adjacent to the two pores and to ensure the current returns to its baseline value between resistive-pulse events. Average pulse amplitudes differ by <2% between the two pores and demonstrate that the fabrication technique is able to produce pores with nearly identical geometries. Because the two nanopores in series sense single particles at two discrete locations, particle properties, e.g., electrophoretic mobility, are determined from the pore-to-pore transit time.
Article
We report characterization of hepatitis B virus (HBV) capsids by resistive-pulse sensing through single track-etched conical nanopores formed in poly(ethylene terephthalate) membranes. The pores were ∼40 nm in diameter at the tip, and the pore surface was covalently modified with triethylene glycol to reduce surface charge density, minimize adsorption of the virus capsids, and suppress electroosmotic flow in the pore. The HBV capsids were assembled in vitro from Cp149, the assembly domain of HBV capsid protein. Assembled T = 3 (90 Cp149 dimer) and T = 4 (120 dimer) capsids are 31 and 36 nm in diameter, respectively, and were easily discriminated by monitoring the change in current as capsids passed through an electrically biased pore. The ratio of the number of T = 3 to T = 4 capsids transiting a pore did not reflect actual concentrations, but favored transport of smaller T = 3 capsids. These results combined with longer transit times for the T = 4 capsids indicated that the capsids must overcome an entropic barrier to enter a pore.
Article
We investigate the translocation of lambda-DNA molecules through resistive-pulse polydimethylsiloxane (PDMS) nanopore sensors. Single molecules of lambda-DNA were detected as a transient current increase due to the effect of DNA charge on ionic current through the pore. DNA translocation was found to deviate from a Poisson process when the interval between translocations was comparable to the duration of translocation events, suggesting that translocation was impeded during the presence of another translocating molecule in the nanopore. Characterization of translocation at different voltage biases revealed that a critical voltage was necessary to drive DNA molecules through the nanopore. Above this critical voltage, frequency of translocation events was directly proportional to DNA concentration and voltage bias, suggesting that transport of DNA from the solution to the nanopore was the rate limiting step. These observations are consistent with experimental results on transport of DNA through nanopores and nanoslits and the theory of hydrodynamically driven polymer flow in pores.