Engineering inlet structures to enhance DNA capture into nanochannels in a polymer nanofluidic device produced via nanoimprint lithography.

Operating nanofluidic biosensors requires threading single molecules to be analyzed from microfluidic networks into nanostructures, mostly nanochannels or nanopores. Different inlet structures have been employed as a means of enhancing the number of the capture events into nanostructures. Here, we systematically investigated the effects of various engineered inlet structures formed at the micro/nanochannel interface on the capture of single λ-DNA molecules into the nanochannels. Different inlet geometries were evaluated and ranked in order of their effectiveness. Adding an inlet structure prior to a nanochannel effectively improved the DNA capture rate by 190 - 700 % relative to that for the abrupt micro/nanochannel interface. The capture of DNA from the microchannel to various inlets was determined mainly by the capture volumes of the inlet structures and the geometrically modified electric field in the inlet structure. However, as the width of the inlet structure increased, the hydrodynamic flow existing in the microchannel negatively influenced the DNA capture by dragging some DNA molecules deep into the inlet structure back to the microchannel. Our results indicate that engineering inlet structures is an effective means of controlling the capture of DNA molecules into nanostructures, which is important for operation of nanofluidic biosensors.

Thermoplastic nanofluidic devices for identifying abasic sites in single DNA molecules.

DNA damage can take many forms such as double-strand breaks and/or the formation of abasic (apurinic/apyrimidinic; AP) sites. The presence of AP sites can be used to determine therapeutic efficacy of many drugs, such as doxorubicin. While there are different assays to search for DNA damage, they are fraught with limitations, such as the need for large amounts of DNA secured from millions of cells. This is challenging due to the growing importance of using liquid biopsies as a source of biomarkers for many in vitro diagnostic assays. To accommodate the mass limits imposed by the use of liquid biopsies, we report a single-molecule DNA damage assay that uses plastic nanofluidic chips to stretch DNA to near its full contour length when the channel dimensions (width and depth) are near the persistence length (∼50 nm) of double-stranded (ds) DNA. The nanofluidic chip consisted of input funnels for high loading efficiency of single DNA molecules, entropic traps to store the DNA and simultaneously load a series of nanochannels for high throughput processing, and an array of stretching nanochannels to read the AP sites. Single dsDNA molecules, which were labeled with an intercalating dye and a biotinylated aldehyde reactive probe (bARP), could be parked in the stretching nanochannels, where the AP sites were read directly using a dual-color fluorescence microscope equipped with an EMCCD camera. One color of the microscope was used to read the DNA length and the second color detected the AP sites. The nanofluidic chip was made from thermoplastics via nanoimprint lithography, which obviated the need for direct writing the devices in glass or quartz using focused ion beam milling. We show that we can read the frequency of AP sites in single dsDNA molecules with the frequency of AP sites determined by associating fluorescently-labeled streptavidin with bARP through a biotin/streptavidin complex.

Thermoplastic nanofluidic devices for biomedical applications.

Microfluidics is now moving into a developmental stage where basic discoveries are being transitioned into the commercial sector so that these discoveries can affect, for example, healthcare. Thus, high production rate microfabrication technologies, such as thermal embossing and/or injection molding, are being used to produce low-cost consumables appropriate for commercial applications. Based on recent reports, it is clear that nanofluidics offers some attractive process capabilities that may provide unique venues for biomolecular analyses that cannot be realized at the microscale. Thus, it would be attractive to consider early in the developmental cycle of nanofluidics production pipelines that can generate devices possessing sub-150 nm dimensions in a high production mode and at low-cost to accommodate the commercialization of this exciting technology. Recently, functional sub-150 nm thermoplastic nanofluidic devices have been reported that can provide high process yield rates, which can enable commercial translation of nanofluidics. This review presents an overview of recent advancements in the fabrication, assembly, surface modification and the characterization of thermoplastic nanofluidic devices. Also, several examples in which nanoscale phenomena have been exploited for the analysis of biomolecules are highlighted. Lastly, some general conclusions and future outlooks are presented.

Electrophoretic Separation of Single Particles Using Nanoscale Thermoplastic Columns.

Phenomena associated with microscale electrophoresis separations cannot, in many cases, be applied to the nanoscale. Thus, understanding the electrophoretic characteristics associated with the nanoscale will help formulate relevant strategies that can optimize the performance of separations carried out on columns with at least one dimension below 150 nm. Electric double layer (EDL) overlap, diffusion, and adsorption/desorption properties and/or dielectrophoretic effects giving rise to stick/slip motion are some of the processes that can play a role in determining the efficiency of nanoscale electrophoretic separations. We investigated the performance characteristics of electrophoretic separations carried out in nanoslits fabricated in poly(methyl methacrylate), PMMA, devices. Silver nanoparticles (AgNPs) were used as the model system with tracking of their transport via dark field microscopy and localized surface plasmon resonance. AgNPs capped with citrate groups and the negatively charged PMMA walls (induced by O2 plasma modification of the nanoslit walls) enabled separations that were not apparent when these particles were electrophoresed in microscale columns. The separation of AgNPs based on their size without the need for buffer additives using PMMA nanoslit devices is demonstrated herein. Operational parameters such as the electric field strength, nanoslit dimensions, and buffer composition were evaluated as to their effects on the electrophoretic performance, both in terms of efficiency (plate numbers) and resolution. Electrophoretic separations performed at high electric field strengths (>200 V/cm) resulted in higher plate numbers compared to lower fields due to the absence of stick/slip motion at the higher electric field strengths. Indeed, 60 nm AgNPs could be separated from 100 nm particles in free solution using nanoscale electrophoresis with 100 µm long columns.

High process yield rates of thermoplastic nanofluidic devices using a hybrid thermal assembly technique.

Over the past decade, thermoplastics have been used as alternative substrates to glass and Si for microfluidic devices because of the diverse and robust fabrication protocols available for thermoplastics that can generate high production rates of the desired structures at low cost and with high replication fidelity, the extensive array of physiochemical properties they possess, and the simple surface activation strategies that can be employed to tune their surface chemistry appropriate for the intended application. While the advantages of polymer microfluidics are currently being realized, the evolution of thermoplastic-based nanofluidic devices is fraught with challenges. One challenge is assembly of the device, which consists of sealing a cover plate to the patterned fluidic substrate. Typically, channel collapse or substrate dissolution occurs during assembly making the device inoperable resulting in low process yield rates. In this work, we report a low temperature hybrid assembly approach for the generation of functional thermoplastic nanofluidic devices with high process yield rates (>90%) and with a short total assembly time (16 min). The approach involves thermally sealing a high T(g) (glass transition temperature) substrate containing the nanofluidic structures to a cover plate possessing a lower T(g). Nanofluidic devices with critical feature sizes ranging between 25-250 nm were fabricated in a thermoplastic substrate (T(g) = 104 °C) and sealed with a cover plate (T(g) = 75 °C) at a temperature significantly below the T(g) of the substrate. Results obtained from sealing tests revealed that the integrity of the nanochannels remained intact after assembly and devices were useful for fluorescence imaging at high signal-to-noise ratios. The functionality of the assembled devices was demonstrated by studying the stretching and translocation dynamics of dsDNA in the enclosed thermoplastic nanofluidic channels.

Surface charge, electroosmotic flow and DNA extension in chemically modified thermoplastic nanoslits and nanochannels.

Thermoplastics have become attractive alternatives to glass/quartz for microfluidics, but the realization of thermoplastic nanofluidic devices has been slow in spite of the rather simple fabrication techniques that can be used to produce these devices. This slow transition has in part been attributed to insufficient understanding of surface charge effects on the transport properties of single molecules through thermoplastic nanochannels. We report the surface modification of thermoplastic nanochannels and an assessment of the associated surface charge density, zeta potential and electroosmotic flow (EOF). Mixed-scale fluidic networks were fabricated in poly(methylmethacrylate), PMMA. Oxygen plasma was used to generate surface-confined carboxylic acids with devices assembled using low temperature fusion bonding. Amination of the carboxylated surfaces using ethylenediamine (EDA) was accomplished via EDC coupling. XPS and ATR-FTIR revealed the presence of carboxyl and amine groups on the appropriately prepared surfaces. A modified conductance equation for nanochannels was developed to determine their surface conductance and was found to be in good agreement with our experimental results. The measured surface charge density and zeta potential of these devices were lower than glass nanofluidic devices and dependent on the surface modification adopted, as well as the size of the channel. This property, coupled to an apparent increase in fluid viscosity due to nanoconfinement, contributed to the suppression of the EOF in PMMA nanofluidic devices by an order of magnitude compared to the micro-scale devices. Carboxylated PMMA nanochannels were efficient for the transport and elongation of λ-DNA while these same DNA molecules were unable to translocate through aminated nanochannels.

Microfluidics for the analysis of membrane proteins: how do we get there?

The development of fully automated and high-throughput systems for proteomics is now in demand because of the need to generate new protein-based disease biomarkers. Unfortunately, it is difficult to identify protein biomarkers that are low abundant when in the presence of highly abundant proteins, especially in complex biological samples such as serum, cell lysates, and other biological fluids. Membrane proteins, which are in many cases of low abundance compared to the cytosolic proteins, have various functions and can provide insight into the state of a disease and serve as targets for new drugs making them attractive biomarker candidates. Traditionally, proteins are identified through the use of gel electrophoretic techniques, which are not always suitable for particular protein samples such as membrane proteins. Microfluidics offers the potential as a fully automated platform for the efficient and high-throughput analysis of complex samples, such as membrane proteins, and do so with performance metrics that exceed their bench-top counterparts. In recent years, there have been various improvements to microfluidics and their use for proteomic analysis as reported in the literature. Consequently, this review presents an overview of the traditional proteomic-processing pipelines for membrane proteins and insights into new technological developments with a focus on the applicability of microfluidics for the analysis of membrane proteins. Sample preparation techniques will be discussed in detail and novel interfacing strategies as it relates to MS will be highlighted. Lastly, some general conclusions and future perspectives are presented.

Immobilization of lambda exonuclease onto polymer micropillar arrays for the solid-phase digestion of dsDNAs.

The process of immobilizing enzymes onto solid supports for bioreactions has some compelling advantages compared to their solution-based counterpart including the facile separation of enzyme from products, elimination of enzyme autodigestion, and increased enzyme stability and activity. We report the immobilization of λ-exonuclease onto poly(methylmethacrylate) (PMMA) micropillars populated within a microfluidic device for the on-chip digestion of double-stranded DNA. Enzyme immobilization was successfully accomplished using 3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling to carboxylic acid functionalized PMMA micropillars. Our results suggest that the efficiency for the catalysis of dsDNA digestion using λ-exonuclease, including its processivity and reaction rate, were higher when the enzyme was attached to a solid support compared to the free solution digestion. We obtained a clipping rate of 1.0 × 10(3) nucleotides s(-1) for the digestion of λ-DNA (48.5 kbp) by λ-exonuclease. The kinetic behavior of the solid-phase reactor could be described by a fractal Michaelis-Menten model with a catalytic efficiency nearly 17% better than the homogeneous solution-phase reaction. The results from this work will have important ramifications in new single-molecule DNA sequencing strategies that employ free mononucleotide identification.