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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Tg (glass transition temperature) substrate containing the nanofluidic structures to a cover plate possessing a lower Tg. Nanofluidic devices with critical feature sizes ranging between 25–250 nm were fabricated in a thermoplastic substrate (Tg = 104 °C) and sealed with a cover plate (Tg = 75 °C) at a temperature significantly below the Tg 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]