Label-Free Identification of Single Mononucleotides by Nanoscale Electrophoresis.

Nanoscale electrophoresis allows for unique separations of single molecules, such as DNA/RNA nucleobases, and thus has the potential to be used as single molecular sensors for exonuclease sequencing. For this to be envisioned, label-free detection of the nucleotides to determine their electrophoretic mobility (i.e., time-of-flight, TOF) for highly accurate identification must be realized. Here, for the first time a novel nanosensor is shown that allows discriminating four 2-deoxyribonucleoside 5'-monophosphates, dNMPs, molecules in a label-free manner by nanoscale electrophoresis. This is made possible by positioning two sub-10 nm in-plane pores at both ends of a nanochannel column used for nanoscale electrophoresis and measuring the longitudinal transient current during translocation of the molecules. The dual nanopore TOF sensor with 0.5, 1, and 5 µm long nanochannel column lengths discriminates different dNMPs with a mean accuracy of 55, 66, and 94%, respectively. This nanosensor format can broadly be applicable to label-free detection and discrimination of other single molecules, vesicles, and particles by changing the dimensions of the nanochannel column and in-plane nanopores and integrating different pre- and postprocessing units to the nanosensor. This is simple to accomplish because the nanosensor is contained within a fluidic network made in plastic via replication.

System Modularity Chip for Analysis of Rare Targets (SMART-Chip): Liquid Biopsy Samples.

Liquid biopsies are becoming popular for managing a variety of diseases due to the minimally invasive nature of their acquisition, thus potentially providing better outcomes for patients. Circulating tumor cells (CTCs) are among the many different biomarkers secured from a liquid biopsy, and a number of efficient platforms for their isolation and enrichment from blood have been reported. However, many of these platforms require manual sample handling, which can generate difficulties when translating CTC assays into the clinic due to potential sample loss, contamination, and the need for highly specialized operators. We report a system modularity chip for the analysis of rare targets (SMART-Chip) composed of three task-specific modules that can fully automate processing of CTCs. The modules were used for affinity selection of the CTCs from peripheral blood with subsequent photorelease, simultaneous counting, and viability determinations of the CTCs and staining/imaging of the CTCs for immunophenotyping. The modules were interconnected to a fluidic motherboard populated with valves, interconnects, pneumatic control channels, and a fluidic network. The SMART-Chip components were made from thermoplastics via microreplication, which lowers the cost of production making it amenable to clinical implementation. The utility of the SMART-Chip was demonstrated by processing blood samples secured from colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) patients. We were able to affinity-select EpCAM expressing CTCs with high purity (0-3 white blood cells/mL of blood), enumerate the selected cells, determine their viability, and immunophenotype the cells. The assay could be completed in <4 h, while manual processing required >8 h.

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.

Microfluidic Device for On-Chip Immunophenotyping and Cytogenetic Analysis of Rare Biological Cells.

The role of circulating plasma cells (CPCs) and circulating leukemic cells (CLCs) as biomarkers for several blood cancers, such as multiple myeloma and leukemia, respectively, have recently been reported. These markers can be attractive due to the minimally invasive nature of their acquisition through a blood draw (i.e., liquid biopsy), negating the need for painful bone marrow biopsies. CPCs or CLCs can be used for cellular/molecular analyses as well, such as immunophenotyping or fluorescence in situ hybridization (FISH). FISH, which is typically carried out on slides involving complex workflows, becomes problematic when operating on CLCs or CPCs due to their relatively modest numbers. Here, we present a microfluidic device for characterizing CPCs and CLCs using immunofluorescence or FISH that have been enriched from peripheral blood using a different microfluidic device. The microfluidic possessed an array of cross-channels (2-4 µm in depth and width) that interconnected a series of input and output fluidic channels. Placing a cover plate over the device formed microtraps, the size of which was defined by the width and depth of the cross-channels. This microfluidic chip allowed for automation of immunofluorescence and FISH, requiring the use of small volumes of reagents, such as antibodies and probes, as compared to slide-based immunophenotyping and FISH. In addition, the device could secure FISH results in <4 h compared to 2-3 days for conventional FISH.

Label-free counting of affinity-enriched circulating tumor cells (CTCs) using a thermoplastic micro-Coulter counter (µCC).

Coulter counters are used for counting particles and biological cells. Most Coulter counters are designed to analyze a sample without the ability to pre-process the sample prior to counting. For the analysis of rare cells, such as circulating tumor cells (CTCs), it is not uncommon to require enrichment before counting due to the modest throughput of µCCs and the high abundance of interfering cells, such as blood cells. We report a microfluidic-based Coulter Counter (µCC) fabricated using simple, low-cost techniques for counting rare cells that can be interfaced to sample pre- and/or post-processing units. In the current work, a microfluidic device for the affinity-based enrichment of CTCs from whole blood into a relatively small volume of ∼10 µL was interfaced to the µCC to allow for exhaustive counting of single CTCs following release of the CTCs from the enrichment chip. When integrated to the CTC affinity enrichment chip, the µCC could count the CTCs without loss and the cells could be collected for downstream molecular profiling or culturing if required. The µCC sensor counting efficiency was >93% and inter-chip variability was ∼1%.

Electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) through thermoplastic nanochannels.

The electrokinetic behavior of molecules in nanochannels (<100 nm in length) have generated interest due to the unique transport properties observed that are not seen in microscale channels. These nanoscale dependent transport properties include transverse electromigration arising from partial electrical double layer overlap, enhanced solute/wall interactions due to the small channel diameter, and field-dependent intermittent motion produced by surface roughness. In this study, the electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) were investigated, including the effects of electric field strength, surface effects, and composition of the carrier electrolyte (ionic concentration and pH). The dNMPs were labeled with a fluorescent reporter (ATTO 532) to allow tracking of the electrokinetic transport of the dNMPs through a thermoplastic nanochannel fabricated via nanoimprinting (110 nm × 110 nm, width × depth, and 100 µm in length). We discovered that the transport properties in plastic nanochannels of the dye-labeled dNMPs produced differences in their apparent mobilities that were not seen using microscale columns. We built histograms for each dNMP from their apparent mobilities under different operating conditions and fit the histograms to Gaussian functions from which the separation resolution could be deduced as a metric to gage the ability to identify the molecule based on their apparent mobility. We found that the resolution ranged from 0.73 to 2.13 at pH = 8.3. Changing the carrier electrolyte pH > 10 significantly improved separation resolution (0.80-4.84) and reduced the standard deviation in the Gaussian fit to the apparent mobilities. At low buffer concentrations, decreases in separation resolution and increased standard deviations in Gaussian fits to the apparent mobilities of dNMPs were observed due to the increased thickness of the electric double layer leading to a partial parabolic flow profile. The results secured for the dNMPs in thermoplastic nanochannels revealed a high identification efficiency (>99%) in most cases for the dNMPs due to differences in their apparent mobilities when using nanochannels, which could not be achieved using microscale columns.

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.

Electrophoretic Transport of Single DNA Nucleotides through Nanoslits: A Molecular Dynamics Simulation Study.

There is potential for flight time based DNA sequencing involving disassembly into individual nucleotides which would pass through a nanochannel with two or more detectors. We performed molecular dynamics simulations of electrophoretic motion of single DNA nucleotides through 3 nm wide hydrophobic slits with both smooth and rough walls. The electric field (E) varied from 0.0 to 0.6 V/nm. The nucleotides adsorb and desorb from walls multiple times during their transit through the slit. The nucleotide-wall interactions differed due to nucleotide hydrophobicities and wall roughness which determined duration and frequency of nucleotide adsorptions and their velocities while adsorbed. Transient association of nucleotides with one, two, or three sodium ions occurred, but the mean association numbers (ANs) were weak functions of nucleotide type. Nucleotide-wall interactions contributed more to separation of nucleotide flight time distributions than ion association and thus indicate that nucleotide-wall interactions play a defining role in successfully discriminating between nucleotides on the basis of their flight times through nanochannels/slits. With smooth walls, smaller nucleotides moved faster, but with rough walls larger nucleotides moved faster due to fewer favorable wall adsorption sites. This indicates that roughness, or surface patterning, might be exploited to achieve better time-of-flight based discrimination between nucleotides.

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.

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.