Detection and identification of single ribonucleotide monophosphates using a dual in-plane nanopore sensor made in a thermoplastic via replication.

We report the generation of ∼8 nm dual in-plane pores fabricated in a thermoplastic via nanoimprint lithography (NIL). These pores were connected in series with nanochannels, one of which served as a flight tube to allow the identification of single molecules based on their molecular-dependent apparent mobilities (i.e., dual in-plane nanopore sensor). Two different thermoplastics were investigated including poly(methyl methacrylate), PMMA, and cyclic olefin polymer, COP, as the substrate for the sensor both of which were sealed using a low glass transition cover plate (cyclic olefin co-polymer, COC) that could be thermally fusion bonded to the PMMA or COP substrate at a temperature minimizing nanostructure deformation. Unique to these dual in-plane nanopore sensors was two pores flanking each side of the nanometer flight tube (50 × 50 nm, width × depth) that was 10 µm in length. The utility of this dual in-plane nanopore sensor was evaluated to not only detect, but also identify single ribonucleotide monophosphates (rNMPs) by using the travel time (time-of-flight, ToF), the resistive pulse event amplitude, and the dwell time. In spite of the relatively large size of these in-plane pores (∼8 nm effective diameter), we could detect via resistive pulse sensing (RPS) single rNMP molecules at a mass load of 3.9 fg, which was ascribed to the unique structural features of the nanofluidic network and the use of a thermoplastic with low relative dielectric constants, which resulted in a low RMS noise level in the open pore current. Our data indicated that the identification accuracy of individual rNMPs was high, which was ascribed to an improved chromatographic contribution to the nano-electrophoresis apparent mobility. With the ToF data only, the identification accuracy was 98.3%. However, when incorporating the resistive pulse sensing event amplitude and dwell time in conjunction with the ToF and analyzed via principal component analysis (PCA), the identification accuracy reached 100%. These findings pave the way for the realization of a novel chip-based single-molecule RNA sequencing technology.

Nano-injection molding with resin mold inserts for prototyping of nanofluidic devices for single molecular detection.

While injection molding is becoming the fabrication modality of choice for high-scale production of microfluidic devices, especially those used for in vitro diagnostics, its translation into the growing area of nanofluidics (structures with at least one dimension <100 nm) has not been well established. Another prevailing issue with injection molding is the high startup costs and the relatively long time between device iterations making it in many cases impractical for device prototyping. We report, for the first time, functional nanofluidic devices with dimensions of critical structures below 30 nm fabricated by injection molding for the manipulation, identification, and detection of single molecules. UV-resin molds replicated from Si masters served as mold inserts, negating the need for generating Ni-mold inserts via electroplating. Using assembled devices with a cover plate via hybrid thermal fusion bonding, we demonstrated two functional thermoplastic nanofluidic devices. The first device consisted of dual in-plane nanopores placed at either end of a nanochannel and was used to detect and identify single ribonucleotide monophosphate molecules via resistive pulse sensing and obtain the effective mobility of the molecule through nanoscale electrophoresis to allow its identification. The second device demonstrated selective binding of a single RNA molecule to a solid phase bioreactor decorated with a processive exoribonuclease, XRN1. Our results provide a simple path towards the use of injection molding for device prototyping in the development stage of any nanofluidic or even microfluidic application, through which rapid scale-up is made possible by transitioning from prototyping to high throughput production using conventional Ni mold inserts.

High Sensitivity Extended Nano-Coulter Counter for Detection of Viral Particles and Extracellular Vesicles.

We present a chip-based extended nano-Coulter counter (XnCC) that can detect nanoparticles affinity-selected from biological samples with low concentration limit-of-detection that surpasses existing resistive pulse sensors by 2-3 orders of magnitude. The XnCC was engineered to contain 5 in-plane pores each with an effective diameter of 350 nm placed in parallel and can provide high detection efficiency for single particles translocating both hydrodynamically and electrokinetically through these pores. The XnCC was fabricated in cyclic olefin polymer (COP) via nanoinjection molding to allow for high-scale production. The concentration limit-of-detection of the XnCC was 5.5 × 103 particles/mL, which was a 1,100-fold improvement compared to a single in-plane pore device. The application examples of the XnCC included counting affinity selected SARS-CoV-2 viral particles from saliva samples using an aptamer and pillared microchip; the selection/XnCC assay could distinguish the COVID-19(+) saliva samples from those that were COVID-19(-). In the second example, ovarian cancer extracellular vesicles (EVs) were affinity selected using a pillared chip modified with a MUC16 monoclonal antibody. The affinity selection chip coupled with the XnCC was successful in discriminating between patients with high grade serous ovarian cancer and healthy donors using blood plasma as the input sample.

Separation of U87 glioblastoma cell-derived small and medium extracellular vesicles using elasto-inertial flow focusing (a spiral channel).

Nanoscale and microscale cell-derived extracellular vesicle types and subtypes are of significant interest to researchers in biology and medicine. Extracellular vesicles (EVs) have diagnostic and therapeutic potential in terms of biomarker and nanomedicine applications. To enable such applications, EVs must be isolated from biological fluids or separated from other EV types. Developing methods to fractionate EVs is of great importance to EV researchers. Our goal was to begin to develop a device that would separate medium EVs (mEVs, traditionally termed microvesicles or shedding vesicles) and small EVs (sEVs, traditionally termed exosomes) by elasto-inertial effect. We sought to develop a miniaturized technology that works similar to and provides the benefits of differential ultracentrifugation but is more suitable for EV-based microfluidic applications. The aim of this study was to determine whether we could use elasto-inertial focusing to re-isolate and recover U87 mEVs and sEVs from a mixture of mEVs and sEVs isolated initially by one round of differential ultracentrifugation. The studied spiral channel device can continuously process 5 ml of sample fluid per hour. Using the channel, sEVs and mEVs were recovered and re-isolated from a mixture of U87 glioma cell-derived mEVs and sEVs pre-isolated by one round of differential ultracentrifugation. Following two passes through the spiral channel, approximately 55% of sEVs were recovered with 6% contamination by mEVs (the recovered sEVs contained 6% of the total mEVs). In contrast, recovery of U87 mEVs and sEVs re-isolated using a typical second centrifugation wash step was only 8% and 53%, respectively. The spiral channel also performed similar to differential ultracentrifugation in reisolating sEVs while significantly improving mEV reisolation from a mixture of U87 sEVs and mEVs. Ultimately this technology can also be coupled to other microfluidic EV isolation methods in series and/or parallel to improve isolation and minimize loss of EV subtypes.

Experiment, theory, and simulation of a flow-electrical-split flow thin particle separation device.

Herein, we describe the simulation of a novel flow-electrical-split flow thin (Fl-El-SPLITT) separation device and validate it using existing theory and experimentation for the first time using polystyrene particles of 28 and 1000 nm diameters. The fraction of particles exiting selected ports with DC El-SPLITT is predicted with existing theory, but the theory does not include AC fields, nor does it incorporate the use of crossflows. Using DC fields the El-SPLITT simulation and theory calculated transition points result in the same values. These calculated values accurately predict the experimentally obtained transition point using a 50:50 outlet splitting plane (OSP). Relative to actual experimentally obtained transition points, the calculated values lag behind for a 90:10 OSP, and lead ahead for a 10:90 OSP. The simulation explains trends seen in AC testing, and reasonably predicts the fraction of particles exiting each port. As DC current increases, the amount of AC current required to scatter the particles away from the DC-intended port decreases. The simulation also models a crossflow in a SPLITT system with a DC current applied in a direction opposite the crossflow with some success. Long term steady-state testing without crossflows shows a DC voltage dependent loss of particles. At 8 V DC, total recovery of 28 and 1000 nm particles was 70% and 26%, respectively. This work effectively models a new Fl-El-SPLITT system via Matlab simulation by demonstrating key experimental results such as the influence of DC, AC, and crossflows on the SPLITT separation of polystyrene particles.

Development and Testing of a Continuous Flow-Electrical-Split-Flow Lateral Transport Thin Separation System (Fl-El-SPLITT).

In this work, a new high-volume, continuous particle separation device that separates based upon size and charge is described. Two continuous flow-electrical-split-flow lateral transport thin (Fl-El-SPLITT) device architectures (a platinum electrode on a porous membrane and a porous graphite electrode under a membrane) were developed and shown to improve particle separations over a purely electrical-SPLITT device. The graphite FL-El-SPLITT device architecture achieved the best separation of approximately 60% of small (28 nm) vs large (1000 nm) polystyrene particles. Fl-El-SPLITT (platinum) achieved a 75% separation on a single pass using these same particles. Fl-El-SPLITT (platinum) achieved a moderate 26% continuous separation of U87 glioma cell-derived small extracellular vesicles (EVs) from medium EVs. Control parameter testing showed that El-SPLITT continuously directed particle motility within a channel to exit a selected port based upon the applied voltage using either direct current or alternating current. The transition from one port to the other was dependent upon the voltage applied. Both large and small polystyrene particles transitioned together rather than separating at each of the applied voltages. These data present the first ever validation of El-SPLITT in continuous versus batch format. The Fl-El-SPLITT device architecture, monitoring, and electrical and fluid interfacing systems are described in detail for the first time. Capabilities afforded to the system by the flow addition include enhanced particle separation as well as the ability to filter out small particles or desalinate fluids. High-throughput continuous separations based upon electrophoretic mobility will be streamlined by this new technique that combines electrical and flow fields into a single device.

Characterization of Human Glioblastoma versus Normal Plasma-Derived Extracellular Vesicles Preisolated by Differential Centrifugation Using Cyclical Electrical Field-Flow Fractionation.

Although many properties for small extracellular vesicles (sEVs, formerly termed "exosomes") isolated at ∼100 000g are known, a wide range of values are reported for their electrophoretic mobility (EM) measurements. This paper reports for the first time the effect of dilution on the EM of U87 glioblastoma cell-derived and plasma-derived sEVs and medium size EVs (mEVs, commonly termed "oncosomes") preisolated by differential centrifugation. Furthermore, the effect of resalting on the EM of sEVs and mEVs was evaluated. The EM of U87 sEVs and U87 mEVs showed an increase as the salt concentration decreased to 0.005% of the initial salt concentration. However, for the plasma sEVs and plasma mEVs, the electrophoretic mobility increased as the salt concentration decreased to 0.01% of the initial salt concentration and then increased to its initial value when the salt concentration decreased to 0.005% of the initial salt concentration. For both U87 and plasma sEVs and mEVs, the EM remained almost constant when the concentration of the particles changed and the salt concentration was kept the same as its initial value. This indicates that the EM of EVs is only a function of the salt concentration of the buffer and is independent of the concentration of the particles. The sEVs and mEVs were separated with cyclical ElFFF for the first time. The results indicate that ElFFF was able to fractionate the EVs, and a crescent-shaped trend was found for the retention time when the applied AC voltage was altered (increased).

Characterization and differential retention of Q beta bacteriophage virus-like particles using cyclical electrical field-flow fractionation and asymmetrical flow field-flow fractionation.

Virus-like particles (VLPs) are widely used in medicine, but can be difficult to characterize and isolate from aggregates. In this research, primarily cyclical electrical field-flow fractionation (CyElFFF) coupled with multi-angle light scattering (MALS), and dynamic light scattering (DLS) detectors, was used for the first time to perform size and electrical characterization of three different types of Q beta bacteriophage virus-like particles (VLPs): a blank Q beta bacteriophage which is denoted as VLP and two conjugated ones with different peptides. The CyElFFF results were verified with transmission electron microscopy (TEM). Asymmetrical flow field-flow fractionation (AF4) coupled with MALS was also applied using conditions similar to those used in the CyElFFF experiments, and the results of the two techniques were compared to each other. Using these techniques, the size and electrophoretic characteristics of the fractionated VLPs in CyElFFF were obtained. The results indicate that CyElFFF can be used to obtain a clear distribution of electrophoretic mobilities for each type of VLP. Accordingly, CyElFFF was able to differentially retain and isolate VLPs with high surface electric charge/electrophoretic mobility from the ones with low electric charge/electrophoretic mobility. Regarding the size characterization, the size distribution of the eluted VLPs was obtained using both techniques. CyElFFF was able to identify subpopulations that did not appear in the AF4 results by generating a shoulder peak, whereas AF4 produced a single peak. Different size characteristics of the VLPs appearing in the shoulder peak and the main peak indicate that CyElFFF was able to isolate aggregated VLPs from the monomers partially. Graphical abstract.

Exosome Isolation: Cyclical Electrical Field Flow Fractionation in Low-Ionic-Strength Fluids.

The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, 1× phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at ∼1.3 V AC. A375 melanoma exosome recovery was approximately 70-80% after each buffer substitution, and recovery was independent of whether the sample was substituted into 1× PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using 1× PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from ∼-1.6 to -0.1 µm cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from ∼-5.5 to -0.1 µm cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.