Nanopore sensing provides an ideal strategy for the label-free detection of single molecules in a variety of application scenarios. Working under the principle of resistive pulse sensing (RPS), nanopores consist of constrictions with sub-100 nm dimensions to enable single-molecule resolution by matching pore size to target dimensions (scaling); the optimal signal-to-noise ratio (SNR) results when the electrically biased pore is comparable in size to the molecule to be analyzed. When single molecules are electrokinetically transported through such remarkably small pores, they temporarily disturb the flux of ions moving through them, generating unique signals. These signals vary based upon the molecules' shape, size, orientation, and other physicochemical properties. Nanopores are generally divided into two main categories owing to their fabrication approach and material: biological and solid state. While biological nanopores have been the dominant sensor format due to their exceptionally small size, solid-state nanopores can demonstrate high performance characteristics attributed to their rigidity, stability, and high versatility in shape, material, and configuration. This review will explore the state-of-the-art in biological and solid-state nanopores and their abilities to detect and identify single biomolecules in a label-free manner. We will also review two topographical configurations of nanopore sensors; in-plane and out-of-plane sensors. The evolution of nanopore sensing will be reviewed, starting with out-of-plane biological sensors and progressing to in-plane sensors fabricated in plastics via replication technologies.

We report a thermoplastic nanofluidic sensor (exonuclease time-of-flight; X-ToF) fabricated via nanoinjection molding that integrates an immobilized nanoscale enzymatic reactor (INER) directly with a dual in-plane nanopore ToF (DNP-ToF) reader, which not only uses the parameters typically used for resistive pulse sensing (RPS)─normalized event amplitude and time width─but the time taken for a single-molecule to travel between two pores in series. This platform enables the label-free monitoring of complex biological reactions at the single-molecule level. A critical hurdle in integrating such bioenzymatic reactions with RPS is reconciling disparate process step requirements, for example salt effects on an enzymatic reaction and high salt needs for RPS. We addressed this through strategic UV/O3 surface engineering; an optimized 3.5 min dose created effectively charge-neutral nanopores that maximized capture rates while sustaining a robust ensemble electroosmotic flow (5.33 ± 0.33 × 10-5 cm2V-1 s-1) and simultaneously preserving enzyme activity. To demonstrate the sensor's utility, exoribonuclease 1 (Xrn1) was used as a model. X-ToF successfully deduced the dissociation constant of an input Cas9 RNA to Xrn1 (2.4 ± 0.02 μM-1) and monitored in real-time ribonucleotide generation from Cas9 with a cleavage rate of 23 nt/s. Ultimately, this platform serves as a highly versatile tool that can be repurposed for DNA, RNA, or protein sequencing simply by changing the identity of the immobilized enzyme.

Extracellular vesicles (EVs) play a crucial role in intercellular communication, signaling pathways, and disease pathogenesis by transporting biomolecules such as DNA, RNA, proteins, and lipids derived from their cells of origin, and they have demonstrated substantial potential in clinical applications. Their clinical significance underscores the need for sensitive methods to fully harness their diagnostic potential. In this comprehensive review, we explore EV heterogeneity related to biogenesis, structure, content, origin, sample type, and function roles; the use of EVs as disease biomarkers; and the evolving landscape of EV measurement for clinical diagnostics, highlighting the progression from bulk measurement to single vesicle analysis. This review covers emerging technologies such as single-particle tracking microscopy, single-vesicle RNA sequencing, and various nanopore-, nanoplasmonic-, immuno-digital droplet-, microfluidic-, and nanomaterial-based techniques. Unlike traditional bulk analysis methods, these methods contribute uniquely to EV characterization. Techniques like droplet-based single EV-counting enzyme-linked immunosorbent assays (ELISA), proximity-dependent barcoding assays, and surface-enhanced Raman spectroscopy further enhance our ability to precisely identify biomarkers, detect diseases earlier, and significantly improve clinical outcomes. These innovations provide access to intricate molecular details that expand our understanding of EV composition, with profound diagnostic implications. This review also examines key research challenges in the field, including the complexities of sample analysis, technique sensitivity and specificity, the level of detail provided by analytical methods, and practical applications, and we identify directions for future research. This review underscores the value of advanced EV analysis methods, which contribute to deep insights into EV-mediated pathological diversity and enhanced clinical diagnostics.

Techniques to analyze proteins often involves complex workflows and/or sophisticated equipment with modest limits-of-detection. While fluorescence spectroscopy can interrogate single molecules, it often requires fluorescence labeling with lasers and microscopes. We report herein a label-free approach for analyzing intact proteins using resistive pulse sensing (RPS). RPS data were secured using a unique RPS device, which we call a dual in-plane nanopore sensor, fabricated in a thermoplastic. The nanopore sensor was produced via nano-injection molding with critical structures of 30 nm, enabling the detection of individual protein molecules and providing an approach toward their identification. Following nano-injection molding, the pore size could be reduced to ∼ 10 nm using thermal fusion bonding of a cover plate to the molded substrate. The device architecture contained two in-plane nanopores flanking a nanochannel (50 × 50 nm width × depth and 5 µm length) that facilitated the measurement of the apparent electrophoretic mobilities of protein molecules in a label free manner via their molecular-dependent time-of-flight (ToF; time-difference between two consecutive RPS events—peak pair). We investigated four model proteins and collected multiple characteristics including RPS peak amplitude and dwell time, as well as an RPS-independent value, which was the ToF. Furthermore, we analyzed the temporal profiles of RPS events revealing distinct peak shapes for spherical and non-spherical proteins that were influenced by their rotational motion when resident within the nanopore.

Understanding cells from complex biological samples is vital to understanding cellular biology and medical applications. One evolving tool for cell sorting is the use of microfluidic devices to achieve higher precision and remove the need for labeling cell subpopulations. However, few microfluidic devices have been translated commercially beyond academic research often due to challenges in larger scale fabrication. Here, we initially investigated a compelling label-free microfluidic device with complex geometries to perform contactless dielectrophoresis (cDEP) for applications in enriching cell subpopulations in oncology, neurology, stem cells, and sample preparation. We began scaling the manufacturing of cDEP devices using Dow Sylgard 184, more commonly referred to as PDMS (polydimethylsiloxane). However, we began observing a new, dynamic bubble formation phenomenon which had significant impacts on device performance. Within just 5 min of exposure at typical experimental values, cell death was nearly 100%. Variables related to manufacturing, environment, equipment, personnel, raw materials sourcing, lithography methods and experimental conditions/parameters were systematically evaluated to find the root cause of the exacerbated bubble formation observed. Further, alternate polymers were sourced for manufacturing and experimental performance comparisons. All variables investigated failed to solve the significant decline in device performance and increase in cell death. Upon completing chemical analysis in this work, we conclude that the decline in device performance was a direct result of changes to the expected PDMS properties and composition. Despite these challenges, our robust quality control combined with experimental protocols to remove bubbles from the cDEP devices achieved consistent experimental performance including 2-3 h run times and >90% cell viability after sorting. These new PDMS behaviors will need to continue to be monitored and controlled to ensure consistency in experimentation, application and commercialization feasibility for a wide variety of microfluidic device designs and applications.

We are developing a unique protein identification method that consists of generating peptides proteolytically from a single protein molecule (i.e., peptide fingerprints) with peptide detection and identification carried out using nanoscale electrochromatography and label-free resistive pulse sensing (RPS). As a step in realizing this technology, we report herein the nanoscale electrochromatography of model peptides using thermoplastic columns with surfaces engineered to identify peptides via their molecularly dependent mobility (i.e., time-of-flight, ToF). ToFs were elucidated using a dual in-plane nanopore sensor, which consisted of two in-plane nanopores placed on either end of the nanoelectrochromatography column. The surface of the nanocolumn, which consisted of poly(methyl methacrylate) (PMMA), was activated with an O2 plasma, creating surface carboxylic acid groups (-COOH) inducing a surface charge on the column wall as well as affecting its hydrophilicity. To understand scaling effects, we carried out microchip and nanochannel electrochromatography of the peptides labeled with an ATTO 532 reporter to allow for single-molecule tracking. Our results indicated that the apparent mobilities of the model peptides did not allow for their separation in a microchannel, but when performed in a nanocolumn, clear differences in their apparent mobilities could be observed especially when operated at high electric field strengths. We next performed label-free detection of peptides using the dual in-plane nanopore sensor with the two pores separated by a 5 μm (length) column with a 50 nm width and depth. When a single peptide molecule passed through an in-plane nanopore, the sensor read a pair of resistive pulses with a time difference equivalent to ToF. We identified the peptides by evaluating their ToF, normalized RPS current transient amplitude (ΔI/I0), and RPS peak dwell time (td). We could identify the model peptides with nearly 100% classification accuracy at the single-molecule level using machine learning with a single molecule measurement requiring <10 ms.

The presence of air bubbles boosts the shear resistance and causes pressure fluctuation within fluid-perfused microchannels, resulting in possible cell damage and even malfunction of microfluidic devices. Eliminating air bubbles is especially challenging in microscale where the adhesive surface tension force is often dominant over other forces. Here, we present an air bubble removal strategy from a novel surface engineering perspective. A microfluidic port-to-port interconnect was fabricated by modifying the peripheral of the microfluidic ports superhydrophobic, while maintaining the inner polymer microchannels hydrophilic. Such a sharp wettability contrast enabled a preferential fluidic entrance into the easy-wetting microchannels over the non-wetting boundaries of the microfluidic ports, while simultaneously filtering out any incoming air bubbles owing to the existence of port-to-port gaps. This bubble-eliminating capability was consistently demonstrated at varying flow rates and liquid analytes. Compared to equipment-intensive techniques and porous membrane-venting strategies, our wettability contrast-governed strategy provides a simple yet effective route for eliminating air bubbles and simultaneously sealing microfluidic interconnects.

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.

An improved, laser-induced fluorescence-based micro-optical biosensor was designed and fabricated, with cyclic olefin copolymer (COC) optical waveguides, a poly(methyl methacrylate) (PMMA) fluidic substrate with an array of microlenses, and a COC coupling prism integrated with the waveguide substrate or cover plate. The double-sided hot embossed fluidic substrate had sampling zone microchannels on the bottom and microlenses on the top. Dissolved COC injected into polydimethylsiloxane (PDMS) lost molds embedded the waveguides in the PMMA cover plate and formed the integrated coupling prism. The embedded COC waveguide was flycut down to 50 μm. The cover plate and shallow, 1:20 aspect ratio, microchannels were thermal fusion bonded using a pressure-assisted boiling point control system, without sagging. The large COC prism coupled better to the waveguide. The highest intensity evanescent excitation of the waveguide was obtained near the critical angle. The maximum signal-to-noise ratio (SNR) was 119 and the lowest detection limit was 7.34 × 11 -20 mol at a SNR of 2 for a 100 μm wide by 50 μm deep waveguide. The microlenses highly focused the fluorescent radiation in the sampling zone.

Existing methods for sealing chip-to-chip (or module-to-motherboard) microfluidic interconnects commonly use additional interconnect components (O-rings, gaskets, and tubing), and manual handling expertise for assembly. Novel gasketless superhydrophobic fluidic interconnects (GSFIs) sealed by transparent superhydrophobic surfaces, forming liquid bridges between the fluidic ports for fluidic passages were demonstrated. Two test platforms were designed, fabricated, and evaluated, a multi-port chip system (ten interconnects) and a modules-on-a-motherboard system (four interconnects). System assembly in less than 3 sec was done by embedded magnets and pin-in-V-groove structures. Flow tests with deionized (DI) water, ethanol/water mixture, and plasma confirmed no leakage through the gasketless interconnects up to a maximum flow rate of $100~\mu \text{L}$ /min for the multi-port chip system. The modules-on-a-motherboard system showed no leakage of water at a flow rate of $20~\mu \text{L}$ /min and a pressure drop of 3.71 psi. Characterization of the leakage pressure as a function of the surface tension of the sample liquid in the multi-port chip system revealed that lower surface tension of the liquid led to lower static water contact angles on the superhydrophobic-coated substrate and lower leakage pressures. The high-density, rapidly assembled, gasketless interconnect technology will open up new avenues for chip-to-chip fluid transport in complex microfluidic modular systems. [2020-0168]

A multi-scale fluidic motherboard, which can be used in a universal molecular processing system (uMPS) integrated with task-specific processing modules, was designed and fabricated in thermoplastics. The motherboard consists of a coverplate and a substrate. The coverplate included fluidic interconnects and thermal grooves on the top side, and the corresponding interconnects on the bottom side. The substrate was comprised of cell lysis microchannels, micromixers, and flow-connecting microchannels on the top side, and reservoirs for sample inputs and waste output, thermal grooves, and valve seats for flow control on the bottom side. The coverplates and substrates were fabricated with double-sided hot embossing of polycarbonate (PC) using four micromilled brass molds, two for the coverplate and another two for the substrate. Evaluation of the relative front-to-backside alignment for the double-sided hot embossing yielded an accuracy of 25 μm ± 14 μm (average ± standard deviation) for the coverplates and 30 μm ± 20 μm for the substrates. Thermal fusion bonding (TFB) of the coverplate and substrate was done using a spring plunger bonding setup with a range of temperatures and pressures. The motherboard bonded at 154 °C and 12.0 psi for 2 hours in a convection oven produced complete bonding with a little deformation of the valve seats. The complete motherboard will be integrated with the task-specific processing modules in the uMPS for investigating circulating markers from whole blood for precision molecular diagnosis of disease at low cost and with high fidelity.

Blood samples from patients with plasma cell disorders were analysed for the presence of circulating plasma cells (CPCs) using a microfluidic device modified with monoclonal anti-CD138 antibodies. CPCs were immuno-phenotyped using a CD38/CD56/CD45 panel and identified in 78% of patients with monoclonal gammopathy of undetermined significance (MGUS), all patients with smouldering and symptomatic multiple myeloma (MM), and none in the controls. The burden of CPCs was higher in patients with symptomatic MM compared with MGUS and smouldering MM (p < 0.05). FISH analysis revealed the presence of chromosome 13 deletions in CPCs that correlated with bone marrow results. Point mutations in KRAS were identified, including different mutations from sub-clones derived from the same patient. The microfluidic assay represents a highly sensitive method for enumerating CPCs and allows for the cytogenetic and molecular characterization of CPCs.

In the context of oncology, liquid biopsies consist of harvesting cancer biomarkers, such as circulating tumor cells, tumor-derived cell-free DNA, and extracellular vesicles, from bodily fluids. These biomarkers provide a source of clinically actionable molecular information that can enable precision medicine. Herein, we review technologies for the molecular profiling of liquid biopsy markers with special emphasis on the analysis of low abundant markers from mixed populations.

A method for the design, construction, and assembly of modular, polymer-based, microfluidic devices using simple micro-assembly technology was demonstrated to build an integrated fluidic system consisting of vertically stacked modules for carrying out multi-step molecular assays. As an example of the utility of the modular system, point mutation detection using the ligase detection reaction (LDR) following amplification by the polymerase chain reaction (PCR) was carried out. Fluid interconnects and standoffs ensured that temperatures in the vertically stacked reactors were within ± 0.2 C° at the center of the temperature zones and ± 1.1 C° overall. The vertical spacing between modules was confirmed using finite element models (ANSYS, Inc., Canonsburg, PA) to simulate the steady-state temperature distribution for the assembly. Passive alignment structures, including a hemispherical pin-in-hole, a hemispherical pin-in-slot, and a plate-plate lap joint, were developed using screw theory to enable accurate exactly constrained assembly of the microfluidic reactors, cover sheets, and fluid interconnects to facilitate the modular approach. The mean mismatch between the centers of adjacent through holes was 64 ± 7.7 μm, significantly reducing the dead volume necessary to accommodate manufacturing variation. The microfluidic components were easily assembled by hand and the assembly of several different configurations of microfluidic modules for executing the assay was evaluated. Temperatures were measured in the desired range in each reactor. The biochemical performance was comparable to that obtained with benchtop instruments, but took less than 45 min to execute, half the time.

Circulating tumor cells consist of phenotypically distinct subpopulations that originate from the tumor microenvironment. We report a circulating tumor cell dual selection assay that uses discrete microfluidics to select circulating tumor cell subpopulations from a single blood sample; circulating tumor cells expressing the established marker epithelial cell adhesion molecule and a new marker, fibroblast activation protein alpha, were evaluated. Both circulating tumor cell subpopulations were detected in metastatic ovarian, colorectal, prostate, breast, and pancreatic cancer patients and 90% of the isolated circulating tumor cells did not co-express both antigens. Clinical sensitivities of 100% showed substantial improvement compared to epithelial cell adhesion molecule selection alone. Owing to high purity (>80%) of the selected circulating tumor cells, molecular analysis of both circulating tumor cell subpopulations was carried out in bulk, including next generation sequencing, mutation analysis, and gene expression. Results suggested fibroblast activation protein alpha and epithelial cell adhesion molecule circulating tumor cells are distinct subpopulations and the use of these in concert can provide information needed to navigate through cancer disease management challenges.

Objectives: Little is known about the role of circulating tumor cells (CTCs) in epithelial ovarian cancer (EOC). Methods available for selecting and enumerating CTCs traditionally analyze cells based on expression of epithelial cell adhesion molecule (EpCAM) only. Fibroblast activation protein α (FAPα), a marker of activated stromal fibroblasts in tumors, is highly expressed in EOC. The goal of this study was to evaluate the sensitivity of adding FAPα as a selection marker for the isolation of CTCs in EOC, and to compare FAPα and EpCAM expressing CTCs in various subgroups of women with EOC.Methods: To isolate CTCs, we used 2 microfluidic chips in series, one with antibodies specific to cells bearing FAPα (CTCFAPα), the other to EpCAM (CTCEpCAM). For analysis, EOC patients were divided into 3 groups: patients with advanced-stage (III/IV) disease undergoing interval debulking after neoadjuvant platinum-based chemotherapy (A-EOC-chemo), patients with advanced-stage disease undergoing primary debulking without prior chemotherapy (A-EOC-no chemo), and patients with early-stage (stage I) disease. Blood specimens from 11 normal donors were also analyzed.Results: Sixteen patients with EOC were enrolled in this study, 8 A-EOC-no chemo, 5 A-EOC-chemo, and 3 stage I (Fig. 1). Median CTCEpCAM and CTCFAPα count was 121 and 31 for stage I, 214 and 58 for A-EOC-no chemo, 48 and 28 for A-EOC-chemo, and 0.1 and 0.3 for normal donors, respectively. EpCAM and FAPα antigens were not co-expressed in single CTCs. Using our dual selection strategy, the sensitivity of CTC detection was 100% for all cohorts, including stage I patients. A 3-fold decrease in median CTCEpCAM count was observed for A-EOC-chemo patients compared with A-EOC-no-chemo patients (P < .007), but no differences were seen in median CTCFAPα counts between these 2 groups.Table 1Average and median CTCFAPα and CTCEpCAM collected from EOC patients and normal donors.Table 1Average and median CTCFAPα and CTCEpCAM collected from EOC patients and normal donors. Objectives: Little is known about the role of circulating tumor cells (CTCs) in epithelial ovarian cancer (EOC). Methods available for selecting and enumerating CTCs traditionally analyze cells based on expression of epithelial cell adhesion molecule (EpCAM) only. Fibroblast activation protein α (FAPα), a marker of activated stromal fibroblasts in tumors, is highly expressed in EOC. The goal of this study was to evaluate the sensitivity of adding FAPα as a selection marker for the isolation of CTCs in EOC, and to compare FAPα and EpCAM expressing CTCs in various subgroups of women with EOC. Methods: To isolate CTCs, we used 2 microfluidic chips in series, one with antibodies specific to cells bearing FAPα (CTCFAPα), the other to EpCAM (CTCEpCAM). For analysis, EOC patients were divided into 3 groups: patients with advanced-stage (III/IV) disease undergoing interval debulking after neoadjuvant platinum-based chemotherapy (A-EOC-chemo), patients with advanced-stage disease undergoing primary debulking without prior chemotherapy (A-EOC-no chemo), and patients with early-stage (stage I) disease. Blood specimens from 11 normal donors were also analyzed. Results: Sixteen patients with EOC were enrolled in this study, 8 A-EOC-no chemo, 5 A-EOC-chemo, and 3 stage I (Fig. 1). Median CTCEpCAM and CTCFAPα count was 121 and 31 for stage I, 214 and 58 for A-EOC-no chemo, 48 and 28 for A-EOC-chemo, and 0.1 and 0.3 for normal donors, respectively. EpCAM and FAPα antigens were not co-expressed in single CTCs. Using our dual selection strategy, the sensitivity of CTC detection was 100% for all cohorts, including stage I patients. A 3-fold decrease in median CTCEpCAM count was observed for A-EOC-chemo patients compared with A-EOC-no-chemo patients (P < .007), but no differences were seen in median CTCFAPα counts between these 2 groups.