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.

Microfluidic Isolation of Neuronal-Enriched Extracellular Vesicles Shows Distinct and Common Neurological Proteins in Long COVID, HIV Infection and Alzheimer's Disease.

Long COVID (LongC) is associated with a myriad of symptoms including cognitive impairment. We reported at the beginning of the COVID-19 pandemic that neuronal-enriched or L1CAM+ extracellular vesicles (nEVs) from people with LongC contained proteins associated with Alzheimer's disease (AD). Since that time, a subset of people with prior COVID infection continue to report neurological problems more than three months after infection. Blood markers to better characterize LongC are elusive. To further identify neuronal proteins associated with LongC, we maximized the number of nEVs isolated from plasma by developing a hybrid EV Microfluidic Affinity Purification (EV-MAP) technique. We isolated nEVs from people with LongC and neurological complaints, AD, and HIV infection with mild cognitive impairment. Using the OLINK platform that assesses 384 neurological proteins, we identified 11 significant proteins increased in LongC and 2 decreased (BST1, GGT1). Fourteen proteins were increased in AD and forty proteins associated with HIV cognitive impairment were elevated with one decreased (IVD). One common protein (BST1) was decreased in LongC and increased in HIV. Six proteins (MIF, ENO1, MESD, NUDT5, TNFSF14 and FYB1) were expressed in both LongC and AD and no proteins were common to HIV and AD. This study begins to identify differences and similarities in the neuronal response to LongC versus AD and HIV infection.

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.

Circulating Tumor Cell Subpopulations Predict Treatment Outcome in Pancreatic Ductal Adenocarcinoma (PDAC) Patients.

There is a high clinical unmet need to improve outcomes for pancreatic ductal adenocarcinoma (PDAC) patients, either with the discovery of new therapies or biomarkers that can track response to treatment more efficiently than imaging. We report an innovative approach that will generate renewed interest in using circulating tumor cells (CTCs) to monitor treatment efficacy, which, in this case, used PDAC patients receiving an exploratory new therapy, poly ADP-ribose polymerase inhibitor (PARPi)-niraparib-as a case study. CTCs were enumerated from whole blood using a microfluidic approach that affinity captures epithelial and mesenchymal CTCs using anti-EpCAM and anti-FAPα monoclonal antibodies, respectively. These antibodies were poised on the surface of two separate microfluidic devices to discretely capture each subpopulation for interrogation. The isolated CTCs were enumerated using immunophenotyping to produce a numerical ratio consisting of the number of mesenchymal to epithelial CTCs (denoted "Φ"), which was used as an indicator of response to therapy, as determined using computed tomography (CT). A decreasing value of Φ during treatment was indicative of tumor response to the PARPi and was observed in 88% of the enrolled patients (n = 31). Changes in Φ during longitudinal testing were a better predictor of treatment response than the current standard CA19-9. We were able to differentiate between responders and non-responders using ΔΦ (p = 0.0093) with higher confidence than CA19-9 (p = 0.033). For CA19-9 non-producers, ΔΦ correctly predicted the outcome in 72% of the PDAC patients. Sequencing of the gDNA extracted from affinity-selected CTC subpopulations provided information that could be used for patient enrollment into the clinical trial based on their tumor mutational status in DNA repair genes.

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.

Assessing Breast Cancer Molecular Subtypes Using Extracellular Vesicles' mRNA.

Extracellular vesicles (EVs) carry RNA cargo that is believed to be associated with the cell-of-origin and thus have the potential to serve as a minimally invasive liquid biopsy marker for supplying molecular information to guide treatment decisions (i.e., precision medicine). We report the affinity isolation of EV subpopulations with monoclonal antibodies attached to the surface of a microfluidic chip that is made from a plastic to allow for high-scale production. The EV microfluidic affinity purification (EV-MAP) chip was used for the isolation of EVs sourced from two-orthogonal cell types and was demonstrated for its utility in a proof-of-concept application to provide molecular subtyping information for breast cancer patients. The orthogonal selection process better recapitulated the epithelial tumor microenvironment by isolating two subpopulations of EVs: EVEpCAM (epithelial cell adhesion molecule, epithelial origin) and EVFAPα (fibroblast activation protein α, mesenchymal origin). The EV-MAP provided recovery >80% with a specificity of 99 ± 1% based on exosomal mRNA (exo-mRNA) and real time-droplet digital polymerase chain reaction results. When selected from the plasma of healthy donors and breast cancer patients, EVs did not differ in size or total RNA mass for both markers. On average, 0.5 mL of plasma from breast cancer patients yielded ∼2.25 ng of total RNA for both EVEpCAM and EVFAPα, while in the case of cancer-free individuals, it yielded 0.8 and 1.25 ng of total RNA from EVEpCAM and EVFAPα, respectively. To assess the potential of these two EV subpopulations to provide molecular information for prognostication, we performed the PAM50 test (Prosigna) on exo-mRNA harvested from each EV subpopulation. Results suggested that EVEpCAM and EVFAPα exo-mRNA profiling using subsets of the PAM50 genes and a novel algorithm (i.e., exo-PAM50) generated 100% concordance with the tumor tissue.

A Simple Micromilled Microfluidic Impedance Cytometer with Vertical Parallel Electrodes for Cell Viability Analysis.

Microfluidic impedance cytometry has been demonstrated as an effective platform for single cell analysis, taking advantage of microfabricated features and dielectric cell sensing methods. In this study, we present a simple microfluidic device to improve the sensitivity, accuracy, and throughput of single suspension cell viability analysis using vertical sidewall electrodes fabricated by a widely accessible negative manufacturing method. A microchannel milled through a 75 µm platinum wire, which was embedded into poly-methyl-methacrylate (PMMA), created a pair of parallel vertical sidewall platinum electrodes. Jurkat cells were interrogated in a custom low-conductivity buffer (1.2 ± 0.04 mS/cm) to reduce current leakage and increase device sensitivity. Confirmed by live/dead staining and electron microscopy, a single optimum excitation frequency of 2 MHz was identified at which live and dead cells were discriminated based on the disruption in the cell membrane associated with cell death. At this frequency, live cells were found to exhibit changes in the impedance phase with no appreciable change in magnitude, while dead cells displayed the opposite behavior. Correlated with video microscopy, a computational algorithm was created that could identify cell detection events and determine cell viability status by application of a mathematical correlation method.

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.

Nanofluidic devices for the separation of biomolecules.

Over the last 30-years, microchip electrophoresis and its applications have expanded due to the benefits it offers. Nanochip electrophoresis, on the other hand, is viewed as an evolving area of electrophoresis because it offers some unique advantages not associated with microchip electrophoresis. These advantages arise from unique phenomena that occur in the nanometer domain not readily apparent in the microscale domain due to scale-dependent effects. Scale-dependent effects associated with nanochip electrophoresis includes high surface area-to-volume ratio, electrical double layer overlap generating parabolic flow even for electrokinetic pumping, concentration polarization, transverse electromigration, surface charge dominating flow, and surface roughness. Nanochip electrophoresis devices consist of channels with dimensions ranging from 1 to 1000 nm including classical (1-100 nm) and extended (100 nm - 1000 nm) nanoscale devices. In this review, we highlight scale-dependent phenomena associated with nanochip electrophoresis and the utilization of those phenomena to provide unique biomolecular separations that are not possible with microchip electrophoresis. We will also review the range of materials used for nanoscale separations and the implication of material choice for the top-down fabrication and operation of these devices. We will also provide application examples of nanochip electrophoresis for biomolecule separations with an emphasis on nano-electrophoresis (nEP) and nano-electrochromatography (nEC).

Microfluidic affinity selection of active SARS-CoV-2 virus particles.

We report a microfluidic assay to select active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles (VPs), which were defined as intact particles with an accessible angiotensin-converting enzyme 2 receptor binding domain (RBD) on the spike (S) protein, from clinical samples. Affinity selection of SARS-CoV-2 particles was carried out using injection molded microfluidic chips, which allow for high-scale production to accommodate large-scale screening. The microfluidic contained a surface-bound aptamer directed against the virus's S protein RBD to affinity select SARS-CoV-2 VPs. Following selection (~94% recovery), the VPs were released from the chip's surface using a blue light light-emitting diode (89% efficiency). Selected SARS-CoV-2 VP enumeration was carried out using reverse transcription quantitative polymerase chain reaction. The VP selection assay successfully identified healthy donors (clinical specificity = 100%) and 19 of 20 patients with coronavirus disease 2019 (COVID-19) (95% sensitivity). In 15 patients with COVID-19, the presence of active SARS-CoV-2 VPs was found. The chip can be reprogrammed for any VP or exosomes by simply changing the affinity agent.

Fluidic operation of a polymer-based nanosensor chip for analysing single molecules.

Most medical diagnostic tests are expensive, involve slow turnaround times from centralized laboratories and require highly specialized equipment with seasoned technicians to carry out the assay. To facilitate realization of precision medicine at the point of care, we have developed a mixed-scale nanosensor chip featuring high surface area pillar arrays where solid-phase reactions can be performed to detect and identify nucleic acid targets found in diseased patients. Products formed can be identified and detected using a polymer nanofluidic channel. To guide delivery of this platform, we discuss the operation of various components of the device and simulations (COMSOL) used to guide the design by investigating parameters such as pillar array loading, and hydrodynamic and electrokinetic flows. The fabrication of the nanosensor is discussed, which was performed using a silicon (Si) master patterned with a combination of focused ion beam milling and photolithography with deep reactive ion etching. The mixed-scale patterns were transferred into a thermoplastic via thermal nanoimprint lithography, which facilitated fabrication of the nanosensor chip making it appropriate for in vitro diagnostics. The results from COMSOL were experimentally verified for hydrodynamic flow using Rhodamine B as a fluorescent tracer and electrokinetic flow using single fluorescently labelled oligonucleotides (single-stranded DNAs, ssDNAs).

Air bubble removal: Wettability contrast enabled microfluidic interconnects.

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.

In-plane Extended Nano-coulter Counter (XnCC) for the Label-free Electrical Detection of Biological Particles.

We report an in-plane extended nanopore Coulter counter (XnCC) chip fabricated in a thermoplastic via imprinting. The fabrication of the sensor utilized both photolithography and focused ion beam milling to make the microfluidic network and the in-plane pore sensor, respectively, in Si from which UV resin stamps were generated followed by thermal imprinting to produce the final device in the appropriate plastic (cyclic olefin polymer, COP). As an example of the utility of this in-plane extended nanopore sensor, we enumerated SARS-CoV-2 viral particles (VPs) affinity-selected from saliva and extracellular vesicles (EVs) affinity-selected from plasma samples secured from mouse models exposed to different ionizing radiation doses.

Modifying surface charge density of thermoplastic nanofluidic biosensors by multivalent cations within the slip plane of the electric double layer.

Thermoplastic nanofluidic devices are promising platforms for sensing single biomolecules due to their mass fabrication capability. When the molecules are driven electrokinetically through nanofluidic networks, surface charges play a significant role in the molecular capture and transportation, especially when the thickness of the electrical double layer is close to the dimensions of the nanostructures in the device. Here, we used multivalent cations to alter the surface charge density of thermoplastic nanofluidic devices. The surface charge alteration was done by filling the device with a multivalent ionic solution, followed by withdrawal of the solution and replacing it with KCl for conductance measurement. A systematic study was performed using ionic solutions containing Mg2+ and Al3+ for nanochannels made of three polymers: poly(ethylene glycol) diacrylate (PEGDA), poly(methyl methacrylate) (PMMA) and cyclic olefin copolymer (COC). Overall, multivalent cations within the slip plane decreased the effective surface charge density of the device surface and the reduction rate increased with the cation valency, cation concentration and the surface charge density of thermoplastic substrates. We demonstrated that a 10-nm diameter in-plane nanopore formed in COC allowed translocation of λ-DNA molecules after Al3+ modification, which is attributed to the deceased viscous drag force in the nanopore by the decreased surface charge density. This work provides a general method to manipulate surface charge density of nanofluidic devices for biomolecule resistive pulse sensing. Additionally, the experimental results support ion-ion correlations as the origin of charge inversion over specific chemical adsorption.

Leakage pressures for gasketless superhydrophobic fluid interconnects for modular lab-on-a-chip systems.

Chip-to-chip and world-to-chip fluidic interconnections are paramount to enable the passage of liquids between component chips and to/from microfluidic systems. Unfortunately, most interconnect designs add additional physical constraints to chips with each additional interconnect leading to over-constrained microfluidic systems. The competing constraints provided by multiple interconnects induce strain in the chips, creating indeterminate dead volumes and misalignment between chips that comprise the microfluidic system. A novel, gasketless superhydrophobic fluidic interconnect (GSFI) that uses capillary forces to form a liquid bridge suspended between concentric through-holes and acting as a fluid passage was investigated. The GSFI decouples the alignment between component chips from the interconnect function and the attachment of the meniscus of the liquid bridge to the edges of the holes produces negligible dead volume. This passive seal was created by patterning parallel superhydrophobic surfaces (water contact angle ≥ 150°) around concentric microfluidic ports separated by a gap. The relative position of the two polymer chips was determined by passive kinematic constraints, three spherical ball bearings seated in v-grooves. A leakage pressure model derived from the Young-Laplace equation was used to estimate the leakage pressure at failure for the liquid bridge. Injection-molded, Cyclic Olefin Copolymer (COC) chip assemblies with assembly gaps from 3 to 240 µm were used to experimentally validate the model. The maximum leakage pressure measured for the GSFI was 21.4 kPa (3.1 psig), which corresponded to a measured mean assembly gap of 3 µm, and decreased to 0.5 kPa (0.073 psig) at a mean assembly gap of 240 µm. The effect of radial misalignment on the efficacy of the gasketless seals was tested and no significant effect was observed. This may be a function of how the liquid bridges are formed during the priming of the chip, but additional research is required to test that hypothesis.

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.

Isolation and analysis methods of extracellular vesicles (EVs).

Extracellular vesicles (EVs) have been recognized as an evolving biomarker within the liquid biopsy family. While carrying both host cell proteins and different types of RNAs, EVs are also present in sufficient quantities in biological samples to be tested using many molecular analysis platforms to interrogate their content. However, because EVs in biological samples are comprised of both disease and non-disease related EVs, enrichment is often required to remove potential interferences from the downstream molecular assay. Most benchtop isolation/enrichment methods require > milliliter levels of sample and can cause varying degrees of damage to the EVs. In addition, some of the common EV benchtop isolation methods do not sort the diseased from the non-diseased related EVs. Simultaneously, the detection of the overall concentration and size distribution of the EVs is highly dependent on techniques such as electron microscopy and Nanoparticle Tracking Analysis, which can include unexpected variations and biases as well as complexity in the analysis. This review discusses the importance of EVs as a biomarker secured from a liquid biopsy and covers some of the traditional and non-traditional, including microfluidics and resistive pulse sensing, technologies for EV isolation and detection, respectively.

Tailoring Thermoplastic In-Plane Nanopore Size by Thermal Fusion Bonding for the Analysis of Single Molecules.

We report a simple method for tailoring the size of in-plane nanopores fabricated in thermoplastics for single-molecule sensing. The in-plane pores were fabricated via nanoimprint lithography (NIL) from resin stamps, which were generated from Si masters. We could reduce the size of the in-plane nanopores from 30 to ∼10 nm during the thermal fusion bonding (TFB) step, which places a cover plate over the imprinted polymer substrate under a controlled pressure and temperature to form the relevant nanofluidic devices. Increased pressures during TFB caused the cross-sectional area of the in-plane pore to be reduced. The in-plane nanopores prepared with different TFB pressures were utilized to detect single-λ-DNA molecules via resistive pulse sensing, which showed a higher current amplitude in devices bonded at higher pressures. Using this method, we also show the ability to tune the pore size to detect single-stranded (ss) RNA molecules and single ribonucleotide adenosine monophosphate (rAMP). However, due to the small size of the pores required for detection of the ssRNA and rAMPs, the surface charge arising from carboxylate groups generated during O2 plasma oxidation of the surfaces of the nanopores to make them wettable had to be reduced to allow translocation of coions. This was accomplished using EDC/NHS coupling chemistry and ethanolamine. This simple modification chemistry increased the event frequency from ∼1 s-1 to >136 s-1 for an ssRNA concentration of 100 nM.

Analytical Technologies for Liquid Biopsy of Subcellular Materials.

Liquid biopsy markers, which can be secured from a simple blood draw or other biological samples, are used to manage a variety of diseases and even monitor for bacterial or viral infections. Although there are several different types of liquid biopsy markers, the subcellular ones, including cell-free DNA, microRNA, extracellular vesicles, and viral particles, are evolving in terms of their utility. A challenge with liquid biopsy markers is that they must be enriched from the biological sample prior to analysis because they are a vast minority in a mixed population, and potential interferences may be present in the sample matrix that can inhibit profiling the molecular cargo from the subcellular marker. In this article, we discuss existing and developing analytical enrichment platforms used to isolate subcellular liquid biopsy markers, and discuss their figures of merit such as recovery, throughput, and purity.

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.