Efficient Delivery of Biological Cargos into Primary Cells by Electrodeposited Nanoneedles via Cell-Cycle-Dependent Endocytosis.

Nanoneedles are a useful tool for delivering exogenous biomolecules to cells. Although therapeutic applications have been explored, the mechanism regarding how cells interact with nanoneedles remains poorly studied. Here, we present a new approach for the generation of nanoneedles, validated their usefulness in cargo delivery, and studied the underlying genetic modulators during delivery. We fabricated arrays of nanoneedles based on electrodeposition and quantified its efficacy of delivery using fluorescently labeled proteins and siRNAs. Notably, we revealed that our nanoneedles caused the disruption of cell membranes, enhanced the expression of cell-cell junction proteins, and downregulated the expression of transcriptional factors of NFκB pathways. This perturbation trapped most of the cells in G2 phase, in which the cells have the highest endocytosis activities. Taken together, this system provides a new model for the study of interactions between cells and high-aspect-ratio materials.

PillarX: A Microfluidic Device to Profile Circulating Tumor Cell Clusters Based on Geometry, Deformability, and Epithelial State.

Circulating tumor cell (CTC) clusters are associated with increased metastatic potential and worse patient prognosis, but are rare, difficult to count, and poorly characterized biophysically. The PillarX device described here is a bimodular microfluidic device (Pillar-device and an X-magnetic device) to profile single CTCs and clusters from whole blood based on their size, deformability, and epithelial marker expression. Larger, less deformable clusters and large single cells are captured in the Pillar-device and sorted according to pillar gap sizes. Smaller, deformable clusters and single cells are subsequently captured in the X-device and separated based on epithelial marker expression using functionalized magnetic nanoparticles. Clusters of established and primary breast cancer cells with variable degrees of cohesion driven by different cell-cell adhesion protein expression are profiled in the device. Cohesive clusters exhibit a lower deformability as they travel through the pillar array, relative to less cohesive clusters, and have greater collective invasive behavior. The ability of the PillarX device to capture clusters is validated in mouse models and patients of metastatic breast cancer. Thus, this device effectively enumerates and profiles CTC clusters based on their unique geometrical, physical, and biochemical properties, and could form the basis of a novel prognostic clinical tool.

Magnetic Ranking Cytometry: Profiling Rare Cells at the Single-Cell Level.

Cellular heterogeneity in biological systems presents major challenges in the diagnosis and treatment of disease and also complicates the deconvolution of complex cellular phenomena. Single-cell analysis methods provide information that is not masked by the intrinsic heterogeneity of the bulk population and can therefore be applied to gain insights into heterogeneity among different cell subpopulations with fine resolution. Over the last 5 years, an explosion in the number of single-cell measurement methods has occurred. However, most of these methods are applicable to pure populations of cultured cells and are not able to handle high levels of phenotypic heterogeneity or a large background of nontarget cells. Microfluidics is an attractive tool for single cell manipulation as it enables individual encasing of single cells, allowing for high-throughput analysis with precise control of the local environment. Our laboratory has developed a new microfluidics-based analytical strategy to meet this unmet need referred to as magnetic ranking cytometry (MagRC). Cells expressing a biomarker of interest are labeled with receptor-coated magnetic nanoparticles and isolated from nontarget cells using a microfluidic device. The device ranks the cells according to the level of bound magnetic nanoparticles, which corresponds to the expression level of a target biomarker. Over the last several years, two generations of MagRC devices have been developed for different applications. The first-generation MagRC devices are powerful tools for the quantitation and analysis of rare cells present in heterogeneous samples, such as circulating tumor cells, stem cells, and pathogenic bacteria. The second-generation MagRC devices are compatible with the efficient recovery of cells sorted on the basis of protein expression and can be used to analyze large populations of cells and perform phenotypic CRISPR screens. To improve analytical precision, newer iterations of the first-generation and second-generation MagRC devices have been integrated with electrochemical sensors and Hall effect sensors, respectively. Both generations of MagRC devices permit the isolation of viable cells, which sets the stage for a wide range of applications, such as generating cell lines from rare cells and in vitro screening for effective therapeutic interventions in cancer patients to realize the promise of personalized medicine. This Account summarizes the development and application of the MagRC and describes a suite of advances that have enabled single-cell tumor cell analysis and monitoring tumor response to therapy, stem cell analysis, and detection of pathogens.

Nanoparticle-Mediated Capture and Electrochemical Detection of Methicillin-Resistant Staphylococcus aureus.

The spread of antibiotic-resistant bacteria poses a global threat to public health. Conventional bacterial detection and identification methods often require pre-enrichment and/or sample preprocessing and purification steps that can prolong diagnosis by days. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most widespread antibiotic-resistant bacteria and is the leading cause of hospital-acquired infections. Here, we have developed a method to specifically capture and detect MRSA directly from patient nasal swabs with no prior culture and minimal processing steps using a microfluidic device and antibody-functionalized magnetic nanoparticles. Bacteria are captured based on antibody recognition of a membrane-bound protein marker that confers ß-lactam antibiotic resistance. MRSA identification is then achieved by the use of a strain-specific antibody functionalized with alkaline phosphatase for electrochemical detection. This approach ensures that only those bacteria of the target strain and resistance profile are measured. The method has a limit of detection of 845 CFU/mL and excellent discrimination against high concentrations of common nontarget nasal flora with a turnaround time of under 4.5 h. This detection method was successfully validated using clinical nasal swab specimens ( n = 30) and has the potential to be tailored to various bacterial targets.

Phenotypic Profiling of Circulating Tumor Cells in Metastatic Prostate Cancer Patients Using Nanoparticle-Mediated Ranking.

The analysis of circulating tumor cells (CTCs) provides a means to collect information about the evolving properties of a tumor during cancer progression and treatment. For patients with metastatic prostate cancer, noninvasive serial measurements of bloodborne cells may provide a means to tailor therapeutic decisions based on an individual patient's response. Here, we used a high-sensitivity profiling approach to monitor CTCs in patients with metastatic castrate-resistant prostate cancer (mCRPC) undergoing treatment with abiraterone and enzalutamide, two drugs used to treat advanced prostate cancer. The capture and profiling approach uses antibody-functionalized magnetic nanoparticles to sort cells according to protein expression levels. CTCs are tagged with magnetic nanoparticles conjugated to an antibody specific for the epithelial cell adhesion molecule (EpCAM) and sorted into four zones of a microfluidic device based on EpCAM expression levels. Our approach was compared to the FDA-cleared CellSearch method, and we demonstrate significantly higher capture efficiency of low-EpCAM cells compared to the commercial method. The nanoparticle-based approach detected CTCs from 86% of patients at baseline, compared to CellSearch which only detected CTCs from 60% of patients. Patients were stratified as prostate specific antigen (PSA) progressive versus responsive based on clinically acceptable definitions, and it was observed that patients with a limited response to therapy had elevated levels of androgen receptor variant 7 (ARV7) and the mesenchymal marker, N-cadherin, expressed on their CTCs. In addition, these CTCs exhibited lower EpCAM expression. The results highlight features of CTCs associated with disease progression on abiraterone or enzalutamide, including mesenchymal phenotypes and increased expression levels of ARV7. The use of a high-sensitivity method to capture and profile CTCs provides more informative data concerning the phenotypic properties of these cells as patients undergo treatment relative to an FDA-cleared method.

Three-Dimensional Nanostructured Architectures Enable Efficient Neural Differentiation of Mesenchymal Stem Cells via Mechanotransduction.

Cell morphology and geometry affect cellular processes such as stem cell differentiation, suggesting that these parameters serve as fundamental regulators of biological processes within the cell. Hierarchical architectures featuring micro- and nanotopographical features therefore offer programmable systems for stem cell differentiation. However, a limited number of studies have explored the effects of hierarchical architectures due to the complexity of fabricating systems with rationally tunable micro- and nanostructuring. Here, we report three-dimensional (3D) nanostructured microarchitectures that efficiently regulate the fate of human mesenchymal stem cells (hMSCs). These nanostructured architectures strongly promote cell alignment and efficient neurogenic differentiation where over 85% of hMSCs express microtubule-associated protein 2 (MAP2), a mature neural marker, after 7 days of culture on the nanostructured surface. Remarkably, we found that the surface morphology of nanostructured surface is a key factor that promotes neurogenesis and that highly spiky structures promote more efficient neuronal differentiation. Immunostaining and gene expression profiling revealed significant upregulation of neuronal markers compared to unpatterned surfaces. These findings suggest that the 3D nanostructured microarchitectures can play a critical role in defining stem cell behavior.

Potential-Responsive Surfaces for Manipulation of Cell Adhesion, Release, and Differentiation.

In living systems, interfacial molecular interactions control many biological processes. New stimuli-responsive strategies are desired to provide versatile model systems that can regulate cell behavior in vitro. Described here are potential-responsive surfaces that control cell adhesion and release as well as stem cell differentiation. Cell adhesion can be modulated dynamically by applying negative and positive potentials to surfaces functionalized with tailored monolayers. This process alters cell morphology and ultimately controls behavior and the fate of the cells. Cells can be detached from the electrode surface as intact clusters with different geometries using electrochemical potentials. Importantly, morphological changes during adhesion guide stem cell differentiation. The higher accessibility of the peptide under a positive applied potential causes phenotypic changes in the cells that are hallmarks of osteogenesis, whereas lower accessibility of the peptide promoted by negative potentials leads to adipogenesis.

A Hierarchical 3D Nanostructured Microfluidic Device for Sensitive Detection of Pathogenic Bacteria.

Efficient capture and rapid detection of pathogenic bacteria from body fluids lead to early diagnostics of bacterial infections and significantly enhance the survival rate. We propose a universal nano/microfluidic device integrated with a 3D nanostructured detection platform for sensitive and quantifiable detection of pathogenic bacteria. Surface characterization of the nanostructured detection platform confirms a uniform distribution of hierarchical 3D nano-/microisland (NMI) structures with spatial orientation and nanorough protrusions. The hierarchical 3D NMI is the unique characteristic of the integrated device, which enables enhanced capture and quantifiable detection of bacteria via both a probe-free and immunoaffinity detection method. As a proof of principle, we demonstrate probe-free capture of pathogenic Escherichia coli (E. coli) and immunocapture of methicillin-resistant-Staphylococcus aureus (MRSA). Our device demonstrates a linear range between 50 and 104 CFU mL-1 , with average efficiency of 93% and 85% for probe-free detection of E. coli and immunoaffinity detection of MRSA, respectively. It is successfully demonstrated that the spatial orientation of 3D NMIs contributes in quantifiable detection of fluorescently labeled bacteria, while the nanorough protrusions contribute in probe-free capture of bacteria. The ease of fabrication, integration, and implementation can inspire future point-of-care devices based on nanomaterial interfaces for sensitive and high-throughput optical detection.

Electrochemical Methods for the Analysis of Clinically Relevant Biomolecules.

Rapid progress in identifying biomarkers that are hallmarks of disease has increased demand for high-performance detection technologies. Implementation of electrochemical methods in clinical analysis may provide an effective answer to the growing need for rapid, specific, inexpensive, and fully automated means of biomarker analysis. This Review summarizes advances from the past 5 years in the development of electrochemical sensors for clinically relevant biomolecules, including small molecules, nucleic acids, and proteins. Various sensing strategies are assessed according to their potential for reaching relevant limits of sensitivity, specificity, and degrees of multiplexing. Furthermore, we address the remaining challenges and opportunities to integrate electrochemical sensing platforms into point-of-care solutions.

Profiling Functional and Biochemical Phenotypes of Circulating Tumor Cells Using a Two-Dimensional Sorting Device.

During cancer progression, tumors shed circulating tumor cells (CTCs) into the bloodstream. CTCs that originate from the same primary tumor can have heterogeneous phenotypes and, while some CTCs possess benign properties, others have high metastatic potential. Deconstructing the heterogeneity of CTCs is challenging and new methods are needed that can sort small numbers of cancer cells according to their phenotypic properties. Here we describe a new microfluidic approach that profiles, along two independent phenotypic axes, the behavior of heterogeneous cell subpopulations. Cancer cells are first profiled according to expression of a surface marker using a nanoparticle-enabled approach. Along the second dimension, these subsets are further separated into subpopulations corresponding to migration profiles generated in response to a chemotactic agent. We deploy this new technique and find a strong correlation between the surface expression and migration potential of CTCs present in blood from mice with xenografted tumors. This system provides an important new means to characterize functional diversity in circulating tumor cells.

Aptamer and Antisense-Mediated Two-Dimensional Isolation of Specific Cancer Cell Subpopulations.

Cancer cells, and in particular those found circulating in blood, can have widely varying phenotypes and molecular profiles despite a common origin. New methods are needed that can deconvolute the heterogeneity of cancer cells and sort small numbers of cells to aid in the characterization of cancer cell subpopulations. Here, we describe a new molecular approach to capturing cancer cells that isolates subpopulations using two-dimensional sorting. Using aptamer-mediated capture and antisense-triggered release, the new strategy sorts cells according to levels of two different markers and thereby separates them into their corresponding subpopulations. Using a phenotypic assay, we demonstrate that the subpopulations isolated have markedly different properties. This system provides an important new tool for identifying circulating tumor cell subtypes.

Beyond the Capture of Circulating Tumor Cells: Next-Generation Devices and Materials.

Over the last decade, significant progress has been made towards the development of approaches that enable the capture of rare circulating tumor cells (CTCs) from the blood of cancer patients, a critical capability for noninvasive tumor profiling. These advances have leveraged new insights in materials chemistry and microfluidics and allowed the capture and enumeration of CTCs with unprecedented sensitivity. However, it has become increasingly clear that simply capturing and counting tumor cells launched into the bloodstream may not provide the information needed to advance our understanding of the biology of these rare cells, or to allow us to better exploit them in medicine. A variety of advances have now emerged demonstrating that more information can be extracted from CTCs with next-generation devices and materials featuring tailored physical and chemical properties. In this Minireview, the last ten years of work in this area will be discussed, with an emphasis on the groundbreaking work of the last five years, during which the focus has moved beyond the simple capture of CTCs and gravitated towards approaches that enable in-depth analysis.

Protein electrocatalysis for direct sensing of circulating microRNAs.

MicroRNAs (miRNAs) are potentially useful biomarkers for diagnosis, classification, and prognosis of many diseases, including cancer. Herein, we developed a protein-facilitated electrocatalytic quadroprobe sensor (Sens(PEQ)) for detection of miRNA signature of chronic lymphocytic leukemia (CLL) in human serum. The developed signal-ON sensor provides a compatible combination of two DNA adaptor strands modified with four methylene blue molecules and electrocatalysis using glucose oxidase in order to enhance the overall signal gain. This enhanced sensitivity provided the response necessary to detect the low-abundant serum miRNAs without preamplification. The developed Sens(PEQ) is exquisitely sensitive to subtle π-stack perturbations and capable of distinguishing single base mismatches in the target miRNA. Furthermore, the developed sensor was employed for profiling of three endogenous miRNAs characteristic to CLL, including hsa-miR-16-5p, hsa-miR-21-5p, and hsa-miR-150-5p in normal healthy serum, chronic lymphocytic leukemia Rai stage 1 (CLL-1), and stage 3 (CLL-3) sera, using a non-human cel-miR-39-3p as an internal standard. The sensor results were verified by conventional SYBR green-based quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis.

Identification of druggable regulators of cell secretion via a kinome-wide screen and high-throughput immunomagnetic cell sorting.

The identification of genetic regulators of cell secretions is challenging because it requires the sorting of a large number of cells according to their secretion patterns. Here we report the development and applicability of a high-throughput microfluidic method for the analysis of the secretion levels of large populations of immune cells. The method is linked with a kinome-wide loss-of-function CRISPR screen, immunomagnetically sorting the cells according to their secretion levels, and the sequencing of their genomes to identify key genetic modifiers of cell secretion. We used the method, which we validated against flow cytometry for cytokines secreted from primary mouse CD4+ (cluster of differentiation 4-positive) T cells, to discover a subgroup of highly co-expressed kinase-coding genes that regulate interferon-gamma secretion by these cells. We validated the function of the kinases identified using RNA interference, CRISPR knockouts and kinase inhibitors and confirmed the druggability of selected kinases via the administration of a kinase inhibitor in an animal model of colitis. The technique may facilitate the discovery of regulatory mechanisms for immune-cell activation and of therapeutic targets for autoimmune diseases.

Three-mode electrochemical sensing of ultralow microRNA levels.

MicroRNAs (miRNAs) are an emerging class of biomarkers that are frequently deregulated in cancer cells and have shown great promise for cancer classification and prognosis. In this work, we developed a three-mode electrochemical sensor for detection and quantitation of ultralow levels of miRNAs in a wide dynamic range of measured concentrations. The sensor facilitates three detection modalities based on hybridization (H-SENS), p19 protein binding (P-SENS), and protein displacement (D-SENS). The combined three-mode sensor (HPD-SENS) identifies as low as 5 aM or 90 molecules of miRNA per 30 µL of sample without PCR amplification, and can be operated within the dynamic range from 10 aM to 1 µM. The HPD sensor is made on a commercially available gold nanoparticles-modified electrode and is suitable for analyzing multiple miRNAs on a single electrode. This three-mode sensor exhibits high selectivity and specificity and was used for sequential analysis of miR-32 and miR-122 on one electrode. In addition, the H-SENS can recognize miRNAs with different A/U and G/C content and distinguish between a fully matched miRNA and a miRNA comprising either a terminal or a middle single base mutation. Furthermore, the H- and P-SENS were successfully employed for direct detection and profiling of three endogenous miRNAs, including hsa-miR-21, hsa-miR-32, and hsa-miR-122 in human serum, and the sensor results were validated by qPCR.

Four-way junction formation promoting ultrasensitive electrochemical detection of microRNA.

MicroRNAs (miRNAs) represent a class of biomarkers that are frequently deregulated in cancer cells and have shown a great promise for cancer classification and prognosis. Here, we endeavored to develop a DNA four-way junction based electrochemical sensor (4J-SENS) for ultrasensitive miRNA analysis. The developed sensor can be operated within the dynamic range from 10 aM to 1 fM and detect as low as 2 aM of miR-122 (∼36 molecules per sample), without PCR amplification. Furthermore, the 4J-SENS was employed to profile endogenouse hsa-miR-122 in healthy human and chronic lymphocyitc leukemia (CLL) patient serum, and the results were validated by qPCR analysis.

Multifunctional electrochemical aptasensor for aptamer clones screening, virus quantitation in blood and viability assessment.

A novel attempt was made to develop a disposable multifunctional sensor for analysis of vaccinia virus (VACV), a promising oncolytic agent that can replicate in and kill tumor cells. Briefly, we developed aptamers specific to VACV that were negatively selected against human serum as well as human and mouse blood to be further utilized for viral analysis directly in serum and blood. In addition, the aptamers were negatively selected against heat-inactivated VACV to enable them to distinguish between viable and nonviable virus particles. The selected aptamers were integrated onto an electrochemical aptasensor to perform multiple functions, including quantification of VACV, viability assessment of the virus, and estimation of the binding affinity between the virus and the developed aptamers. The aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and a VACV-specific aptamer onto a gold nanoparticles modified screen-printed carbon electrode (GNPs-SPCE). Square wave voltammetry was employed to quantify VACV in serum and blood within the range of 150-900 PFU, with a detection limit of 60 PFU in 30 µL. According to the electrochemical affinity measurements, three virus specific aptamer clones, V-2, V-5, and V-9 exhibited the highest affinity to VACV. Furthermore, flow cytometry was employed to estimate the dissociation constants of the clones which were found to be 26.3, 40.9, and 24.7 nM, respectively. Finally, the developed aptasensor was able to distinguish between the intact virus and the heat-inactivated virus thanks to the tailored selectivity of the aptamers that was achieved via negative selection.

A magneto-activated nanoscale cytometry platform for molecular profiling of small extracellular vesicles.

Exosomal PD-L1 (exoPD-L1) has recently received significant attention as a biomarker predicting immunotherapeutic responses involving the PD1/PD-L1 pathway. However, current technologies for exosomal analysis rely primarily on bulk measurements that do not consider the heterogeneity found within exosomal subpopulations. Here, we present a nanoscale cytometry platform NanoEPIC, enabling phenotypic sorting and exoPD-L1 profiling from blood plasma. We highlight the efficacy of NanoEPIC in monitoring anti-PD-1 immunotherapy through the interrogation of exoPD-L1. NanoEPIC generates signature exoPD-L1 patterns in responders and non-responders. In mice treated with PD1-targeted immunotherapy, exoPD-L1 is correlated with tumor growth, PD-L1 burden in tumors, and the immune suppression of CD8+ tumor-infiltrating lymphocytes. Small extracellular vesicles (sEVs) with different PD-L1 expression levels display distinctive inhibitory effects on CD8 + T cells. NanoEPIC offers robust, high-throughput profiling of exosomal markers, enabling sEV subpopulation analysis. This platform holds the potential for enhanced cancer screening, personalized treatment, and therapeutic response monitoring.

Isolation of tumour-reactive lymphocytes from peripheral blood via microfluidic immunomagnetic cell sorting.

The clinical use of tumour-infiltrating lymphocytes for the treatment of solid tumours is hindered by the need to obtain large and fresh tumour fractions, which is often not feasible in patients with unresectable tumours or recurrent metastases. Here we show that circulating tumour-reactive lymphocytes (cTRLs) can be isolated from peripheral blood at high yield and purity via microfluidic immunomagnetic cell sorting, allowing for comprehensive downstream analyses of these rare cells. We observed that CD103 is strongly expressed by the isolated cTRLs, and that in mice with subcutaneous tumours, tumour-infiltrating lymphocytes isolated from the tumours and rapidly expanded CD8+CD103+ cTRLs isolated from blood are comparably potent and respond similarly to immune checkpoint blockade. We also show that CD8+CD103+ cTRLs isolated from the peripheral blood of patients and co-cultured with tumour cells dissociated from their resected tumours resulted in the enrichment of interferon-γ-secreting cell populations with T-cell-receptor clonotypes substantially overlapping those of the patients' tumour-infiltrating lymphocytes. Therapeutically potent cTRLs isolated from peripheral blood may advance the clinical development of adoptive cell therapies.