A Triple Signal Amplification Strategy for Accurate and Ultrasensitive miRNA-21 Detection.

A triple signal amplification strategy was integrated with a built-in double electrode and external energy storage device to fabricate a novel self-powered biosensor for ultrasensitive detection of miRNA-21. Specifically, DNA tetrahedra and haripin2-glucose oxidase are modified on the surface of the biocathode and bioanode by catalytic hairpin assembly (CHA) to achieve dual signal amplification. Moreover, triple signal amplification is realized by including an external capacitor. Consequently, the as-constructed self-powered biosensor demonstrates a low detection limit of 0.06 fM toward the miRNA-21 assay within the range of 0.1 fM to 10 pM. This study presents a practical and sensitive approach to timely cancer detection.

Integration of Lateral Filter Arrays with Immunoaffinity for Circulating-Tumor-Cell Isolation.

Circulating tumor cells (CTCs) are an important biomarker for cancer prognosis and treatment monitoring. However, the heterogeneity of the physical and biological properties of CTCs limits the efficiency of various approaches used to isolate small numbers of CTCs from billions of normal blood cells. To address this challenge, we developed a lateral filter array microfluidic (LFAM) device to integrate size-based separation with immunoaffinity-based CTC isolation. The LFAM device consists of a serpentine main channel, through which most of a sample passes, and an array of lateral filters for CTC isolation. The unique device design produces a two-dimensional flow, which reduces nonspecific, geometric capture of normal cells as typically observed in vertical filters. The LFAM device was further functionalized by immobilizing antibodies that are specific to the target cells. The resulting devices captured pancreatic cancer cells spiked in blood samples with (98.7±1.2) % efficiency and were used to isolate CTCs from patients with metastatic colorectal cancer.

A Capillary-Force-Driven, Single-Cell Transfer Method for Studying Rare Cells.

The characterization of individual cells within heterogeneous populations (e.g., rare tumor cells in healthy blood cells) has a great impact on biomedical research. To investigate the properties of these specific cells, such as genetic biomarkers and/or phenotypic characteristics, methods are often developed for isolating rare cells among a large number of background cells before studying their genetic makeup and others. Prior to using real-world samples, these methods are often evaluated and validated by spiking cells of interest (e.g., tumor cells) into a sample matrix (e.g., healthy blood) as model samples. However, spiking tumor cells at extremely low concentrations is challenging in a standard laboratory setting. People often circumvent the problem by diluting a solution of high-concentration cells, but the concentration becomes inaccurate after series dilution due to the fact that a cell suspension solution can be inhomogeneous, especially when the cell concentration is very low. We report on an alternative method for low-cost, accurate, and reproducible low-concentration cell spiking without the use of external pumping systems. By inducing a capillary force from sudden pressure drops, a small portion of the cellular membrane was aspirated into the reservoir tip, allowing for non-destructive single-cell transfer. We investigated the surface membrane tensions induced by cellular aspiration and studied a range of tip/tumor cell diameter combinations, ensuring that our method does not affect cell viability. In addition, we performed single-cell capture and transfer control experiments using human acute lymphoblastic leukemia cells (CCRF-CEM) to develop calibrated data for the general production of low-concentration samples. Finally, we performed affinity-based tumor cell isolation using this method to generate accurate concentrations ranging from 1 to 15 cells/mL.

Mitochondrial Probe for Glutathione Depletion Reveals NME3 Essentiality for Mitochondrial Redox Response.

Maintenance of the mitochondrial thiol redox state is essential for cell survival. However, we lack a comprehensive understanding of the redox response to mitochondrial glutathione depletion. We developed a mitochondria-penetrating peptide, mtCDNB, to specifically deplete mitochondrial glutathione. A genome-wide CRISPR/Cas9 screen in tandem with mtCDNB treatment was employed to uncover regulators of the redox response to mitochondrial glutathione depletion. We identified nucleoside diphosphate kinase 3 (NME3) as a regulator of mitochondrial dynamics. We show that NME3 is recruited to the mitochondrial outer membrane when under redox stress. In the absence of NME3, there is impaired mitophagy, which leads to the accumulation of dysfunctional mitochondria. NME3 knockouts depleted of mitochondrial glutathione have increased mitochondrial ROS production, accumulate mtDNA lesions, and present a senescence-associated secretory phenotype. Our findings suggest a novel role for NME3 in selecting mitochondria for degradation through mitophagy under conditions of mitochondrial redox stress.

A Micropillar Array Based Microfluidic Device for Rare Cell Detection and Single-Cell Proteomics.

Advancements in single-cell-related technologies have opened new possibilities for analyzing rare cells, such as circulating tumor cells (CTCs) and rare immune cells. Among these techniques, single-cell proteomics, particularly single-cell mass spectrometric analysis (scMS), has gained significant attention due to its ability to directly measure transcripts without the need for specific reagents. However, the success of single-cell proteomics relies heavily on efficient sample preparation, as protein loss in low-concentration samples can profoundly impact the analysis. To address this challenge, an effective handling system for rare cells is essential for single-cell proteomic analysis. Herein, we propose a microfluidics-based method that offers highly efficient isolation, detection, and collection of rare cells (e.g., CTCs). The detailed fabrication process of the micropillar array-based microfluidic device is presented, along with its application for CTC isolation, identification, and collection for subsequent proteomic analysis.

Lateral Filter Array Microfluidic Devices for Detecting Circulating Tumor Cells.

Circulating tumor cells (CTCs) are an important liquid biopsy biomarker for next-generation cancer diagnosis and prognosis. However, their clinical usage is hindered by the rarity of CTCs in patient's peripheral blood. Microfluidics has shown unique advantages in CTC isolation and detection. We have developed lateral filter array microfluidic (LFAM) devices for highly efficient CTC isolation. In this chapter, we describe in detail the design and fabrication of the LFAM devices and their applications for CTC enumeration from clinical blood samples.

Gold Nanoparticle-Based Microfluidic Chips for Capture and Detection of Circulating Tumor Cells.

Liquid biopsy has progressed to its current use to diagnose and monitor cancer. Despite the recent advances in investigating cancer detection and diagnosis strategies, there is still a room for improvements in capturing CTCs. We developed an efficient CTC detection system by integrating gold nanoparticles with a microfluidic platform, which can achieve CTC capture within 120 min. Here, we report our development of a simple and effective way to isolate CTCs using antibodies attached on gold nanoparticles to the surface of a lateral filter array (LFA) microdevice. Our method was optimized using three pancreatic tumor cell lines, enabling the capture with high efficiency (90% ± 3.2%). The platform was further demonstrated for isolating CTCs from patients with metastatic pancreatic cancer. Our method and platform enables the production of functionalized, patterned surfaces that interact with tumor cells, enhancing the selective capture of CTCs for biological assays.

Microfluidics-Enabled Isolation and Single-Cell Analysis of Circulating Tumor Cells.

Microfluidic platforms enable the enrichment and analysis of circulating tumor cells (CTCs), a potential biomarker for cancer diagnosis, prognosis, and theragnosis. Combined with immunocytochemistry/immunofluorescence (ICC/IF) assays for CTCs, microfluidics-enabled detection presents a unique opportunity to study tumor heterogeneity and predict treatment response, both of which can help cancer drug development. In this chapter, we detail the protocols and methods employed to fabricate and use a microfluidic device for the enrichment, detection, and analysis of single CTCs from the blood samples of sarcoma patients.

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.

Comparison of sample preparation methods for rare cell isolation in microfluidic devices.

The analysis of circulating tumor cells (CTCs) is important for cancer diagnosis and prognosis. Microfluidics has been employed for CTC analysis due to its scaling advantages and high performance. However, pre-analytical methods for CTC sample preparation are often combined with microfluidic platforms because a large sample volume is required to detect extremely rare CTCs. Among pre-analytical methods, Ficoll-Paque™, OncoQuick™, and RosetteSep™ are commonly used to separate cells of interest. To compare their performance, we spiked L3.6pl pancreatic cancer cells into healthy blood samples and then employed each technique to prepare blood samples, followed by using a microfluidic platform to capture and detect L3.6pl cells. We found these three methods have similar performance, though the slight edge of RosetteSep™ over Ficoll-Paque™ is statistically significant. We also studied the effects of the tumor cell concentrations on the performance of the frequently used Ficoll-Paque™ method. Furthermore, we examined the repeatability and variability of each pre-analytical technique and the microfluidics-enabled detection. This study will provide researchers and clinicians with comparative data that can influence the choice of sample preparation method, help estimate CTC loss in each pre-analytical method, and correlate the results of clinical studies that employ different techniques.

L'analyse des cellules tumorales circulantes (CTC) est une pierre angulaire du diagnostic et du pronostic du cancer. On recourt à la microfluidique pour l'analyse des CTC en raison des avantages qu'elle offre pour la mise à l'échelle et de sa grande performance. Par ailleurs, les méthodes préanalytiques pour la préparation d'échantillons de CTC font souvent appel à des plateformes microfluidiques, car il faut un grand volume d'échantillon pour détecter des CTC extrêmement rares. Parmi les méthodes préanalytiques couramment utilisées pour séparer les cellules sanguines d'intérêt, notons Ficoll-PaqueMC, OncoQuickMC et RosetteSepMC. Afin de comparer les performances de ces méthodes, nous avons additionné de cellules de cancer du pancreas L3,6pl des échantillons de sang sains, puis nous avons utilisé les trois méthodes pour préparer les échantillons sanguins, que nous avons ensuite soumis à une plateforme microfluidique pour isoler et détecter les cellules L3,6pl. Nos résultats montrent que les performances de ces trois méthodes sont similaires, bien que le léger avantage de RosetteSepMC par rapport à Ficoll-PaqueMC soit statistiquement significatif. Nous avons également étudié les effets des concentrations de cellules tumorales sur la performance de la méthode Ficoll-PaqueMC, qui est la plus fréquemment utilisée. En outre, nous avons examiné la répétabilité et la variabilité de chaque méthode préanalytique et les caractéristiques de détection que permet d'obtenir la microfluidique. Cette étude fournit aux chercheurs et aux cliniciens des données comparatives qui peuvent influencer leur choix de la méthode de préparation des échantillons, et permet d'estimer la perte de CTC propre à chaque méthode préanalytique et de corréler les résultats des études cliniques qui utilisent différentes techniques. [Traduit par la Rédaction].

Ultrathroughput immunomagnetic cell sorting platform.

High-throughput phenotypic cell sorting is critical to the development of cell-based therapies and cell screening discovery platforms. However, current cytometry platforms are limited by throughput, number of fractionated populations that can be isolated, cell viability, and cost. We present an ultrathroughput microfluidic cell sorter capable of processing hundreds of millions of live cells per hour per device based on protein expression. This device, a next-generation microfluidic cell sorter (NG-MICS), combines multiple technologies, including 3D printing, reversible clamp sealing, and superhydrophobic treatments to create a reusable and user-friendly platform ready for deployment. The utility of such a platform is demonstrated through the rapid isolation of mature natural killer cells from peripheral blood mononuclear cells, for use in CAR-NK therapies at clinically-relevant scale.

nuPRISM: Microfluidic Genome-Wide Phenotypic Screening Platform for Cellular Nuclei.

Genome-wide loss-of-function screens are critical tools to identify novel genetic regulators of intracellular proteins. However, studying the changes in the organelle-specific expression profile of intracellular proteins can be challenging due to protein localization differences across the whole cell, hindering context-dependent protein expression and activity analyses. Here, we describe nuPRISM, a microfluidics chip specifically designed for large-scale isolated nuclei sorting. The new device enables rapid genome-wide loss-of-function phenotypic CRISPR-Cas9 screens directed at intranuclear targets. We deployed this technology to identify novel genetic regulators of ß-catenin nuclear accumulation, a phenotypic hallmark of APC-mutated colorectal cancer. nuPRISM expands our ability to capture aberrant nuclear morphological and functional traits associated with distinctive signal transduction and subcellular localization-driven functional processes with substantial resolution and high throughput.

Genome-wide in vivo screen of circulating tumor cells identifies SLIT2 as a regulator of metastasis.

Circulating tumor cells (CTCs) break free from primary tumors and travel through the circulation system to seed metastatic tumors, which are the major cause of death from cancer. The identification of the major genetic factors that enhance production and persistence of CTCs in the bloodstream at a whole genome level would enable more comprehensive molecular mechanisms of metastasis to be elucidated and the identification of novel therapeutic targets, but this remains a challenging task due to the heterogeneity and extreme rarity of CTCs. Here, we describe an in vivo genome-wide CRISPR knockout screen using CTCs directly isolated from a mouse xenograft. This screen elucidated SLIT2-a gene encoding a secreted protein acting as a cellular migration cue-as the most significantly represented gene knockout in the CTC population. SLIT2 knockout cells are highly metastatic with hypermigratory and mesenchymal phenotype, resulting in enhanced cancer progression in xenograft models.

Phage-Based Profiling of Rare Single Cells Using Nanoparticle-Directed Capture.

Advances in single-cell level profiling of the proteome require quantitative and versatile platforms, especially for rare cell analyses such as circulating tumor cell (CTC) profiling. Here we demonstrate an integrated microfluidic chip that uses magnetic nanoparticles to capture single tumor cells with high efficiency, permits on-chip incubation, and facilitates in situ cell-surface protein expression analysis. Combined with phage-based barcoding and next-generation sequencing technology, we were able to monitor changes in the expression of multiple surface markers stimulated in response to CTC adherence. Interestingly, we found fluctuations in the expression of Frizzled2 (FZD2) that reflected the microenvironment of the single cells. This platform has a high potential for in-depth screening of multiple surface antigens simultaneously in rare cells with single-cell resolution, which will provide further insights regarding biological heterogeneity and human disease.

Incorporation of lateral microfiltration with immunoaffinity for enhancing the capture efficiency of rare cells.

The methods for isolating rare cells such as circulating tumor cells (CTCs) can be generally classified into two categories: those based on physical properties (e.g., size) and methods based on biological properties (e.g., immunoaffinity). CellSearch, the only FDA-approved method for the CTC-based cancer prognosis, relies on immunoaffinity interactions between CTCs and antibodies immobilized on magnetic particles. Immunoaffinity-based CTC isolation has also been employed in microfluidic devices, which show higher capture efficiency than CellSearch. We report here our investigation of combining size-based microfiltration into a microfluidic device with immunoaffinity for enhanced capture efficiency of CTCs. The device consists of four serpentine main channels, and each channel contains an array of lateral filters that create a two-dimensional flow. The main flow is through the serpentine channel, allowing the majority of the sample to pass by while the secondary flow goes through the lateral filters. The device design is optimized to make all fluid particles interact with filters. The filter sizes range from 24 to 12 µm, being slightly larger than or having similar dimension of CTCs. These filters are immobilized with antibodies specific to CTCs and thus they function as gates, allowing normal blood cells to pass by while forcing the interactions between CTCs and antibodies on the filter surfaces. The hydrodynamic force experienced by a CTC was also studied for optimal experimental conditions to ensure immunoaffinity-enabled cell capture. The device was evaluated by capturing two types of tumor cells spiked in healthy blood or a buffer, and we found that their capture efficiency was between 87.2 and 93.5%. The platform was further validated by isolating CTCs from blood samples of patients with metastatic pancreatic cancer.

Microfluidic Isolation of Circulating Tumor Cells and Cancer Stem-Like Cells from Patients with Pancreatic Ductal Adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) requires multimodal therapeutic approaches and disease monitoring for effective treatment. Liquid biopsy biomarkers, including circulating tumor cells (CTCs) and cancer stem-like cells (CSCs), hold promise for evaluating treatment response promptly and guiding therapeutic modifications. Methods: From 24 patients with metastatic PDAC (stage IV, M1) undergoing active systemic treatment, we collected 78 blood samples at different time points for CTC and CSC isolation using a microfluidic platform functionalized with antibodies against a CTC biomarker, epithelial cell adhesion molecule (EpCAM), or a CSC biomarker, CD133. These isolated cells were further verified, via fluorescent staining and imaging, using cytokeratin (CK), CD45, and nucleic acid stain 4',6-diamidino-2-phenylindole (DAPI). Results: The majority (84.4%) of patient blood samples were positive for CTCs (EpCAM+CK+CD45-DAPI+) and 70.8% of patient blood samples were positive for CSCs (CD133+CK+CD45-DAPI+), using the highest baseline value of healthy samples as threshold. The CTC subtypes (EpCAM+CK+CD45-DAPI+CD133+ and EpCAM+CK+CD45-DAPI+CD133-) and CSC subtypes (CD133+CK+CD45-DAPI+EpCAM+ and CD133+CK+CD45-DAPI+EpCAM-) were also analyzed using immunochemical methods. In several cases, CSCs exhibited cytokeratin expression that did not express EpCAM, indicating that they will not be detected using EpCAM-based isolation. Conclusion: The microfluidic platform enabled the reliable isolation of CTCs and CSCs from PDAC patient samples, as well as their subtypes. Complementary assessment of both CTCs and CSCs appears advantageous to assess the profile of tumor progressing in some cases. This research has important implications for the application and interpretation of approved methods to detect CTCs.

Free-standing 2D nanorafts by assembly of 1D nanorods for biomolecule sensing.

Novel materials from self-assembled nanocrystals hold great promise for applications ranging from inorganic catalysis to bio-imaging. However, because of the inherent anisotropic properties, it is challenging to assemble one-dimensional (1D) nanorods into higher-order structures (e.g. 2D sheets or 3D networks) without any support. Here, we have developed a facile strategy for the direct self-assembly of 1D nanorods into free-standing 2D nanorafts with lateral dimensions up to several micrometers. As a general approach, 2D nanorafts with diverse compositions, e.g. MgF2, WO2, CdS, ZnS, and ZnSe nanorafts, have been fabricated from the assembly of their 1D building blocks. More importantly, these nanorafts show high stability even when dispersed in different solvents, making them suitable for various applications. Because of their high porosity and strong adsorption capability, MgF2 nanorafts were investigated to illustrate the collective advantages generated from the assembly platform. Moreover, flexibility in the composition and structure of the building blocks demonstrated in this work will lead to next generation materials with rich functionalities.

3D halos assembled from Fe3O4/Au NPs with enhanced catalytic and optical properties.

3D structures assembled from multiple components have attracted increasing research interest based on their enriched functionalities and broadened applications. Here, we report a bottom-up strategy to fabricate 3D halos through the co-assembly of Fe3O4 and Au nanoparticles (NPs). Typically, Fe3O4 NPs assemble into a 3D core (size around 500 nm) with simultaneous growth of Au NPs on the 3D surface during the assembly process. As a general approach, a variety of 3D halos were fabricated from the co-assembly of Fe3O4 and Au NPs of different sizes and shapes. To demonstrate the advantages of these 3D halo structures, their catalytic activity to mimic natural enzymes was investigated. Compared with Fe3O4 NP building blocks, enhanced catalytic efficiency was achieved by the 3D halos. In addition, the optical behavior of the 3D halos was simulated using a three-dimensional finite-difference time-domain (3D-FDTD) method. As shown in the results, the 3D halos attached to 90 nm Au NPs could absorb more incident light owing to high electric field intensities, making these structures promising for applications in energy harvesting and detection-related fields.

Free-Floating 2D Nanosheets with a Superlattice Assembled from Fe3O4 Nanoparticles for Peroxidase-Mimicking Activity.

The organization of nanoparticles (NPs) with controlled chemical composition and size distribution into well-defined sheets will find many practical applications, but the chemistry remains problematic. Therefore, we report a facile method to assemble NPs to free-floating two-dimensional (2D) nanosheets with a superlattice and thicknesses reaching 22.8 nm. The ligand oleic acid is critical in the formation of nanosheets. As assembled, these free-floating 2D nanosheets remain intact in both polar and nonpolar solvents, e.g., deionized water, ethanol, N,N-dimethylformamide, dimethyl sulfoxide, toluene, hexane, and chloroform, without any disassembly. Compared to Fe3O4 NP building blocks, these 2D nanosheets show more favorable catalytic properties and enhanced catalytic reactivity, which can be exploited to mimic natural enzymes. Our work is expected to open up a new avenue for synthesizing free-floating 2D supersheets by NP assembly, leading to a new generation of materials with enriched functions and broader applications.

A Novel Microfluidic Device for Isolation of Circulating Tumor Cells from Pancreatic Cancer Blood Samples.

Enumeration of circulating tumor cells (CTCs) can provide valuable prognostic information to guide cancer treatment as well as help monitor disease progression. Analysis of these rare malignant cells has the potential to further our understanding of cancer metastasis by gaining insights into CTC characteristics and properties. Microfluidics presents a unique platform to isolate and study CTCs. In this chapter, we describe the detailed procedures for the fabrication and use of a microfluidic device to detect CTCs from the blood of pancreatic cancer patients.