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Point-of-care testing (POCT) of clinical biomarkers is critical to health monitoring and timely treatment, yet biosensing assays capable of detecting biomarkers without the need for costly external equipment and reagents are limited. Blood-based assays are, specifically, challenging as blood collection is invasive and follow-upprocessing is required. Here, we report a versatile assay that employs hydrogel microneedles (HMNs) to extract interstitial fluid (ISF), in a minimally invasive manner integrated with graphene oxide-nucleic acid (GO.NA)-based fluorescence biosensor to sense the biomarkers of interest in situ. The HMN-GO.NA assay is supplemented with a portable detector, enabling a complete POCT procedure. Our system could successfully measure four clinically important biomarkers (glucose, uric acid (UA), insulin, and serotonin) ex vivo, in addition, to accurately detecting glucose and UA in vivo.
Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , Hidrogéis , Glucose , Biomarcadores , Sondas de Ácido NucleicoRESUMO
In immunobead-based assays, micro/nanobeads are functionalized with antibodies to capture the target analytes, which can significantly improve the assay's performance. The immunobead-based assays have been recently combined with microfluidic mixing devices and customized for a variety of applications. However, device design and process optimization to achieve the best performance remain a substantial technological challenge. Here, we introduce a computational model that enables the rational design and optimization of the immunobead-based assay in a microfluidic mixing channel. We use numerical methods to examine the effect of the flow rates, channel geometry, bead's trajectory, and the analyte and reagent characteristics on the efficiency of analyte capture on the surface of microbeads. This model accounts for different bead movements inside the microchannel, with the goal of simulating an actual active binding environment. The model is further validated experimentally where different microfluidic channels are tested to capture the target analytes. Our experimental results are shown to meet theoretical predictions. While the model is demonstrated here for the analysis of IgG capture in simple and herringbone-structured microchannels, it can be readily adapted to a broad range of target molecules and different device designs.
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Técnicas Analíticas Microfluídicas , Microfluídica , Dispositivos Lab-On-A-Chip , Microfluídica/métodos , Microesferas , Modelos TeóricosRESUMO
Conventional microneedles (MNs) have been extensively reported and applied toward a variety of biosensing and drug delivery applications. Hydrogel forming MNs with the added ability to electrically track health conditions in real-time is an area yet to be explored. The first conductive hydrogel microneedle (HMN) electrode that is capable of on-needle pH detection with no postprocessing required is presented here. The HMN array is fabricated using a swellable dopamine (DA) conjugated hyaluronic acid (HA) hydrogel, and is embedded with poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) to increase conductivity. The catechol-quinone chemistry intrinsic to DA is used to measure pH in interstitial fluid (ISF). The effect of PEDOT:PSS on the characteristics of the HMN array such as swelling capability and mechanical strength is fully studied. The HMN's capability for pH measurement is first demonstrated using porcine skin equilibrated with different pH solutions ranging from 3.5 to 9. Furthermore, the HMN-pH meter is capable of in vivo measurements with a 93% accuracy compared to a conventional pH probe meter. This HMN technology bridges the gap between traditional metallic electrochemical biosensors and the direct extraction of ISF, and introduces a platform for the development of polymeric wearable sensors capable of on-needle detection.
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Hidrogéis , Agulhas , Suínos , Animais , Condutividade Elétrica , Eletrodos , Concentração de Íons de HidrogênioRESUMO
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.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , Nanoestruturas/química , Neurônios , Técnicas de Cultura de Células/métodos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fatores de TempoRESUMO
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.
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Quimiotaxia , Dispositivos Lab-On-A-Chip , Neoplasias/patologia , Células Neoplásicas Circulantes/patologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Separação Celular/instrumentação , Desenho de Equipamento , Feminino , Humanos , Masculino , Camundongos SCID , Neoplasias da Próstata/patologiaRESUMO
A chip-based approach for electrochemical characterization and detection of microsomes and exosomes based on direct electro-oxidation of metal nanoparticles (MNPs) that specifically recognize surface markers of these vesicles is reported. It is found that exosomes and microsomes derived from prostate cancer cells can be identified by their surface proteins EpCAM and PSMA, suggesting the potential of exosomes and microsomes for use as diagnostic biomarkers.
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Exossomos/metabolismo , Nanopartículas Metálicas/química , Microssomos/metabolismo , Linhagem Celular Tumoral , Eletroquímica , Exossomos/ultraestrutura , Humanos , Masculino , Microssomos/ultraestrutura , Neoplasias da Próstata/sangueRESUMO
The electrochemical detection of nucleic acids using an electrocatalytic reporter system and nanostructured microelectrodes is a powerful approach to ultrasensitive biosensing. In this report we systematically study for the first time the behavior of an electrocatalytic reporter system at nucleic acid-modified electrodes with varying structures and sizes. [Ru(NH3)6](3+) is used as a primary electron acceptor that is electrostatically attracted to nucleic acid-modified electrodes, and [Fe(CN)6](3-) is introduced into the redox system as a secondary electron acceptor to regenerate Ru(3+) after electrochemical reduction. We found that the electrode structure has strong impact on mass transport and electron-transfer kinetics, with structures of specific dimensions yielding much higher electrochemical signals and catalytic efficiencies. The electrocatalytic signals obtained when gold sensors were electrodeposited in both circular and linear apertures were studied, and the smallest structures plated in linear apertures were found to exhibit the best performance with high current densities and turnover rates. This study provides important information for optimal assay performance and insights for the future design and fabrication of high performance biomolecular assays.
Assuntos
Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Microeletrodos , Ácidos Nucleicos/químicaRESUMO
Circulating tumor cells (CTCs) can be collected noninvasively and provide a wealth of information about tumor phenotype. For this reason, their specific and sensitive detection is of intense interest. Herein, we report a new, chip-based strategy for the automated analysis of cancer cells. The nanoparticle-based, multi-marker approach exploits the direct electrochemical oxidation of metal nanoparticles (MNPs) to report on the presence of specific surface markers. The electrochemical assay allows simultaneous detection of multiple different biomarkers on the surfaces of cancer cells, enabling discrimination between cancer cells and normal blood cells. Through multiplexing, it further enables differentiation among distinct cancer cell types. We showcase the technology by demonstrating the detection of cancer cells spiked into blood samples.
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Técnicas Biossensoriais/métodos , Nanopartículas/química , Células Neoplásicas Circulantes/química , Biomarcadores Tumorais , Linhagem Celular Tumoral , Eletroquímica , HumanosRESUMO
Continuous health monitoring aims to reduce hospitalization and the need for constant supervision of the patients. For an outpatient monitoring device to be effective, it must meet certain criteria: it should demand minimal patient involvement, be reliable, be connected, remain stable with infrequent replacements, be cost-efficient, be compatible with humans, and ultimately be self-powered. Microneedle (MN) technology, designed for transdermal biosensing, offers a promising solution for meeting a wide range of these demands in the field of continuous health monitoring. A variety of MN platforms have been developed to facilitate this crucial function. Our focus in this Perspective is on the significant challenges linked to MN-based biosensors. These challenges include ensuring skin compatibility, the effective integration of biorecognition elements into the MN systems, and the durability concerns of these sensors in enabling extended periods of continuous monitoring. Tackling these hurdles could pave the way for more effective and reliable MN-based health monitoring solutions in the future.
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Técnicas Biossensoriais , Agulhas , Humanos , Pele , Administração CutâneaRESUMO
During the multistep process of metastasis, cancer cells encounter various mechanical forces which make them deform drastically. Developing accurate in-silico models, capable of simulating the interactions between the mechanical forces and highly deformable cancer cells, can pave the way for the development of novel diagnostic and predictive methods for metastatic progression. Spring-network models of cancer cell, empowered by our recently proposed identification approach, promises a versatile numerical tool for developing experimentally validated models that can simulate complex interactions at cellular scale. Using this numerical tool, we presented spring-network models of breast cancer cells that can accurately replicate the experimental data of deformation behavior of the cells flowing in a fluidic domain and passing narrow constrictions comparable to microcapillary. First, using high-speed imaging, we experimentally studied the deformability of breast cancer cell lines with varying metastatic potential (MCF-7 (less invasive), SKBR-3 (medium-high invasive), and MDA-MB-231 (highly invasive)) in terms of their entry time to a constricted microfluidic channel. We observed that MDA-MB-231, that has the highest metastatic potential, is the most deformable cell among the three. Then, by focusing on this cell line, experimental measurements were expanded to two more constricted microchannel dimensions. The experimental deformability data in three constricted microchannel sizes for various cell sizes, enabled accurate identification of the unknown parameters of the spring-network model of the breast cancer cell line (MDA-MB-231). Our results show that the identified parameters depend on the cell size, suggesting the need for a systematic procedure for identifying the size-dependent parameters of spring-network models of cells. As the numerical results show, the presented cell models can simulate the entry process of the cell into constricted channels with very good agreements with the measured experimental data.
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Diabetic ketoacidosis (DKA), a severe complication of type 1 diabetes (T1D), is triggered by production of large quantities of ketone bodies, requiring patients with T1D to constantly monitor their ketone levels. Here, a skin-compatible hydrogel microneedle (HMN)-continuous ketone monitoring (HMN-CKM) device is reported. The sensing mechanism relies on the catechol-quinone chemistry inherent to the dopamine (DA) molecules that are covalently linked to the polymer structure of the HMN patch. The DA serves the dual-purpose of acting as a redox mediator for measuring the byproduct of oxidation of 3-beta-hydroxybutyrate (ß-HB), the primary ketone bodies; while, also facilitating the formation of a crosslinked HMN patch. A universal approach involving pre-oxidation and detection of the generated catechol compounds is introduced to correlate the sensor response to the ß-HB concentrations. It is further shown that real-time tracking of a decrease in ketone levels of T1D rat model is possible using the HMN-CKM device, in conjunction with a data-driven machine learning model that considers potential time delays.
Assuntos
Dopamina , Eletrodos , Hidrogéis , Dopamina/análise , Animais , Ratos , Hidrogéis/química , Agulhas , Cetonas/química , Catecóis/química , Catecóis/análise , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Cetoacidose Diabética/diagnóstico , Ácido 3-Hidroxibutírico/química , Diabetes Mellitus Tipo 1/sangue , Oxirredução , Diabetes Mellitus ExperimentalRESUMO
Continuous monitoring of clinically relevant biomarkers within the interstitial fluid (ISF) using microneedle (MN)-based assays, has the potential to transform healthcare. This study introduces the Wearable Aptalyzer, an integrated system fabricated by combining biocompatible hydrogel MN arrays for ISF extraction with an electrochemical aptamer-based biosensor for in situ monitoring of blood analytes. The use of aptamers enables continuous monitoring of a wide range of analytes, beyond what is possible with enzymatic monitoring. The Wearable Aptalyzer is used for real-time and multiplexed monitoring of glucose and lactate in ISF. Validation experiments using live mice and rat models of type 1 diabetes demonstrate strong correlation between the measurements collected from the Wearable Aptalyzer in ISF and those obtained from gold-standard techniques for blood glucose and lactate, for each analyte alone and in combination. The Wearable Aptalyzer effectively addresses the limitations inherent in enzymatic detection methods as well as solid MN biosensors and the need for reliable and multiplexed bioanalytical monitoring in vivo.
Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Ácido Láctico , Agulhas , Dispositivos Eletrônicos Vestíveis , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Camundongos , Ácido Láctico/análise , Ácido Láctico/sangue , Ratos , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Aptâmeros de Nucleotídeos/química , Glucose/análise , Glicemia/análise , Líquido Extracelular/química , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/diagnósticoRESUMO
Biomarker detection in whole blood enables understanding of the cause, progression, relapse or outcome of treatment of a disease. Conventional biomarker detection techniques, such as enzyme-linked immunosorbent assay, polymerase chain reaction, and immunofluorescence, require long assay time, costly laboratory instruments, large reagent volume and sample pre-processing. Hence, there is an unmet need for reliable capture and detection of biomarkers in unprocessed blood which are adaptable to point-of-care (POC) testing. Here, we present a simple, low-cost, and rapid protein detection device from whole blood samples which has the potential to be employed in a POC setting. The platform consists of two components: a plasma separation device that extracts plasma from whole blood without the application of any external active forces and a SPR sensor chip that uses a label-free optical technique for the detection of biomarkers in the extracted plasma. We have demonstrated the detection of IgG and IgM biomolecules in unprocessed blood at concentrations lower than the physiological value within 15 min. The proposed technique has the potential for improving the diagnosis and screening of many diseases, including cancer, influenza, human immunodeficiency virus, and SARS-Cov2 at POC.
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Técnicas Biossensoriais , COVID-19 , Humanos , Ressonância de Plasmônio de Superfície , Microfluídica , RNA Viral , SARS-CoV-2 , Técnicas Biossensoriais/métodos , BiomarcadoresRESUMO
Continuous glucose meters (CGMs) have tremendously boosted diabetes care by emancipating millions of diabetic patients' need for repeated self-testing by pricking their fingers every few hours. However, CGMs still suffer from major deficiencies regarding accuracy, precision, and stability. This is mainly due to their dependency on an enzymatic detection mechanism. Here a low-cost hydrogel microneedle (HMN)-CGM assay fabricated using swellable dopamine (DA)-hyaluronic acid (HA) hydrogel for glucose interrogation in dermal interstitial fluid (ISF) is introduced. Platinum and silver nanoparticles are synthesized within the 3D porous hydrogel scaffolds for nonenzymatic electrochemical sensing of the glucose. Incorporation of a highly water dispersible conductive polymer, poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) enhances the electrical properties of HMN array, making the patch suitable as the working electrode of the sensor. The in vitro and ex vivo characterization of this newly developed HMN patch is fully studied. The performance of the HMN-CGM for real-time measurement of glucose is also shown using a rat model of type 1 diabetes. The device introduces the first HMN-based assay for tracking important disease biomarkers and expect to pave the way for next generation of polymeric-based sensors.
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Glucose , Nanopartículas Metálicas , Animais , Ratos , Glucose/química , Hidrogéis , PrataRESUMO
The requirement for rapid, in-field detection of cyanotoxins in water resources necessitates the developing of an easy-to-use and miniaturized system for their detection. We present a novel bead-based, competitive fluorescence assay for multiplexed detection of two types of toxins: microcystin-LR (MC-LR) and okadaic acid (OA). To automate the detection process, a reusable microfluidic device, termed toxin-chip, was designed and validated. The toxin-chip consists of a micromixer where the target toxins were efficiently mixed with a reagent solution, and a detection chamber for magnetic retainment of beads for downstream analysis. Quantum dots (QDs) were used as the reporter molecules to enhance the sensitivity of the assay and the emitted fluorescence signal from QDs was reversely proportional to the amount of toxins in the solution. An image analysis program was also developed to further automate the detection and analysis steps. Two toxins were simultaneously analyzed on a single microfluidic chip, and the device exhibited a low detection limit of 10-4 µg ml-1 for MC-LR and 4 × 10-5 µg ml-1 for OA detection. The bead-based, competitive assay also showed remarkable chemical specificity against potential interfering toxins. We also validated the device performance using natural lake water samples from Sunfish Lake of Waterloo. The toxin-chip holds promise as a versatile and simple quantification tool for cyanotoxin detection, with the potential of detecting more toxins.
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Toxinas Marinhas , Microfluídica , Poluentes Químicos da Água , Poluentes Químicos da Água/análise , Ácido Okadáico/análise , Toxinas Marinhas/análiseRESUMO
Cervical cancer (CC) is a major health care problem in low- and middle-income countries, necessitating the development of low-cost and easy-to-use assays for CC detection at point-of-care (POC) settings. An integrated microfluidic electrochemical assay for CC detection, named IMEAC, is presented that has the potential for identifying CC circulating DNA in whole blood samples. The IMEAC consists of two main modules: a plasma separator device that isolates plasma from whole blood with high purity and without the need for any external forces connected to a graphene oxide-based electrochemical biosensor that uses specific probe molecules for the detection of CC circulating DNA molecules. We fully characterize the performance of the individual modules and show that the integrated assay can be utilized for target DNA detection in whole blood samples, thus potentially transforming CC detection and screening at remote locations.
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Técnicas Biossensoriais , Ácidos Nucleicos Livres , Neoplasias do Colo do Útero , Técnicas Eletroquímicas , Feminino , Humanos , Microfluídica , Sistemas Automatizados de Assistência Junto ao Leito , Testes Imediatos , Neoplasias do Colo do Útero/diagnósticoRESUMO
During cancer progression, tumors shed different biomarkers into the bloodstream, including circulating tumor cells (CTCs), extracellular vesicles (EVs), circulating cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA). The analysis of these biomarkers in the blood, known as 'liquid biopsy' (LB), is a promising approach for early cancer detection and treatment monitoring, and more recently, as a means for cancer therapy. Previous reviews have discussed the role of CTCs and ctDNA in cancer progression; however, ctDNA and EVs are rapidly evolving with technological advancements and computational analysis and are the subject of enormous recent studies in cancer biomarkers. In this review, first, we introduce these cell-released cancer biomarkers and briefly discuss their clinical significance in cancer diagnosis and treatment monitoring. Second, we present conventional and novel approaches for the isolation, profiling, and characterization of these markers. We then investigate the mathematical and in silico models that are developed to investigate the function of ctDNA and EVs in cancer progression. We convey our views on what is needed to pave the way to translate the emerging technologies and models into the clinic and make the case that optimized next-generation techniques and models are needed to precisely evaluate the clinical relevance of these LB markers.
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Spectroscopy in the sub-terahertz (sub-THz) range of frequencies has been utilized to study the picosecond dynamics and interaction of biomolecules. However, widely used free-space THz spectrometers are typically limited in their functionality due to low signal-to-noise ratio and complex setup. On-chip spectrometers can revolutionize THz spectroscopy allowing integration, compactness, and low-cost fabrication. In this paper, a low-loss silicon-based platform is proposed for on-chip sub-THz spectroscopy. Through functionalization of silicon chip and immobilization of bio-particles, we demonstrate the ability to characterize low-loss nano-scale biomolecules across the G-band (0.14-0.22 THz). We also introduce an electromagnetic thin-film model to account for the loading effect of the immobilized biomolecules, i.e. dehydrated streptavidin and immunoglobulin antibody, as two key molecules in the biosensing discipline. The proposed platform was fabricated using a single mask micro-fabrication process, and then measured by a vector network analyzer (VNA), which offers high dynamic range and high spectral resolution measurements. The proposed planar platform is general and paves the way towards low-loss, cost-effective and integrated sub-THz biosensors for the detection and characterization of biomolecules.
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Silício , Espectroscopia Terahertz , Estreptavidina , Espectroscopia Terahertz/métodosRESUMO
Analyzing interstitial fluid (ISF) via microneedle (MN) devices enables patient health monitoring in a minimally invasive manner and in point-of-care settings. However, most MN-based diagnostic approaches require complicated fabrication processes and postprocessing of the extracted ISF or are limited to detection of electrochemically active biomarkers. Here, we show on-needle measurement of target analytes by integrating hydrogel microneedles with aptamer probes as the recognition elements. Fluorescently tagged aptamer probes are chemically attached to the hydrogel matrix using a simple and novel approach, while a cross-linked patch is formed. For reagentless detection, we employ a strand displacement strategy where fluorophore-conjugated aptamers are hybridized with a DNA competitor strand conjugated to a quencher molecule. The assay is utilized for rapid (2 min) measurement of glucose, adenosine triphosphate, l-tyrosinamide, and thrombin ex vivo. Furthermore, the system enables specific and sensitive quantification of rising and falling concentrations of glucose in an animal model of diabetes to track hypoglycemia, euglycemia, and hyperglycemia conditions. Our assay can be applied for rapid measurement of a diverse range of biomarkers, proteins, or small molecules, introducing a generalizable platform for biomolecule quantification, and has the potential to improve the quality of life of patients who are in need of close monitoring of biomarkers of health and disease.
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Hidrogéis , Qualidade de Vida , Animais , Biomarcadores , Fluorescência , Glucose , OligonucleotídeosRESUMO
Loss of photoreceptors due to retinal degeneration is a major cause of untreatable visual impairment and blindness. Cell replacement therapy, using retinal stem cell (RSC)-derived photoreceptors, holds promise for reconstituting damaged cell populations in the retina. One major obstacle preventing translation to the clinic is the lack of validated markers or strategies to prospectively identify these rare cells in the retina and subsequently enrich them. Here, we introduce a microfluidic platform that combines nickel micromagnets, herringbone structures, and a design enabling varying flow velocities among three compartments to facilitate a highly efficient enrichment of RSCs. In addition, we developed an affinity enrichment strategy based on cell-surface markers that was utilized to isolate RSCs from the adult ciliary epithelium. We showed that targeting a panel of three cell surface markers simultaneously facilitates the enrichment of RSCs to 1 : 3 relative to unsorted cells. Combining the microfluidic platform with single-cell whole-transcriptome profiling, we successfully identified four differentially expressed cell surface markers that can be targeted simultaneously to yield an unprecedented 1 : 2 enrichment of RSCs relative to unsorted cells. We also identified transcription factors (TFs) that play functional roles in maintenance, quiescence, and proliferation of RSCs. This level of analysis for the first time identified a spectrum of molecular and functional properties of RSCs.