Detachable acoustofluidic droplet-sorter.

BACKGROUND: Cell sorting is crucial in isolating specific cell populations. It enables detailed analysis of their functions and characteristics and plays a vital role in disease diagnosis, drug discovery, and regenerative medicine. Fluorescence-activated cell sorting (FACS) is considered the gold standard for high-speed single-cell sorting. However, its high cost, complex instrumentation, and lack of portability are significant limitations. Additionally, the high pressure and electric fields used in FACS can harm cell integrity. In this work, an acoustofluidic device was developed in combination with surface acoustic wave (SAW) and droplet microfluidics to isolate single-cell droplets with high purity while maintaining high cell viability. RESULT: Human embryonic kidney cells, transfected with fluorescent reporter plasmids, were used to demonstrate the targeted droplet sorting containing single cells. The acoustofluidic sorter achieved a recovery rate of 81 % and an accuracy rate higher than 97 %. The device maintained a cell viability rate of 95 % and demonstrated repeatability over 20 consecutive trials without compromising efficiency, thus underscoring its reliability. Thermal image analysis revealed that the temperature of the interdigital transducer (IDT) during SAW operation remained within the permissible range for maintaining cell viability. SIGNIFICANCE: The findings highlighted the sensitivity and effectiveness of the developed acoustofluidic device as a tool for single-cell sorting. The detachable microfluidic chip design enables the reusability of the expensive IDT, making it cost-effective and reducing the risk of cross-contamination between different biological samples. The results underscore its capability to accurately isolate individual cells on the basis of specific criteria, showcasing its potential to advance research and clinical applications requiring precise cell sorting methodologies.

Magnetic Stirring Device for Limiting the Sedimentation of Cells inside Microfluidic Devices.

In experiments considering cell handling in microchannels, cell sedimentation in the storage container is a key problem because it affects the reproducibility of the experiments. Here, a simple and low-cost cell mixing device (CMD) is presented; the device is designed to prevent the sedimentation of cells in a syringe during their injection into a microfluidic channel. The CMD is based on a slider crank device made of 3D-printed parts that, combined with a permanent magnet, actuate a stir bar placed into the syringe containing the cells. By using A549 cell lines, the device is characterized in terms of cell viability (higher than 95%) in different mixing conditions, by varying the oscillation frequency and the overall mixing time. Then, a dedicated microfluidic experiment is designed to evaluate the injection frequency of the cells within a microfluidic chip. In the presence of the CMD, a higher number of cells are injected into the microfluidic chip with respect to the static conditions (2.5 times), proving that it contrasts cell sedimentation and allows accurate cell handling. For these reasons, the CMD can be useful in microfluidic experiments involving single-cell analysis.

An Integrated Inertial-Magnetophoresis Microfluidic Chip Online-Coupled with ICP-MS for Rapid Separation and Precise Detection of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are recognized as promising targets for liquid biopsy, which play an important role in early diagnosis and efficacy monitoring of cancer. However, due to the extreme scarcity of CTCs and partial size overlap between CTCs and white blood cells (WBCs), the separation and detection of CTCs from blood remain a big challenge. To address this issue, we fabricated a microfluidic chip by integrating a passive contraction-expansion array (CEA) inertial sorting zone and an active magnetophoresis zone with the trapezoidal groove and online coupled it with inductively coupled plasma mass spectrometry (ICP-MS) for rapid separation and precise detection of MCF-7 cells (as a model CTC) in blood samples. In the integrated microfluidic chip, most of the small-sized WBCs can be rapidly removed in the circular CEA inertial sorter, while the rest of the magnetically labeled WBCs can be further captured in the trapezoidal groove under the magnetic field. As a result, the rapid separation of MCF-7 cells from blood samples was achieved with an average recovery of 91.6% at a sample flow rate of 200 µL min-1. The developed online integrated inertial-magnetophoresis microfluidic chip-ICP-MS system has been applied for the detection of CTCs in real clinical blood samples with a fast analysis speed (5 min per 1 mL blood). CTCs were detected in all 24 blood samples from patients with different types of cancer, exhibiting excellent application potential in clinical diagnosis.

Efficient separation of large particles and giant cancer cells using an isosceles trapezoidal spiral microchannel.

Polyploid giant cancer cells (PGCCs) contribute to the genetic heterogeneity and evolutionary dynamics of tumors. Their size, however, complicates their isolation from mainstream tumor cell populations. Standard techniques like fluorescence-activated cell sorting (FACS) rely on fluorescent labeling, introducing potential challenges in subsequent PGCC analyses. In response, we developed the Isosceles Trapezoidal Spiral Microchannel (ITSµC), a microfluidic device optimizing the Dean drag force (FD) and exploiting uniform vortices for enhanced separation. Numerical simulations highlighted ITSµC's advantage in producing robust FD compared to rectangular and standard trapezoidal channels. Empirical results confirmed its ability to segregate larger polystyrene (PS) particles (avg. diameter: 50 µm) toward the inner wall, while directing smaller ones (avg. diameter: 23 µm) outward. Utilizing ITSµC, we efficiently isolated PGCCs from doxorubicin-resistant triple-negative breast cancer (DOXR-TNBC) and patient-derived cancer (PDC) cells, achieving outstanding purity, yield, and viability rates (all greater than 90%). This precision was accomplished without fluorescent markers, and the versatility of ITSµC suggests its potential in differentiating a wide range of heterogeneous cell populations.

Combining hybrid cell membrane modified magnetic nanoparticles and inverted microfluidic chip for in situ CTCs capture and inactivation.

Circulating tumor cells (CTCs) serve as crucial indicators for tumor occurrence, progression, and prognosis monitoring. However, achieving high sensitivity and high purity capture of CTCs remains challenging. Additionally, in situ capture and synchronous clearance hold promise as methods to impede tumor metastasis, but further exploration is needed. In this study, biomimetic cell membrane-coated magnetic nanoparticles (NPs) were designed to address the issue of nonspecific adsorption of capture probes by the immune system during blood circulation. Membranes from human breast cancer cells (tumor cell membranes, TMs) and leukocytes (white blood cell membranes, WMs) were extracted and fused to form a hybrid membrane (HM), which was further modified onto the surface of porous magnetic NPs loaded with indocyanine green (ICG). The incorporation of TM enhanced the material's target specificity, thus increasing capture efficiency, while WM coating reduced interference from homologous white blood cells (WBCs), further enhancing capture purity. Additionally, in conjunction with our novel inverted microfluidic chip, this work introduces the first use of polymer photonic crystals as the capture interface for CTCs. Besides providing an advantageous surface structure for CTC attachment, the 808 nm photonic bandgap effectively amplifies the 808 nm excitation light at the capture surface position. Therefore, upon capturing CTCs, the ICG molecules in the probes facilitate enhanced photothermal (PTT) and photodynamic (PDT) synergistic effects, directly inactivating the captured CTCs. This method achieves capture efficiency and purity exceeding 95% and permits in situ inactivation post-capture, providing an important approach for future research on impeding tumor metastasis in vivo.

High-throughput viable circulating tumor cell isolation using tapered-slit membrane filter-based chipsets in the differential diagnosis of ovarian tumors.

OBJECTIVE: To evaluate the diagnostic performance of circulating tumor cells (CTCs) using tapered-slit membrane filter (TSF)-based chipsets for the differential diagnosis of adnexal tumors. METHODS: A total of 230 women with indeterminate adnexal tumors were prospectively enrolled. The sensitivity, specificity, and accuracy of the CTC-detecting chipsets were analyzed according to postoperative pathological results and compared with those of cancer antigen (CA)-125 and imaging tests. RESULTS: Eighty-one (40.3%) benign tumors, 31 (15.4%) borderline tumors, and 89 (44.3%) ovarian cancers were pathologically confirmed. The sensitivity, specificity, and accuracy of CTC-detecting chipsets (75.3%, 58.0%, and 67.1%) for differentiating ovarian cancer from benign tumors were similar to CA-125 (78.7%, 53.1%, and 66.5%), but lower than CT/MRI (94.2%, 77.9%, and 86.5%). "CTC or CA125" showed increased sensitivity (91.0%) and "CTC and CA-125" revealed increased specificity (77.8%), comparable to CT/MRI. CTC detection rates in stage I/II and stage III/IV ovarian cancers were 69.6% and 81.4%, respectively. The sensitivity to detect high-grade serous (HGS) cancer from benign tumors (84.6%) was higher than that to detect non-HGS cancers (68.0%). CONCLUSION: Although the diagnostic performance of the TSF platform to differentiate between ovarian cancer and benign tumors did not yield significant results, the combination of CTC and CA-125 showed promising potential in the diagnostic accuracy of ovarian cancer.

[Study on Circulating Tumor Cell Detection System Based on Microfluidic Chip].

Objective: To achieve high throughput and high detection rate of circulating tumor cells (CTCs) in human peripheral blood, and to provide efficient and accurate early screening for cancer patients. Methods: A microfluidic chip with the integration of sorting, enrichment and detection was designed, and CTCs at the single cell level were detected by fluorescence detection system to obtain the number of CTCs in samples. Results: The peripheral blood samples after lysed red blood cells were used for 6 experiments. When the injection rate reached 0.2 mL/h, CTCs could reach the best detection rate of 78.6%, and the correlation coefficient within the group was above 0.8. Conclusion: CTCs detection system can achieve high detection rate and has good reliability, which can provide a reliable reference for clinical research in related fields.

A High-Throughput Circular Tumor Cell Sorting Chip with Trapezoidal Cross Section.

Circulating tumor cells are typically found in the peripheral blood of patients, offering a crucial pathway for the early diagnosis and prediction of cancer. Traditional methods for early cancer diagnosis are inefficient and inaccurate, making it difficult to isolate tumor cells from a large number of cells. In this paper, a new spiral microfluidic chip with asymmetric cross-section is proposed for rapid, high-throughput, label-free enrichment of CTCs in peripheral blood. A mold of the desired flow channel structure was prepared and inverted to make a trapezoidal cross-section using a micro-nanotechnology process of 3D printing. After a systematic study of how flow rate, channel width, and particle concentration affect the performance of the device, we utilized the device to simulate cell sorting of 6 µm, 15 µm, and 25 µm PS (Polystyrene) particles, and the separation efficiency and separation purity of 25 µm PS particles reached 98.3% and 96.4%. On this basis, we realize the enrichment of a large number of CTCs in diluted whole blood (5 mL). The results show that the separation efficiency of A549 was 88.9% and the separation purity was 96.4% at a high throughput of 1400 µL/min. In conclusion, we believe that the developed method is relevant for efficient recovery from whole blood and beneficial for future automated clinical analysis.

Enhanced microfluidic multi-target separation by positive and negative magnetophoresis.

We introduce magnetophoresis-based microfluidics for sorting biological targets using positive Magnetophoresis (pM) for magnetically labeled particles and negative Magnetophoresis (nM) for label-free particles. A single, externally magnetized ferromagnetic wire induces repulsive forces and is positioned across the focused sample flow near the main channel's closed end. We analyze magnetic attributes and separation performance under two transverse dual-mode magnetic configurations, examining magnetic fields, hydrodynamics, and forces on microparticles of varying sizes and properties. In pM, the dual-magnet arrangement (DMA) for sorting three distinct particles shows higher magnetic gradient generation and throughput than the single-magnet arrangement (SMA). In nM, the numerical results for SMA sorting of red blood cells (RBCs), white blood cells (WBCs), and prostate cancer cells (PC3-9) demonstrate superior magnetic properties and throughput compared to DMA. Magnetized wire linear movement is a key design parameter, allowing device customization. An automated device for handling more targets can be created by manipulating magnetophoretic repulsion forces. The transverse wire and magnet arrangement accommodate increased channel depth without sacrificing efficiency, yielding higher throughput than other devices. Experimental validation using soft lithography and 3D printing confirms successful sorting and separation, aligning well with numerical results. This demonstrates the successful sorting and separating of injected particles within a hydrodynamically focused sample in all systems. Both numerical and experimental findings indicate a separation accuracy of 100% across various Reynolds numbers. The primary channel dimensions measure 100 µm in height and 200 µm in width. N52 permanent magnets were employed in both numerical simulations and experiments. For numerical simulations, a remanent flux density of 1.48 T was utilized. In the experimental setup, magnets measuring 0.5 × 0.5 × 0.125 inches and 0.5 × 0.5 × 1 inch were employed. The experimental data confirm the device's capability to achieve 100% separation accuracy at a Reynolds number of 3. However, this study did not explore the potential impact of increased flow rates on separation accuracy.

Microfluidic Biochips for Single-Cell Isolation and Single-Cell Analysis of Multiomics and Exosomes.

Single-cell multiomic and exosome analyses are potent tools in various fields, such as cancer research, immunology, neuroscience, microbiology, and drug development. They facilitate the in-depth exploration of biological systems, providing insights into disease mechanisms and aiding in treatment. Single-cell isolation, which is crucial for single-cell analysis, ensures reliable cell isolation and quality control for further downstream analyses. Microfluidic chips are small lightweight systems that facilitate efficient and high-throughput single-cell isolation and real-time single-cell analysis on- or off-chip. Therefore, most current single-cell isolation and analysis technologies are based on the single-cell microfluidic technology. This review offers comprehensive guidance to researchers across different fields on the selection of appropriate microfluidic chip technologies for single-cell isolation and analysis. This review describes the design principles, separation mechanisms, chip characteristics, and cellular effects of various microfluidic chips available for single-cell isolation. Moreover, this review highlights the implications of using this technology for subsequent analyses, including single-cell multiomic and exosome analyses. Finally, the current challenges and future prospects of microfluidic chip technology are outlined for multiplex single-cell isolation and multiomic and exosome analyses.

Development of a thermotaxis and rheotaxis microfluidic device for motile spermatozoa sorting.

Male infertility is a pervasive global reproductive challenge, primarily attributed to a decline in semen quality. Addressing this concern, there has been a growing focus on spermatozoa sorting in assisted reproductive technology. This study introduces a groundbreaking development in the form of a thermotaxis and rheotaxis microfluidic (TRMC) device designed for efficient motile spermatozoa sorting within a short 15-min timeframe. The TRMC device mimics the natural sperm sorting mechanism of the oviduct, selecting spermatozoa with superior motility and DNA integrity. The experimental outcomes demonstrate a remarkable enhancement in the percentage of progressive spermatozoa following sorting, soaring from 3.90% to an impressive 96.11% when subjected to a temperature decrease from 38 °C to 35 °C. Notably, sperm motility exhibited a substantial 69% improvement. The TRMC device exhibited a commendable recovery rate of 60.93%, surpassing current clinical requirements. Furthermore, the sorted spermatozoa displayed a notable reduction in the DNA fragmentation index to 6.94%, signifying a substantial 90% enhancement in DNA integrity. This remarkable advancement positions the TRMC device as highly suitable for applications in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), offering a promising solution to male infertility challenges.

Microchip for Immunomagnetic Sorting of Circulating Tumor Cells (CTCs).

Circulating tumor cells (CTCs) isolated directly from whole blood opens new perspectives for cancer monitoring and the development of personalized treatments. However, due to their rarity among the multitude of blood cells, it remains a challenge to recover them alive with high level of purity, i.e., with few remaining white blood cells, and in a time frame compatible with the clinical context. Microfluidic chips have emerged as promising tools to address these challenges. We propose a two-step workflow including a pre-enrichment step, performed by a size-based pre-enrichment system, and a purification step, performed by an immunomagnetic chip. Here, we describe the protocol for the fabrication of the immunomagnetic microchip, the preparation of the sample, and the procedure for injection into the microchip allowing the sorting of the CTCs.

Micropump integrated white blood cell separation platform for detection of chronic granulomatous disease.

White blood cells (WBCs) are robust defenders during antigenic challenges and prime immune cell functioning indicators. High-purity WBC separation is vital for various clinical assays and disease diagnosis. Red blood cells (RBCs) are a major hindrance in WBC separation, constituting 1000 times the WBC population. The study showcases a low-cost micropump integrated microfluidic platform to provide highly purified WBCs for point-of-care testing. An integrated user-friendly microfluidic platform was designed to separate WBCs from finger-prick blood (⁓5 µL), employing an inertial focusing technique. We achieved an efficient WBC separation with 86% WBC purity and 99.99% RBC removal rate in less than 1 min. In addition, the microdevice allows lab-on-chip colorimetric evaluation of chronic granulomatous disease (CGD), a rare genetic disorder affecting globally. The assay duration, straight from separation to disease detection, requires only 20 min. Hence, the proposed microfluidic platform can further be implemented to streamline various clinical procedures involving WBCs in healthcare industries.

CellMag-CARWash: A High Throughput Droplet Microfluidic Device for Live Cell Isolation and Single Cell Applications.

The recent push toward understanding an individual cell's behavior and identifying cellular heterogeneity has created an unmet need for technologies that can probe live cells at the single-cell level. Cells within a population are known to exhibit heterogeneous responses to environmental cues. These differences can lead to varied cellular states, behavior, and responses to therapeutics. Techniques are needed that are not only capable of processing and analyzing cellular populations at the single cell level, but also have the ability to isolate specific cell populations from a complex sample at high throughputs. The new CellMag-Coalesce-Attract-Resegment Wash (CellMag-CARWash) system combines positive magnetic selection with droplet microfluidic devices to isolate cells of interest from a mixture with >93% purity and incorporate treatments within individual droplets to observe single cell biological responses. This workflow is shown to be capable of probing the single cell extracellular vesicle (EV) secretion of MCF7 GFP cells. This article reports the first measurement of ß-Estradiol's effect on EV secretion from MCF7 cells at the single cell level. Single cell processing revealed that MCF7 GFP cells possess a heterogeneous response to ß-Estradiol stimulation with a 1.8-fold increase relative to the control.

Repurposing discarded leukodepletion filters as a source of mononuclear cells for advanced in vitro research.

In light of advancements in the field of immuno-oncology, the demand for obtaining mononuclear cells for in vitro assays has surged. However, obtaining these cells from healthy donors remains a challenging task due to difficulties in donor recruitment and the requirement for substantial blood volumes. Here, we present a protocol for isolating peripheral blood mononuclear cells (PBMCs) from leukodepletion filters used in whole blood and erythrocytes by apheresis donations at the Hemonucleus of the Barretos Cancer Hospital, Brazil. The method involves rinsing the leukodepletion filters and subsequent centrifugation using a Ficoll-Paque concentration gradient. The isolated PBMCs were analyzed by flow cytometry, which allowed the identification of various subpopulations, including CD4+ T lymphocytes (CD45+CD4+), CD8+ T lymphocytes (CD45+CD8+), B lymphocytes (CD45+CD20+CD19+), non-classical monocytes (CD45+CD64+CD14-), classical monocytes (CD45+CD64+CD14+), and granulocytes (CD45+CD15+CD14-). In our comparative analysis of filters, we observed a higher yield of PBMCs from whole blood filters than those obtained from erythrocytes through apheresis. Additionally, fresh samples exhibited superior viability when compared to cryopreserved ones. Given this, leukodepletion filters provide a practical and cost-effective means to isolate large quantities of pure PBMCs, making it a feasible source for obtaining mononuclear cells for in vitro experiments. SUMMARY: Here, we provide a detailed protocol for the isolation of mononuclear cells from leukodepletion filters, which are routinely discarded at the Barretos Cancer Hospital's Hemonucleus.

High-Density Microporous Drainage-Integrating Sheath Flow Generator for Streamlining Microfluidic Cell Sorting Systems.

Tremendous efforts have been made to develop practical and efficient microfluidic cell and particle sorting systems; however, there are technological limitations in terms of system complexity and low operability. Here, we propose a sheath flow generator that can dramatically simplify operational procedures and enhance the usability of microfluidic cell sorters. The device utilizes an embedded polydimethylsiloxane (PDMS) sponge with interconnected micropores, which is in direct contact with microchannels and seamlessly integrated into the microfluidic platform. The high-density micropores on the sponge surface facilitated fluid drainage, and the drained fluid was used as the sheath flow for downstream cell sorting processes. To fabricate the integrated device, a new process for sponge-embedded substrates was developed through the accumulation, incorporation, and dissolution of PMMA microparticles as sacrificial porogens. The effects of the microchannel geometry and flow velocity on the sheath flow generation were investigated. Furthermore, an asymmetric lattice-shaped microchannel network for cell/particle sorting was connected to the sheath flow generator in series, and the sorting performances of model particles, blood cells, and spiked tumor cells were investigated. The sheath flow generation technique developed in this study is expected to streamline conventional microfluidic cell-sorting systems as it dramatically improves versatility and operability.

Isolation of acute myeloid leukemia blasts from blood using a microfluidic device.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and associated with poor prognosis. Unfortunately, most of the patients that achieve clinical complete remission after the treatment will ultimately relapse due to the persistence of minimal residual disease (MRD), that is not measurable using conventional technologies in the clinic. Microfluidics is a potential tool to improve the diagnosis by providing early detection of MRD. Herein, different designs of microfluidic devices were developed to promote lateral and vertical mixing of cells in microchannels to increase the contact area of the cells of interest with the inner surface of the device. Possible interactions between the cells and the surface were studied using fluid simulations. For the isolation of leukemic blasts, a positive selection strategy was used, targeting the cells of interest using a panel of specific biomarkers expressed in immature and aberrant blasts. Finally, once the optimisation was complete, the best conditions were used to process patient samples for downstream analysis and benchmarking, including phenotypic and genetic characterisation. The potential of these microfluidic devices to isolate and detect AML blasts may be exploited for the monitoring of AML patients at different stages of the disease.

Microfluidics as an emerging paradigm for assisted reproductive technology: A sperm separation perspective.

Millions of people are subject to infertility worldwide and one in every six people, regardless of gender, experiences infertility at some period in their life, according to the World Health Organization. Assisted reproductive technologies are defined as a set of procedures that can address the infertility issue among couples, culminating in the alleviation of the condition. However, the costly conventional procedures of assisted reproduction and the inherent vagaries of the processes involved represent a setback for its successful implementation. Microfluidics, an emerging tool for processing low-volume samples, have recently started to play a role in infertility diagnosis and treatment. Given its host of benefits, including manipulating cells at the microscale, repeatability, automation, and superior biocompatibility, microfluidics have been adopted for various procedures in assisted reproduction, ranging from sperm sorting and analysis to more advanced processes such as IVF-on-a-chip. In this review, we try to adopt a more holistic approach and cover different uses of microfluidics for a variety of applications, specifically aimed at sperm separation and analysis. We present various sperm separation microfluidic techniques, categorized as natural and non-natural methods. A few of the recent developments in on-chip fertilization are also discussed.

Portable platform for leukocyte extraction from blood using sheath-free microfluidic DLD.

Leukocyte count is routinely performed for diagnostic purposes and is rapidly emerging as a significant biomarker for a wide array of diseases. Additionally, leukocytes have demonstrated considerable promise in novel cell-based immunotherapies. However, the direct retrieval of leukocytes from whole blood is a significant challenge due to their low abundance compared to erythrocytes. Here, we introduce a microfluidic-based platform that isolates and recovers leukocytes from diluted whole blood in a single step. Our platform utilizes a novel, sheathless method to initially sediment and focus blood cells into a dense stream while flowing through a tubing before entering the microfluidic device. A hexagonal-shaped structure, patterned at the device's inlet, directs all the blood cells against the channel's outer walls. The focused cells are then separated based on their size using the deterministic lateral displacement (DLD) microfluidic technique. We evaluated various parameters that could influence leukocyte separation, including different focusing structures (assessed both computationally and experimentally), the orientation of the tubing-chip interface, the effects of blood sample hematocrit (dilution), and flow rate. Our device demonstrated the ability to isolate leukocytes from diluted blood with a separation efficiency of 100%, a recovery rate of 76%, and a purity of 80%, while maintaining a cell viability of 98%. The device operates for over 30 min at a flow rate of 2 µL min-1. Furthermore, we developed a handheld pressure controller to drive fluid flow, enhancing the operability of our platform outside of central laboratories and enabling near-patient testing. Our platform can be integrated with downstream cell-based assays and analytical methods that require high leukocyte purity (80%), ranging from cell counting to diagnostics and cell culture applications.

Optoelectronic Tweezers Micro-Well System for Highly Efficient Single-Cell Trapping, Dynamic Sorting, and Retrieval.

Single-cell arrays have emerged as a versatile method for executing single-cell manipulations across an array of biological applications. In this paper, an innovative microfluidic platform is unveiled that utilizes optoelectronic tweezers (OETs) to array and sort individual cells at a flow rate of 20 µL min-1. This platform is also adept at executing dielectrophoresis (DEP)-based, light-guided single-cell retrievals from designated micro-wells. This presents a compelling non-contact method for the rapid and straightforward sorting of cells that are hard to distinguish. Within this system, cells are individually confined to micro-wells, achieving an impressive high single-cell capture rate exceeding 91.9%. The roles of illuminating patterns, flow velocities, and applied electrical voltages are delved into in enhancing the single-cell capture rate. By integrating the OET system with the micro-well arrays, the device showcases adaptability and a plethora of functions. It can concurrently trap and segregate specific cells, guided by their dielectric signatures. Experimental results, derived from a mixed sample of HepG2 and L-O2 cells, reveal a sorting accuracy for L-O2 cells surpassing 91%. Fluorescence markers allow for the identification of sequestered, fluorescence-tagged HepG2 cells, which can subsequently be selectively released within the chip. This platform's rapidity in capturing and releasing individual cells augments its potential for future biological research and applications.