Development of a biosensing microfluidic platform based on dielectrophoresis and impedance technique for cancer cell detection (tt)

Block diagram of the proposed dielectrophoresis microfluidic enrichment platform with an integrated impedance sensor for detecting circulating tumor cells: A Biological cells initially d

INTRODUCTION AND LITERATURE REVIEW

Motivation and research overview

1.1.1 Overview of cancer situations domestically and internationally

Cancer is the second leading cause of death, following cardiovascular diseases, characterized by uncontrolled cell growth and a failure to follow the body's regulatory mechanisms Its ability to spread and recur complicates treatment, requiring a multifaceted approach that may include surgery, radiation therapy, chemotherapy, and immunotherapy Early detection and timely intervention are crucial for improving treatment outcomes and enhancing the overall quality of life for patients.

According to Vietnam's Ministry of Health, GLOBOCAN reports that only 185 out of 204 countries provide cancer statistics, highlighting a global increase in cancer incidence and mortality rates In Vietnam, there are approximately 182,563 new cancer cases and 122,690 cancer-related deaths annually, translating to 159 new diagnoses and 106 deaths per 100,000 people Vietnam ranks 50th in cancer mortality and 91st in new cases among 185 countries, showing a significant rise from its 2018 rankings of 56th and 99th, respectively This trend reflects a broader global pattern, as many countries, including developed nations like the UK, France, and the US, experience rising cancer rates despite a decline in mortality.

In Vietnam, the most prevalent cancers among men include liver, lung, stomach, colorectal, and prostate cancers, which account for approximately 65.8% of all cases, while women commonly face breast, lung, colorectal, stomach, and liver cancers, representing around 59.4% of cases Both genders frequently encounter liver, lung, breast, stomach, and colorectal cancers Notably, lung and bronchial cancers, although second in incidence, lead in mortality rates, contributing to about 20% of all cancer-related deaths Early detection of lung cancer, typically when tumors range from 2-10 cm, is crucial for effective treatment, prompting recent research into molecular diagnostics for early-stage cancer detection Detecting cancer early, especially lung cancer, allows healthcare professionals to select the most appropriate and effective treatment options.

Figure 1 The 2020 GLOBOCAN statistics on the cancer situation in Vietnam show that the incidence and mortality rates are on the rise

Metastasis is the leading cause of death in cancer-related diseases, and the spread of tumor cells via the circulatory system plays a critical role in the metastatic process

Early detection and analysis of circulating tumor cells (CTCs) are vital for the diagnosis, prognosis, and treatment of cancer, as precise techniques for isolating and identifying CTCs are necessary for understanding cancer progression Metastasis, responsible for nearly 90% of cancer-related deaths, involves tumor cells breaking away from the primary tumor, infiltrating blood vessels, and traveling through the bloodstream to form new tumors at distant sites CTCs can adhere to vessel walls and exit the bloodstream, leading to secondary tumor development Some metastatic cells may enter a dormant state, while others can establish new tumors, with their behavior influenced by the metastatic microenvironment, resulting in different expression patterns compared to the original tumor cells.

The increasing incidence of cancer highlights the critical need for effective screening and early detection, as many patients receive their diagnosis at advanced stages Timely diagnosis significantly enhances treatment efficacy and improves the likelihood of successful recovery.

Figure 2 The process of tumor metastasis [8]

Figure 2 outlines the metastasis process involving circulating tumor cells (CTCs)

During metastasis, tumor cells detach from the primary tumor and enter the bloodstream as circulating tumor cells (CTCs), marking their transition from epithelial to mesenchymal cells While most CTCs succumb to programmed cell death or necrosis, releasing cellular debris and materials like circulating tumor microemboli (CTM), these CTMs are less common but pose a greater threat The unique microenvironment within CTMs protects the tumor cells, allowing them to proliferate and potentially cause vessel rupture In contrast, CTCs typically need to exit the bloodstream to establish metastases.

The discovery of circulating tumor cells (CTCs) dates back to 1869, but research has been limited due to their extreme rarity in the bloodstream, with only one CTC present for every 10⁶ to 10⁷ white blood cells in cancer patients This scarcity poses significant challenges for the enrichment and detection of CTCs However, recent advancements in science and technology have sparked global interest in CTC research, particularly in the development of devices for their detection and analysis, emphasizing the importance of isolating and concentrating these cells.

The advancements in detection and measurement techniques are crucial for enhancing the efficiency of patient sample analysis These improvements lead to greater accuracy and effectiveness in cancer treatment, allowing for the assessment of patients' cancer status with minimal intervention.

Lung cancer is primarily categorized into two types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) NSCLC accounts for the majority of lung cancer cases and generally progresses more slowly, offering higher recovery chances when detected and treated early Treatment options for NSCLC include surgery, radiation therapy, and chemotherapy In contrast, SCLC is a fast-growing cancer that often metastasizes quickly and is usually diagnosed at a later stage, with chemotherapy as the main treatment option The A549 cell line, a common epithelial cancer cell, is recognized as circulating tumor cells (CTCs) in the early stages of NSCLC.

1.1.2 Cancer detection procedures and the urgency of cellular tests

Cancer diagnosis today involves a combination of methods, starting with a thorough assessment of the patient's family, personal, and medical history, alongside presenting symptoms Clinical examinations play a crucial role in identifying tumors through visual and tactile techniques, allowing doctors to evaluate tumor size, characteristics, and lymph node involvement To gain deeper insights, para-clinical investigations are employed, which include hematological tests, tumor marker assessments, and advanced imaging techniques such as X-rays, ultrasounds, CT scans, and MRIs These methods are essential for detecting both superficial and deep-seated tumors, enhancing the overall diagnostic process.

Cytological diagnostic methods, including smear, scrape, brush, cell block, and fine needle aspiration, provide several advantages over traditional histopathological diagnosis, such as being faster, simpler, more cost-effective, and less invasive, while still allowing for immunohistochemical staining Despite these benefits, histopathology remains the "gold standard" for cancer confirmation, utilizing tissue sampling techniques like needle biopsy, excisional biopsy, endoscopic biopsy, and open surgery This approach is essential for determining whether tumors are benign or malignant, identifying cancer types and grades, and complementing other diagnostic tests such as immunohistochemistry and molecular biology However, biopsy procedures can carry risks, including bleeding, thrombosis, pain, infection, and pneumonia.

Figure 3 Routine cancer diagnosis procedure [15]

Detection of circulating tumor cells (CTCs) in the bloodstream primarily utilizes commercial technologies, including flow cytometers, the CellSearch system, high-resolution fluorescence scanning microscopes, fiber-optic array scanning technology (FAST), isolation by epithelial tumor cell size (ISET), and laser scanning cytometry devices.

Advancements in microfabrication technology have made the integration of physical methods on microfluidic platforms a pivotal focus for developing lab-on-a-chip systems for point-of-care diagnostics This integration promotes benefits like miniaturization, cost efficiency, and reduced chemical usage Microfluidic platforms also enable the direct incorporation of ancillary processes, such as sample pre-processing, within the chip Furthermore, disposable microfluidic chips ensure a sterile environment, minimizing the risk of cross-contamination and safely handling hazardous biological samples By combining cell-based diagnostic systems with microfluidic technologies, testing time and costs are significantly reduced, while also creating new avenues for biomedical research The evolution of microfluidic flow cytometry devices enhances laboratory capabilities, supporting critical biological research areas such as cancer studies, drug development, and genetic research, ultimately benefiting patients through expedited testing.

6 reducing the number of samples needed, and lowering the economic burden of medical testing

Cell assays are essential for measuring and analyzing the biological responses of cells to various external stimuli, including chemical and physical factors These responses encompass changes in cell membrane properties, movement, and growth characteristics, aiding in the identification of different cell phenotypes Traditionally conducted in culture dishes and multi-well plates, such as 96 or 384 wells, these assays have evolved with the advent of microfluidic devices, also known as on-chip cell assays While culture dishes require larger volumes of solutions, multi-well plates allow for smaller volumes and the simultaneous analysis of multiple cell types Additionally, plate readers, which utilize technologies like fluorescence intensity and absorbance, have become crucial for medium and high-throughput screening in cell and molecular biology assays.

Figure 4 Point-of-care testing [15]

Point-of-care (PoC) tests are straightforward medical assessments conducted at the patient's bedside, designed to provide immediate diagnostic results This convenience enhances the likelihood of rapid findings for patients, physicians, and care teams, facilitating quicker clinical decision-making Common examples of PoC tests include blood glucose monitoring, blood gas and electrolyte analysis, rapid coagulation tests, cardiovascular marker diagnostics, drug abuse screenings, urine dipstick tests, and pregnancy tests.

Background and literature review

Current commercial methods for isolating circulating tumor cells (CTCs) are either labor-intensive or reliant on costly automated equipment, and they struggle to isolate individual CTCs from blood samples Recent advancements in microelectromechanical systems (MEMS) and microfluidic technology have led to the development of more effective techniques for single CTC isolation These innovative methods provide enhanced performance, reproducibility, and functional integration The integration of microfluidic platforms has significantly miniaturized on-chip analytical techniques, facilitating more automated, precise, and high-throughput testing, while also reducing production costs and allowing for precise control over the isolation process.

The Lab-on-a-Chip technology for liquid biopsy offers significant advantages in disease monitoring and genetic profiling of lung cancer This innovative approach operates with just 15 key parameters, utilizing a closed architecture to prevent sample loss and allowing for the use of small sample volumes Additionally, it reduces reagent consumption and minimizes waste generation, making it a highly efficient and minimally invasive solution for cancer diagnostics.

Figure 8 Illustrates the applications of Microelectromechanical Systems

In the last two decades, microfluidic technologies for circulating tumor cell (CTC) isolation have gained prominence due to their ability to integrate multiple physical isolation methods These platforms leverage the biological differences between CTCs and normal cells, enabling precise separation from millions of background cells The low shear stress in microchannels, resulting from laminar flow, allows for the preservation of intact CTCs for subsequent testing and culturing By combining biological and physical isolation principles with advanced nanotechnologies on-chip, these multistep processes enhance the efficiency of CTC isolation Furthermore, the mass production of microfluidic devices offers a cost-effective solution for CTC research and diagnostics.

Microfluidic systems provide significant advantages over traditional methods due to their simplicity and rapid analysis capabilities They utilize smaller blood sample volumes and allow whole blood analysis without prior preparation Their compact design reduces workflow complexity, making them suitable for point-of-care testing and urgent applications By integrating multiple analytical steps into a single platform, microfluidic devices deliver faster and more reliable results These characteristics position microfluidic systems as a valuable option for diagnostics and research, particularly in personalized medicine and disease monitoring.

Microfluidic platforms provide significant advantages for the isolation and detection of circulating tumor cells (CTCs) from peripheral blood, allowing for direct processing without the need for dilution or labeling These low-cost chips demonstrate high sensitivity, capturing approximately 70% of CTCs at concentrations as low as 3 to 5 cells per milliliter Isolated CTCs can be utilized for further phenotypic identification and molecular analysis, enhancing their role in clinical diagnostics and cancer research The ability to conduct detailed downstream analyses on CTCs emphasizes the importance of microfluidic technologies in advancing personalized medicine and improving patient outcomes.

A CTC assay involves three key steps: sample preparation and tumor cell isolation, tumor cell staining or oncogene probing, and the detection of tumor cells.

Microfluidic technology is increasingly used by scientists to develop diagnostic chips for analyzing cell samples and solutions This technology facilitates the creation of essential structures like channels and incubation chambers for biochips, particularly in cell assays Typically, cell assays, especially those targeting circulating tumor cells (CTCs), involve three main steps Initially, blood samples are prepared by lysing red blood cells and filtering out white blood cells Next, tumor cells are separated using techniques such as antigen-antibody interactions or based on their physical properties like size and density Finally, these tumor cells are stained with specific markers and detected through various analysis methods.

17 such as flow cytometry, microscopy, optofluidic techniques, or molecular biology methods like polymerase chain reaction (PCR), as outlined in Figure 9

Recent studies on live cell assay systems utilizing microfluidic platforms focus on the design and fabrication of systems that integrate biological and physical interactions for the targeted isolation and analysis of specific cells Biochip systems, particularly for the separation and detection of circulating tumor cells (CTCs), are interdisciplinary innovations that merge nanotechnology, electronics, sensors, biology, fluid mechanics, and microfabrication The design of these separation systems demands high precision, a requirement that microfluidic chip systems are uniquely positioned to fulfill due to their exceptional advantages.

1.2.2 Research on CTCs isolation encompasses both domestic and international perspectives

Most cancers arise from epithelial cells, and techniques for isolating circulating tumor cells (CTCs) from blood samples leverage their histological origin Blood cells belong to connective tissue, which contrasts with epithelial cells in terms of size, shape, and nuclear characteristics Additionally, these cell types exhibit different surface receptors; notably, epithelial cells lack CD45, a marker specific to white blood cells.

Epithelial cells possess specific markers absent in blood cells, including EpCAM, E-cadherin, cytokeratin, zonula occludens, and ESPR1 Evaluating the expression levels of these surface receptors enables the differentiation of normal and abnormal epithelial cells For example, while EpCAM is minimally expressed in healthy epithelial cells, it is significantly overexpressed in certain cancer cells Additionally, elevated N-cadherin and reduced E-cadherin levels are indicative of cancer cells and circulating tumor cells (CTCs) in the bloodstream E-cadherin is crucial for maintaining cell-cell adhesion in epithelial cells, whereas N-cadherin is linked to epithelial-to-mesenchymal transition and is more prevalent in cancerous tissues.

Many commercial devices have been developed to isolate and detect CTCs from blood, as shown in Figure 10

Several commercial products leverage microfluidic systems for the isolation and detection of circulating tumor cells (CTCs) from blood, including MACS® by Miltenyi Biotec, Dynabeads™ from ThermoFisher Scientific, C1 by Fluidigm, and the DEPArray System from Menarini-Silicon Biosystems, each accompanied by their respective microfluidic devices.

Microstructures offer an efficient method for capturing individual cells due to their straightforward design and operational efficiency The C1 system by Fluidigm automates this process by isolating cells into 800 distinct microchambers within an integrated fluidic circuit (IFC) Although the system necessitates sample preparation to purify target cells and does not allow for the isolation of individual captured cells, it enables automatic staining within the IFC for monitoring cell viability, labeling surface markers, or observing reporter genes microscopically Furthermore, the captured cells can be automatically separated to facilitate the preparation of single-cell samples for subsequent qPCR or sequencing analysis.

Dielectrophoresis (DEP) is an innovative liquid biopsy technique that enables the separation of particles with varying polarizations in a non-uniform electric field Utilizing DEP, microcircuits have been developed to effectively isolate circulating tumor cells (CTCs) through a series of integrated electrodes, generating an alternating electric field within liquid environments ApoCell's ApoStream™ system stands out as the first commercial application of DEP forces for differentiating CTCs from white blood cells based on their conductivity differences This advanced device achieves a capture rate exceeding 70%, with a remarkable viability rate above 97%, and processes a 10 mL blood sample in under one hour However, it is important to note that the purity of the isolated CTCs remains below 1%.

Figure 11 Schematic diagram of the ApoStream device; inset shows cell flow and separation in the flow chamber [53]

The DEPArray, developed by Menarini-Silicon Biosystems, is an advanced microfluidic device that utilizes dielectrophoresis (DEP) to trap individual cells within 16,000 electrode cages This innovative system allows for the identification of trapped cells through integrated fluorescence microscopy, enabling the controlled transfer of selected cells into adjacent cages, encapsulation into droplets, and release into a chamber as single or multiple cells To effectively analyze circulating tumor cells (CTCs), the DEPArray requires prior sample enrichment, often achieved through systems like CellSearch, which isolates CTCs from blood for mutation identification via sequencing Numerous studies have successfully employed DEP principles within microfluidic structures to separate CTCs from normal cells, such as the method developed by Jen et al., which efficiently isolates cervical cancer cells (HeLa) from blood samples using circular microelectrodes to create a stepped electric field.

HeLa cells exhibit a separation efficiency approximately seven times greater than red blood cells (RBCs) when subjected to a specific electric field distribution in a sucrose medium This allows for the effective isolation of HeLa cells from normal blood cells, concentrating them at the central microelectrode Experimental findings demonstrate the viability of extracting HeLa cells from blood samples.

Figure 12 Schematic diagram of a two-stage enrichment chip for selective isolation of CTCs [56]

RESEARCH METHODS

Proposed platform for detecting lung cancer cells using impedance

Cell enrichment and detection play a vital role in biomedical applications, particularly in early disease diagnosis and treatment monitoring This study introduces an innovative microfluidic chip aimed at enhancing the capture and detection of A549 lung cancer cells, integrating a dielectrophoresis (DEP) platform with impedance measurement using interdigitated electrodes (IDE) The chip employs a chemical interaction-based binding method utilizing cell-specific aptamers to boost capture efficiency and specificity Constructed with Indium-Tin-Oxide (ITO) and a self-assembled monolayer of gold nanoparticles (AuNP), the microelectrode structure facilitates targeted cell movement through hydrodynamic traction and positive dielectrophoretic forces Impedance spectroscopy at varying cell concentrations confirms the sensor's ability to detect A549 cells at low concentrations, while electric field simulations offer insights into the dynamics of captured cells The integration of DEP and impedance measurements enhances sensitivity and specificity, effectively concentrating cancer cells at the sensors and minimizing background noise This label-free method simplifies sample preparation and maintains the natural state of cells for reliable results, enabling real-time analysis for immediate diagnostic insights With high throughput capabilities for large-scale screenings and a modular design for flexibility in adapting to various cancer cells, this platform broadens its diagnostic applications beyond lung cancer.

The proposed dielectrophoresis microfluidic enrichment platform features an integrated impedance sensor designed for the detection of circulating tumor cells Initially, biological cells are randomly distributed, but the innovative chip structure facilitates targeted manipulation through DEP electrodes This process effectively captures the cells, followed by impedance spectroscopy sensing for accurate detection.

This study presents a novel technique that boasts high sensitivity, specificity, and accuracy for targeted applications The method is simple, rapid, label-free, and cost-effective, making it a promising advancement for improving cell diagnostic systems in biomedical fields.

Materials and methods

This study focused on gold nanoparticles measuring 13 nm in diameter, sourced from TANBead in Taoyuan City, Taiwan, at a concentration of 179 ppm The surface functionalization was achieved using (3-aminopropyl) triethoxysilane (APTES) along with alkoxysilane molecules for glass silanization, procured from Sigma-Aldrich in St Louis.

Cell staining dyes such as calcein green AM and calcein red-orange AM, along with phosphate-buffered saline (PBS, 10X), bovine serum albumin (BSA), and yeast tRNA, were obtained from Thermo Fisher Scientific in Eugene, OR, USA The full-length DNA aptamer sequence, 5’-ACGC TCGG ATGC CACT ACAG GGTT GCAT GCCG TGGG GAGG GGGG TGGG TTTT ATAG CGTA CTCA GCTC ATGG ACGT GCTG GTGA C-3’-modified 5’-thiol (5’-C6SH), was supplied by MdBio, Inc in Taiwan This aptamer has been shown to effectively bind to A549 cells in multiple studies.

For the study, two solutions were prepared: a binding buffer for aptamer treatment and an 8.62 wt% sucrose solution for cell-related processes, including DEP manipulation and washing steps The binding buffer contained 15 mg of BSA, 0.72 mg of MgCl2, and 150 µL of yeast tRNA (10 mg/mL), all diluted in 15 mL of PBS (1X, pH 7.4), using ultrapure water (18.2 MΩ cm at 25°C) from a Direct-Q system (Milli-Q, Millipore Simplicity, Billerica, MA, USA) A549 cells were obtained from Sigma-Aldrich Chemical Co (St Louis, MO, USA), and indium tin oxide (ITO) was selected as the electrode material for its high transparency and excellent electrical conductivity, sourced from Indium Corporation®.

The approach's effectiveness in targeting and manipulating specific cell types is highlighted by its advanced materials and precise methodologies The detection system's sensitivity and specificity are greatly improved through the use of well-characterized aptamers, along with the careful selection of nanoparticles and functionalized surfaces.

To clarify the preparation procedures for chemicals, equipment, and the measurement system, the detailed processes are presented as follows: a) Culture of human lung adenocarcinoma cells (A549 cells)

A549 cells, derived from human alveolar basal epithelial cells, play a crucial role in lung cancer research due to their unique characteristics as non-small cell adenocarcinoma These naturally squamous cells facilitate the diffusion of water and electrolytes across the alveolar wall and form a tightly adherent monolayer in laboratory cultures With an average size of about 25 μm, A549 cells are distinguished by their genetic profile and transcriptional gene expression changes They are extensively utilized to investigate the mechanisms of lung cancer development, evaluate treatment efficacy, and study carcinogenic agents, as well as in various research fields including molecular biology, genetics, physiology, and microbiology.

The A549 cell culture process is a vital technique widely used in Medicine and Molecular Biology, particularly for studying lung cancer and respiratory diseases Derived from lung cancer, A549 cells play a significant role in research on cell development and functions The procedure involves essential steps, starting with the careful preparation of a culture medium that contains glucose, metabolic reactants, vitamins, and necessary proteins for A549 cell growth and maintenance This medium is then placed in sterilized test tubes, inoculated, and allowed to solidify at room temperature, ensuring optimal conditions for cell culture.

A549 cells are retrieved from storage and carefully introduced into the medium The injection must be performed with precision to maintain the cells' viability

Figure 21 The incubator for cell culture

The A549 cells are incubated under controlled temperature and humidity conditions, with continuous monitoring throughout the cultivation process Regular evaluations of cell quantity, sensitivity, and other characteristics are crucial for maintaining their stability and quality Moreover, it is important to monitor environmental factors such as pH, humidity, and temperature to ensure optimal growth conditions.

Continuous monitoring and adjustment of CO2 levels during the cell culture process are crucial, as depicted in Figure 21 These parameters directly impact cell growth and can greatly influence the success of the cultivation process if not properly managed.

Figure 22 A549 cells are cultured on a Petri dish

This study utilizes human lung adenocarcinoma cells (A549 cells), which necessitate specific nutrients and an optimal growth environment for ideal development A549 cells are cultured in 10 cm dishes using Dulbecco's Modified Eagle Medium (DMEM) as the culture medium, and they are incubated at 37°C with 5% CO2.

During the preparation of DMEM, 3.7 g/L of NaHCO3 is thoroughly mixed, and the pH is adjusted to 7.2-7.4 using HCl or NaOH to create an optimal environment for cell growth To prevent bacterial and fungal contamination, the medium is supplemented with 1% (v/v) penicillin/streptomycin (Gibco, Grand Island, NY, USA), and to supply essential nutrients, 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) is added.

As A549 cells grow, they generate metabolic waste and eventually die, making it essential to wash them with phosphate-buffered saline (PBS, Biochrome, pH 7.4) to eliminate these byproducts Typically, A549 cells adhere to and cover the culture dish within 2-3 days If they have not yet achieved full coverage, it's important to refresh the medium by washing with 1.5 mL of PBS to remove waste and then adding 8-10 mL of fresh medium to ensure optimal growth conditions.

Figure 23 A549 cells, after being successfully cultured

When cells reach confluence, subculturing is essential to maintain healthy growth This process begins by detaching the cells from the dish surface using 1.5 mL of Trypsin-0.5% EDTA, which disrupts adhesion proteins The enzyme is incubated for five minutes to activate the trypsin Once detached, the cells are suspended and diluted with fresh medium to prevent damage from the enzyme The cell suspension is then transferred to a centrifuge tube and centrifuged at 500 rpm for five minutes to pellet the cells After centrifugation, the supernatant is discarded, and the cells are resuspended for further use.

41 a fresh medium before being transferred to a new culture dish to complete the subculturing process

Figure 23 illustrates the successful cultivation of A549 cells, with the left side displaying a microscopic view at a scale of 100 µm, revealing a dispersed arrangement with some clusters, indicative of early growth stages The right side provides a closer observation at 50 µm, highlighting individual cells and small clusters, which showcases their morphology and distribution in the culture medium Both images confirm the health and stability of the A549 cell culture, reflecting a well-maintained environment Additionally, cell counting was performed using a hemocytometer.

This study utilizes a hemocytometer with dimensions of 0.05 x 0.05 mm² to accurately count and quantify A549 cells post-cultivation for experimental analysis The hemocytometer features two parallel striped glass plates that create a grid-like counting area at their intersections This counting area comprises 16 small squares, each measuring 0.0025 mm², with the central square further subdivided into 25 smaller squares, each having an area of 0.0001 mm².

Figure 24 Preparing the hemocytometer for A549 cell counting by injecting the cell and Trypan Blue mixture into the counting chambers

To accurately count cells using a hemocytometer, first sterilize the device with 95% ethanol Prepare a cell solution by mixing 10 µL from a centrifuge tube with 10 µL of Trypan Blue solution in a 1:1 ratio Inject 10 µL of this mixture into the designated areas of the hemocytometer and examine it under an inverted microscope Dead cells will absorb the dye and appear stained due to their ruptured membranes, whereas live cells will remain unstained Finally, calculate the total cell count by dividing the number of counted cells by the eight counting areas of the hemocytometer, and multiply by the dilution factor.

42 factor (equal volumes of cell and dye mixture), and converting to the appropriate volume unit to determine the number of cells per milliliter of cell suspension

The cell count is calculated using the formula shown in Equation 1:

Figure 25 The distribution of A549 cells in the counting area of a hemocytometer

Microchip design and fabrication

This study utilizes a microfluidic chip featuring ITO electrodes to generate dielectrophoretic forces that facilitate the capture of A549 cells by SH aptamers The chip is composed of three primary components: ITO capture electrodes, a self-assembled monolayer of gold nanoparticles, and a microfluidic channel designed with a height of 100 µm to ensure smooth cell flow It includes three inlets for sucrose washing and cell samples, along with one outlet for uncaptured cells The ITO microelectrodes, known for their excellent electrical conductivity, are patterned on an ITO-coated glass substrate through photolithography To improve capture efficiency, eight pairs of electrodes are strategically designed, and a gold nanoparticle monolayer enhances the contact area for aptamer modification The microfluidic channel is constructed from biocompatible PDMS and is bonded via oxygen plasma treatment, with the actual chip illustrated in accompanying figures.

Figure 29 Schematic diagram of chip design

Figure 30 (a) Schematic diagram of ito electrode dimensions (b) Cross-sectional schematic diagram of aptamer-modified self-assembled electrode boundaries

Figure 31 This image shows the actual microfluidic chip used in the experiment

This study utilizes indium tin oxide (ITO) conductive glass as the substrate for its superior conductivity, transparency, and affordability The fabrication process involves defining patterns and dimensions with a photomask, followed by the creation of electrode patterns on the ITO glass through standard photolithography Additionally, microfluidic channel patterns are developed on a silicon wafer, with the channels molded using polydimethylsiloxane (PDMS), known for its high affinity.

The chip fabrication process consists of four steps:

2.3.2 Surface modification of the chip and gold nanoparticle self-assembly 2.3.3 Microfluidic channel fabrication

2.3.4 Bonding of the PDMS microchannel to the chip

The flowchart in Figure 32 outlines the comprehensive process for fabricating ITO electrode chips, which involves several key stages: spin-coating, exposure, development, and etching Each of these steps plays a crucial role in accurately creating electrode patterns on an ITO-coated glass substrate.

The process involves using S1813 positive photoresist on an ITO conductive glass chip Initially, the chip is thoroughly cleaned with acetone, methanol, and deionized water Next, an appropriate amount of S1813 photoresist is applied to the chip's surface using a spin coater This spin coater operates in a two-stage process: the first stage spins at 700 rpm for 10 seconds to eliminate excess photoresist, while the second stage spins at 1700 rpm for 20 seconds to achieve a uniform distribution of the photoresist across the chip.

3 àm thick photoresist layer Finally, the chip is soft-baked at 90°C for 4 minutes to remove residual moisture and enhance the adhesion of the photoresist to the chip surface b) Exposure

The soft-baked chip is exposed through a plastic photomask that features the electrode pattern Prior to exposure, a power meter measures the exposure power (mW/cm²) of the machine to determine the necessary exposure time, as detailed in Equation (9).

In this study, an exposure dose of 90 mJ/cm² with a UV light source at a wavelength of 365 nm is used to expose the electrode pattern onto the photoresist To

53 further increase the adhesion strength of the photoresist to the chip, a post-exposure bake is performed by heating the chip at 90°C for 4 minutes

Exposure Dose (mJ/cm 2 ) = Exposure intensity (mW/cm 2 ) ×

Figure 32 ITO electrode chip fabrication flowchart c) Development

The S1813 positive photoresist is developed using MP351 developer, which is diluted with deionized (DI) water at a 1:4 ratio The chip is immersed in this developer mixture, allowing the UV-exposed S1813 photoresist layer to be removed during the development process After rinsing the chip with DI water, a post-development bake at 120°C for 4 minutes is performed to stabilize the resist layer, preparing it for the subsequent electrode etching.

This study utilizes wet etching, where the chip is immersed in ITO etchant at 65°C for 1 minute to effectively etch the ITO glass substrate Following the etching process, the chip is rinsed with deionized water, and a multimeter measures the conductivity of the patterned electrodes to ensure the removal of coating outside the electrode areas Finally, a microscope is employed to examine the completeness of the electrode patterns, marking the conclusion of the etching procedure.

2.3.2 Surface modification of the chip and gold nanoparticle self-assembly

Recent studies have shown that gold nanoparticles (AuNPs) can be effectively immobilized on glass surfaces using an APTES layer APTES contains three alkoxy functional groups that interact with hydroxyl groups on the glass, resulting in the formation of siloxane linkages This process modifies the surface charge from negative to positive by introducing amino groups Meanwhile, the AuNPs acquire a negative charge due to their inherent properties and the presence of citrate groups formed during synthesis As a result, the substrate exhibits strong adhesion to AuNPs, enhancing their immobilization on the glass surface.

The SH-aptamer, a full-size DNA aptamer modified with a thiol group (−SH) at the 5′-end, specifically targets the A549 human lung carcinoma cell line, as demonstrated by previous research This aptamer simplifies the preparation process by requiring only a single modification step after the folding procedure, enhancing capture efficiency The strong Au–S bond immobilizes one end of the SH-aptamer to a gold surface, while the other end is designed to capture A549 cells These cells are effectively attracted by gold nanoparticles (AuNPs) through dielectrophoresis (DEP), showcasing the dual benefits of the thiol functional group and targeted cell capture.

This study utilized pre-synthesized gold nanoparticles (AuNPs) with a diameter of 13 nm to achieve a high-density and uniform distribution on the desired self-assembled monolayer (SAM) area The small size of these nanoparticles provides a high surface-to-volume ratio, which is essential for maximizing interaction with target molecules or cells, thereby enhancing the sensitivity and uniformity of the biosensor Additionally, AuNPs exhibit unique optical properties, particularly strong surface plasmon resonance (SPR), which amplifies local electromagnetic fields upon excitation This characteristic facilitates the detection of molecular interactions on their surfaces, directly correlating with the presence of target analytes such as cancer cells.

Despite employing chemical modifications and DEP manipulations that generated an electric current on the electrode surface, no flaking or destruction of gold nanoparticles (AuNPs) occurred during the experiment This remarkable stability is vital, as AuNPs are chemically resilient and can be easily modified with various functional groups In this case, they are anchored to the substrate through APTES (3-Aminopropyltriethoxysilane) bridges, which effectively link the glass surface to the AuNPs, ensuring their adherence under electrical and mechanical stresses.

The SAM area was specifically designed with a width of 350 μm and a length of 4500 μm to ensure complete coverage of the cell enrichment region by AuNPs While the SAM area incorporates the IDE structure, AuNPs are immobilized solely on the glass surface through APTES bridges SH-aptamers can be functionalized on both the electrode structure and the AuNPs, with the primary objective being the capture of target A549 lung cancer cells at the SAM of AuNPs This immobilization not only utilizes the chemical properties of the aptamers but also enhances binding interactions through the localized electromagnetic fields around the AuNPs, significantly improving the specificity and efficiency of capturing target A549 cells.

The detailed implementation steps are outlined below: a) Silanization surface modification

In this study, a solution of 3-Aminopropyltriethoxysilane (APTES) is mixed with deionized water at a 1:1000 ratio to modify the ITO chip surface A pipette is used to apply 20 μL of the solution to the electrode boundaries on the ITO substrate, allowing the glass surface to be treated for 1 minute, which results in the formation of an amino (NH2) monolayer The final steps involve rinsing the chip with deionized water and drying it with nitrogen gas to eliminate excess moisture, thereby completing the silanization modification process.

Gold nanoparticles are concentrated at 0.9 mM to enhance modification opportunities After centrifugation, these nanoparticles self-assemble onto the modified patterned electrode boundaries and are allowed to bond chemically with amino (NH2) groups for 90 minutes Following this bonding period, the chip is rinsed with deionized water to eliminate any non-assembled nanoparticles The final step involves using acetone, methanol, and deionized water to remove the S1813 photoresist layer from the electrode surface, successfully completing the self-assembly of gold nanoparticles on the ITO electrode boundaries.

The process of modifying an indium tin oxide (ITO) electrode involves silanization, where 3-Aminopropyltriethoxysilane is used to create an amino (NH2) monolayer on the silicon surface of the glass substrate This layer provides reactive sites for further modifications Following this, gold nanoparticles undergo self-assembly onto the modified patterned electrode boundaries, forming chemical bonds with the amino groups on the surface.

56 resulting in a stable and functionalized electrode suitable for bioanalytical applications

Numerical calculations

This study utilized COMSOL Multiphysics 6.0 to analyze the electric field distribution in the capture electrode area of the chip, enabling precise cell manipulation while preventing damage from high-intensity electric fields.

The CM factor is a key determinant of particle movement in dielectrophoresis (DEP), influenced by the polarizability of particles compared to their surrounding medium When particles are more polarizable than the medium, a positive CM factor leads to positive DEP (pDEP) forces, while less polarizable particles experience negative DEP (nDEP) forces The polarizability of particles can be adjusted by varying the applied frequency, allowing for control over DEP force characteristics This study utilized the protoplast model to analyze the DEP force on A549 cells, based on their physical properties and those of the sucrose medium, as detailed in the corresponding table.

Table 3 The dielectric properties of A549 cells and the sucrose medium (8.62 wt%) [106], [122]

Radius (𝛍m) Cytoplasm Membrane Cytoplasm Membrane

Numerical simulations are essential for predicting and optimizing device design by accurately representing electric field distribution This study utilized COMSOL Multiphysics 6.0 to simulate the electric field generated by the proposed device during dielectrophoresis (DEP) A 3D model was created to reflect the actual chip size, ensuring alignment between simulation results and experimental data The configuration included an interdigitated electrode (IDE) structure situated in a straight channel filled with a sucrose solution, as illustrated in the accompanying figures.

Figure 35 Establishing the electrode simulation model

Figure 36 Simulation parameter setup diagram

The meshing step is essential for achieving accurate simulations, as reducing the mesh element size increases the maximum electric field, particularly at the sharp points of the electrode However, this effect is localized, with the electric field in other regions remaining largely unaffected In this study, we utilized the finest element size available in the software, enhancing both the accuracy of our simulation and the smoothness of the distribution results.

The study employs mesh partitioning techniques and carefully selected refined mesh sizes to achieve precise physical control simulations, ensuring accurate results This setup is illustrated in Figure 37.

The investigation focused on the electrostatics interface in AC/DC physics, specifically for calculating electric field, electric displacement field, and potential distributions Utilizing a Dirichlet boundary condition within a two-electrode system, the study applied voltages while immersed in a conductive solution The simulation equations established a relationship between the electric field (E) and electric potential (V).

Besides, equation (11) relates the space charge density to the divergence of the electric field, showing how charge distribution affects the electric field in the medium

𝜌 =∇⋅ D (11) where D is the electric displacement field

The selected materials for the electrode, channel, and glass substrate include ITO electrodes, water, and glass, respectively, all derived from a software library that reflects the actual materials utilized in the device To accurately represent the environment of the sucrose solution (8.62 wt%), several electrical parameters of the water, such as permittivity and conductivity, have been modified, as detailed in Table 3.

Experiment setup

After overnight incubation of the aptamer, the chip was rinsed with a sucrose solution at a flow rate of 1 µL/min for 30 minutes to remove unbound aptamers and minimize nonspecific cell adhesion A function generator (SFG-2004, GW Instek, New Taipei, Taiwan) was set up to produce dielectrophoretic (DEP) force within a fluidic flow, with a frequency range of up to 4 MHz A549 cells were transferred from their culture medium to a sucrose solution for consistency and stained with calcein green AM dye The green-stained A549 cells were diluted and injected into the channel using a syringe pump system (KDS Model 101, KD Scientific Inc., Holliston, MA, USA) at a rate of 2 µL/min Once the cell sample entered the chip, the function generator was activated to apply positive DEP force on the target cells The cell trapping process was continuously observed using an inverted fluorescence microscope (CKX41, Olympus, Tokyo, Japan) connected to a CCD camera (DP71, Olympus, Tokyo, Japan) and a computer with Olympus DP Controller image software Following DEP manipulation, the channel was washed with a sucrose solution at 5 µL/min for 10 minutes to eliminate nonspecific adsorption from the electrode surface.

The experimental setup for capturing target A549 cells utilizing dielectrophoresis (DEP) includes essential components such as a function generator for DEP signal generation, an inverted fluorescence microscope for visual observation, and a syringe pump system for precise sample injection.

The DEP force improved the interaction between cells and aptamers, leading to the effective capture of A549 cells in the SAM area through specific aptamer binding, as illustrated in Figure 38.

Figure 39 (A) Schematic diagram of syringe pump operation; (B) Schematic diagram of cell accumulation

To improve the capture efficiency of the microfluidic chip and reduce errors from cell sedimentation, a reverse pump clamp was developed This experimental setup utilizes a syringe pump that secures the sample-containing syringe in place, employing a slider to transport the sample into the channel along a guide rail, as shown in Figure 39 (A).

Figure 40 Schematic diagram of reverse pump clamp design

In the fabrication method outlined in section 2.3.4, the PDMS inlet, created using a hole punch, is positioned at a 90° angle to the glass substrate This setup can lead to sedimentation of cells at the inlet if the fluid shear force from the pump is lower than the combined effects of cell mass and gravity, which negatively affects experimental accuracy, as illustrated in Figure 39 (B) To mitigate this issue, SolidWorks software was employed to design a clamp model, which was subsequently produced using a 3D printer, as demonstrated in Figure 40.

Figure 41 (A) Schematic diagram of reverse pump operation direction; (B)

Schematic diagram of reverse pump extraction

The clamp model, when mounted on the syringe pump, effectively minimized cell accumulation at the inlet by reducing the size of the cell sample inlet This innovative design, illustrated in Figure 41, significantly decreased experimental errors.

The research methods section details the strategies and techniques employed in the development of the microfluidic device, focusing on material selection, theoretical operation principles, and the careful design and fabrication of the microfluidic chip It emphasizes the integration of gold nanoparticles and SH-aptamers, which are essential for improving the device’s functionality The precise fabrication methods are critical for optimizing the device for effective cell capture, which is vital for subsequent experimental validation.

RESULTS AND DISCUSSIONS

The working mechanism of the chip for the DEP-based cell trapping 64 3.2 Effects of applied voltage and duration on cell trapping by DEP

The dependence of the CM factor was calculated using a MATLAB program, as illustrated in Figure 42 This factor was derived from Equation (4) based on the protoplast model, incorporating parameters such as 𝜏 m, 𝜏 𝑚 ∗, 𝜏 c, and 𝜏 𝑐 ∗, which were calculated from the dielectric properties listed in Table 3 Consequently, 𝑓CM emerges as a complex function of frequency The cell membrane's extremely low electrical conductivity, primarily due to its phospholipid bilayer structure acting as a semi-porous barrier, means that the cell's overall conductivity is mainly attributed to the cytoplasm, rendering the membrane's contribution negligible and excluded from this model Therefore, the membrane conductivity parameter is not included in Table 3 and does not significantly influence the DEP force.

The research findings reveal that the real part of the CM factor becomes positive at frequencies exceeding 10 3.5 Hz, also known as the crossover frequency This indicates a significant change in the behavior of the CM factor, particularly in relation to the DEP manipulation conducted at this frequency.

1 × 10 6 Hz, which exhibits a pDEP force Consequently, the A549 cells will be directed to areas with a high electric field gradient

Figure 42 The Clausius–Mossotti factor plot: real and imaginary components as a function of frequency for A549 cells in sucrose medium

This study evaluated the effects of four alternating voltage levels—5, 7.5, 10, and 12.5 Vpp—on electric field strength COMSOL simulations indicated that at the maximum voltage of 12.5 Vpp, the electric field strength reached 2.12 x 10^5 V/m at the electrode edges The intensity distribution showed that the area between the electrodes, where immobilized AuNPs are located, experienced high electric field strength, while the IDE surface exhibited low intensity This configuration effectively concentrates and traps cells in the region between the electrodes, particularly around the immobilized AuNPs, rather than on the electrode surface The midsection of the electrode, especially at the corners of the comb structures, demonstrated low electric field gradients, which likely hindered A549 cell accumulation in those areas These observations were corroborated by actual cell distribution results from the experiments.

Figure 43 Diagram of electric field strength

A549 cells were observed to aggregate in regions with elevated electric field gradients, aligning with simulation findings The maximum electric field strength recorded was around 2.12 × 10^5 V/m near the electrodes Research indicates that cell viability is effectively preserved in low conductivity solutions and in the presence of toxic metal ions, even at electric field strengths reaching approximately 5 kV/cm or 10 kV/cm for durations of one hour.

Figure 44 Electric field distribution and cell capture result diagram

The study demonstrates that the peak electric field area is minimal compared to the total capture area, leading to a rapid decrease in the electric field strength away from the electrodes Consequently, this has a negligible impact on cell viability, as evidenced by the observation of cells captured by dielectrophoresis (DEP) for 15 minutes without significant cell death These results underscore the effectiveness of the generated electric field and lay the groundwork for future cell capture applications utilizing modified aptamers on gold nanoparticles (AuNPs).

3.2 Effects of applied voltage and duration on cell trapping by DEP

This study evaluated three distinct microfluidic chip designs for their effectiveness in capturing lung adenocarcinoma cells The first design featured a basic chip without self-assembled layers, gold nanoparticles, or aptamers The second design included a self-assembled layer of gold nanoparticles, enhancing surface properties for potential interactions, but lacked aptamers The third and most advanced design incorporated both gold nanoparticles and aptamers in the self-assembled layer, aiming to maximize cell capture specificity and efficiency.

The study investigated lung adenocarcinoma cell capture using various chip designs that employed dielectrophoresis for cell manipulation The effectiveness of each chip in isolating target cells was influenced by the presence of gold nanoparticles and aptamers Quantitative assessments of cell capture efficiency were performed using fluorescence detection and analyzed with Image J software This method facilitated a comprehensive comparison of the impact of different chip configurations on capture performance, yielding valuable insights.

68 into the potential benefits of integrating gold nanoparticles and aptamers into microfluidic devices for enhanced cancer cell detection

3.2.1 Electric field application time comparison

This study investigates the impact of varying electric field application times on cell capture using chips devoid of a self-assembled layer, relying exclusively on dielectrophoresis To assess the influence of application duration on nonspecific cell capture, voltages were applied for intervals of 5, 10, and 15 minutes.

Figure 45 Diagrams of cell capture experiments with voltage applied for (A) 5 minutes; (B) 10 minutes; (C) 15 minutes

Figure 46 Diagrams of cell capture experiments after releasing the voltage following (A) 5 minutes, (B) 10 minutes, and (C) 15 minutes of voltage application

Figure 47 Graph of cell capture efficiency at various voltage application times

After deactivating the AC signal, the microchannel was rinsed with sucrose, and fluorescence intensity was measured using Image J software (Figure 45) Applying a 12.5 Vpp AC signal for 5 minutes resulted in a capture rate of 24.9% ± 1.2% of cells, with 7.2% ± 2.4% remaining at the electrode edges post-voltage release (Figure 46 A) Extending the AC signal to 10 minutes increased cell capture to 54.6% ± 3.5%, with 16.1% ± 2.1% remaining (Figure 46 B) A 15-minute application further improved capture to 81.4% ± 3.1%, but 18.2% ± 1.9% of nonspecific cells were still present after voltage release (Figure 46 C) The findings indicate that longer electric field application times enhance nonspecific cell capture; therefore, a 10-minute application time was selected for subsequent experiments to optimize capture efficiency while reducing nonspecific errors and minimizing electric field-induced cell damage.

3.2.2 Cell capture results for chips without self-assembled layer modification

In this experiment, microfluidic chips devoid of gold nanoparticles and aptamers were utilized to capture cells exclusively through dielectrophoretic force by applying an AC signal The objective was to demonstrate that while dielectrophoretic force can facilitate cell capture, it does not provide stable and firm retention of cells at the electrode edges The efficiency of cell capture was quantified using Image J for fluorescence intensity detection, as illustrated in Figure 50.

Figure 48 Diagrams of cell capture experiments for chips without self- assembled layers under applied voltages of (A) 5 Vpp; (B) 7.5 Vpp; (C) 10 Vpp; and (D) 12.5 Vpp

Figure 49 Diagrams of cell capture experiments for chips without self- assembled layers under applied AC signals of (A) 5 Vpp; (B) 7.5 Vpp; (C) 10

Vpp; and (D) 12.5 Vpp, after releasing the voltage

Figure 50 Graph of cell capture efficiency at different voltages (chips without self-assembled layers)

Experimental data revealed that at 5 Vpp AC voltage, 11.9% ± 2.0% of cells were captured, with only 2.4% ± 0.8% remaining post-voltage release At 7.5 Vpp, 30.6% ± 5.4% of cells were captured, leaving 9.2% ± 0.9% after release With a 10 Vpp AC signal, 32.6% ± 2.2% of cells were captured, and 13.2% ± 1.3% remained afterward At 12.5 Vpp, 54.6% ± 3.5% of cells were captured, with 16.1% ± 2.1% still present after the voltage was removed These findings indicate that following the release of the dielectrophoretic force, most cells are affected by fluid shear force, leading them to move towards the outlet, while only a small fraction remains at the electrode edges due to the applied voltage and duration.

3.2.3 Cell capture results for chips with self-assembled gold nanoparticles

The experiment utilized microfluidic chips embedded with gold nanoparticles, intentionally excluding aptamers An alternating current (AC) signal was employed to create a dielectrophoretic force on the surface of the gold nanoparticles, facilitating the capture of A549 cells The capture efficiency at various voltage levels was assessed and quantified through Image J fluorescence intensity detection, as illustrated in Figure 53.

Figure 51 Diagrams of cell capture experiments at (A) 5 Vpp; (B) 7.5 Vpp; (C)

Figure 52 Cell capture experiments with applied AC signals of (A) 5 Vpp; (B)

Figure 53 Cell capture efficiency graph at various voltages (self-assembled gold nanoparticle chips)

The study revealed that applying a 5 Vpp AC voltage resulted in the capture of 32.4% ± 0.9% of cells, with only 9.1% ± 0.7% of cells remaining at the electrode edges after the voltage was released In contrast, using a 7.5 Vpp AC signal increased cell capture to 46.4% ± 1.1%, while only 1.9% ± 1.3% of nonspecific cells persisted post voltage release.

10 Vpp AC voltage, 58.7% ± 3.5% of cells were captured, as shown in Figure 51 (C), with 20.3% ± 0.2% of cells remaining after voltage release, as shown in Figure 52 (C)

At 12.5 Vpp AC voltage, 70.5% ± 3.1% of cells were captured, as shown in Figure 51 (D), with 24.4% ± 1.1% of cells remaining after voltage release, as shown in Figure 52 (D) The quantification data indicate that the self-assembled gold nanoparticle layer increases the likelihood of cells being influenced by dielectrophoretic force to approach the capture area, without causing excessive nonspecific adhesion to the electrode surface

3.2.4 Cell capture results for aptamer-modified self-assembled layer chips

This study focuses on the modification of thiol-based aptamers on self-assembled gold nanoparticle chips, establishing a cell capture zone between the edges of ITO electrodes By applying a 1 MHz AC signal, a positive dielectrophoretic force is generated, facilitating the effective capture of lung adenocarcinoma cells by the aptamers.

Figure 54 Diagrams of cell capture experiments for aptamer-modified self- assembled layer chips under applied voltages of (A) 5 Vpp; (B) 7.5 Vpp; (C) 10

Figure 55 Diagrams of cell capture experiments for aptamer-modified self- assembled layer chips under applied AC signals of (A) 5 Vpp; (B) 7.5 Vpp; (C)

10 Vpp; and (D) 12.5 Vpp, after releasing the voltage

Figure 56 Cell capture efficiency graph at various voltages (aptamer-modified chips)

A549 cell detection platform based on impedance analysis combined

3.3.1 Effects of applied voltage and duration on cell trapping via DEP

Dielectrophoresis (DEP) is crucial for effectively guiding A549 cells towards the surfaces in the midsection area of a microfluidic chip The interaction between aptamers and target cells takes several minutes, leading to nearly zero capture efficiency without DEP at the flow rate used in this study After determining an optimal frequency of 1 MHz for trapping A549 cells using positive DEP force, further experiments were conducted to assess the time and voltage parameters to ensure device performance Prior to injecting a concentration of 2 × 10^4 stained A549 cells/mL at a flow rate of 2 µL/min, the microfluidic chip was integrated with pumping and fluorescence microscopy systems, allowing efficient cell transport to the capture area while minimizing adhesion to the channel walls.

Figure 58 Images displayed the fluorescence of calcein green AM-labeled A549 cells positioned centrally within the midsection region

Figure 58 illustrates the trapping of A549 cells through positive dielectrophoresis (pDEP) at different applied voltages and durations The study employed a peak-to-peak voltage of 12 Vp-p, with investigation periods varying accordingly.

Using ImageJ software from the National Institutes of Health, a quantitative assessment of trapping effectiveness was performed by analyzing integrated density (IntDen) values IntDen, which reflects the cumulative sum of pixel values in an image, is calculated by multiplying the area by the mean gray value, providing a measure proportional to the number of trapped cells across the midsection area along the electrodes Consistent resolution and brightness were maintained during image capture with a fluorescence microscope.

Figure 59 The efficacy of capturing A549 cells versus DEP implementation time was calculated by measuring trapping density

Within the initial 5 minutes, minimal cell trapping was observed at a voltage of 12

The low concentration of the cell sample and delays in the pumping system can lead to challenges in cell capture during DEP manipulation Notably, significant cell capture was observed after 15 minutes of applying DEP techniques.

Figure 58 illustrates the fluorescence image of cell capture, revealing a higher concentration of cells at the front and fewer at the rear This distribution is primarily influenced by dielectrophoresis (DEP), which affects cells upon entering areas with high electric field intensity, causing them to be predominantly trapped at the front The trapping density effectively represents the capture of A549 cells in the analyzed scenarios, as shown in the figure.

3.3.2 Capturing cells and conducting electrical impedance spectroscopy measurements

Following the implementation of DEP-based cell collection procedures, the target cells were successfully captured in the SAM region During the experiment, a specific volume of the cell sample was introduced into the channel, with multiple repetitions performed using varying cell sample volumes to ensure consistency in results.

This study involved impedance measurements of cells at a concentration of 2 × 10^4 cells/mL, conducted over time intervals of 5, 10, and 15 minutes to correlate with fluorescence data To ensure stabilization, fluid flow was paused for three minutes, and dielectrophoresis (DEP) was turned off during the impedance measurement A sinusoidal alternating potential of 50 mV peak-to-peak was applied in a sucrose buffer medium, with wires from the sensing microelectrodes connected to an impedance analyzer Utilizing electrical impedance spectroscopy (EIS), the research provided insights into cellular properties by analyzing impedance across a logarithmic frequency range from 1 kHz to 1 MHz This method enabled a detailed examination of cell behavior and characterization of various cellular parameters, highlighting EIS's effectiveness as a versatile tool for biological and medical research into cellular dynamics and responses.

Figure 60 Results of micro-electrical impedance spectroscopy: A) Measurements of resistance changes; B) Measurements of reactance changes

The study examines real impedance, or resistance, and imaginary impedance, known as reactance, highlighting their behavior as frequency increases across various cell numbers Both resistance and reactance decrease significantly within the low-frequency range of 1 to 400 kHz, particularly as the number of cells captured by pDEP increases At 400 kHz, the resistance changes from 4,069 KΩ to 0,617 KΩ over 15 minutes of DEP application, while reactance decreases from 7,03 KΩ to 0,506 KΩ in the same period The most significant reductions in both parameters occur after 15 minutes, attributed to Joule heating, which results from electric current passing through electrodes and converting electrical energy into thermal energy Consequently, longer DEP application durations lead to increased heat generation, impacting impedance and reactance measurements.

The application of DEP for 15 minutes indicates that Joule heating significantly influences the electrical properties of the system When the heat flux density exceeds a specific threshold, boiling occurs, leading to the formation of air bubbles This occurrence reflects the substantial thermal energy produced within the system.

Thermal energy disrupts cell structures and accelerates cell death, while dielectrophoresis (DEP) increases the likelihood of cell lysis and irreversible electroporation on a microscale The combined effects of thermal energy and DEP significantly affect cellular integrity and viability, highlighting the complex interaction between electrical and thermal phenomena in biological systems Studies have shown that even modest voltages can create high electric fields capable of rupturing cell membranes, as documented by D C Chang et al These electric fields induce structural changes in the lipid bilayer, resulting in pore formation and cell lysis Understanding these processes is crucial for biomedical and biotechnological applications, as applying the right potential for a moderate duration is essential for cell survival and subsequent analyses Ultimately, the DEP technique enhances the number of captured cells and improves capture efficiency.

The results and discussions section highlights the experimental findings that showcase the enhanced capabilities of the microfluidic platform in trapping and detecting A549 cells A critical analysis reveals the performance improvements achieved through the integration of gold nanoparticles and SH-aptamers The discussions further compare different chip configurations, emphasizing the significant advantages of aptamer-modified setups regarding specificity and efficiency in cell capture.

CONCLUSIONS

This study successfully developed a microfluidic device that enhances the capture of circulating tumor cells (CTCs) through dielectrophoresis-assisted self-assembly of nanoparticle and aptamer layers Utilizing standard photolithography techniques, the device features gold nanoparticles (AuNPs) and thiol-based aptamers (SH-aptamer) on indium tin oxide (ITO) interdigitated electrodes Experiments employed a reverse clamp and injection pump to introduce cell samples into the flow channel, while an AC electric field applied to the capture electrodes facilitated the specific adsorption of aptamers using dielectrophoretic forces.

Numerical simulations confirmed the strength and distribution of the electric field, ensuring its effectiveness over time without compromising the experiment's integrity Higher voltages significantly improved cell capture efficiency, with aptamer-modified, gold nanoparticle-integrated chips achieving a capture efficiency of 66.6% ± 1.4% at 12.5 Vpp In contrast, chips containing only gold nanoparticles without aptamers reached a capture efficiency of 24.4% ± 1.1%, while those lacking any modifications recorded only 16.1% ± 2.1%.

Self-assembled gold nanoparticles and thiol-based aptamers play a crucial role in enhancing cell contact opportunities and capture efficiency through aptamer specificity This device's adaptability allows for the selection of different aptamers to target various cancer cell types, such as breast, prostate, or leukemia cells, each with unique biomarkers Its versatility positions it as a valuable tool for early detection, diagnosis, and monitoring in clinical settings, offering increased speed, convenience, and selectivity The study's findings indicate significant advancements in rapid diagnostics and point-of-care testing for cancer cells Furthermore, this microfluidic platform supports personalized medicine and improves therapeutic outcomes, marking a pivotal contribution to modern oncology and patient care strategies.

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