Indium Tin Oxide Microelectrodes Research Articles

Toward development of dual optical and electrical cell‐based biosensor: An investigation on electrode geometry and transparent conductive material function

AbstractHerein is explored a dual optical and electrical cell based biosensor that can provide insights into cellular events. The fabrication steps along with the electrical characterization of the microelectrodes is described. Initial experiments show that the ability of indium tin oxide (ITO) to detect biological cells at the electrode‐cell layer interface mainly depends on the size of sensing area. Following this, the impact of conductivity was also explored to assess the real time impedance signal during the adhesion of a non‐confluent layer of the cells on different substrates. Electrodes with a higher conductivity than ITO gave much higher senstivity of impedance changes which allowed the dynamics of subtle cellular morphology changes to be monitored at densities far lower than a confluent layer of cells. Finally, the capability of ITO and gold microelectrode as a functional readout for G protein couple receptor activation were determined. These set of analyses highlighted the challenges and opportunities of ITO substrate as a dual cell‐based assay for quantitative analysis of subtle changes in cell morphology.

Fabrication of ITO microelectrodes and electrode arrays using a low-cost CO2 laser plotter.

Good electronic (Rs = ∼5 Ω sq-1) and optical properties (transmittance: >83%) make indium tin oxide (ITO) an attractive electrode substrate. Despite the commercial availability of high-quality ITO and some low-cost methods for direct deposition being in use by now, the definition of patterns is still a concern. Putting their popularity and extensive use aside, the manufacturing of ITO electrodes so far lacks a rapid, highly reproducible, flexible, cost-effective, easy patterning process that could surpass difficult, time-consuming techniques such as lithography. Herein, we present a low-cost method based on CO2 laser irradiation for preparing ITO microelectrodes and electrode arrays. Electrodes of different sizes and shapes were examined to identify the performance of the proposed methods. Direct ablation of the ITO layer was optimized for rectangular electrodes of 25, 50, and 100 μm in width, while laser cutting of scotch tape stencils and subsequent wet etching were used to create circular electrodes with a diameter of 1.75 mm. A multielectrode array system consisting 8 of these circular electrodes was fabricated on a (25 × 25) mm2 plate, characterized electrochemically through cyclic voltammetry (CV) and scanning electrochemical microscopy (SECM), and as an example application used for monitoring the anchoring behavior of HeLa and HepG2 cell cultures through cell-based electrochemical impedance spectroscopy. Together, the direct ablation method and preparation of laser cut stencils form a complete toolbox, which allows for low-cost and fast fabrication of ITO electrodes for a wide variety of applications. To demonstrate the general availability of the method, we have also prepared a batch of electrodes using a laser plotter in a local printing shop, achieving high intra-workshop reproducibility.

A portable and affordable aligner for the assembly of microfluidic devices

The incorporation of sophisticated capabilities within microfluidic devices often requires the assembly of different layers in a correct arrangement. For example, when it is desired to include electrodes inside microfluidic channels or to create 3D microfluidic structures. However, the alignment between different substrates at the microscale requires expensive equipment not available for all research groups. In this work, we present an affordable, compact and portable aligner for assembling multilayered composite microfluidic chips. The instrument is composed of aluminum machined pieces combined with precision stages and includes a digital microscope with a LED illumination system for monitoring the alignment process. An interchangeable holder was created for substrate fixing, allowing the bonding of PDMS with other materials. Microscopic visualization is achieved through any device with internet access, avoiding the need of a computer attached to the aligner. To test the performance of the aligner, the center of an indium tin oxide microelectrode on a glass substrate was aligned with the center of a microchannel in a PDMS chip. The accuracy and precision of the instrument are suited for many microfluidic applications. The small and inexpensive design of the aligner makes it a cost-effective option for small groups working in microfluidics.

Low-Cost ITO Microelectrodes for Bio-Sensing and Impedance Based Cellular Assays

Excellent electronic and optical properties make Indium tin oxide (ITO) an attractive electrode substrate.1 It is an alloy of indium oxide (In2O3) and tin oxide (SnO2) in a weight ratio of 90:10. It is transparent as thin film, so, it is also good to be viewed under different types of microscopes used in cell-culture studies. Despite the commercial availability of finest quality ITO and some low-cost methods for direct deposition being in use by now, definition of patterns on them is still a concern. Placing its popularity and extensive usage aside, the manufacturing of ITO electrodes so far lacks a rapid, proficient, highly reproducible, flexible, cost-effective, easy patterning process that could surpass non-trivial, time consuming techniques as lithography. A promising advancement towards achieving these limitations was the use of lasers for the fabrication of the ITO electrodes and related structure. Even though several laser techniques are explored, with the exception of CO2 lasers, none of the others provides the benefit of being widely available and comparatively inexpensive means for fabrication of microelectrode sensors. Presently, these are the highest-power continuous wave lasers available. With their high efficiency, they are usually employed in laser cutting, welding, drilling and surface treatment.2 A cost-effective method based on CO2 laser irradiation for the preparation of ITO microelectrodes to find applications in the bio-sensing, impedance-based analysis, and more, is presented. Electrodes of different sizes and shapes were examined to identify the performance of the samples. They were later optimized to suit our applications for studies in three dimensional cell-cultures. For rectangular shaped electrodes, the smallest sizes optimized were 25, 50 and 100 µm wide. The figure attached shows cyclic voltammetry results illustrating the reproducibility of 100 µm electrodes with ferrocenedimethanol as the redox probe in potassium nitrate. However, smaller electrodes are not always desirable. For the cell-culture work, electrodes with area 1.75mm2 were fabricated and characterized to get reproducible results. Initial measurements of impedance based cellular assays have been conducted first in DMEM medium and then in the cell culture medium to understand the parameters and study the adhesion of the cells on to the electrodes. Analysis of glucose sensing properties of the system have also been attempted. Further modifications of the electrode structure and characterizations for validation will be discussed.References Stadler A. et al. J.Materials, 2012, 5, 661-683Wang Y. et al. J Energy Chem. 2021, 59, 642-665 Figure 1

Resonance-Like Impedance Measurement Technique for Life Science Applications

In this paper, we propose a novel real-time method to convert standard impedance spectroscopy of an N-shape Negative Differential Resistance (NDR) system into resonance-like pseudo-impedance spectroscopy. This method relies on the creation of a singularity in the impedance spectra with the frequency where the real and imaginary parts of the impedance are equal to zero and pure capacitive, respectively. This resonance-like impedance spectrum allows us to monitor a unique frequency related to the electrochemical events at the interface between electrode and sample. The proposed method was validated using an NDR created by an indium tin oxide (ITO) microelectrode array exposed to mouse neuroblastoma cells with two different concentrations 1×10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sup> and 2×10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sup> cells/mL. Based on the results and discussions in this paper, resonance-like impedance spectroscopy offers advantages for the rapid detection and real-time monitoring of biological and/or chemical NDR systems.

Label-free mechanoelectrical investigation of single cancer cells by dielectrophoretic-induced stretch assay

It is well-known that cancerous transformation induces many disruptions in the chemical, mechanical, and electrical functions of cells. However, limited data have been reported on their correlative behavior, such as mechanoelectrical responses. In this work, we have applied dielectrophoretic stimulation using transparent Indium Tin Oxide (ITO) microelectrodes to analyze the mechanical deformation of the cells. Our studies have shown high electrodeformation response in non-cancer cells (e.g., for the breast cell line (MCF-10A) ∼21 %). In contrast, the stretch response to the same applied dielectrophoretic stimulation was extremely weak in malignant cells (breast cell line (MDA-MB-231) ∼6 %). This observation points to a new cancer investigation technique based on the real-time correlation between dielectric properties and mechanical behaviors of single cells. Using the smaller electro-deformative ability of cancer cells compared to healthy ones, we have designed and proposed a label-free mechanoelectrical chip to detect metastatic cancer cells in unprocessed tumor samples. The methods of conventional pathology and immunofluorescence assays confirmed the results obtained from our "Dielectrophoretic Stretch Assay" (DSA). This method could help develop protocols for detecting micro invasions in small samples of suspicious masses or cytological samples diagnosis without requirement to pretreatment or labeling and in single-cell resolutions.

(Invited) Transparent and Flexible PEDOT:PSS/ITO/Ag/ITO Microelectrodes for Electrophysiology Recording

High-resolution microelectrodes made of transparent conducting materials are advantageous for electrophysiology recordings when used in combination with optogenetic stimulation or optical imaging. Existing transparent electrodes made of indium tin oxide (ITO), ultrathin metal, graphene, poly-(3, 4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT:PSS), gold nanomesh, or silver nanowire network present tradeoffs with optical transmittance, resistivity, mechanical flexibility, and biocompatibility. In this work, we designed and constructed a flexible, fully transparent microelectrode array using a PEDOT:PSS/ITO/Ag/ITO multilayer assembly. Nanometer-thick Ag provides high electrical conductivity while being transparent over a broad spectrum. ITO offers excellent transparency as well as chemical and biological stability to protect Ag from corrosion in the biological environments. PEDOT:PSS further reduces electrochemical impedance by increasing the active surface area with the same electrode size. The thickness of each layer of the multilayer assembly was optimized at a specific wavelength using admittance loci analysis to minimize the reflectance at the inhomogeneous material interfaces. As a proof of concept, an electrode array was designed to have two sub panels, each of which consisted of 16 microelectrodes on a 3x3 mm2 area, with various electrode diameters of 25 µm to 50 µm. The fabrication of such an array started from Parylene C deposition on a 3-inch silicon wafer. Photoresist (PR) was spun on the substrate and photolithographically patterned as a sacrificial mask. After that, ITO (20 nm), Ag (9.5 nm) and ITO (24 nm) thin films were sputtered consecutively in a radio-frequency (RF) magnetron sputtering system (Denton Explorer-14, Denton Vacuum, Inc), and then patterned using the liftoff process to form microelectrodes, interconnection wires, and contact pads. Next 2 μm Parylene C was deposited on the substrate as an insulating layer and selectively etched using oxygen plasma dry etching (RIE-1701 plasma system, Nordson March, Inc) to expose the recording sites and contact pads. After that, another PR mask was patterned to expose only the microelectrode sites followed by coating diluted 0.55% PEDOT:PSS. Finally, PR was rinsed off with acetone, IPA and DI water to remove unwanted PEDOT:PSS, leaving PEDOT:PSS only on top of the ITO-Ag-ITO microelectrodes.The optical, electrical, electrochemical, and mechanical properties of the PEDOT:PSS-ITO-Ag-ITO multilayer assembly was characterized using various experimental methods. In particular, a Filmetrics thin film analyzer was utilized to measure the transmittance of thin films in a wavelength range of 300 nm - 800 nm (F20-UVX, Filmetrics, Inc) and a four-point probe (SRM-232, Bridge Technology, Inc) analyzer was used to measure the sheet resistances of the samples. Compared to a single ITO film of the same thickness, the PEDOT:PSS/ITO/Ag/ITO assembly exhibits a combination of high broad-band transmission (>80%) and significantly improved conductivity. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) analyses demonstrated the significantly enhanced electrical impedance of the multilayer film by 91.85%. The electrochemical impedance of the films remained stable after 12 weeks of storage in room temperature air, with less than 12% change. Meanwhile, peel-off tests verified the excellent adhesion of the ITO-Ag-ITO assembly on the Parylene C substrate, and bending test results demonstrated the mechanical flexibility of the combined thin films over a large surface area. In addition, the noise performance of the transparent microelectrodes was analyzed under exposure to optical illumination of various wavelengths, confirming the negligible photon-induced artifacts suitable for electrophysiology recording in combination with optical imaging or stimulation. The optical transparency of the microelectrode array was further demonstrated under both the fluorescent and bright-field illumination conditions. Finally, the ability to use the transparent microelectrodes for recording neurophysiology and electrocardiography signals was demonstrated in vivo using rat models or in vitro using heart organoids.

Application of a Thin-Film Transistor Array for Cellular-Resolution Electrophysiology and Electrochemistry

The constant development and improvement of microelectromechanical systems (MEMS) have been given a great opportunity to develop new reliable microsystems devices for biomedical research. This article presents a locally addressable 2-D arrayed indium-tin oxide microelectrode platform with integrated thin-film transistors (TFTs) for biological and chemical sensing. Microelectrode arrays (MEAs) using TFT have the advantage of being transparent with a high density of microelectrodes on a large surface. In vitro 2-D electrical measurements on 28 parallel-connected lines selected from a 22 500 MEA were successfully performed with heart cells for the first time. Observation of cell contraction was performed simultaneously with an inverted microscope. Voltammetry and amperometry measurements were also demonstrated using the transparent microelectrodes as working electrodes and an integrated Ag/AgCl reference electrode. Due to its unique features, we believe that the TFT platform can provide more understanding of the key communication between heart cells, and large-scale cardiovascular and nervous systems. As a result, the platform offers unique access to versatile lab-on-a-chip devices that integrate many measurement techniques on one chip for the study of cell cultures, tissues, and organoids.

Dielectrophoresis-Based Method for Measuring the Multiangle Mechanical Properties of Biological Cells.

The mechanical properties of cells are closely related to their physiological functions and states. Analyzing and measuring these properties are beneficial to understanding cell mechanisms. However, most measurement methods only involve the unidirectional analysis of cellular mechanical properties and thus result in the incomplete measurement of these properties. In this study, a microfluidic platform was established, and an innovative microfluidic chip was designed to measure the multiangle cellular mechanical properties by using dielectrophoresis (DEP) force. Three unsymmetrical indium tin oxide (ITO) microelectrodes were designed and combined with the microfluidic chip, which were utilized to generate DEP force and stretch cell from different angles. A series of experiments was performed to measure and analyze the multiangle mechanical properties of red blood cells of mice. This work provided a new tool for the comprehensive and accurate measurement of multiangle cellular mechanical properties. The results may contribute to the exploration of the internal physiological structures of cells and the building of accurate cell models.

A patterned polystyrene-based microelectrode array for in vitro neuronal recordings.

Substrate-integrated microelectrode arrays (MEAs) are non-invasive platforms for recording supra-threshold signals, i.e. action potentials or spikes, from a variety of cultured electrically active cells, and are useful for pharmacological and toxicological studies. However, the MEA substrate, which is often fabricated using semiconductor processing technology, presents some challenges to the user. Specifically, the electrode encapsulation, which may consist of a variety of inorganic and organic materials, requires a specific substrate preparation protocol to optimize cell adhesion to the surface. Often, these protocols differ from and are more complex than traditional protocols for in vitro cell culture in polystyrene petri dishes. Here, we describe the fabrication of an MEA with indium tin oxide microelectrodes and a patterned polystyrene electrode encapsulation. We demonstrate the electrochemical stability of the electrodes and encapsulation, and show viable cell culture and in vitro recordings.

DAQ based Impedance Measurement System for Low Cost and Portable Electrical Cell-Substrate Impedance Sensing

Electric cell-substrate impedance sensing (ECIS), a technique for label-free and real-time detection of cells, is emerging as an alternative or assistive method to traditional biochemical assays for diagnostic and pharmaceutical applications. In this work, we developed a DAQ based impedance measurement system for low cost and portable ECIS applications. The alternating input signal for ECIS was generated from the digital-to-analog converter (DAC) channel of the DAQ board and applied through the current limited resistor to the cells on the electrode. The responding signal was recorded by the analog-to-digital converter (ADC) channel of the DAQ board, and its amplitude and phase were analyzed by software lock-in amplifier. The feasibility of the developed ECIS system was evaluated by impedance measurement and DAPI analysis of C2C12 cell growth on the indium tin oxide microelectrode, and by impedimetric and MTT cell viability assay of HEK293 cells exposed to H2O2.

Characteristics of Transparent, PEDOT:PSS Coated Indium-Tin-Oxide (ITO) Microelectrodes.

This paper reports on electrochemical and optical characteristics of flexible, transparent microelectrodes, which consist of thin poly-(3, 4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT:PSS) spun onto indium-tin-oxide (ITO) electrodes for potential applications in biomedical optoelectronic devices. Although PEDOT:PSS/ITO combined films have been extensively investigated for applications in optical devices, such as solar cells and LEDs, PEDOT:PSS/ITO films for use in electrophysiological recording have not been well-characterized yet. In this work, PEDOT:PSS coated ITO microelectrodes with various diameters of 10 μm, 37 μm, 50 μm and 80 μm were microfabricated and characterized, and their properties were compared with plain ITO microelectrodes. Experimental results demonstrate that PEDOT:PSS coated ITO electrodes exhibit decreased electrochemical impedance, well-performed stability in saline, and increased charge storage capacity while preserving excellent optical transparency and mechanical flexibility. Equivalent circuit models were fitted to the experimental results to analytically extract interface capacitance, charge transfer resistance and solution resistance at the electrode-electrolyte interface.

Impedance Monitoring of SH-SY5Y Cell Adhesion and Responses to As2O3Treatment by Indium Tin Oxide Microelectrode Arrays

For most of the cells, cell adhesion is one of the essential factors for cell growth in vitro . In this paper, the impedance was used to monitor SH-SY5Y cell adhesion and evaluate the responses to As2O3 in a real-time, noninvasive, and label-free manner with indium tin oxide microelectrode arrays. Due to the transparency of the electrodes, cell activities can be observed simultaneously when impedance monitoring proceeded. The results indicated that the impedance responses had a good correlation with microscopic analysis of cell activities. Moreover, As2O3 at different concentrations were added for the evaluation of its effect on cell adhesion. $40~\mu $ mol/L As2O3 could detach almost all of the cells from the microelectrode surface, leading to an obvious decrease in impedance responses and declined cell survival rate after 9 h of treatment. Importantly, the impedance monitoring can provide the information about cell adhesion changes much earlier than commonly used cell microphotographs determination. This paper indicated the convenience and sensitivity of impedance for the direct and real-time monitoring of cell behaviors and drug-induced cell activities in vitro . It may be a driving force for the study of As2O3 effect on neuroblastoma and provides a useful and non-invasive analytical approach for cancer research.

Microfluidic single-cell array platform enabling week-scale clonal expansion under chemical/electrical stimuli

Single-cell culture represents the most straightforward method for investigating cellular heterogeneity. In this paper, we present a novel microfluidic platform that can individually array and culture hundreds of cells under chemical and electrical stimuli for week-scale characterization. Single cells can be deterministically and gently captured in a microchamber array on the proposed platform. The size of the microchamber can be adjusted to fit different cell culture times, and this characteristic enables remarkable scalability. Transparent indium tin oxide microelectrodes were integrated with the single-cell array platform for on-chip electrical stimuli. The platform exhibited nearly 90% single-cell efficiency and facilitated week-scale clonal expansion of different types of single cells. Chemical and electrical stimuli affected proliferation and differentiation of MC 3T3-E1 cells were examined on the chip prototype that contained 416 (32 rows × 13 columns) microchambers, and each microchamber had 1 mm diameter. By tracking clonal expansion of cells under chemical/electrical stimuli for relatively long periods, the proposed platform can facilitate the screening of the cell subpopulation with a favorable growth phenotype for drug testing and cell therapy.

A gold nanoparticle-modified indium tin oxide microelectrode for in-channel amperometric detection in dual-channel microchip electrophoresis

A modified indium tin oxide (ITO) microelectrode detector integrated with a dual-channel polydimethylsiloxane (PDMS) microchip is proposed for in-channel amperometric detection in microchip electrophoresis (ME).

Label-free and direct detection of C-reactive protein using reduced graphene oxide-nanoparticle hybrid impedimetric sensor

For label-free and direct detection of C-reactive protein (CRP), an impedimetric sensor based on an indium tin oxide (ITO) electrode array functionalized with reduced graphene oxide-nanoparticle (rGO-NP) hybrid was fabricated and evaluated. Analytical measurements were performed to examine the properties of rGO-NP-modified ITO microelectrodes and to determine the influence upon sensory performance of using nanostructures modified for antibody immobilization and for recognition of CRP binding events. Impedimetric measurements in the presence of the redox couple [Fe(CN)6]3−/4− showed significant changes in charge transfer resistance upon binding of CRP. The impedance measurements were highly target specific, linear with logarithmic CRP concentrations in PBS and human serum across a 1ngmL−1 and 1000ngmL−1 range and associated with a detection limits of 0.06 and 0.08ngmL−1 respectively.

A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces.

Metal-reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. This process is one of the earliest forms of respiration on earth and has significant environmental and technological implications. By performing electron transfer to electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. Understanding such a complex biotic-abiotic interaction necessitates the development of tools capable of probing extracellular electron transfer down to the level of single cells. Here, we describe an experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect the respiration current correlated with cell-electrode contact. We demonstrate the system with single cell measurements of the dissimilatory-metal reducing bacterium Shewanella oneidensis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer-membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. In addition to the application for extracellular electron transfer studies, the ability to electronically measure cell-specific respiration rates may provide answers for a variety of fundamental microbial physiology questions.

Preparation and Experimental Study on Dielectrophoresis-Based Microfluidic Chip for Cell Patterning

Abstract A dielectrophoresis (DEP)-based microfluidic chip for cell patterning was designed and fabricated to achieve non-contact and batch manipulation of cells. The microfluidic chip employed a polydimethylsiloxane (PDMS) microchannel and two indium tin oxide (ITO) electrodes which were designed as a “step” structure. The distribution of the electric field caused by the microelectrodes was simulated by finite element simulation software COMSOL. The position of the maximum intensity of the electric field was also determined. The ITO microelectrodes and PDMS microchannel were fabricated using the micro-electro-mechanical system (MEMS) technique. After oxygen plasma surface treatment, the PDMS microchannel and glass substrate with the ITO microelectrodes were aligned and bonded to form the experimental microfluidic chip. Through DEP experiments with varying frequencies, the DEP behavior of yeast cells was examined, and the electric field frequency of both positive and negative DEP responses were determined. The results showed that yeast cells with solution conductivity of 60 μS cm−1 exhibited negative DEP movement in a frequency range of 1–10 kHz, positive DEP movement from 100 kHz to 10 MHz, and no DEP movement at 50 kHz. Under a sinusoidal potential of 8Vp-p with frequency of 5 MHz, the yeast cells were aligned into chains along the “step” edge of microelectrodes.

Voltammetry of Micro-Liter Electrolyte Samples on ITO Microelectrodes for Analyte Recognition

Obtaining information on the nature of ionic species in electrolyte samples is in demand. The examination process must take less than ~1 min, and the test is expected to require small volume (micro-liter) samples. Here, we report electrolyte examination results with a simple voltammetry process carried out on thin film indium tin oxide micro-electrodes deposited on polyethylene terephthalate substrates and micromachined by CO2 laser ablation. The sample volume required is 2 μl, which is dropped manually on the electrodes with a sampler. The voltage waveform applied between the electrodes is a combination of positive and negative pulses of different magnitudes. The transient electric current passing through the electrolyte is monitored and plotted versus time. These plots are utilized as the finger prints of the related electrolyte samples. Pattern recognition methods are employed for information extraction from the obtained patterns. The diagnostic information extracted from these patterns proved sufficient for discrimination among KF, KCl and KI samples at various concentrations.

DNA Methylation Detection and Inhibitor Screening Based on the Discrimination of the Aggregation of Long and Short DNA on a Negatively Charged Indium Tin Oxide Microelectrode

On the basis of the different diffusivity and existence of electrostatic repulsion between long and short DNA on the negatively changed indium tin oxide (ITO) microelectrode, a simple but sensitive immobilization free solution-phase electrochemical method for DNA methylation detection and inhibitor screening has been developed. Electroactive substance (methylene blue) tagged at the penultimate base T close to the 3'-terminal first, in the absence of DNA methylation, methylene blue-labeled electroactive fragments cannot be generated by Dpn I and results in a weak electrochemical response being detected on the ITO electrode. On the contrary, a remarkable electrochemical response can be achieved by the cleavage in the presence of DNA methylation since methylene blue-labeled electroactive fragments can be generated and aggregate on the ITO electrode. The proposed system does not need complex operation procedures such as bisulfite treatment, PCR amplification, and electrode immobilization. Six ITO microelectrodes had been assembled on the same microchip, which can achieve the parallel detection of the same sample and improve the experimental efficiency of drug screening. The system was used to conveniently and specifically monitor the change of the DNA methylation level with high sensitivity and selectivity. The proposed system has the potential application to screen the drugs as inhibitors on the activity of methyltransferase in the clinic.