Rapid label-free impedimetric detection of progesterone enhanced by immunomagnetic bead-based competitive immunoreaction.

Detecting progesterone (P4) concentration in cow serum is essential for monitoring the pregnancy progress after fertilization and is significant for the dairy farming industry and veterinary medicine. This study reports enzyme-free immunomagnetic beads (IMBs)-based competitive immunoassay for detecting P4 by P4-bovine serum albumin (BSA)-modified biosensors. The anti-P4 antibody-conjugated IMBs serve as collectors to capture P4 in undiluted serum samples to prevent the biosensor surface from biosample contamination and as insulated labels to report the electron-transfer resistance signal of electrochemical impedance spectroscopy (EIS) measurement. The IMBs and P4-containing samples were mixed for 15-30 min, capable of obtaining stable P4@IMB complexes. The 0.2-kGauss pulsed magnetic field (PMF) of the 20-s pulse width and 20-s relaxation time applied for 5 min can shorten the immunoreaction time between the P4@IMBs and the P4-BSA-modified biosensor and reduce the IMB's nonspecific adsorption on the biosensor surface. This competitive immunoassay's cut-off value and detection limit were 7.71 ng/mL and 7.33 ng/mL, respectively, which is lower than the serum's P4 plateau concentration (over 8 ng/mL) of dairy cows on days 6-16 of estrus cycles and that in pregnancy. The IMB-based immunoassay combining the PMF attraction and the label-free EIS measurement exhibits promising potential for rapidly detecting P4 in undiluted serum.

A Label-Free Droplet Sorting Platform Integrating Dielectrophoretic Separation for Estimating Bacterial Antimicrobial Resistance.

Antimicrobial resistance (AMR) has become a crucial global health issue. Antibiotic-resistant bacteria can survive after antibiotic treatments, lowering drug efficacy and increasing lethal risks. A microfluidic water-in-oil emulsion droplet system can entrap microorganisms and antibiotics within the tiny bioreactor, separate from the surroundings, enabling independent assays that can be performed in a high-throughput manner. This study presents the development of a label-free dielectrophoresis (DEP)-based microfluidic platform to sort droplets that co-encapsulate Escherichia coli (E. coli) and ampicillin (Amp) and droplets that co-encapsulate Amp-resistant (AmpR) E. coli with Amp only based on the conductivity-dependent DEP force (FDEP) without the assistance of optical analyses. The 9.4% low conductivity (LC) Luria-Bertani (LB) broth diluted with 170 mM mannitol can maintain E. coli and AmpR E. coli growth for 3 h and allow Amp to kill almost all E. coli, which can significantly increase the LCLB conductivity by about 100 µS/cm. Therefore, the AmpR E. coli/9.4%LCLB/Amp where no cells are killed and the E. coli/9.4%LCLB/Amp-containing droplets where most of the cells are killed can be sorted based on this conductivity difference at an applied electric field of 2 MHz and 100 Vpp that generates positive FDEP. Moreover, the sorting ratio significantly decreased to about 50% when the population of AmpR E. coli was equal to or higher than 50% in droplets. The conductivity-dependent DEP-based sorting platform exhibits promising potential to probe the ratio of AmpR E. coli in an unknown bacterial sample by using the sorting ratio as an index.

A Critical Review on Detection of Foodborne Pathogens Using Electrochemical Biosensors.

An outbreak of foodborne pathogens would cause severe consequences. Detecting and diagnosing foodborne diseases is crucial for food safety, and it is increasingly important to develop fast, sensitive, and cost-effective methods for detecting foodborne pathogens. In contrast to traditional methods, such as medium-based culture, nucleic acid amplification test, and enzyme-linked immunosorbent assay, electrochemical biosensors possess the advantages of simplicity, rapidity, high sensitivity, miniaturization, and low cost, making them ideal for developing pathogen-sensing devices. The biorecognition layer, consisting of recognition elements, such as aptamers, antibodies and bacteriophages, and other biomolecules or polymers, is the most critical component to determine the selectivity, specificity, reproducibility, and lifetime of a biosensor when detecting pathogens in a biosample. Furthermore, nanomaterials have been frequently used to improve electrochemical biosensors for sensitively detecting foodborne pathogens due to their high conductivity, surface-to-volume ratio, and electrocatalytic activity. In this review, we survey the characteristics of biorecognition elements and nanomaterials in constructing electrochemical biosensors applicable for detecting foodborne pathogens during the past five years. As well as the challenges and opportunities of electrochemical biosensors in the application of foodborne pathogen detection are discussed.

Recent Advances in Electrochemical Immunosensors with Nanomaterial Assistance for Signal Amplification.

Electrochemical immunosensors have attracted immense attention due to the ease of mass electrode production and the high compatibility of the miniature electric reader, which is beneficial for developing point-of-care diagnostic devices. Electrochemical immunosensors can be divided into label-free and label-based sensing strategies equipped with potentiometric, amperometric, voltammetric, or impedimetric detectors. Emerging nanomaterials are frequently used on electrochemical immunosensors as a highly rough and conductive interface of the electrodes or on nanocarriers of immobilizing capture antibodies, electroactive mediators, or catalyzers. Adopting nanomaterials can increase immunosensor characteristics with lower detection limits and better sensitivity. Recent research has shown innovative immobilization procedures of nanomaterials which meet the requirements of different electrochemical immunosensors. This review discusses the past five years of advances in nanomaterials (metal nanoparticles, metal nanostructures, carbon nanotubes, and graphene) integrated into the electrochemical immunosensor. Furthermore, the new tendency and endeavors of nanomaterial-based electrochemical immunosensors are discussed.

Application of aminobenzoic acid electrodeposited screen-printed carbon electrode in the beta-amyloid electrochemical impedance spectroscopy immunoassay.

Alzheimer's disease (AD) is one of the important neurodegenerative diseases, in the modern aging society, it has become an issue people need to work on. Of the pathogenic factor which leads to AD, beta-amyloid (Aß) is the most important one. It can form the senile plaque which aggregates in the neuron and interrupts the signal transmission. This research is based on the electrochemical system and screen-printed carbon electrode (SPCE) incorporated with pretreatment, electrodeposition, electrochemical impedance spectroscopy (EIS), antibody, and blocking agent. This immunosensor is applied to detect the different concentrations of Aß. The standard curve between electrical impedance and concentration of Aß is calculated. The specificity of the immunosensor is tested. This survey optimizes the electrodeposition condition for 4-aminobenzoic acid (4-ABA) and the parameter for antibody and blocking agents. This study fabricates a more dense, uniform, and stable film of 4-ABA. This sensor presents a range of detection from 1 fg/ml to 100 pg/ml and a limit of detection to 3.84 fg/ml. This sensor can identify the isoform of Aß. This research shortens the fabricating time to 3.5 h. This study fabricates a label-free and low-cost immunosensor for Aß with a short fabricating time, high stability, wide range of detection, low limit of detection, and good specificity. The impedance of the carbon printed electrodes is very high and is always measured by its current but this study provides a fabrication technique for high-efficiency carbon printed electrodes for electrochemical impedance spectroscopy sensing.

Dissolved Oxygen-Sensing Chip Integrating an Open Container Connected with a Position-Raised Channel for Estimation of Cellular Mitochondrial Activity.

The measurement of oxygen consumption of adherent cells is a profoundly important issue for estimating the bioenergetic health and metabolism activity of cells. The study describes the construction of a microfluidic chip consisting of an open container connected with a position-raised channel and dissolved oxygen (DO)-sensing gold ultramicroelectrodes for quantifying the oxygen consumption rate (OCR) of adherent cells. The microfluidic chip design can reduce the action of shear force on the adherent cells during medium replacement. The residual concentration of analytes in the open container was only 4.4% after solution replacement via the position-raised channel. The DO reduction current measured by ultramicroelectrodes averaged in the range of 40-60 s presented high reproducibility with a 1.1% relative standard deviation suitable for OCR calculation. After short-term (90 min) cultivation, the microfluidic chip can monitor the time-dependent change in the OCR of 3T3-L1 cells for several hours by repeatedly replacing the culture medium or with the stimulation of different mitochondrial inhibitors. The presented microfluidic cell-based chip has great promise for drug screening and chemosensitivity testing by measuring OCR to evaluate the mitochondrial function of adherent cells.

A Label-Free Electrochemical Impedimetric Immunosensor with Biotinylated-Antibody for SARS-CoV-2 Nucleoprotein Detection in Saliva.

The timely detecting of SARS-CoV-2 coronavirus antigens for infection validation is an urgent request for COVID-19 pandemic control. This study constructed label-free electrochemical impedance spectroscopy (EIS)-based immunosensors based on gold nanostructured screen-printed carbon electrodes (AuNS/SPCEs) to detect the SARS-CoV-2 nucleocapsid protein (N-protein) in saliva. Using short-chain 3-mercaptopropionic acid (MPA) as a linker to covalently bond streptavidin (SA) and bovine serum albumin (BSA) for controlling the oriented immobilization of the biotinylated anti-N-protein antibody (BioAb) can offer a greater sensitivity, a lower limit of detection (LOD), and better reproducibility of immunosensors (defined as BioAb/SA-BSA/MPA/AuNS/SPCEs) than the antibody randomly immobilized immunosensors and the long-chain 11-mercaptoundecanoic acid (MUA)-modified immunosensors (BioAb/SA-BSA/MUA/AuNS/SPCEs). The BioAb/SA-BSA/MPA/AuNS/SPCE-based immunosensors presented good linearity from 0.01 ng/mL to 100 ng/mL and a low LOD of 6 pg/mL in a phosphate buffer solution (PBS) and PBS-diluted saliva. Moreover, the immunosensor exhibited little cross-activity with other viral antigens such as MERS-CoV N-protein, influenza A N-protein, influenza B N-protein, and SARS-CoV-2 spike protein, indicating the high specificity of the immunosensors. The disposable label-free EIS-based immunosensors have promising potential in facilitating the rapid and sensitive tests of saliva-based COVID-19 diagnostics.

A Novel Biocompatible Herbal Extract-Loaded Hydrogel for Acne Treatment and Repair.

A novel herbal extract-loaded gel containing several biofunctional extracts, including green tea, Zingiber officinale Rosc, Phyllanthus emblica, and salicylic acid, was developed for acne vulgaris. These natural raw materials were blended with suitable dosages of gelatin and carboxymethyl cellulose (CMC) to produce a biocompatible herbal gel. The physical chemistry properties of the hydrogel were determined by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), rheometry, and scanning electron microscopy (SEM), and the hydrogel showed good mechanical and morphological characteristics. The herbal extract-loaded hydrogel mimicked extracellular matrix properties and showed good antioxidant and anti-inflammatory properties and various advantages, serving as a potential wound dressing material because of its high moisture retention ability, wound exudate absorption behavior, and biocompatibility. It exhibited moderate-high antioxidative and anti-inflammatory qualities that were important for dermis wound closure. The clinical trial results showed that most patients experienced moderate to high healing rates, and four of twenty-four individuals (16.67%) had recovery area ratios greater than 80%. This herbal extract-loaded hydrogel has effective ingredients and excellent mechanical properties as a bioactive dressing agent for acne treatment.

A light-up "G-quadruplex nanostring" for label-free and selective detection of miRNA via duplex-specific nuclease mediated tandem rolling circle amplification.

MicroRNA (miRNA) has emerged as a promising genetic marker for cancer diagnosis and therapy because its expression level is closely related to the progression of malignant diseases. Herein, a label-free and selective fluorescence platform was proposed for miRNA based on light-up "G-quadruplex nanostring" via duplex-specific nuclease (DSN) mediated tandem rolling circle amplification (RCA). First, a long DNA generated from upstream RCA was designed with the antisense sequences for miR-21 and downstream RCA primer. Upon recognizing miR-21, the resulting DNA-RNA permitted DSN digestion and triggered downstream two-way RCA, and generation of abundant "G-quadruplex nanostring" binding with ZnPPIX for label-free fluorescent responses. In our strategy, the strong preference of DSN for perfectly matched DNA/RNA ensures its excellent selectivity. The developed method generated wide linear response with LOD of 1.019 fM. Additionally, the miR-21 levels in cell extracts have been evaluated, revealing the utility of this tool for biomedical research and clinical diagnosis.

A Label-Free Impedimetric Genosensor for the Nucleic Acid Amplification-Free Detection of Extracted RNA of Dengue Virus.

Developing rapid and sensitive diagnostic methods for dengue virus (DENV) infection is of prime priority because DENV infection is the most prevalent mosquito-borne viral disease. This work proposes an electrochemical impedance spectroscopy (EIS)-based genosensor for the label-free and nucleic acid amplification-free detection of extracted DENV RNA intended for a sensitive diagnosis of DENV infection. A concentration ratio of 0.04 mM 6-mercaptohexanoic acid (MHA) to 1 mM 6-mercapto-1-hexanol (MCH) was selected to modify thin-film gold electrodes as a link to control the coverage of self-designed probe DNA (pDNA) at a density of 4.5 ± 0.4 × 1011 pDNA/cm2. The pDNA/MHA/MCH-modified genosensors are proven to improve the hybridization efficiency of a synthetic 160-mer target DNA (160mtDNA) with a 140-mer electrode side overhang as compared to other MHA/MCH ratio-modified genosensors. The MHA(0.04 mM)/MCH(1 mM)-modified genosensors also present good hybridization efficiency with the extracted DENV serotype 1 (DENV1) RNA samples, having the same electrode side overhangs with the 160mtDNA, showing a low detection limit of 20 plaque forming units (PFU)/mL, a linear range of 102-105 PFU/mL and good selectivity for DENV1. The pDNA density-controlled method has great promise to construct sensitive genosensors based on the hybridization of extracted DENV nucleic acids.

Label-Free Impedimetric Immunosensors Modulated by Protein A/Bovine Serum Albumin Layer for Ultrasensitive Detection of Salbutamol.

The sensing properties of immunosensors are determined not only by the amount of immobilized antibodies but also by the number of effective antigen-binding sites of the immobilized antibody. Protein A (PA) exhibits a high degree of affinity with the Fc part of IgG antibody to feasibly produce oriented antibody immobilization. This work proposes a simple method to control the PA surface density on gold nanostructure (AuNS)-deposited screen-printed carbon electrodes (SPCEs) by mixing concentration-varied PA and bovine serum albumin (BSA), and to explore the effect of PA density on the affinity attachment of anti-salbutamol (SAL) antibodies by electrochemical impedance spectroscopy. A concentration of 100 µg/mL PA and 100 µg/mL BSA can obtain a saturated coverage on the 3-mercaptoproponic acid (MPA)/AuNS/SPCEs and exhibit a 50% PA density to adsorb the amount of anti-SAL, more than other concentration-varied PA/BSA-modified electrodes. Compared with the randomly immobilized anti-SAL/MPA/AuNS/SPCEs and the anti-SAL/PA(100 µg/mL):BSA(0 µg/mL)/MPA/AuNS/SPCE, the anti-SAL/PA(100 µg/mL): BSA(100 µg/mL)/MPA/AuNS/SPCE-based immunosensors have better sensing properties for SAL detection, with an extremely low detection limit of 0.2 fg/mL and high reproducibility (<2.5% relative standard deviation). The mixture of PA(100 µg/mL):BSA(100 µg/mL) for the modification of AuNS/SPCEs has great promise for forming an optimal protein layer for the oriented adsorption of IgG antibodies to construct ultrasensitive SAL immunosensors.

Effect of Poly-l-Lysine Polycation on the Glucose Oxidase/Ferricyanide Composite-Based Second-Generation Blood Glucose Sensors.

Second-generation glucose biosensors are presently the mainstream commercial solution for blood glucose measurement of diabetic patients. Screen-printed carbon electrodes (SPCEs) are the most-used substrate for glucose testing strips. This study adopted hydrophilic and positively charged α-poly-l-lysine (αPLL) as the entrapment matrix for the immobilization of negatively charged glucose oxidase (GOx) and ferricyanide (FIC) on SPCEs to construct a disposable second-generation glucose biosensor. The αPLL modification is shown to facilitate the redox kinetics of FIC and ferrocyanide on the SPCEs. The SPCEs coated with 0.5 mM GOx, 99.5 mM FIC, and 5 mM αPLL had better sensitivity for glucose detection due to the appreciable effect of protonated αPLL on the promotion of electron transfer between GOx and FIC. Moreover, the SPCEs coated with 0.5 mM GOx, 99.5 mM FIC, and 5 mM αPLL were packaged as blood glucose testing strips for the measurement of glucose-containing human serum samples. The glucose testing strips had good linearity from 2.8 mM to 27.5 mM and a detection limit of 2.3 mM. Moreover, the 5 mM αPLL-based glucose testing strips had good long-term stability to maintain GOx activity in aging tests at 50 °C.

A Microchip Electrophoresis Device Integrated with the Top-bottom Antiparallel Electrodes of Indium Tin Oxide to Detect Inorganic Ions by Contact Conductivity.

A microchip electrophoresis (ME) device with off-channel contact conductivity detection (C2D) was constructed using top-bottom antiparallel indium tin oxide (ITO) electrodes and a cross-type microchannel. The 500-m wide top-bottom antiparallel decouplers were found to effectively decrease the interference of the electrophoretic current. The cross-type microchannel was formed by bonding the patterned negative photoresist microstructures and the two top-bottom opposed ITO-deposited glass substrates. Five seconds of 150 V/cm injection field and the 100 V/cm separation field equipped with the C2D of AC 200 mV excitation voltage provided adequate ME operational parameters to obtain the K+ and Na+ peaks separation. The ME devices obtained good coefficients in a range of 11000 µM for the K+ and Na+ detection. The calculated limit of detection was 1 µM. This design for off-channel and top-bottom antiparallel electrodes shows that C2D ME devices have great potential for the measurement of inorganic ions.

An electrooxidative technique to fast fabricate copper phosphate electrodes capable of integrating high performance liquid chromatography for the label-free detection of fish freshness.

A simple and fast one-step electrooxidative method has been developed to monolithically produce a copper phosphate (Cu3(PO4)2) compound on a disposable copper tape, which can be integrated with high performance liquid chromatography (HPLC) for the estimation of fish freshness. The Cu3(PO4)2 compound of flake-like nanostructures was formed by applying a first anodic peak potential at the copper tape for 10 min in a 1 M sodium dihydrogen phosphate (NaH2PO4) (pH 5.0) solution. The Cu3(PO4)2 electrodes can detect the oxidative reaction of histidine and histamine in 20 mM NaH2PO4 solutions with pH 5.0-8.5. When integrating the electrodes with a flow injection system, the linear range and the calculated detection limit of histamine were respectively 2.5-250 ppm and 0.15 ppm. The electrodes integrated to HPLC can specifically detect the histamine concentrations in fish samples in the pH 7.5 NaH2PO4 solution, achieving an accuracy rate of 95.3% and a recovery rate of 101.1%.

An impedimetric bioaffinity sensing chip integrated with the long-range DC-biased AC electrokinetic centripetal vortex produced in a high conductivity solution.

Immunoreaction of specific antibodies to antigens is widely used in numerous immunoanalysis applications. However, diffusion-dominated transport in stationary solutions limits the rate and binding density of immunoreaction. This research describes the construction of chip-type concentric multi-double ring electrodes and single central disk electrode. A +1 V-biased 6 Vpp voltage was applied to the multi-double ring electrodes to induce a long-range DC-biased AC electrokinetic flow (ACEKF). The immunoreaction was quantified by electrochemical impedance spectrum (EIS). Fluorescence-labeled secondary antibody (FLSA) and protein A were exemplified as an immunoreacting model to demonstrate the effect of ACEKF on immunoreaction efficiency. The results showed that FLSA binding can reach a plateau in 8 min with the DC-biased ACEKF vortex, and the increment of electron transfer resistance is 2.26 times larger than that obtained in the unstirred solution. The sensitivity of the calibration curves obtained by EIS detection with the aid of DC-biased ACEKF vortex is 1.51 times larger than that obtained in an unstirred solution. The label-free EIS-based sensing chip integrated with the long-range DC-biased ACEKF vortex promises to facilitate immunoreaction efficiency, which is beneficial for the development of a miniature and fast-detection in vitro diagnostic device.

Effect of the Chain Length of a Modified Layer and Surface Roughness of an Electrode on Impedimetric Immunosensors.

An ultrasensitive label-free impedimetric immunosensor is constructed by modifying a 3-mercaptoproponic acid (MPA) monolayer on highly rough gold nanostructure (AuNS)-electrodeposited screen printed carbon electrodes (SPCEs) for the detection of small molecular weight drugs (SMWDs), such as salbutamol (SAL). The SPCEs preoxidized in a 0.1 M H2SO4 solution (called po-SPCEH2SO4) are electrodeposited with the AuNS to increase the roughness factor to 23.64 ± 1.76, larger than the AuNS/po-SPCENaOH or the AuNS/po-SPCEPBS. Furthermore, the MPA modified layer as a link for the anti-SAL immobilization to give the immunosensors an impedimetric signal-to-noise ratio larger than the 11-mercapto-undecanoic acid- and 16-mercaptohexadecanoic acid-modified layer, due to the lower interfacial impedance of the MPA monolayer. The MPA/AuNS/po-SPCEH2SO4-based immunosensors have a wide linear range of 1 fg mL-1 to 1 ng mL-1 and a limit of detection of 0.6 fg mL-1. Moreover, the immunosensors can practically quantify the SAL concentrations in 1000 times-diluted serum samples with good recovery.

Enhanced Hydrophilicity and Biocompatibility of Dental Zirconia Ceramics by Oxygen Plasma Treatment.

Surface properties play a critical role in influencing cell responses to a biomaterial. The objectives of this study were (1) to characterize changes in surface properties of zirconia (ZrO2) ceramic after oxygen plasma treatment; and (2) to determine the effect of such changes on biological responses of human osteoblast-like cells (MG63). The results indicated that the surface morphology was not changed by oxygen plasma treatment. In contrast, oxygen plasma treatment to ZrO2 not only resulted in an increase in hydrophilicity, but also it retained surface hydrophilicity after 5-min treatment time. More importantly, surface properties of ZrO2 modified by oxygen plasma treatment were beneficial for cell growth, whereas the surface roughness of the materials did not have a significant efficacy. It is concluded that oxygen plasma treatment was certified to be effective in modifying the surface state of ZrO2 and has the potential in the creation and maintenance of hydrophilic surfaces and the enhancement of cell proliferation and differentiation.

A label-free impedimetric DNA sensing chip integrated with AC electroosmotic stirring.

AC electroosmosis (ACEO) flow and label-free electrochemical impedance spectroscopy are employed to increase the hybridization rate and specifically detect target DNA (tDNA) concentrations. A low-ionic-strength solution, 6.1µS/cm 1mM Tris (pH 9.3), was used to produce ACEO and proved the feasibility of hybridization. Adequate voltage parameters for the simultaneous ACEO driving and DNA hybridization in the 1mM Tris solution were 1.5 Vpp and 200Hz. Moreover, an electrode set with a 1:4 ring width-to-disk diameter ratio exhibited a larger ACEO velocity above the disk electrode surface to improve collecting efficiency. The ACEO-integrated DNA sensing chips could reach 90% saturation hybridization within 117s. The linear range and detection limit of the sensors was 10aM-10pM and 10aM, respectively. The label-free impedimetric DNA sensing chips with integrated ACEO stirring can perform rapid hybridization and highly-sensitive detections to specifically measure tDNA concentrations.

Electroosmotic flow control in microfluidic chips using a self-assembled monolayer as the insulator of a flow field-effect transistor.

A novel concept for electroosmotic flow (EOF) control in a microfluidic chip is presented by using a self-assembled monolayer as the insulator of a flow field-effect transistor. Bidirectional EOF control with mobility values of 3.4 × 10(-4) and -3.1 × 10(-4) cm(2)/V s can be attained, corresponding to the applied gate voltage at -0.8 and 0.8 V, respectively, without the addition of buffer additives. A relatively high control factor (approximately 400 × 10(-6) cm(2)/V(2) s) can be obtained. The method presented in this study offers a simple strategy to control the EOF.

The open container-used microfluidic chip using IrO(x) ultramicroelectrodes for the in situ measurement of extracellular acidification.

The measurement of metabolic activity based on the extracellular acidification rate has attracted wide interests in the field of biochemical detection. In the study, the chip comprising a microfluid-controlled open container and iridium oxide (IrO(x)) pH ultramicroelectrodes (UMEs) was constructed for the purpose of in situ measurement of extracellular acidification rate. The feasible anodic depositing parameters of IrO(x) film were in the range of +0.53 to +0.8 V by means of exploring the electrochemical properties of alkaline Ir(IV) deposition solution. The IrO(x) pH UMEs electrodeposited for 300 cycles between 0 V and +0.6 V exhibited the near-super-Nernstian sensitivity of -68 to -76 mV/pH and the good stability with potential drifting of 11.7 mV within 24h. The design of the open container connected with a position-raised microchannel improved the sensing stability of IrO(x) pH UMEs, with the potential deviation of as low as 0.1 mV under the flow rate of 20 µl/min. The acidification rate of HeLa cells (2160 cells/mm(2)) repeatedly measured 5 times in the microfluidic chip showed the good reproducibility of 0.021±0.002 pH/min. Moreover, the chip can decrease the acidosis occurrence, a decrease of only 0.13-0.17 pH unit in 8 min interval, during the measurement of cellular metabolic activity.