Development of the Sensing Platform for Protein Tyrosine Kinase Activity.

A miniature tyrosinase-based electrochemical sensing platform for label-free detection of protein tyrosine kinase activity was developed in this study. The developed miniature sensing platform can detect the substrate peptides for tyrosine kinases, such as c-Src, Hck and Her2, in a low sample volume (1-2 µL). The developed sensing platform exhibited a high reproducibility for repetitive measurement with an RSD (relative standard deviation) of 6.6%. The developed sensing platform can detect the Hck and Her2 in a linear range of 1-200 U/mL with the detection limit of 1 U/mL. The sensing platform was also effective in assessing the specificity and efficacies of the inhibitors for protein tyrosine kinases. This is demonstrated by the detection of significant inhibition of Hck (~88.1%, but not Her2) by the Src inhibitor 1, an inhibitor for Src family kinases, as well as the significant inhibition of Her2 (~91%, but not Hck) by CP-724714 through the platform. These results suggest the potential of the developed miniature sensing platform as an effective tool for detecting different protein tyrosine kinase activity and for accessing the inhibitory effect of various inhibitors to these kinases.

Development of flexible electrochemical impedance spectroscopy-based biosensing platform for rapid screening of SARS-CoV-2 inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cells through the binding of its spike protein (S-protein) to the cell surface-expressing angiotensin-converting enzyme 2 (ACE2). Thus, inhibition of S-protein-ACE2 binding may impede SARS-CoV-2 cell entry and attenuate the progression of Coronavirus disease 2019 (COVID-19). In this study, an electrochemical impedance spectroscopy-based biosensing platform consisting of a recombinant ACE2-coated palladium nano-thin-film electrode as the core sensing element was fabricated for the screening of potential inhibitors against S-protein-ACE2 binding. The platform could detect interference of small analytes against S-protein-ACE2 binding at low analyte concentration and small volume (0.1 µg/mL and ~1 µL, estimated total analyte consumption < 4 pg) within 21 min. Thus, a few potential inhibitors of S-protein-ACE2 binding were identified. This includes (2S,3aS,6aS)-1-((S)-N-((S)-1-Carboxy-3-phenylpropyl)alanyl)tetrahydrocyclopenta[b] pyrrole-2-carboxylic acid (ramiprilat) and (2S,3aS,7aS)-1-[(2S)-2-[[(2S)-1-Carboxybutyl]amino]propanoyl]-2,3,3a,4,5,6,7,7a-octahydroindole-2-carboxylic acid (perindoprilat) that reduced the binding affinity of S-protein to ACE2 by 72% and 67%; and SARS-CoV-2 in vitro infectivity to the ACE2-expressing human oral cavity squamous carcinoma cells (OEC-M1) by 36.4 and 20.1%, respectively, compared to the PBS control. These findings demonstrated the usefulness of the developed biosensing platform for the rapid screening of modulators for S-protein-ACE2 binding.

The Development of CMOS Amperometric Sensing Chip with a Novel 3-Dimensional TiN Nano-Electrode Array.

An electrochemical sensing chip with an 8 × 8 array of titanium nitride three-dimensional nano-electrodes (TiN 3D-NEA) was designed and fabricated via a standard integrated complementary metal oxide semiconductor process. Each nano-electrode in 3D-NEA exhibited a pole-like structure with a radius of 100 nm and a height of 35 nm. The numeric simulation showed that the nano-electrode with a radius of around 100 nm exhibited a more uniformly distributed electric field and a much higher electric field magnitude compared to that of the microelectrode. Cyclic voltammetry study with Ru(NH3)63+ also revealed that the TiN 3D-NEA exhibited a much higher current density than that obtained from the microelectrode by two orders of magnitude. Further studies showed that the electrocatalytical reduction of hydrogen peroxide (H2O2) could occur on a TiN 3D-NEA-based sensing chip with a high sensitivity of 667.2 mA⋅mM-1⋅cm-2. The linear detection range for H2O2 was between 0.1 µM and 5 mM with a lowest detection limit of 0.1 µM. These results indicated that the fabricated TiN 3D-NEA exhibited high catalytic activity and sensitivity to H2O2 and could be a promising sensor for H2O2 measurement.

Development of CpG-oligodeoxynucleotides for effective activation of rabbit TLR9 mediated immune responses.

CpG-oligodeoxynucleotides (CpG-ODN) are potent immune stimuli being developed for use as adjuvants in different species. Toll-like receptor 9 (TLR9) is the cellular receptor for CpG-ODN in mammalian cells. The CpG-ODN with 18-24 deoxynucleotides that are in current use for human and mouse cells, however, have low activity with rabbit TLR9. Using a cell-based activation assay, we developed a type of CpG-ODN containing a GACGTT or AACGTT motif in 12 phosphorothioate-modified deoxynucleotides with potent stimulatory activity for rabbit TLR9. The developed CpG-ODN have higher activities than other developed CpG-ODN in eliciting antigen-nonspecific immune responses in rabbit splenocytes. When mixed with an NJ85 peptide derived from rabbit hemorrhagic disease virus, they had potent activities to boost an antigen-specific T cell activation and antibody production in rabbits. Compared to Freund's adjuvant, the developed CpG-ODN are capable of boosting a potent and less toxic antibody response. The results of this study suggest that both the choice of CpG-motif and its length are important factors for CpG-ODN to effectively activate rabbit TLR9 mediated immune responses.

A general strategy to achieve ultra-high gene transfection efficiency using lipid-nanoparticle composites.

Gene therapy provides a new hope for previously "incurable" diseases. Low gene transfection efficiency, however, is the bottle-neck to the success of gene therapy. It is very challenging to develop non-viral nanocarriers to achieve ultra-high gene transfection efficiencies. Herein, we report a novel design of "tight binding-but-detachable" lipid-nanoparticle composite to achieve ultrahigh gene transfection efficiencies of 60∼82%, approaching the best value (∼90%) obtained using viral vectors. We show that Fe@CNPs nanoparticles coated with LP-2000 lipid molecules can be used as gene carriers to achieve ultra-high (60-80%) gene transfection efficiencies in HeLa, U-87MG, and TRAMP-C1 cells. In contrast, Fe@CNPs having surface-covalently bound N,N,N-trimethyl-N-2-methacryloxyethyl ammonium chloride (TMAEA) oligomers can only achieve low (23-28%) gene transfection efficiencies. Similarly ultrahigh gene transfection/expression was also observed in zebrafish model using lipid-coated Fe@CNPs as gene carriers. Evidences for tight binding and detachability of DNA from lipid-nanoparticle nanocarriers will be presented.

Programmable redox state of the nickel ion chain in DNA.

DNA is a nanowire in nature which chelates Ni ions and forms a conducting chain in its base-pairs (Ni-DNA). Each Ni ion in Ni-DNA exhibits low (Ni(2+)) or high (Ni(3+)) oxidation state and can be switched sequentially by applying bias voltage with different polarities and writing times. The ratio of low and high oxidation states of Ni ions in Ni-DNA represents a programmable multistate memory system with an added capacitive component, in which multistate information can be written, read, and erased. This study also indicates that the biomolecule-based self-organized nanostructure can be used as a template for nanodevice fabrication.

The APE1 Asp/Asp genotype and the combination of APE1 Asp/Asp and hOGG1-Cys variants are associated with increased p53 mutation in non-small cell lung cancer.

BACKGROUND: The hOGG1 Ser326Cys polymorphism is associated with lung cancer risk, but there are limited data regarding an association between the APE1 Asp148Glu polymorphism and lung cancer. Biological evidence shows that the hOGG1-Cys allele results in less DNA repair activity; however, this is not associated with p53 mutation in lung cancer. Therefore, we investigated whether an interaction between hOGG1 and APE1 is associated with the frequency of p53 mutation in lung cancer. METHODS: We studied 217 Taiwanese adults with primary lung cancer. DNA polymorphisms of hOGG1 and APE1 were determined by polymerase chain reaction (PCR)-based restriction fragment length polymorphism. Mutations in p53 exons 5-8 were detected by direct sequencing. Multiple logistic regression was used to estimate odds ratios (ORs) and 95% CIs for the risk of p53 mutation associated with polymorphisms of hOGG1 and APE1 in lung cancer. RESULTS: As expected, no association between hOGG1 polymorphism and p53 mutation was observed in this population. However, a higher risk of p53 mutation was found in participants with the APE1 Asp/Asp genotype than in those with the APE1-Glu allele (OR, 2.15; 95% CI, 1.19-3.87; P = 0.011). The risk of p53 mutation was also higher in participants with APE1 Asp/Asp plus hOGG1-Cys than in those with APE1-Glu plus hOGG1 Ser/Ser (OR, 3.72; 95% CI, 1.33-10.40; P = 0.012). CONCLUSIONS: These results suggest that the APE1 Asp/Asp genotype and the combination of the APE1 Asp/Asp and hOGG1-Cys variants are associated with increased risk of p53 mutation in non-small cell lung cancer.

Activation of rabbit TLR9 by different CpG-ODN optimized for mouse and human TLR9.

Synthetic CpG-oligodeoxynucleotides (CpG-ODN) are potent adjuvants that accelerate and boost antigen-specific immune responses. Toll-like receptor 9 (TLR9) is the cellular receptor for these CpG-ODN. Previous studies have shown species-specific activation of mouse TLR9 (mTLR9) and human TLR9 (hTLR9) by their optimized CpG-ODN. The interaction between rabbit TLR9 (rabTLR9) and CpG-ODN, however, has not been previously investigated. Here, we cloned and characterized rabTLR9 and comparatively investigated the activation of the rabbit, mouse, and human TLR9 by CpG-ODN. The complete open reading frame of rabTLR9 encodes 1028 amino acid residues, which share 70.6% and 75.5% of the identities of mTLR9 and hTLR9, respectively. Rabbit TLR9 is preferentially expressed in immune cells rich tissues, and is localized in intracellular vesicles. While mTLR9 and hTLR9 displayed species-specific recognition of their optimized CpG-ODN, rabbit TLR9 was activated by these CpG-ODN without any preference. This result suggests that rabTLR9 has a broader ligand-recognition profile than mouse and human TLR9.

Reusable amperometric biosensor for measuring protein tyrosine kinase activity.

This work presents a simple, low-cost and reusable label-free method for detecting protein tyrosine kinase activity using a tyrosinase-based amperometric biosensor (tyrosine kinase biosensor). This method is based on the observation that phosphorylation can block the tyrosinase-catalyzed oxidation of tyrosine or tyrosyl residue in peptides. Therefore, the activity of p60c-src protein tyrosine kinase (Src) on the developed tyrosine kinase biosensor could be quickly determined when its specific peptide substrate, p60c-src substrate I, was used. The tyrosine kinase biosensor was highly sensitive to the activity of Src with a linear dynamic range of 1.9-237.6 U/mL and the lowest detection limit of 0.23 U/mL. Interestingly, the tyrosine kinase activity can be measured using the developed tyrosine kinase biosensor repetitively without regeneration. The inhibitory effect of various kinase inhibitors on the Src activity could be determined on the tyrosine kinase biosensor. Src-specific inhibitors, PP2 and Src inhibitor I, effectively suppressed Src activity, whereas PD153035, an inhibitor of the epidermal growth factor receptor, was ineffective. Staurosporine, a universal kinase inhibitor, inhibited Src activity in an ATP concentration-dependent manner. These results suggests that the activities of tyrosine kinases and their behaviors toward various reagents can be effectively measured using the developed tyrosine kinase biosensor.

Development of single-vector Tet-on inducible systems with high sensitivity to doxycycline.

Single-vector Tet-on systems were developed to enable the tight regulation of transgenes in mammalian cells with a low dosage of doxycycline. Both the regulatory and the responsive units were integrated in a single vector and separated by a short DNA segment (214 bp). In the developed single-vector Tet-on systems, a high level of expression of the transgene can be induced by doxycycline at a concentration of as low as 1 ng/ml, which is 500-1,000 times lower than that usually utilized in other Tet-on systems. The single-vector Tet-on system developed here exhibited 3.5-10.8 times greater inducibility of the transgene in response to doxycycline than did a dual-vector system from a commercial source. Further studies indicate that the basal activity of Tet-on systems depends greatly on the strength of the promoter that controls the transactivator. The basal activity of Tet-on systems was high when the transactivator that was directed by the human cytomegalovirus promoter, and it was almost undetectable when the transactivator was placed under the control of a moderate strength mouse mammary tumor virus promoter. Moreover, the introduction of selectable markers allows the developed single-vector Tet-on systems to facilitate the generation of conditional transgenic cells and animals with high inducibility, low basal activity and detrimental effects of the long-term administration of doxycycline.

Mammalian Ste20-like protein kinase 3 plays a role in hypoxia-induced apoptosis of trophoblast cell line 3A-sub-E.

Mammalian Ste20-like protein kinase 3 (Mst3) is a key player in inducing apoptosis in a variety of cell types and has recently been shown to participate in the signaling pathway of hypoxia-induced apoptosis of human trophoblast cell line 3A-sub-E (3A). It is believed that oxidative stress may occur during hypoxia and induce the expression of Mst3 in 3A cells via the activation of c-Jun N-terminal protein kinase 1 (JNK1). This hypothesis was demonstrated by the suppressive effect of dl-α-lipoic acid, a reactive oxygen species scavenger, in hypoxia-induced responses of 3A cells such as Mst3 expression, nitrotyrosine formation, JNK1 activation and apoptosis. Similar results were also observed in trophoblasts of human placental explants in both immunohistochemical studies and immunoblot analyses. These suggested that the activation of Mst3 might trigger the apoptotic process in trophoblasts by activating caspase 3 and possibly other apoptotic pathways. The role of nitric oxide synthase (NOS) and NADPH oxidase (NOX) in hypoxia-induced Mst3 up-regulation was also demonstrated by the inhibitory effect of N(G)-nitro-l-arginine and apocynin, which inhibits NOS and NOX, respectively. Oxidative stress was postulated to be induced by NOS and NOX in 3A cells during hypoxia. In conclusion, hypoxia induces oxidative stress in human trophoblasts by activating NOS and NOX. Subsequently, Mst3 is up-regulated and plays an important role in hypoxia-induced apoptosis of human trophoblasts.

Fabrication of a carbon fiber paper as the electrode and its application toward developing a sensitive unmediated amperometric biosensor.

Carbon fiber paper (CFP), a material frequently used as the diffusion layer in fuel cells, was found recently to exhibit a potential as an electrode for the development of sensitive, unmediated biosensors. After nitrogen plasma treatment, the CFP exhibited a quasi-reversible behavior to the redox couple (e.g., ferricyanide) with an electron transfer rate constant of 7.2 × 10(-3)cms(-1). This rate constant is approximately double that of a Pt-electrode and is much higher than that of many carbon-based electrodes. The unmediated CFP-based tyrosinase biosensor fabricated for this study exhibited an optimal working potential and operating pH value of -0.2V and 6.5, respectively. Compared to other unmediated tyrosinase biosensors, the CFP-based tyrosinase biosensor offers a high sensitivity for the monitoring of phenolic compounds (17.8, 7.1, 5.2 and 3.7 µA µM(-1)cm(-2) for catechol, phenol, bisphenol and 3-aminophenol, respectively). The lowest detection limit for catechol, phenol, bisphenol and 3-aminophenol was 2, 5, 5 and 12 nM, respectively. Furthermore, this biosensor exhibited a good repeatability, a fast response time (around 10s), and a wide linear dynamic range of detection for phenolic compounds.

Neutron capture nuclei-containing carbon nanoparticles for destruction of cancer cells.

HeLa cells were incubated with neutron capture nuclei (boron-10 and gadolinium)-containing carbon nanoparticles, followed by irradiation of slow thermal neutron beam. Under a neutron flux of 6 x 10(11) n/cm(2) (or 10 min irradiation at a neutron flux of 1 x 10(9) n/cm(2) s), the percentages of acute cell death at 8 h after irradiation are 52, 55, and 28% for HeLa cells fed with BCo@CNPs, GdCo@CNPs, and Co@CNPs, respectively. The proliferation capability of the survived HeLa cells was also found to be significantly suppressed. At 48 h after neutron irradiation, the cell viability further decreases to 35 +/- 5% as compared to the control set receiving the same amount of neutron irradiation dose but in the absence of carbon nanoparticles. This work demonstrates "proof-of-concept" examples of neutron capture therapy using (10)B-, (157)Gd-, and (59)Co-containing carbon nanoparticles for effective destruction of cancer cells. It will also be reported the preparation and surface functionalization of boron or gadolinium doped core-shell cobalt/carbon nanoparticles (BCo@CNPs, GdCo@CNPs and Co@CNPs) using a modified DC pulsed arc discharge method, and their characterization by various spectroscopic measurements, including TEM, XRD, SQUID, FT-IR, etc. Tumor cell targeting ability was introduced by surface modification of these carbon nanoparticles with folate moieties.

Mammalian Ste20-like protein kinase 3 induces a caspase-independent apoptotic pathway.

In this study, it was shown that the mammalian sterile 20-like serine/threonine protein kinase 3 (Mst3) plays an essential role in the staurosporine-induced apoptosis of HeLa cells. The staurosporine-induced apoptosis was reduced by around 65% by the selective knockdown of Mst3 in stable clones, HeLa(siMst3). Although caspases were shown to be involved in the Mst3-mediated apoptosis, only 15-20% of staurosporine-induced apoptosis was suppressed by the caspase inhibitor, z-DEVD-fmk. Accordingly, Mst3 was proposed to trigger a caspase-independent apoptotic pathway in response to staurosporine. Interestingly, staurosporine greatly induced the mitochondrial membrane potential transition in HeLa cells, but had no effect in Hela(siMst3). The role of Mst3 in controlling the mitochondrial integrity was therefore proposed, presumably through the regulation of Bax. Furthermore, it was shown that staurosporine promoted the nuclear translocation of apoptosis-inducing factor and endonuclease G in HeLa cells. The nuclease activity associated with endonuclease G was also enhanced in response to staurosporine. However, both staurosporine-induced nuclear translocation of apoptosis-inducing factor and endonuclease G and the nuclease activity associated with endonuclease G were markedly reduced in Hela(siMst3). These results suggest that Mst3 may respond to staurosporine to trigger the caspase-independent apoptotic pathway by regulating the nuclear translocation of apoptosis-inducing factor and endonuclease G, and the nuclease activity associated with endonuclease G.

Improving the detection of hydrogen peroxide of screen-printed carbon paste electrodes by modifying with nonionic surfactants.

Nonionic surfactants, such as Triton X-100 and Tween-20, were shown in this study to improve the electrocatalytic activity of screen-printed carbon paste electrodes (SPCE). The electrochemical response of SPCE to hydrogen peroxide increased 8-10-fold with the modification of nonionic surfactants. In addition, the glucose biosensors fabricated from nonionic surfactant-modified SPCE exhibited 6.4-8.6-fold higher response to glucose than that fabricated from unmodified SPCE. A concentration effect is proposed for nonionic surfactant to bring neutral reactants to the surface of electrode. Moreover, nonionic surfactant-modified SPCE exhibits a capability of repetitive usage and good reproducibility (R.S.D.<5%) in the measurement of H(2)O(2). Interestingly, the nonionic surfactant-modified SPCE exhibited an opposite effect to ascorbic acid, a common electroactive agent, which causes interference during clinical diagnosis. The differential responses of nonionic surfactant-modified SPCE to H(2)O(2) and ascorbic acid suggest its potential in the development of biosensors for clinical diagnosis.

Biosignaling of mammalian Ste20-related kinases.

Sterile 20 (ste20) protein is an upstream ser/thr kinase in yeast, and several mammalian Ste20-like (MST) kinases have been identified. This review focuses on the signal transduction, interacting proteins, and potential biological function of MST1, 2, 3, and 4 kinases, since several novel signal pathways of these kinases have been characterized recently. MST1 and MST2 kinases play an important role in cell growth and apoptosis, and the signal pathways involves many important molecules including RAS, AKT, and FOXO3. MST3 and MST4 have similar kinase domain, but have opposite effects on apoptosis and transformation. The downstream signaling molecules of these two kinases are beginning to be elucidated. Based on the expression pattern and signal pathways, we will discuss the perspective biological functions of four MST family kinases in cancer, immune, cardiovascular, and brain function.

Mammalian Ste20-like protein kinase 3 mediates trophoblast apoptosis in spontaneous delivery.

The placenta is essential in transferring gases and nutrients from the mother to the developing fetus. Trophoblast apoptosis may cause labor or other pregnancy-related disorders. This study demonstrated the essential role of Mst3, a human Ste20-like protein kinase, in the oxidative stress-induced apoptosis of trophoblasts of term placenta in normal spontaneous delivery. Oxidative stress, but not hormones released during labor such as prostaglandin E1, oxytocin or angiotensin II, induces the expression of Mst3 and apoptosis of human term placenta after elective Cesarean section without labor pain. The role of Mst3 in oxidative stress-induced apoptosis was further demonstrated in the 3A-sub-E, a human trophoblast cell line. The H2O2-induced apoptosis of 3A-sub-E cells was largely suppressed by overexpressed Mst3KR, the kinase-dead mutant or by selective knockdown of endogenous Mst3. Further studies showed that Jun N-terminal kinase (JNK) may participate in the signaling pathway of H2O2-induced apoptosis by mediating the level of Mst3. Subsequently, caspase 3 and other downstream apoptotic components may be activated by Mst3 and trigger the apoptotic process in human trophoblasts.

Fabrication of a miniature CMOS-based optical biosensor.

This work presents a novel, miniature optical biosensor by immobilizing horseradish peroxidase (HRP) or the HRP/glucose oxidase (GOx) coupled enzyme pair on a CMOS photosensing chip with a detection area of 0.5 mm x 0.5 mm. A highly transparent TEOS/PDMS Ormosil is used to encapsulate and immobilize enzymes on the surface of the photosensor. Interestingly, HRP-catalyzed luminol luminescence can be detected in real time on optical H(2)O(2) and glucose biosensors. The minimum reaction volume of the developed optical biosensors is 10 microL. Both optical H(2)O(2) and glucose biosensors have an optimal operation temperature and pH of 20-25 degrees C and pH 8.4, respectively. The linear dynamic range of optical H(2)O(2) and glucose biosensors is 0.05-20 mM H(2)O(2) and 0.5-20 mM glucose, respectively. The miniature optical glucose biosensor also exhibits good reproducibility with a relative standard deviation of 4.3%. Additionally, ascorbic acid and uric acid, two major interfering substances in the serum during electrochemical analysis, cause only slight interference with the fabricated optical glucose biosensor. In conclusion, the CMOS-photodiode-based optical biosensors proposed herein have many advantages, such as a short detection time, a small sample volume requirement, high reproducibility and wide dynamic range.

Comparison of amperometric biosensors fabricated by palladium sputtering, palladium electrodeposition and Nafion/carbon nanotube casting on screen-printed carbon electrodes.

Different strategies, including palladium electrodeposition (Pd(CV)), Pd sputtering (Pd(S)) and Nafion-solubilized carbon nanotube casting (Nafion/CNT), were used to modify screen-printed carbon electrodes (SPCEs) for the fabrication of amperometric enzyme biosensors. The electrochemical properties of the bare and modified SPCEs and the optimal conditions for surface modification were determined. The electrochemical response of the bare SPCE to H(2)O(2) under the potential of 0.3 V could be improved about 100-fold by Pd modification by electrodeposition or sputtering. By contrast, the electrochemical response of the bare SPCE was enhanced by only about 11-fold by Nafion/CNT casting. Moreover, the Pd(CV)-SPCEs exhibited better reproducibility of electrochemical response (a relative standard deviation (R.S.D.)<6.0%) than freshly prepared Pd(S)-SPCEs (R.S.D.>10%). The glucose biosensor fabricated from Pd-modified electrodes could be stored for up to 108 days without loosing significant activity. The Pd(CV)-SPCE also showed very reliable signal characteristics upon 50 consecutively repeated measurements of ascorbic acid. The electrocatalytic detection of the Pd-SPCE was combined with additional advantages of resistance to surface fouling and hence good stability. In conclusion, this study demonstrated that deposition of Pd thin film on SPCEs by electrodeposition or sputtering provided superior enhancement of electrochemical properties compared to Nafion/CNT-SPCEs. Despite their high electrochemical response, Pd(S)-SPCEs required an activation process to improve stability and Pd(CV)-SPCEs suffered from poor between electrode reproducibility.

Series quartz crystal sensor for remote bacteria population monitoring in raw milk via the Internet.

A remote monitoring system based on a piezoelectric quartz crystal (SPQC) sensor was developed for the determination of the bacteria population in raw milk. The system employs the Windows XP server operating system, and its programs for data acquisition, display and transmission were developed using the LabVIEW 7.1 programming language. The circuit design consists of a circuit with a piezoelectric quartz crystal (SPQC) and a pair of electrodes. This system can provide dynamic data monitoring on a web-page via the Internet. Immersion of the electrodes in a cell culture with bacteria inoculums resulted in a change of frequency caused by the impedance change due to microbial metabolism and the adherence of bacteria on the surface of the electrodes. The calibration curve of detection times against density of bacteria showed a linear correlation coefficient (R(2) = 0.9165) over the range of 70-10(6) CFU ml(-1). The sensor could acquire sufficient data rapidly (within 4 h) and thus enabled real-time monitoring of bacteria growth via the Internet. This system has potential application in the detection of bacteria concentration of milk at dairy farms.