Microfaradaic Electrochemical Biosensors for the Study of Anticancer Action of DNA Intercalating Drug: Epirubicin 341 3.2 DPV analysis of epirubicin-DNA interaction at bare GCFE The first set i.e. without DNA, produced a DPV oxidation peak for epirubicin at +0.54V, which shifted to more electro-positive potential with increasing DNA concentration and the peak current shortened. The shift in Ep value and shortening of peak current may be explained on the basis of change of species that is oxidized at the GCFE surface, i.e. due to the formation of drug-DNA complex. Although, the above experimental results confirm the formation of Epirubicin-DNA complex, but, to have a clear-cut understanding on the mechanism of the drug-DNA interaction at charged surfaces, the GCFE has been modified in three different ways: 3.3 Epirubicin-DNA interaction at epirubicin adsorbed GCFE It showed a big peak at +0.54V due to oxidation of adsorbed epirubicin and the other peaks may be due to oxidation of purine bases of DNA. This explanation of the observed voltammogram is based on the presumption that DNA diffuses from bulk of the solution to electrode surface and the chemisorbed epirubicin is intercalated into its double helix. As such, the distortion of double strand takes place, which allows the oxidation of purine bases. However, after the first scan if a potential of -0.60V was applied for 60 s, and then the voltammogram was recorded, it produced a peak at +0.45V (Figure 7). The appearance of this peak is due to the interaction of epirubicin with ds-DNA through guanine rich region. Fig. 7. Differential Pulse Voltammogram for 80µg/ml ds-DNA solution in 0.1M acetate buffer at pH 4.5±0.1, after applying a potential of -0.60V during 60 s, at epirubicin modified GCFE. 3.4 Epirubicin-DNA interaction at thin layer ds-DNA modified GCFE The DPV for the oxidation of epirubicin, showed a well defined peak with peak potential +0.54V. The peak may be attributed to the oxidation of 6,11-dihydroquinone group of epirubicin molecule. However, after recording the oxidation peak, a negative potential of -0.60V was applied on the modified electrode for 60 s, followed by recording of DP Voltammogram with positive potential scanning of the working electrode. The resulting voltammogarm showed two new peaks in addition to the epirubicin oxidation peak. The peak at +0.90V (Figure 8) may be Biosensors for Health, Environment and Biosecurity 342 attributed as due to 8-oxo-Guanine (8-oxo-G) oxidation and that at +0.40V may be due to the oxidation of purine bases of DNA. A clear separation of the peak due to 8-oxo-G and epirubicin can be explained on the basis of non-uniform coverage of the GCFE surface by DNA and adsorption of epirubicin at these uncovered surfaces. [The results are in good agreement with those observed using thick layer DNA modified GCFE]. This shift of 8-oxo- G peak to less positive potential informs about the DNA-epirubicin interaction (damage to DNA). Fig. 8. Differential Pulse Voltammogram in 0.1M acetate buffer at pH 4.5±0.1, obtained with a thin layer ds-DNA modified GCFE after being immersed in 20µg/ml epirubicin solution during 180 s, after applying a potential -0.60V during 60 s. 3.5 Epirubicin-DNA interaction at thick layer ds-DNA modified GCFE Epirubicin produced a well-defined voltammetric oxidation peak with Ep value +0.54V. The height of the epirubicin oxidation peak with respect to the time of immersion of the thick layer ds-DNA modified GCFE in epirubicin solution was investigated. The results showed a linear relationship between the peak height and time of immersion of the electrode in epirubicin solution i.e.0.00 to 60 min, and then it attained a constant value. Thus, indicating the preconcentration of epirubicin at the thick layer ds-DNA modified electrode surface. It is important to note that reproducible peak currents were observed for the similar time of immersion of the thick layer ds-DNA modified GCFE in epirubicin solution for the first scan only. However, if the differential pulse voltammogram is recorded using the same modified electrode, an abrupt decrease in the peak current was observed. This suggests a fast consumption of the neoplasic drug at the modified electrode surface. However, on performing the above voltammetric experiments separately using bare GCFE and thick layer ds-DNA modified GCFE as working electrode and scanning the potential from -0.70V to -0.00V, the resulting DPV curve with bare GCFE produced only one peak at -0.56V. Whereas, using thick layer ds-DNA modified GCFE two peaks were observed at -0.60V and -0.45V, respectively. The observed new peak at -0.45V speaks of a different interaction mechanism of epirubicin-DNA, at the modified GCFE surface. Since, epirubicin is irreversibly adsorbed at the bare GCFE surface, it becomes necessary to clean the electrode each time before use. Whereas, the thick layer ds-DNA modified GCFE Microfaradaic Electrochemical Biosensors for the Study of Anticancer Action of DNA Intercalating Drug: Epirubicin 343 did not require cleaning. This clearly reveals that the epirubicin is intercalated inside ds- DNA film and could not reach the electrode surface. On the basis of above observations it could be concluded that the voltammetric peaks are observed due to epirubicin which is intercalated into thick layer of ds-DNA. Since, the voltammograms were recorded in acetate buffer supporting electrolyte solution only, the possibility of any contribution to the voltammetric peaks from epirubicin present in solution is ruled out. As such, the observed new peak at -0.45V may be attributed to the epirubicin-guanine site (in DNA) interaction leading to a charge transfer reaction to from epirubicin semiquinone and guanine radical cation. However, the peak at -0.60V may be attributed to the reduction of the epirubicin. As mentioned earlier, epirubicin at bare GCFE produces a peak at -0.56V, the shift in the peak potential for epirubicin reduction at the two different electrode surfaces may be explained due to the change in the electrode surfaces. However, if the ds-DNA modified GCFE after being dipped in epirubicin for 300s, rinsed and immersed in a buffer solution at pH 4.5±0.1, was subjected to a potential of -0.60V for about 60s and then the voltammogram was recorded by positive potential scanning of the modified electrode, the resulting voltammogram produced two new peaks, one at +0.80V and other at +1.1V (Figure 9). The former peak may be attributed to guanine oxidation and the later due to adenine oxidation. Fig. 9. Differential Pulse Voltammogram in 0.1M acetate buffer at pH4.5±0.1 obtained with a thick later ds-DNA modified GCFE after being immersed in 20µg/ml epirubicin solution for 60 s at potential -0.60V. 4. Mechanism Epirubicin transfers an electron to its quinone portion (Perry, 1996) to generate a free radical. The highly reactive free radical formed at -0.60V may oxidize the guanine site of ds- DNA in which it is intercalated within the double helix, forming drug-DNA complex. Besides, the study on drug-DNA interaction at bare GCFE showed that the peak at +0.54V as observed in case of pure epirubicin oxidation, at bare GCFE shifts to less positive side i.e.+0.45V, on its complexation with ds-DNA, which may be explained as due to interaction between epirubicin and 8-oxo-G which is formed as a result of interaction of epirubicin with Biosensors for Health, Environment and Biosecurity 344 guanine rich region of ds-DNA. As such, one electron transfer from guanine moiety to quinone leading to guanine cation formation appears to be the probable reaction. However, due to the tendency of guanine cation to undergo hydrolysis, finally the semiquinone is further reduced to form epirubicin and 8-oxo-G. Mechanism model C G C G Guanine Redical Cation +e - +H + - 0.6 V H 2 O C G C G 8-Oxo-Guanine N N N N N O H O H H N N N H H O O o 3' 5' O O o 3' 5' O O CH 3 NH 3 OH + OH O OCH 3 C C HO HO H H O O HO Epirubicin N N N N N O H O H H N N N H H O O o 3' 5' O O o 3' 5' + O O CH 3 NH 3 OH + OH O OCH 3 C CHO HO H H O HO HO O O CH 3 NH 3 OH + OH OCH 3 C C HO HO H H O HO O N N N N N O H O H H N N N H H O O o 3' 5' O O o 3' 5' OH Epirubicin Semi Quinone N N N N N O H O H H N N N H H O O o 3' 5' O O o 3' 5' O O CH 3 NH 3 OH + OH O OCH 3 C C HO HO H H O HO HO Mechanism model : Mechanism of electrochemical epirubicin oxidative damage to DNA 5. Conclusion Voltammetric in-situ sensing of DNA oxidative damage caused by reduced epirubicin intercalated into DNA is possible using ds-DNA modified GCFE microfaradaic biosensor. The results show that epirubicin intercalated in double helix of DNA can undergo oxidation Microfaradaic Electrochemical Biosensors for the Study of Anticancer Action of DNA Intercalating Drug: Epirubicin 345 or reduction and react specifically with the guanine moiety and thus forms mutagenic 8- oxo-G residue. A mechanism model for the reaction may be proposed. The fabricated microfaradaic biosensors are of utmost relevance because the mechanism of interaction of DNA-epirubicin at charged interfaces is parallel to in-vivo DNA-drug complex reaction, where DNA is in close contact with charged phospholipid membranes and proteins rather then when intercalation is in solution. It also promises the use of voltammetric techniques for in situ generation of reaction intermediates. As such, is a complementary tool for the study of biomolecular interaction mechanism of medicinal relevance. 6. Acknowledgment University Grants Commission, New Delhi, India, for financial support under its special assistance program (SAP) level-1. 7. References Blackburn, GM. & Gair, MJ. (1996). Nucleic acids in chemistry and biology, Oxford University Press, UK. Brett. OM.; Serrano, SP., & Piedade, JP. (1999). Comprehensive chemical kinetics compton, R.G. Hancock (Eds), Elsevier, Amsterdam. Bousse, L. (1996). Whole cell biosensors. Sensors Actuators, Vol. B34, pp. 270–275. Clark, LC. & Lyons, C. (1962). Electrode systems for continuous monitoring of cardiovascular surgery. Ann. NY. Acad. Sci., Vol. 102, pp. 29–35. Erdem, A.; Kosmider, B.; Osiecka, R.; Zyner, E.; Ochocki, J., & Ozsoz, M. (2005). Electrochemical genosensing of the interaction between the potential chemotherapeutic agent, cis-bis (3-aminoflavone) dichloroplatinum (II) and DNA in comparison with cis-DDP. J. Pharm. 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Application of the electrochemical concepts and techniques to amperometric biosensor devices. J. Electroceram., Vol. 16, pp. 79-91. Martínez, R. & Chacón-García, L. (2005). The search of DNA-intercalators as antitumoral drugs: What it worked and what did not work. Curr. Med. Chem., Vol. 12, pp. 127- 151. Meadows, D. (1996). Recent developments with biosensing technology and applications in the pharmaceutical industry. Adv. Drug Deliv. Rev., Vol. 21, pp. 179–189. Biosensors for Health, Environment and Biosecurity 346 Nakamura, H. & Karube, I. (2003). Current research activity in biosensors. Anal. Bioanal. Chem., Vol. 377, pp. 446-468. Niu, S.; Li, F.; Zhang, S.; Wang, L.; Li, X., & Wang, S. (2006). Studies on the interaction mechanism of 1,10-phenanthroline cobalt (II) complex with DNA and preparation of electrochemical DNA biosensor. Sensor, Vol. 6, pp. 1234-1244. Ozkan, A. & Fiskin, K. (2003). Cytotoxicity of low dose epirubicin-HCI combined with lymphokine activated killer cells against hepatocellular carcinoma cell line hepatoma G2. Turk. J. Med. Sci., Vol. 34, pp. 11-19. Ozkan, D.; Karadeniz, H.; Erdem, A.; Mascini, M., & Ozsoz, M. (2004). Electrochemical genosensor for Mitomycin C–DNA interaction based on guanine signal. J. Pharm. Biomed. Anal., Vol. 35, pp. 905-912. Paddle, BM. (1996). Biosensors for chemical and biological agents of defence interest. Biosens. Bioelectron., Vol. 11, pp. 1079–1113. Palacek, E. (1983). Modern polarographic (voltammetric) techniques part (ii) in biochemistry and molecular biology, In: Topics in Bioelectrochemistry and Bioenergetics, G. Milazzo (Eds), John Wiley & Sons, New York. Pang, DW. & Abruna, HD. (2000). Interactions of benzyl viologen with surface-bound single and double-stranded DNA. Anal. Chem., Vol. 72, pp. 4700-4706. Perry, MC. (1996). The Chemotherapy Source Book, Williams and Wilkins, Baltimore, USA. Rauf, S.; Gooding, JJ.; Akhtar, K.; Ghauri, MA.; Rahman, M.; Anwar, MA., & Khalid, AM. (2005). Electrochemical approach of anticancer drugs–DNA interaction. J. Pharm. Biomed. Anal., Vol. 37, pp. 205-217. Ravishankara, MN.; Pillai, AD., & Handral, RD. (2001) Biosensor and its application. East. Pharm., Vol. 44, pp. 21-25. Shrivastava, AK. (2004). Electrochemical sensors based on macrocyclic compounds in International Conference on electroanalytical chemistry and allied topics, January 18-23, 2004 Dona Paula, Goa (India), Indian Soc. Electroanal. Chem., Mumbai (India). Silley, P. & Forsythe, S. (1996). Impedance microbiology: a rapid change for microbiologists. J. Appl. Bacteriol., Vol. 80, pp. 233–243. Yuqing, M.; Jianquo, G., & Jianrong C. (2003). Ion sensitive field effect transducer-based biosensors. Biotechnol. Adv., Vol. 21, pp. 527–534. Yuqing, M.; Jianrong, C., & Keming, F. (2005). New technology for the detection of pH. J. Biochem. Biophys. Methods, Vol. 63, pp. 1–9. Ziegler, C. & Göpel, W. (1998). Biosensor development. Curr. Opin. Chem. Biol., Vol. 2, pp. 585–591. 16 Light Addressable Potentiometric Sensor as Cell-Based Biosensors for Biomedical Application Hui Yu, Qingjun Liu and Ping Wang * Biosensor National Special Lab, Key Lab of Biomedical Engineering of Ministry of Education, Department of Biomedical Engineering, Zhejiang University China 1. Introduction One of most enduring topics in the field of biosensors and bioelectronics is cell-based biosensors, which are able to convert cellular biological effects to electrical signals, via living cells. As the archetypal interface between a biological and an electronic system, the research and development of cell-based biosensors are essentially dependent on the combined knowledge of engineers, physicists, chemists and biologists. In recent years, the miniaturization and expanding applications of cell-based biosensors in biology, environment and medicine fields, have drawn extensive attention. Light addressable potentiometric sensor (LAPS) is a semiconductor device proposed by Hafeman in 1988, and it is now as commonly used as ISFET (Hafeman et al., 1988). LAPS indicates a heterostructure of silicon/silicon oxide/silicon nitride, excited by a modulated light source to obtain a photocurrent. The amplitude of this light induced photocurrent is sensitive to the surface potential and thus LAPS is able to detect the potential variation caused by an electrochemical even. Therefore, in principle, any event that results in the change of surface potential can be detected by LAPS, including the change of ion concentration (Parce et al., 1989), redox effect (Piras et al., 1996), etc. LAPS shows some advantages comparing to ISFET while constructing cell-based biosensor. The easier fabrication process of LAPS is fully compatible with the standard microelectronics facilities. The encapsulation of LAPS is much less critical since no metal contact is formed on the surface. Besides, the extremely flat surface makes it compatible to incorporate into very small volume chamber, which is important for small dose measurement. Therefore, LAPS seems promising for biomedical application. Due to the spatial resolving power, LAPS also has an advantage for array sensing application (Shimizu et al., 1994). Usually, no additional sensor structure is needed to realize the LAPS array sensing. In fact, LAPS is an integrated sensor itself, whose integration level is defined by the spatial resolution and the illuminating system. Thus, miniaturization with high integration level can be achieved. Many efforts have been drawn on the integration of LAPS (Men et al., 2005; Wang et al., 2005). Among these attempts, most are focused on the * Corresponding address: cnpwang@zju.edu.cn Biosensors for Health, Environment and Biosecurity 348 multi-sensing of different ions. Our lab proposed an electronic nose with MLAPS for environmental detection, which can detect H + , Fe 3+ and Cr 6+ simultaneously (Men et al., 2005). Schooning et al. proposed a 16-channel handheld pen-shaped LAPS which can detect pH of 16 spots on the surface (Schooning et al., 2005). For biomedical sensing, our lab reported a novel microphysiometer to detect several different ions in cell metabolism (Wu et al., 2001a). Besides integrating LAPS to detect different ions, other possible attempts are also performed to integrate both abilities of ion concentration detection and extracellular potential signal detection, although it is still a long term from realistic application (Yu et al., 2009). While constructing cell-based biosensors, one of the biggest obstacles is that the target cells are required to be deposited on predetermined electrodes. Due to the light addressing ability, the light addressable potentiometric sensor (LAPS) can overcome this geometrical restrict, which makes LAPS an outstand candidate among various cell-based biosensors. LAPS show great potential for constructing miniaturized and integrated biosensors. One promising solution is the LAPS array for integrated cell-based biosensors. By combining the IC techniques, mechanisms, and signal sampling methods, the LAPS array system has been greatly improved and miniaturized for biomedical applications. LAPS as cell-based biosensors are able to perform longtime monitoring of several different cell physiology parameters, including extracellular acidification rate, various metabolites in extracellular microenvironment and action potential. These distinguish functions provide LAPS some promising applications in biomedical fields, such as cell biology, pharmacology, toxicology, etc (Parce et al., 1989; Mcconnell et al., 1992; Wada et al., 1992; Hafner, 2000; Wille et al., 2003). Furthermore, the multi functions of LAPS array as integrated cell-based biosensors makes the LAPS array system a good platform for drug analysis. This chapter covers design and fabrication rules, systems and applications of LAPS. LAPS as cell-based biosensors are described in details, including principle, developments, and applications. Promising aspects and developments in miniaturization of LAPS array systems are introduced for cell-based biosensors. Applications of LAPS as cell-based biosensors are provided in biomedical fields, including the interaction of ligands and receptors, drug analysis, etc. Some future developments of LAPS as cell-based biosensors are proposed in the last part of this chapter. 2. Principle LAPS is a semiconductor based potential sensitive device that usually consists of the metal- insulator-semiconductor (MIS) or electrolyte-insulator-semiconductor (EIS) structure. As for constructing cell-based biosensor, electrolyte is needed for cells living, thus LAPS with EIS structure is always adopted. LAPS with EIS structure is schematically shown in Figure 1A. The LAPS consists of the heterostructure of Si/SiO 2 /Si 3 N 4 . An external DC bias voltage is applied to scan the EIS structure to form accumulation, depletion and inversion layer at the interface of the insulator (SiO 2 ) and semiconductor (Si), sequentially. When a modulated light pointer illuminates the bulk silicon, light induced charge carriers are separated by the internal electric field and thus photocurrent can be detected by the peripheral circuit. The amplitude of the photocurrent depends on the local surface potential. By detecting the photocurrent of LAPS, localized surface potential can be obtained (Hafeman et al., 1988). The basic function of LAPS is for pH detection. Usually, a layer of Si 3 N 4 is fabricated on the surface of LAPS as the H + -sensitive layer. According to the site-binding theory, a potential Light Addressable Potentiometric Sensor as Cell-Based Biosensors for Biomedical Application 349 difference which is related to the concentration of H + in the electrolyte forms at the interface of insulator (Si 3 N 4 /SiO 2 ) and solution (Siu et al., 1979; Bousse et al., 1982). This potential is coupled to the bias voltage applied to the sensor. Larger concentration of H + provides a larger value of this potential difference, causing the I-V curve to shift along the axis of bias voltage (Figure 1B). When the bias voltage keeps constant in the middle region, change of the photocurrent indicates the pH change of the electrolyte. With the microchamber specified for biological assay, the extracellular acidification rate of cells can be monitored in the microenvironment by the commercialized Cytosensor TM Microphysiomter system. Fig. 1. (A) Working principle of the LAPS. (B) Characteristic I-V curves of n-type LAPS Beside the pH detection, attempt has been made for the extracellular detection of electrical signals. LAPS is a surface potential detector with spatial resolution. Light pointer used for LAPS detection can be focused by microscope and optical lens, which suggests the LAPS possible for cell analysis on any non-predetermined testing site. After cells are cultured on the LAPS, a focused laser, 10 μm in diameter, is used to illuminate the front side of the chip to address the cells to be monitored. Excitable cells such as cardiac myocytes or neuron cells can generate extracellular action potential. This potential is coupled to the bias voltage applied to the LAPS, which correspondingly changes the amplitude of the photocurrent. Thus, by monitoring the photocurrent at a constant bias voltage, extracellular potential signals can be detected (Xu et al., 2005). Illuminating different sensing areas with modulated lights of different frequencies generates a photocurrent signal, from which corresponding information of each testing site can be obtained by FFT (Fast Fourier Transform) methods (Cai et al., 2007). Comparing with conventional surface potential detectors such as FET or MEA, integration of LAPS array has many advantages. The most important feature of LAPS array is the great reduction of the required leads. For MEA, the number of required leads is the same as the number of electrodes, while for LAPS array, only one lead is necessary, regardless of the number of testing sites, which is important for high level integration (George et al., 2000). Besides, LAPS can detect extracellular potential as well as ion concentrations (Wu et al., 2001b), Biosensors for Health, Environment and Biosecurity 350 which makes it suitable for multi-functional integration. Sensing surface of LAPS is extremely flat and free of metal contact. Thus it’s easy for encapsulation of LAPS array and fabrication of micro testing chamber. 3. Device and system The LAPS device is a typical EIS structure. Fabrication procedure is easy and fully compatible with standard microelectronics facilities. We have introduced in our publications the most commonly used LAPS device and system (Xu et al., 2005). In this section, we mainly introduce the devices and fabrication process of LAPS array sensors. 3.1 Devices As mentioned before, the LAPS can be treated as an array sensor with no extra structures due to the spatial resolution. However, since only a little part of the LAPS surface is illuminated with the modulated light pointer, unilluminated parts, where no photocurrent flows, act as stray capacitance and cause noises. Therefore, the smaller the total capacitance of the device is, the better the potential sensitivity will be. Small effective areas as well as a thick insulating layer reduce the total capacitance, and thus improve the potential sensitivity. Nevertheless, by reducing the effective LAPS surface to small areas, the advantage of the LAPS against surface potential detectors with discrete active sites is lost (George et al., 2000). According to our experience in cell experiments, we found 200μm×200μm a compromised size between cell culture and the noise level (Xu et al., 2006). One typical structure of the integrated LAPS array sensor reported for multifunctional detection of extracellular pH detection and extracellular potential signals is schematically shown in Fig.2 (a). (Yu et al., 2009) The chip has a total area of about 1cm×2cm. Testing areas of two different sizes are fabricated on the same silicon chip by heavily doping the silicon between the testing areas. For extracellular potential signal detection, about 400 small square wells were fabricated in size of 200μm×200μm and the plateau between two adjacent testing areas was 100μm in width. Cells were cultured on the areas with small wells for potential detection. The depth of the well shaped structure was about several hundred nanometers, and we found that cells are more likely to grow on the testing areas of the arrays, which had lower altitude. Four larger wells for detection of cell acidification were 3mm×3mm in size and 1mm away from each other. The fabrication process of such LAPS array structures was shown in Fig.2(b). A p-type silicon wafer (thickness of 450μm) with <100> crystal orientation was used. First, a thick layer of silicon oxide was thermally grown on the surface. Then, after the pattern was transferred to the surface using photolithography, all silicon oxide, except that grown on testing areas (acting as a protector of substrate at testing areas from being doped in the following step), was removed by etching. Thermal diffusion doping was then carried out. As the silicon wafer is p-type, boron was selected as the impurity. There were two steps in doping process. First, a glass layer containing boron was pre-deposited on the sensing surface. Then pre-diffusion doped the surface of silicon to a small depth. After pre-diffusion, the glass layer was removed, followed by the redistribution step. During the redistribution step, a thick layer of silicon oxide about several hundred nanometers formed on the surface of doped areas, which participated in forming a well shaped structure. The doped part of the semiconductor was several micrometers in depth to cut off the depletion layer of adjacent detection sites. After the doping procedure, silicon oxide layer on testing areas was [...]... Electrochemical Biosensors for Clinical Analysis Table 1 Important domains for biosensors usage 371 372 Biosensors for Health, Environment and Biosecurity Table 1 Important domains for biosensors usage cont’ Sea food, Sol-Gel Technology in Enzymatic Electrochemical Biosensors for Clinical Analysis Table 1 Important domains for biosensors usage cont’ 373 374 Biosensors for Health, Environment and Biosecurity For. .. for demodulation Therefore, the result of the lock-in amplifier includes the amplitude and phase information of the photocurrent signal, which reflects the change of the surface potential signal of the 352 Biosensors for Health, Environment and Biosecurity LAPS chip After signal demodulation by lock-in amplifier, data is then sampled by a 16-bit acquisition card to the computer for data screening and. .. detection and control of a large number of analytes with important applications in health, agriculture, food industry, environmental monitoring etc With an annual growth rate estimated at 60% (Chaplin & Bucke, 1990), biosensors and analytical techniques in which they are involved will play an increasingly important role in the future technology Some of the 370 Biosensors for Health, Environment and Biosecurity. .. Light Addressable Potentiometric Sensor as Cell-Based Biosensors for Biomedical Application 353 Fig 3 Schematic diagram of the line-scanning light sources based on microlens array Fig 4 Multi-functional LAPS system for simultaneously detecting extracellular acidification and extracellular potential signals 354 Biosensors for Health, Environment and Biosecurity Basically, single spike of action potential... 1998 Biosensors & Bioelectronics 13, 613-618 Stenger, D.A., Gross, G.W., Keefer, E.W., Shaffer, K.M., Andreadis, J.D., Ma, W., Pancrazio, J.J., 2001 Detection of physiologically active compounds using cell-based biosensors Trends Biothchnol 19: 304-309 362 Biosensors for Health, Environment and Biosecurity Squibb, K.S., Fowler, B.A., 1981 Relationship between metal toxicity to subcellular systems and. .. instruments for Light Addressable Potentiometric Sensor as Cell-Based Biosensors for Biomedical Application 355 biological detection To solve this problem, getting more information about the multifunctional cellular processing of input- and output-signals in different cellular plants is essential for basic research as well as for various fields of biomedical applications Therefore, research work with LAPS for. .. decrease of amplitude and duration, however a slight 358 Biosensors for Health, Environment and Biosecurity progressive increase of frequency was observed On the contrary, the three parameters all increased in Hg2+ solution There were no apparent trends with regard to Cu2+ and Zn2+ toxic effects on measured parameters (only duration on Zn2+ showed a slight increase) Comparing with biosensors using pure... scanning electron microscope 360 Biosensors for Health, Environment and Biosecurity Fig 9 Responses of the extracellular potential changes of olfactory mucosa tissue to odors of butanedione (A) and acetic acid (B), with different discharge models (C) 5 Conclusion In this chapter, we engaged the light addressable potentiometric sensor (LAPS) as the cellbased biosensors for biomedical application The main... opens a simple route to produce materials like glasses, monoliths, powders, thin films in mild conditions Inorganic and hybrid organic-inorganic micro and nanostructered matrices based mainly on silica gels will be briefly described Enzymes 364 Biosensors for Health, Environment and Biosecurity entrapment in silica gels by sol-gel route is now history (Avnir et al., 1994; Gill & Ballesteros, 2000;... backbones, mixing with the electrode material (e.g carbon paste) 366 Biosensors for Health, Environment and Biosecurity or entrapment into a polymeric matrix by ion exchange) to ensure a suitable electron transfer pathway (Castillo et al., 2004; Singh & Choi, 2009) Fig 1 Biosensor device Sol-Gel Technology in Enzymatic Electrochemical Biosensors for Clinical Analysis 367 The analyte molecules may comprise . due to interaction between epirubicin and 8-oxo-G which is formed as a result of interaction of epirubicin with Biosensors for Health, Environment and Biosecurity 344 guanine rich region. address: cnpwang@zju.edu.cn Biosensors for Health, Environment and Biosecurity 348 multi-sensing of different ions. Our lab proposed an electronic nose with MLAPS for environmental detection,. Deliv. Rev., Vol. 21, pp. 179–189. Biosensors for Health, Environment and Biosecurity 346 Nakamura, H. & Karube, I. (2003). Current research activity in biosensors. Anal. Bioanal. Chem.,