- Email: [email protected]
USA — Identification of receptor ligands and receptor subtypes
Biosensors & Bioelectronics Vol. 11 No. 4 (1996)
Biosensors & Bioelectronics Abstracts Valerie M O w e n
This international update on new publications in biosensors and related activities includes: antagonists as identifier tools, in vivo electrochemical monitoring, low concentration electrochemical detection, needle-type biosensors, novel microbial sensor monitoring, on-line monitoring, and flow-injection biosensors.
China - Biosensor using novel L-glutamate oxidase In J. BIOTECHNOL. (42/1 (45-52) 1995) B. -C. Ye, Q.-S. Li, Y.-R. Li, X.-B. Li & J.-T. Yu of East China Univ. report on 'L-Glutamate biosensor using a novel L-glutamate oxidase and its application to flow injection analysis system'. Streptomyces P-106, isolated from soil samples, produced extracellular L-glutamate oxidase in liquid culture. About 10 units enzyme activity per ml medium could be reached after 48-60 h incubation at 28°C. A micro-enzyme electrode was prepared by cross linking the purified novel L-glutamate oxidase, produced in the author's lab, with glutaraldehyde on an aminopropyl-platinized-platinum wire (6p 0.5 mm). It was used for the determination of L-glutamate in a flow injection analysis system with good performance: accuracy (CV = 0.4%), fast response (< 40 s) and stability (> 30 d). The system showed linear response to the L-glutamate concentration ranging from 3.0 mg 1~ to 300 mg 1-1 (approx. 0.02 mol-ll to 2.0 mmdl 1 ). The system was applied to determine the concentration of L-glutamic acid during synthesis. Good correlations were achieved between results obtained with the system and with Warburg method. Contact: Research Inst. Biochemical Engin., East China Univ. Sc./Technology, Shanghai 200237, China.
0956-5663/96/$15.0001996 Elsevier Science Ltd
USA - Identification of receptor ligands and receptor subtypes In PROC. NATL. ACAD. SCI. U. S. A. (92/17 (7877-7881) 1995) H.A. Fishman, O. Orwar, R.H. Scheller & R.N. Zare of Stanford University report on 'Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system'. The authors have used a capillary electrophoresis system with single-cell biosensors as a detector to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B 2 receptor subtype (BzBK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms
vii
Biosensors & Bioelectronics Vol, 11 No. 4 (1996)
of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes. Contact: Department of Chemistry, Stanford University, Stanford, CA 94305, USA. F r a n c e - In vivo e l e c t r o c h e m i c a l m o n i t o r i n g o f serotonin
N-mCFEs PCA induced a rapid increase of peak 3N (137 _+6% at 90 min). The xanthine oxidase inhibitor allopurinol (10 mg/kg i.p.) produced a decrease (30 _+ 3% at 180 min) in peak 3 (mCFEs), but peak 3N (N- mCFEs) was not affected (106% at 180 min). After pretreatment with allopurinol, PCA also produced an increase (135 _ 6% at 90 min) in peak 3N. These in vitro and in vivo data provide evidence for a highly preferential detection of 5-HT versus 5-HIAA and UA by N-mCFEs, which could be used to follow the extracellular 5-HT concentration within very discrete structures throughout the CNS. Contact: INSERM U. 161, 2, rue d'Alesia, F75014 Paris, France.
In J. NEUROCHEM. (65/3 (1257-1263) 1995) J.P. Rivot, R. Cespuglio, S. Puig, M. Jouvet, J.-M. Besson of INSERM U. report on 'In vivo electrochemical monitoring of serotonin in spinal dorsal horn with Nation-coated multi-carbon fiber electrodes'. The authors constructed biosensors, sensitive for in vivo monitoring of serotonin (5-HT) in the CNS by differential normal pulse voltammetry, by coating treated multicarbon fiber electrodes (mCFEs) with Nation (N-mCFE). In vitro sensitivities of mCFE and N-mCFE were compared in solutions ranging from 5 nM to 20 ~M of uric acid (UA), 5-hydroxyindoleacetic acid (5-HIAA), and 5-HT. The mCFEs were three to seven times less sensitive for 5-HIAA or UA than for 5-HT. Nation treatment dramatically decreased sensitivity for 5-HIAA and UA of N-mCFEs (- 103 times), whereas it remained in the nanomolar range for 5- HT. In vivo, in the dorsal horn of the lumbar spinal cord of anaesthetized rats, the monoamine oxidase inhibitor clorgyline (10 mg/kg i.p.) produced a reduction (55 _ 3% at 180 min) of peak 3 of oxidation current (characteristic of 5-hydroxyindoles) monitored with mCFEs, but with N-mCFEs (in this latter case the peak was termed 3N) peak 3N increased to 135 +__5% at 180 min. The 5-HT release-inducer p-chloroamphetamine (PCA; 6 mg/kg i.p.) induced a slight (12 _+3% at 150 min) decrease in peak 3 measured with mCFEs, whereas with
In NATURE (376/6542 (672-675) 1995) A. Riklin, E. Katz, I. Willner, A. Stocker & A.F. Buckmann of The Hebrew University of Jerusalem report on 'Improving enzyme-electrode contacts by redox modification of cofactors'. Efficient electron transfer of redox proteins to and from their environment is essential for the use of such proteins in biotechnological applications such as amperometric biosensors and photosynthetic biocatalysts. But most redox enzymes lack pathways that can transport an electron from their embedded redox site to an electrode or a diffusing photoexcited species. Electrical communication between redox proteins and electrode surfaces has been improved by aligning proteins on chemically modified electrodes, by attaching electron-transporting groups and by immobilizing proteins in polymer matrices tethered by redox. Generally these methods involve contacting the groups enzymes at random with electron relay units. The authors report an approach that allows site-specifi, positioning of electron-mediating units in redox proteins. They strip glucose oxidase of its flavin adenine dinucleotide (FAD) cofactors, modify the latter with redox-active ferrocene-containing groups, and then reconstitute the apoprotein with these modified cofactors. In this way, electrical contact between an electrode and the resulting enzyme in solution is greatly enhanced in a controlled and reproducible way. Contact: hzstitute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
viii
Israel - I m p r o v i n g e n z y m e - e l e c t r o d e c o n t a c t s
Biosensors & Bioelectronics Abstracts Valerie M O w e n
This international update on new publications in biosensors and related activities includes: antagonists as identifier tools, in vivo electrochemical monitoring, low concentration electrochemical detection, needle-type biosensors, novel microbial sensor monitoring, on-line monitoring, and flow-injection biosensors.
China - Biosensor using novel L-glutamate oxidase In J. BIOTECHNOL. (42/1 (45-52) 1995) B. -C. Ye, Q.-S. Li, Y.-R. Li, X.-B. Li & J.-T. Yu of East China Univ. report on 'L-Glutamate biosensor using a novel L-glutamate oxidase and its application to flow injection analysis system'. Streptomyces P-106, isolated from soil samples, produced extracellular L-glutamate oxidase in liquid culture. About 10 units enzyme activity per ml medium could be reached after 48-60 h incubation at 28°C. A micro-enzyme electrode was prepared by cross linking the purified novel L-glutamate oxidase, produced in the author's lab, with glutaraldehyde on an aminopropyl-platinized-platinum wire (6p 0.5 mm). It was used for the determination of L-glutamate in a flow injection analysis system with good performance: accuracy (CV = 0.4%), fast response (< 40 s) and stability (> 30 d). The system showed linear response to the L-glutamate concentration ranging from 3.0 mg 1~ to 300 mg 1-1 (approx. 0.02 mol-ll to 2.0 mmdl 1 ). The system was applied to determine the concentration of L-glutamic acid during synthesis. Good correlations were achieved between results obtained with the system and with Warburg method. Contact: Research Inst. Biochemical Engin., East China Univ. Sc./Technology, Shanghai 200237, China.
0956-5663/96/$15.0001996 Elsevier Science Ltd
USA - Identification of receptor ligands and receptor subtypes In PROC. NATL. ACAD. SCI. U. S. A. (92/17 (7877-7881) 1995) H.A. Fishman, O. Orwar, R.H. Scheller & R.N. Zare of Stanford University report on 'Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system'. The authors have used a capillary electrophoresis system with single-cell biosensors as a detector to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B 2 receptor subtype (BzBK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms
vii
Biosensors & Bioelectronics Vol, 11 No. 4 (1996)
of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes. Contact: Department of Chemistry, Stanford University, Stanford, CA 94305, USA. F r a n c e - In vivo e l e c t r o c h e m i c a l m o n i t o r i n g o f serotonin
N-mCFEs PCA induced a rapid increase of peak 3N (137 _+6% at 90 min). The xanthine oxidase inhibitor allopurinol (10 mg/kg i.p.) produced a decrease (30 _+ 3% at 180 min) in peak 3 (mCFEs), but peak 3N (N- mCFEs) was not affected (106% at 180 min). After pretreatment with allopurinol, PCA also produced an increase (135 _ 6% at 90 min) in peak 3N. These in vitro and in vivo data provide evidence for a highly preferential detection of 5-HT versus 5-HIAA and UA by N-mCFEs, which could be used to follow the extracellular 5-HT concentration within very discrete structures throughout the CNS. Contact: INSERM U. 161, 2, rue d'Alesia, F75014 Paris, France.
In J. NEUROCHEM. (65/3 (1257-1263) 1995) J.P. Rivot, R. Cespuglio, S. Puig, M. Jouvet, J.-M. Besson of INSERM U. report on 'In vivo electrochemical monitoring of serotonin in spinal dorsal horn with Nation-coated multi-carbon fiber electrodes'. The authors constructed biosensors, sensitive for in vivo monitoring of serotonin (5-HT) in the CNS by differential normal pulse voltammetry, by coating treated multicarbon fiber electrodes (mCFEs) with Nation (N-mCFE). In vitro sensitivities of mCFE and N-mCFE were compared in solutions ranging from 5 nM to 20 ~M of uric acid (UA), 5-hydroxyindoleacetic acid (5-HIAA), and 5-HT. The mCFEs were three to seven times less sensitive for 5-HIAA or UA than for 5-HT. Nation treatment dramatically decreased sensitivity for 5-HIAA and UA of N-mCFEs (- 103 times), whereas it remained in the nanomolar range for 5- HT. In vivo, in the dorsal horn of the lumbar spinal cord of anaesthetized rats, the monoamine oxidase inhibitor clorgyline (10 mg/kg i.p.) produced a reduction (55 _ 3% at 180 min) of peak 3 of oxidation current (characteristic of 5-hydroxyindoles) monitored with mCFEs, but with N-mCFEs (in this latter case the peak was termed 3N) peak 3N increased to 135 +__5% at 180 min. The 5-HT release-inducer p-chloroamphetamine (PCA; 6 mg/kg i.p.) induced a slight (12 _+3% at 150 min) decrease in peak 3 measured with mCFEs, whereas with
In NATURE (376/6542 (672-675) 1995) A. Riklin, E. Katz, I. Willner, A. Stocker & A.F. Buckmann of The Hebrew University of Jerusalem report on 'Improving enzyme-electrode contacts by redox modification of cofactors'. Efficient electron transfer of redox proteins to and from their environment is essential for the use of such proteins in biotechnological applications such as amperometric biosensors and photosynthetic biocatalysts. But most redox enzymes lack pathways that can transport an electron from their embedded redox site to an electrode or a diffusing photoexcited species. Electrical communication between redox proteins and electrode surfaces has been improved by aligning proteins on chemically modified electrodes, by attaching electron-transporting groups and by immobilizing proteins in polymer matrices tethered by redox. Generally these methods involve contacting the groups enzymes at random with electron relay units. The authors report an approach that allows site-specifi, positioning of electron-mediating units in redox proteins. They strip glucose oxidase of its flavin adenine dinucleotide (FAD) cofactors, modify the latter with redox-active ferrocene-containing groups, and then reconstitute the apoprotein with these modified cofactors. In this way, electrical contact between an electrode and the resulting enzyme in solution is greatly enhanced in a controlled and reproducible way. Contact: hzstitute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
viii
Israel - I m p r o v i n g e n z y m e - e l e c t r o d e c o n t a c t s