Micro Bioactivity Analyzer

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1 Micro Bioactivity Analyzer AMIS-11 Application Data Bio-X Inc. October 28

2 Micro Bioactivity Analyzer MIS-11 enables the real-time base quantitative measurement of bioactive reactions such as enzyme reaction, without complicated coloring process. Since this analyzer minimizes the sample amount, it is suitable to analyze the metabolism of protein, having low yield to obtain the sample. This book contains the data with reagent base as analysis example to show the basic performance of this analyzer, which indicates the possibility of wide range application. Substrate Enzyme Co-enzyme Substrate Sensitivity (µm) Enzyme Sensitivity (Aboslute amount in 2µ) Sensing Object Glucose Glucose Oxidase 1µg/ml (55µM).5µg/ml (1ng) Gluconic acid Glucose Glucose Dehydrogenase NAD 2µg/ml (111µM) Proton Glycerol Glycerol Kinase + Glycerophosphate Oxidase ATP 1.5µg/ml (16µM) Peroxide Ethanol Alcohol Dehydrogenase NAD Formaldehyde Formaldehyde Dehydrogenase NAD Acetaldehyde Aldehydel Dehydrogenase NAD 1µg/ml (217µM).3µg/ml (1µM) 5ng/ml (.1µM) Proton Proton + Formic Acid Proton + Acetic Acid ATP Alkaline Phosphatase 5µg/ml (1µM).1unit/ml (.2unit) Phosphoric Acid Urea Urease 2µg/ml (33µM) Ammonia Creatinine Creatinine Deiminase 1µg/ml (8.8µM) 1μg/ml (2ng) Ammonis Olive Oil Lipoprotein Lipase 1µg/ml Oleic Acid Cholesteryl Linoreate Cholesterol Esterase 2µg/ml (3µM) Lonleic Acid DL-BAPNA Tripsin 6µg/ml Carboxylicc Acid Since the all protocols contained in this data book are based on purified reagents, suitable pretreatment and reagent design to each sample condition may be required.

3 1) Glucose (1) Buffer: Glucoseoxidase Glucose 1mM tris-hcl ph mM NaCl Glucoseoxidase β-d-glucose + O 2 + H 2 O D-Glucono- δ-lactone + H 2 O 2 1. Enzyme, Glucoseoxidase, was dissolved with Buffer to.1mg/ml. 2. Substrate, Glucose, was dissolved with Buffer to the designated concentration µl of enzyme solution prepared in 1. was placed into the sensor A and B. 4. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 2. was placed in the sensor A, and Buffer (reference) into 5. measured the value by subtracting in Sensor A from in Sensor B as Reaction. Analytical Curve (Linear) 1mg/dl 5mg/dl 25mg/dl 1mg/dl Sec mg/dl

4 2) Glucose (2) Coenzyme: Buffer: Glucosedehydrogenase NAD Glucose 1mM PBS-KOH ph7 + 1mM NaCl Glucose Dehydrogenase β-d-glucose + NAD + D-Glucono- δ-lactone + NADH + H + 1. Enzyme, Glucosedehydrogenase, was dissolved with Buffer to 1.4mg/ml, 2. Co-Enzyme, NAD, was dissolved with Buffer to 3.5mg/ml, 3. Substrate, Glucose, was dissolved with Buffer to the designated concentration, 4. 9µl of enzyme solution prepared in 1. was placed into the sensor A and B, 5. 9µl of Co-Enzyme solution prepared in 2. was placed into the sensor A and B, 6. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 3. was added in the sensor A, and Buffer (reference) into 7. measured the value by subtracting in Sensor A from in Sensor B as Reaction. Analytical Curve (End Point) Sec. m y = x R 2 =.9975 mg/dl

5 3) Glycerol Buffer: Glycerol Kinase, Glycerol Phosphate Oxidase Glycerol 1mM ATP-NaOH ph7. Glycerol Kinase Glycerophosphate Oxidase Glucerol + ATP Glycerol-3phosphate + O2 Dihydroxyacetonephosphate + H 2 O 2 1. Enzyme 1, Glycerol Kinase, was dissolved with Buffer to.2mg/ml, 2. Enzyme 2, Glycerol Phosphate Oxidase, was dissolved with Buffer to.2mg/ml, 3. Substrate, Glycerol, was dissolved with Buffer to the designated concentration, 4. 9µl of enzyme solution prepared in 1. was placed into the sensor A and B, 5. 9µl of enzyme solution prepared in 2. was placed into the sensor A and B, 6. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 3. was added in the sensor A, and Buffer (reference) into 7. measured the value by subtracting in Sensor A from in Sensor B as Reaction. Analytical Curve (End Point) mg 3.2mg 6.5mg SEC m mg/dl

6 4) Ethanol Alcohol Dehydrogenase Co- NAD Ethanol Buffer: 1mM K-PBS ph7. + NaCl 5mM Alcohol Dehydrogenase Ethanol + NAD + Acetaldehyde + NADA + H + 1. Enzyme, Alcohol Dehydrogenase, was dissolved with Buffer to.1mg/ml, 2. Co-Enzyme, NAD, was dissolved with Buffer to 1.8mg/ml, 3. Substrate, Ethanol, was dissolved with Buffer to the designated concentration, 4. 9µl of enzyme solution prepared in 1. was placed into the sensor A and B, 5. 9µl of Co-Enzyme solution prepared in 2. was placed into the sensor A and B, 6. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 3. was added to the sensor A, and Buffer (reference) into 7. measured the value by subtracting in Sensor A from in Sensor B as Reaction. Analytical Curve (End Point) m Sec. -5 g/l

7 5) Formaldehyde Formaldehyde Dehydrogenase Co- NAD Formaldehyde Buffer: 1mM Tris-HCl ph8.5 Formaldehyde Dehydrogenase Formaldehyde + NAD + Formic Acid + NADH + H + 1. Enzyme, Formaldehyde Dehydrogenase, was dissolved with Buffer to.1mg/ml, 2. Co-Enzyme, NAD, was dissolved with Buffer to 1.8mg/ml, 3. Substrate, Formaldehyde, was dissolved with Buffer to the designated concentration, 4. 9µl of enzyme solution prepared in 1. was placed into the sensor A and B, 5. 9µl of Co-Enzyme solution prepared in 2. was placed into the sensor A and B, 6. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 3. was added in the sensor A, and Buffer (reference) into 7. measured the value by subtracting in Sensor A from in Sensor B as Reaction. Analytical Curve (Linear).5mM.2mM.1mM.5mM.1mM m min Formaldehyde mm

8 6) Acetaldehyde Aldehyde Dehydrogenase Co- NAD Acetaldehyde Buffer: 1mM K-PBS ph7. Formaldehyde Dehydrogenase Acetaldehyde + NAD + Acetic Acid + NADH + H + 1. Enzyme, Aldehyde Dehydrogenase, was dissolved with Buffer to.1mg/ml, 2. Co-Enzyme, NAD, was dissolved with Buffer to 1.8mg/ml, 3. Substrate, Acetaldehyde, was dissolved with Buffer to the designated concentration µl of enzyme solution prepared in 1. was placed into the sensor A and B, µl of Co-Enzyme solution prepared in 2. was placed into the sensor A and B, 6. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 3. was added in the sensor A, and Buffer (reference) into the sensor B, each 5µL for measurement, and 7. measured the value by subtracting in Sensor A from in Sensor B as Reaction. Analytical Curve (End Point) Sec. m y = x R 2 =.9952 g/l

9 7) ATP (Adenosine Triphosphate) Alkaline Phosphatase ATP(Li) Buffer: 1mM Mops-Tris ph mM MgCl 2 ATP Alkaline Phosphatase MgCl 2 ADP, AMP 1. Enzyme, Alkaline Phosphatase, was dissolved with Buffer to 6u/ml, 2. Substrate, ATP(Li), was dissolved with Buffer to the designated concentration, 3. 18µl of enzyme solution prepared in 1. was placed into the sensor A and B, 4. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 2. was placed in the sensor A, and Buffer (reference) into 5. measured the value by subtracting in Sensor A from in Sensor B as Reaction. Analytical Curve (Linear) 5.2mM 3mM 2mM 1mM 1mM(2).5mM.2mM m min ATP (mm)

10 8) Urea Buffer: Urease Urea 1mM Tris-HCl ph8. + 5mM KCl Urease (NH 2 ) 2 CO + H 2 O + 2H + 2NH4 + + CO 2 1. Enzyme, Urease, was dissolved with Buffer to.75mg/ml, 2. Substrate, Urea, was dissolved with Buffer to the designated, 3. 15µl of enzyme solution prepared in 1. was placed into the sensor A and B, 4. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 2. was added in the sensor A, and Buffer (reference) into the sensor B, each 5µL for measurement, and 5. measured the value by subtracting in Sensor A from in Sensor B as Reaction Analytical Curve (End Point) 1mg/dl 8mg/dl 5mg/dl 3mg/dl 2mg/dl 1mg/dl Sec. m y = x R 2 = mg/dl

11 9) Creatinine Buffer: Creatinine Deiminase Creatinine 1mM Tris-HCl ph7.5 5mM NaCl Creatinine Deiminase Creatinine + H 2 O + H + N-Methylhydrantoin + NH Enzyme, Creatinine Deiminase, was dissolved with Buffer to.1mg/ml 2. Substrate, Creatinine, was dissolved with Buffer to the designated concentration, 3. 18µl of enzyme solution prepared in 1. was placed into the sensor A and B, 4. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 2. was added in e sensor A, and Buffer (reference) into 5. measured the value by subtracting in Sensor A from in Sensor B as Reaction Analytical Curve (End Point) 1mg/dl 7mg/dl 5mg/dl 3mg/dl 1mg/dl y = x R 2 = Sec mg/dl

12 1) Olive Oil Buffer: Lipoprotein Lipase Olive Oil 5mM K-PBS ph7. Detecting oleic acid formed by hydrolytic reaction of Olive oil with Lipoprotein Lipase 1. Enzyme, Lipoprotein Lipase, was dissolved with Buffer to.1mg/ml, 2. Substrate, Olive Oil, was dissolved with Buffer to the designated concentration, and sonicated, 3. 18µl of enzyme solution prepared in 1. was placed into the sensor A and B, 4. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 2. was placed in the sensor A, and Buffer (reference) into 5. measured the value by subtracting in Sensor A from in Sensor B as Reaction. Analytical Curve (Linear) 1mg/ml 5mg/ml 1mg/ml.5mg/ml.1mg/ml X E sec. -1 mg/ml

13 11) Cholestryl Linoreate Buffer: Cholestrol Esterase Cholestryl Linoreate 1mM Tris-HCl ph7.5 Detecting linoleic acid formed by decomposition reaction of Cholestryl Linoreate with Cholestrol Esterase 1. Enzyme, Cholestrol Esterase, was dissolved with Buffer to.1mg/ml, 2. Substrate, Cholestryl Linoreate, was dissolved with IPA first and dissolved with buffer to the designated concentration, and subsequently heated up for evaporating IPA and sonicated, 3. 18µl of enzyme solution prepared in 1. was placed into the sensor A and B, 4. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 2. was placed in the sensor A, and Buffer (reference) into 5. measured the value by subtracting in Sensor A from in Sensor B as Reaction. Analytical Curve (Linear) 3mg/dl 2mg/dl 1mg/dl 5mg/dl 1mg/dl Sec y = -.914x R 2 =.9765 mg/dl

14 12) DN-BAPNA Buffer: Tripsin Nα-Benzoyl L-arginine 4-nitroanilide hydrochloride (DL-BAPNA) 2.5mM Tris-HCl ph8.3 Detecting carboxylic acid formed by decomposition reaction of DL-BAPNA with Tripsin. 1. Enzyme, Tripsin, was dissolved with Buffer to 1mg/ml, 2. Substrate, DL-BAPNA, was diluted with Buffer to the designated concentration r, 3. 18µl of DL-BAPNA solution prepared in 2. was placed into the sensor A and B, 4. The sensor was set to AMIS-11, with subsequent stabilized temperature, the substrate prepared in 2. was placed in the sensor A, and Buffer (reference) into 5. measured the value by subtracting in Sensor A from in Sensor B as Reaction. Analytical Curve (End Point) SEC -.6 mg/ml

16 . Bio-X Inc. 12 Kuze Tonoshiro-cho, Minami-ku, Kyoto Phone: Fax : info@bio-x.co.jp Web

APPENDIX Heparin 2 mg heparin was dissolved in 0.9 % NaCl (10 ml). 200 µl of heparin was added to each 1 ml of blood to prevent coagulation.

APPENDIX Heparin 2 mg heparin was dissolved in 0.9 % NaCl (10 ml). 200 µl of heparin was added to each 1 ml of blood to prevent coagulation. APPENDIX 1 Preparation of reagents 1.1. Preparation of dosing solution Nonylphenol 15 mg of Nonylphenol was dissolved in olive oil (10 ml) and used as stock solution. The stock solution was serially diluted