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Syed Ali Process Analytical Science Group (PASG) Pfizer Inc., Peapack, New Jersey June, 17,2011
Objective
Analytical methods for bacteria and pathogens detection Theory about analytical technique Expert and institution on different analytical techniques Commercial available Instrumentation Sample preparation
Biosensors
A biosensor is a biologically sensitive material or device works as a suitable transducing system which converts the biochemical signal into a quantifiable and process able electrical signal by contacting with analyte. The biosphere contains a huge number of substances which can influence, inhibit, aggravate or stimulate various aspects of the health and behavior of whole living organisms or systems isolated from them.3 Cheap and reliable in vitro sensors are required for monitoring biological activity.
Instrumentation
Instrumentation: A) Prism couplers/Grating couplers/Optical fibers/integrated optical wavelength B) Sensor surface C) Detector D) Light sources
Bio-luminescence Process
Luciferase encoding genes are incorporated in the bacteriophase. The luciferase genes remains unchanged in the bacteriophase due to the absence of transcriptional and translational machinery. When this bacteriophase are infected , the luciferase is expressed. In the firely luc luminescence system, its luciferin , a heterocarboxylic acid, is oxidized in an ATP dependent manner. Luciferin + ATP+ Mg+2 + O2 Oxyluciferin + AMP + PPi + light
Chemiluminescence Process
CL is differentthe from BL. Because CL is production of light occurred in biological system catalyzed by enzymes. Several chemiluminescent compound can be for this type of reaction such as luminol[5-amino-2,3 dihydro-1,2 phthalazine dione], Lucigenin.
CL has been used
mainly for the detection of food borne pathogens in combination with immunoassays.
Principle of TSM
TSM consists of quartz crystal which is connected with two metallic electrodes( gold, silver, palladium). The electrodes are coated with high affinity bioreceptors. When an electrical potential is created across the electrodes, deformed quartz can induces a transverse , standing wave of resonance oscillation in the quartz because of its piezoelectric properties. A special type of cut in the quartz can displace the oscillation parallel to the resonator surface. Any type of the changes in the resonance frequency of the crystal can indicate the added mass due to binding at the active area of the electrodes. Sample coupled with bioreceptors can attribute to a mass change that can be converted to signal output.
Commercial MALDI-TOP
a) Intertek (MALDI-TOF mass spectrometry can provide high mass range (up to 100,000 D), high mass resolution and high mass accuracy) b) JEOL USA ( mass range up to 30,000 D) c) Shimadzu (Axima Assurance) d) AB SCIEX e) Waters Corporation
Flow Cytometry
Flow cytometry is a popular method for analysis of suspended cells , bacteria and microorganism. Monoclonal antibody and fluorescent probe made flow cytometry more popular. This is a useful technique to examine cells by suspending them in a stream of fluid and passing them by an electronic detection apparatus. Profiling, sorting and measurement of various physical properties of cells and bacterial for biomedical application can be performed with flow cytometric approach.
Instrumentation
Fluorescent leveled cells and bacteria are hydrodynamically focused by surrounding sheath flow into a narrow stream to pass through a region where fluorescence emission or scattered light is collected by several sophisticated optical detection instrument.
Reference
1. 2. 3. 4. 5. Zourob, M., Principle of bacterial Detection. Springer: 2008; p 83. Using surface plasmon resonance (SPR). (09/2011) Lowe, C. R., An introduction to the concepts and technology of biosensors. Biosensors 1985, 1, (1), 3-16. Moldenhauer, J., proteotypic Identification Method-A Change in Identification Methods. Microbiology 2011, 34-37. Andral, J. r.; Bolhling, A.; Gronewold, T. M. A.; Schlecht, U.; Perpeet, M.; Gutsmann, T., Surface Acoustic Wave Biosensor as a Tool to Study the Interaction of Antimicrobial Peptides with Phospholipid and Lipopolysaccharide Model Membranes. Langmuir 2008, 24, (16), 91489153. Mauritz, K. MALDI-TOF Mass Spectrometry. http://www.psrc.usm.edu/mauritz/maldi.html (May,16,2011).
6.
Acknowledgement
Bradley Diehl Manager, PAT Projects, Pfizer Global Manufacturing.
Thank You