Analytical Techniques for Bacteria Detection

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

Microbiological Method for Bacteria Detection

As science move forward, new technique are evolving. The following technique are popular to detect bacteria and pathogens: Biosensors and Immunosensors Luminescence Technique (Bioluminescence, Adenylate Kinase, Autofluorescence, Direct Epifluorescent Filter Technique, Fluorescent probe detection ) Enzyme linked immunosorbend assay , Western Blotting Flow Cytometry Fourier Transformed Infrared spectroscopy (FTIR), NIR,Raman Mass spectrometry (Matrix-Assisted Laser Desorption Time of Flight) Turbidimetry

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.

Surface Plasmon Resonance Sensors

Surface plasmons (SPs), are coherent electron oscillations that exist at the interface between any two materials.1 The surface plasmon resonance (SPR) phenomenon occurs when polarized light, under conditions of total internal reflection, strikes an electrically conducting gold layer at the interface between media of different refractive index: the glass of a sensor surface (high refractive index) and a buffer (low refractive index).2 This technology can quantify the kinetics, affinity and concentration of surface interaction.

Instrumentation
Instrumentation: A) Prism couplers/Grating couplers/Optical fibers/integrated optical wavelength B) Sensor surface C) Detector D) Light sources

Expert in SPR technology

Prominent researcher : a. Allen D. Taylor, Jon Ladd, Shaoyi Jiang, department of Chemical engineering, University of Washington, Seattle, Washington, USA Company: A. Biacore B. Biosensing Instrument Inc. C. Plexera LLC D. BioNavis Ltd E. SensiQ F. Nanofilm Technologie GmbH (Germany) G. Reichert Inc H. Jandratek

Luminescence Technique for the Detection of Bacterial Pathogens

The primary advantages of all luminescence based assays is their rapidity and sensitivity. Bioluminescence (BL) and Chemiluminescence (CL) have been commercially adapted for bacterial detection. BL is a naturally occurring process by which living organism covert chemical energy into light. CL is defined the as the production of light by chemicals during an exothermic reaction.

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.

Fig. 1. Schematic representation for chemiluminescent process.

Fluorescence Peptide Biosensors

Nek2 enzyme has been found to be abnormally expressed in many tumar cells. We can identify these enzyme by fluorescence biosensors. These type of sensors can be preapred by solid phase peptide synthesis where the peptide substrate contain fluorophore. We can determine the express level of Nek2 enzyme by observing fluorescence response.

Experimental Procedure of Peptide

Peptide or Protein substrate can be prepared by Fmoc solid phase peptide synthesis (SPPS). The general principle of SPPS is one of repeated cycles of coupling and Fmoc deprotection. Example of peptide biosensors:

Expert in Luminescence Techniques

Prominent Researcher: Leigh Farris, Mussie Y. Habteselassie et al.,Department of Food Science, Prude University. Company: a. Calbiochem-Novabiochem Corporation ( ATP Luminescence Assay Kit) b. Molecular Probes ( ATP Determination Luminescence Kit) c. PerkinElmer ( APT lite Cell Viability Assay) d. Promega (Enlighten Total ATP Rapid , Biocontaminatio Detection Kit, Enlighten ATP Luminescence Assay Kit, BacTiter-Glo Luminrscent Cell Viability Assay Kit) e. Biothema(Microbial ATP kit HS)

Expert in Luminescence Techniques (Cont.)

f. Roache Applied Science ( ATP Biouminescence Assay Kit, CLS II, ATP biouminescence Assay Kit, HS II. g. Sigma-Aldrich ( ATP Bioluminescent Assay Kit) h. Celsius ( AKuScreen i. Thermo Scientific ( ATP Luninescence Assay Kit) j. Oxford Biochemical Research Inc. (ATP Luminescence Assay Kit) k. Cambrex bio ( ViaLight MDA Plus Cytotoxicity and Celll Proliferation Bioassay) l. New Horizons Diagnostics ( Profile 1 Bacterial Detection Kit) m. Kikkoman ( CheckLite 25- Plus)

Porous Silicon Sensors

Silicon is inexpensive, biocompatible and easily derivatized with a broad range of capture agents such as peptides, protein and nucleic acids. Silicon (Si) can be easily porous when it is subjected to electrochemical etching process. The size and density of these pores can be controlled by etching condition and type of silicon .

Process for Porous Silicon Sensors

Mesoporous microcavity is derivativzed with TWTCP (tetratryptophan- ter-cyclopentane) TWTCP is a synthetic receptor for bacterial lipid A A mixed solution of TWTCP and glycine methyl ester ( a spacer molecule) is used to prepare the sensor This process show photoluminescence response

Expert in Porous and Planar Silicon Sensors

Prominent Researcher: a. Charles R. Mace and Benjamin L. Miller, University of Rochester. b. Vladimir V. Plashnitsaa, Taro Uedab, Perumal Elumalaia and Norio Miuraa, Science and Technology Center for Cooperative Research (KASTEC), Kyushu University, Japan

Thickness Shear Mode (TSM) Biosensors

TSM biosensors is known as quartz crystal microbalance (QSM). Crystal has piezoelectricity property. Piezoelectricity property makes TSM a highly sensitive analytical balance machine that is capable of measuring extremely small mass deposition. It is an excellent analytical tool for the study of specific molecular interaction at the solid liquid interface in air and under vacuum or aqueous condition.

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 TSM Microbalances

a) b) c) d) e) f) g) h) i) j) k) l) m) n) Maxtek Inc. Q-Sense Universal Sensors Inc. Seiko EG&G Princeton Applied Research QCM research Tectra KSV Instrument , LTD SRS Masscal Faraday Labs Initium, Inc. Sigma Instruments Tangidyne and Technochip

Matrix-assisted laser desorption/ionization (MALDI)

MALDI-TOF stands for Matrix-assisted laser desorption/ionization time of flight. Time of flight is the place where m/z separation is occurred. This technique is used to study the analysis of biomolecules (biopolymers such as proteins, peptides and sugars) and large organic molecules and other macromolecules. Matrix assisted refers to the use of an organic solution of a matrix compound to penetrate the bacterial cell walls and extract the proteins and other intracellular material. When the matrix solution dried, it creates a crystalline matrix. This dried sample is placed in the source region of a time of flight mass spectrometer and irradiated with focused light.

MALDI TOF (Cont.)

The matrix absorbs the laser light causing the energetic ejection of a small volume of the matrix into the gas phase above the sample. Desorption and ionization refers to the process where Fig. 2. MALDI-TOF.Time of flight mass separation is where the charged proteins the proteins are released are accelerated by high electric fields and and become charged in a they drift up to the vacuum tube towards the detector.6 gaseous state.

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 for Flow Cytometer

The main components of a flow cytometer: Flow cell - liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing Measuring system - commonly used are measurement of impedance (or conductivity) and optical systems - lamps (mercury, xenon); high-power water-cooled lasers (argon, krypton, dye laser). Detector and Analogue-to-Digital Conversion (ADC) system which generates fluorescence signals from light into electrical signals that can be processed by a computer Computer for analysis of the signals.

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.

Fig.3. Flow Cytometry

Expert in Flow Cytometry

Reseaccher: a) Wayne Green, Ph.D. , Flow Cytometry Facility, University of Utah. b) Sung-Yi and Gwo-Bin Lee, Department of Engineering Science , National Cheng Kung University, Taiwan. Commerially avialbale Flow Cyotmeter: a) Aber Instruments (they market the Optoflow Microcyte flow cytometer) b) Amnis (ImageStream imaging flow cytometer) c) Beckman Coulter (flow cytometers and sorters) d) BD Biosciences (flow cytometers and sorters) e) GCAT (distributor of Partec flow cytometers in the USA) f) Guava Technologies (a personal flow cytometer!) g) Hamamatsu (manufacturers of PMTs for flow cytometers) h) OptoFlow AS (manufacturers of the portable Microcyte flow cytometer) i) Partec (a German flow cytometer manufacturer)

Fourier-Transform (FTIR) Spectroscopy

Microbial cell can be analyzed by using FTIR spectra and provide a type of biochemical fingerprint that can be compared to a reference database of known microbial isolates. People are interested in this technology because the small sample is required and sample preparation is simple. Staphylococcus microbes, Enterococci, Listeria , Brucella and Candida species can be easily identified by FT-IR.

Sample Preparation for FTIR

The cells from a pure culture are inoculated directly onto a solid media and incubated (24 hours) These cells are suspended in water. After that samples are put into the sample carrier in array. After the sample has dried on the carrier, the FTIR analysis can be conducted Genotypic method such as PCR are performed to endure the accuracy of the analyzed sample.

Expert in FTIR Technology in Microbiology

Mandana Veiseh, Omid Veise, Michael C. Martin, and Miqin Zhang, Department of Materials Science & Engineering, University of Washington, United States Carolyn Bertozzi ,University of California at Berkeley, United States Laboratory for Intensive Care Research and Optical Spectroscopy, Department of General Surgery 10M, Erasmus MC, University Medical Center Rotterdam, Netherlands Joint Institute for Food Safety and Applied Nutrition, Marryland

Future of Microbiology in Bacterial Detection revolutioned Development of microbiological methods

pharmaceutical industry. Significant work still require to apply these work in the industry. Scientists are examining the unique nanoparticles of exotic elements formed by bacteria with an eye towards industrial and scientific applications. Small chip based on nanotechnology and magnetic nanoparticles which can bound to a suitable antibody, are used to label specific molecules, structures or microorganisms. In future , nano technology will apply in the microbiology field and will improve analytical technique for bacterial detection.

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).

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Acknowledgement
Bradley Diehl Manager, PAT Projects, Pfizer Global Manufacturing.

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