Analytical Techniques for Bacteria Detection

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Objective

Analytical methods for bacteria and pathogensdetection

Theory about analytical technique

Expert and institution on different analyticaltechniques

Commercial available Instrumentation

Sample preparation


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Microbiological Method for BacteriaDetection

As science move forward, new technique are evolving. The followingtechnique 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 DesorptionTime of Flight) Turbidimetry


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Biosensors

A biosensor is a biologically sensitive material ordevice 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 wholeliving organisms or systems isolated from them.3

Cheap and reliable in vitro sensors are required for

monitoring biological activity.


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Surface Plasmon Resonance Sensors

Surface plasmons (SPs), are coherent electron oscillations thatexist 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 theinterface 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 andconcentration of surface interaction.


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Instrumentation

Instrumentation:

A) Prism couplers/Grating couplers/Optical

fibers/integrated optical wavelength

B) Sensor surfaceC) Detector

D) Light sources


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Luminescence Technique for theDetection of Bacterial Pathogens

The primary advantages of all luminescence basedassays is their rapidity and sensitivity.

Bioluminescence (BL) and Chemiluminescence (CL)

have been commercially adapted for bacterialdetection.

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.


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Bio-luminescence Process

Luciferase encoding genes are incorporated in thebacteriophase.

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 dependentmanner.

Luciferin + ATP+ Mg+2 + O2 Oxyluciferin + AMP + PPi + light


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Chemiluminescence Process

CL is differentthe from BL. Because CL is production of light occurred inbiological system catalyzed by enzymes.

Several chemiluminescent compound can be for this type of reaction such

as luminol[5-amino-2,3dihydro-1,2phthalazine dione], Lucigenin.

Fig. 1. Schematic representation for chemiluminescentprocess.

CL has been used

mainly for the

detection of food

borne pathogens in

combination with

immunoassays.


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Fluorescence Peptide Biosensors

Nek2 enzyme has been found to be abnormally expressed inmany 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.


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Experimental Procedure of Peptide

Peptide or Protein substrate can be prepared by Fmoc solidphase peptide synthesis (SPPS).

The general principle of SPPS is one of repeated cycles of

coupling and Fmoc deprotection.

Example of peptide biosensors:


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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)


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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)


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Porous Silicon Sensors

Silicon is inexpensive, biocompatible and easilyderivatized with a broad range of capture agents

such as peptides, protein and nucleic acids.

Silicon (Si) can be easily porous when it is subjectedto electrochemical etching process.

The size and density of these pores can be controlled

by etching condition and type of silicon .


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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


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Expert in Porous and Planar SiliconSensors

Prominent Researcher:

a. Charles R. Mace and Benjamin L. Miller, University

of Rochester.

b. Vladimir V. Plashnitsaa, Taro Uedab, PerumalElumalaia and Norio Miuraa, Science and Technology

Center for Cooperative Research (KASTEC), Kyushu

University, Japan


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Principle of TSM

TSM consists of quartz crystal which is connected with two metallicelectrodes( 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.


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Commercial TSM Microbalancesa) Maxtek Inc.

b) Q-Sense

c) Universal Sensors Inc.

d) Seiko EG&G

e) Princeton Applied Research

f) QCM researchg) Tectra

h) KSV Instrument , LTD

i) SRS

j) Masscal

k) Faraday Labs

l) Initium, Inc.

m) Sigma Instruments

n) Tangidyne and Technochip


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Matrix-assisted laserdesorption/ionization (MALDI)

MALDI-TOF stands for Matrix-assisted laserdesorption/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 largeorganic 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.


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MALDI TOF (Cont.)The matrix absorbs thelaser light causing theenergetic ejection of a smallvolume of the matrix into thegas phase above the

sample.Desorption and ionizationrefers to the process wherethe proteins are released

and become charged in agaseous state.

Fig. 2. MALDI-TOF.Time of flight massseparation is where the charged proteinsare accelerated by high electric fields and

they drift up to the vacuum tube towardsthe detector.6


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Commercial MALDI-TOP

a) Intertek (MALDI-TOF mass spectrometry canprovide 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


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Flow Cytometry

Flow cytometry is a popular method for analysis of suspendedcells , bacteria and microorganism.

Monoclonal antibody and fluorescent probe made flow

cytometry more popular.

This is a useful technique to examine cells by suspendingthem 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 canbe performed with flow cytometric approach.


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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 ofimpedance (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.


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Instrumentation

Fluorescent leveled cells andbacteria are hydrodynamicallyfocused by surrounding sheathflow into a narrow stream to

pass through a region wherefluorescence emission orscattered light is collected byseveral sophisticated opticaldetection instrument.

Fig.3. Flow Cytometry


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Expert in Flow CytometryReseaccher:

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)


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Fourier-Transform (FTIR)Spectroscopy

Microbial cell can be analyzed by using FTIR spectraand 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.


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Sample Preparation for FTIR

The cells from a pure culture are inoculated directlyonto a solid media and incubated (24 hours)

These cells are suspended in water.

After that samples are put into the sample carrier inarray.

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.


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Expert in FTIR Technology inMicrobiology

Mandana Veiseh, Omid Veise, Michael C. Martin, and MiqinZhang, 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


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Future of Microbiology in BacterialDetection

Development of microbiological methods revolutioned

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 ormicroorganisms.

In future , nano technology will apply in the microbiology

field and will improve analytical technique for bacterial

detection.


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Reference

1. Zourob, M., Principle of bacterial Detection. Springer: 2008; p 83.2. Using surface plasmon resonance (SPR). (09/2011)

3. Lowe, C. R., An introduction to the concepts and technology ofbiosensors. Biosensors 1985, 1, (1), 3-16.

4. Moldenhauer, J., proteotypic Identification Method-A Change in

Identification Methods. Microbiology2011, 34-37.5. Andral, J. r.; Bolhling, A.; Gronewold, T. M. A.; Schlecht, U.; Perpeet, M.;

Gutsmann, T., Surface Acoustic Wave Biosensor as a Tool to Study theInteraction of Antimicrobial Peptides with Phospholipid andLipopolysaccharide Model Membranes. Langmuir2008, 24, (16), 9148-9153.

6. 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.

Thank You

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Analytical Techniques for Bacteria Detection