1

Presented By : Laleh Mehryar (871101007)

Dear Professor : Dr. Rezazadeh

Department of Food Science, Urmia UniversityDepartment of Food Science, Urmia University

Seminar 1

2

Father of the Biosensor

Professor Leland C Clark Jnr 1918–2005

3

Review

4

What is a Biosensor?

in : Biosensor (www.google.com)

International Union of Pure and Applied Chemistry (IUPAC) defines biosensor as a “device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals”.

5

INTRODUCTION

What Is a Biosensor?

Biosensor = bioreceptor + transducer

The bioreceptor is a biomolecule that recognizes the target analyte whereas the transducer converts the recognition event into a measurable signal.

Enzyme is a Bioreceptor

in : Biosensors (www.egtoget.com)

6

Block Diagram of a Biosensor

Sample (Analyte

or Substrate)

BiorecognitionElement

Transducer

Signal Processing

Device

Olfactory Olfactory MembraneMembrane

Olfactory Olfactory Nerve CellNerve Cell

Introduction to Biophotonics – Prasad - John Wiley & Sons © 2003

odor

BrainBrain

7

Block Diagram of a Biosensor

a) Biocatalyst

b) Transducer

c) Amplifier

d) Processor

e) Monitor

8

•SpecificityWith biosensors, it is possible to measure specific analytes with great accuracy.

•Speedanalyte tracers or catalytic products can be directly and instantaneously measured

• Simplicity receptor and transducer are integrated into one single sensor& the measurement of target analytes without using reagents is possible

• Continuous monitoring capabilityBiosensors regenerate and reuse the immobilized biological recognition element

in : Biosensors (www.egtoget.com)

9

Applications of Biosensor

Health Care Measurement of Metabolites Market Potential Diabetes Insulin Therapy Artificial Pancreas

Industrial Process Control Bioreactor Control

Military Application

Environmental Monitoring Air and Water Monitoring

in : Biosensors (www.egtoget.com)

10

1 2

3 4

5 6

7 8

9

analyte

BMSignal transducer

Affinity reaction

How it works ???

11

The ability to determine the occurrence of food contaminationdue to foodborne pathogens at every stage

of food production, processing, and distribution iscrucial to improving the safety of our food supply.

There are more than 250 known food borne diseasescaused by bacterial and viral infections in the United

States. Annually, these foodborne diseases result in anestimated 76 million illnesses, 325,000 hospitalizations,

5,000 deaths, and 6 billions dollars in unneededexpenditure. Bacterial contamination accounts for 91%

of total foodborne diseases. Salmonella sp.,Escherichia coli, Listeria monocytogenes, Staphylococcus

aureus, Campylobacter jejuni, Campylobacter coliand Bacillus cereus were found to be main source of

bacterial contaminations in our food supply.

12

13

14

15

16

17

18

A microbial biosensor consists of a transducer in conjunction with immobilised viable or non-viable microbial cells. Non-viable cells obtained after permeabilisation or whole cells containing periplasmic enzymes have mostly been used as an economical substitute for enzymes. Viable cells make use of the respiratory and metabolic functions of the cell, the analyte to be monitored being either a substrate or an inhibitor of these processes. Bioluminescence-based microbial biosensors have also been developed using genetically engineered microorganisms constructed by fusing the lux gene with an inducible gene promoter for toxicity and bioavailability testing (7).

19

Advantages• able to metabolise a wide range of chemical compounds• have a great capacity to adapt to adverse conditions and to develop

the ability to degrade new molecules with time.• are also amenable for genetic modifications through mutation or

through recombinant DNA technology and serve as an economical source of intracellular enzymes.

20

عناصر انواعبیولوژیکی

1 آنزیم های خالص شده .

2 سلولهای کامل زنده و غیر .زنده

21

آنزیم های خالص شده

الیت وMورها بعلت فعMاخت بیوسنسMده در سMالص شMای خMزیم هMآنقMرار اسMتفاده مMورد متMداول بطMور بMاال اختصاصMی آنالیزهMای

کاربردهMای آنهMا در زمینMه ناپایMداری و گMرانیمیگیرنMد. کMه بعلت % آنMزیم هMای شMناخته 90بیش از بیوسنسMورها محMدود می شMود.

شMده درون سMلولی هسMتند بMه این تMرتیب اسMتفاده از سMلول هMای فراینMدهای در سMلولی داخMل هMای آنMزیم منبMع بعنMوان کامMل فراینMد مMانع کMه باشMد می آلترنMاتیو بهMترین مختلMف صMنعتی طMوالنی و گMرانقیمت خMالص سMازی آنMزیم شMده آنMزیم هMا در داخMل سMلول بMاقی مانMده و بMا ترکیبMات سMمی از جملMه فلزهMای سMنگین

(. 7واکنش نمیدهند )

22

سلولهای کامل

دهMده و هم غیرزنMرم زنMل هم در فMسلولهای کاممیگیرنMد. قMرار اسMتفاده زنMده مMورد سMلولهای

اهمMیت قابMلM مالحظMه اMی در سMاخت MبیوسنسMورهMا M.دMMدارنMM M کMMارگانی MایMMیبهMترک MدهMMزن میکروMبهMMاMی

یMا متMابMولیMز MمMیکMننMد. MبصMوMرتM MهMواMزMی را MیMتلفMمخاز MجMملMه MیMوالتMحصMم بMه MتولیMد مMنجMر غMیرMهواMزMی اMکMسMید کMربنM،M اسMیMدMهMا وM.M.M M. میMشMود آMموMنیMاMکM،M دی هMایM MمMختMلMف MمونMیMتMور MلMدMمب MزMاM MادهMفMتMسMاM MاMبM MهMکمیMشMونMد.M MسMلوMلهMای MزMنMدMه زMمMانMیM MاسMتMفMاده مMیشMوند M MطMMسMتوMM راMتMMبسMسوMM MهMMهم جMMذMب MرفیتMMظMM MهMMک

مMیMکرMوارMگاMنیMسMم MبعنMوان MشMاخصM MفMعMالMیت MمMتMابMولMیکی تMنMفسMی دMرM نظMر گMرفتMه شMوMد.در MمMوارMدMی Mاز جMملMه

محاسبه نیاز اکسیژن بیولوژیکی .

23

محدودیتهای سلولهای کامل

1ترینMمهم کامMل محMدودیت . از سMلولهای اسMتفاده کMه سMلولها دیMواره از محصMوالت و سوبسMترا نفMوذ بMا مقایسMه در پاسMخ زمMان بMه طMوالنی شMدن منجMر

سنسورهای آنزیمی میشود.

2 ائینMپ کامMل از محMدودیتهای سMلولهای دیگMر یکی .بMMا مقایسMMه در آنهMMا اختصاصMMی عملکMMرد بMMودن شMده خMالص هMای آنMزیم از اسMت بیوسنسMورهایی اسMتفاده شMده کMه اساسMا بMه علت واکنشMهای کاتMالیز در موجMود هMای آنMزیم دیگMر توسMط ناخواسMته شMده

(.7سلول میباشد)

24

یکی از روشMهای غلبMه بMر این مشMکل اسMتفاده از . 1روشMMهای از کMMه اسMMت نفوذپMMذیر سMMلولهای و ارگانیMک( شMیمیایی)حاللهMای فMیزیکی)انجمMاد(، آنMزیمی)لMیزوزوم یMا پاپMایین( اسMتفاده میشMود. رایج جملMه از ارگانیMک حاللهMای اسMتفاده روش تMرین این بوتMانول میباشMد. اتMانول و تولMوئن، کلروفMرم، کMوچکی منفMذهای ایجMاد بMاعث شMیمیایی تیمMار اMز سMطح سMلول MمیشMود لیپیMدMها بوسMیMله جداسMاMزی کMهM بMاعMثM نفMوMذ سMریع سMوبسMترMا یMا MمحصMوالت ازM این آنMزیم MروجMخ Mاز و شMدMه غشMایی MکMکوچ MذهایMمنفM امMانج MدMر چنMود.هMشMمی MیریMوگMجلM لولیMون سMای درMه

MکMان یMوMنMبع MاMمMد اMوMشMمی MولMلMرگ سMعث مMاMب MدMنMایMرMین فMامنMبMع MازM آنMزیم هMاMی دMرون MسMلوMلی MعمMلM میکنMد.برای عملکردهMای سMاده بیوسنسMورها مMورد اسMتفاده قMرار

میگیرند.

25

روشMهایی بMرای کMاهش این واکنشMها بررسMی شMده . 2اسMت. نفوذپMذیری سMلول بMاعث خMارج شMدن تعMدادی از کوفاکتورهMا بMا وزن مولکMولی کم شMده کMه این عمMل یMک گرچMه میدهMد. کMاهش را ناخواسMته واکنشMهای داخMل بتاگالکتوزیMداز شMامل مخمMر کامMل سMلول

اتMانول و تبMدیل میکنMد در co2سMلولی الکتMوز را بMه را نفوذپMذیرالکتوز در شMرایط حالیکMه همMان سMلول رفتن دسMت از بعلت گMاالکتوز و گلMوکز بMه فقMط روشMهای از دیگMر یکی میکنMد. تبMدیل کوفاکتورهMا روشMMهای از اسMMتفاده آنزیمهMMا کMMردن غیرفعMMال اسMتفاده مMرده از سMلولهای زمانیکMه بMوده فMیزیکی شMود.روش دیگMر در کMاهش واکنشMهای ناخواسMته در

زنMده متMابولیکی سMلولهای راههMای کMردن بلوکMه است.سیستم انتقال یا ناخواسته

26

تثبیت مواد بیولوژیکی

روشهای شیمیایی پیوندکوواالنسی

اتصاالت عرضی

روشهای فیزیکی جذبی

تله اندازی

27

پیوند کوواالنسی

بیتMتث بMرای رایج تکنیMک یMک کوواالنسMی پیونMد آنMزیم هMا و آنMتی بMادی هMا اسMت کMه بMرای تثMبیت از مشMکالت سMلول روش مناسMبی نیسMت. یکی اساسMی این تکنیMک اینسMت کMه سMلولها در معMرض گروههMای عامMل واکنش دهنMده قMوی و شMرایط آسMیب بMاعث کMه گرفتMه قMرار واکنش سMخت همینطMور و شMده زنMده سMلولهای بMه رسMیدن

آنزیمهای درون سلولی کاهش پیدا میکند.

28

روشهای فیزیکی

روشMهای جMذب و بMه دام انMداختن وقتیکMه اMز سMلMولهایM MزنMدهM اسMتفMاده MمMیشMوند MمفیMد میبMاشMند.راMه معمMولM نگهMداری سMلولها در Mاز اسMMتMفاده مبMMدل سMMطMح نMMزدMیکی در M.دMشMاMمیب دیMالMیز غشMا مثMل MیMاهایMغشحMاMلت کلMی غشMاM بMایMد MاMز لحMاظ شMیمMیایی و

μm 15-10مکMاMنیکی MپاMیMداMرM بMوده ضMخامتM آن باشد. M μm 0.1-1 و انMدازMه Mمنافذ آMن

29

تله اندازیپلیمرهای سنتزی

پلی آکریل آمید-هیدروژلهای بر اساس پلی اورتان و پلی وینیل الکل میباشد.یکی از مهمترین محدودیتهای این ترکیبات احتمال

آسیب رسیدن به سلولهای زنده است.

پلیمرهای طبیعی

مورد استفاده برای به دام انداختن سلولها شامل آلژینات- کاراگینان-آگارز با نقطه ذوب پائین-کیتوزان ... میباشد که این

پلیمرها برای تثبیت سلولهای زنده بسیار مفید هستند.

30

کاربردهای بیوسنسورهای میکروبی در غذا

ریع وMالیتیکی سMای آنMرای ابزارهMا بMیر تقاضMالهای اخMدر ساMختصاصMی بMرای MآنMاMلیز غMذMا و فرMمانتاسMیون افMزایش یافتMه

پارامترهMای MمغMذی دادMن MانMنش آناMلیزهMا MبMرای ، اسMت. آلودMگیهMا M، غMذایی و افMزودنیهMای شMمارش MمیکMروبی ،

بویMایی MیاتMر خصوصMدیگ و زمMان MنگهMداMری مMدت تMعMیین .Mاز استMوردنیMم

31

کنترل کیفیت شیر

تMاس مهمی پMارامتر شMیریک کیفیت کMردن کنMترل در شیرومحصMوالت شMیری off- flavourراMنسیداسMیون MیMا

( C4- C12بعلت آزMاد شMدنM اسMیدهای چMرب کوMتMاه زنجMیر) هMMا میکروارگانیسMMم سMMایر میشMMود.بر MMخالف ایMجMMاد بتMا نMوع از هMاMیی MزیمMآن دارای نیکوMتیMان آرتMروبMاکتر اکسیداMسMیون بMوده کMه نسMبت بMه اMسMیدهاMی چMرب کوMتMاه زنMجMیر اختصاMصMی عمMل میکMنMد. MبنMابMر گMزMارش اشMمیت و هMمکMارانM یMک بیMوسنسMور میMکMروبMی بMر اMسMاس تMکنولMوژی Mفیلم MضMخMیم MکMه ازM آرنروMبMاکتر نیMکوتMیMان تMثMبیت MشMده در اکسMیژMن MترودMالک روی MسMطح بMر MاتMینMآلژ کلسMیم اسMتفاده شMده اسMت.این MسنسMورM در سMیسMتم ناپیMوسMته مMورد شMیMر در Mآزاد اسMید MچMرب MیزانMم بMرای MتعMییMن پیش هیچ Mروش این اسMت.دMر گرMفتMه قMرار اMسMتفادMه تMیمMاری MبMرای MنمونMه هMا انMجMام نگرفتMه وM سنسMور نتیجMه

دMقیقه نشاMن میMدهد.3راM در

32

تعیین اسیدآمینه ها

یینMرای تعMروبی بMورهای میکMدادی از بیوسنسMتعتیروزین) جمله از phenologensآمینواسیدها

Aeromonas (انMMMتریپتوف )Ps.fluorescens و ) اسMید) دارد.تعMیین B.subtilisگلوتامیMک وجMود )

فنیMل تخمMیر فراینMد کنMترل بMرای آالنین فنیMل بیوسنسMور یMک کMه اسMت نیMاز مMورد آالنین پروتئMMوس سMMلولهای اسMMاس بMMر میکMMروبی بMر آلژینMات کلسMیم در شMده تثMبیت وولگMاریس فنیMل تعMیین منظMور بMه اکسMیژن الکMترود روی دآمینMاز آالنین اسMت.فنیل شMده تهیMه آالنین موجMود در سMلول فنیMل آالنین را بMه فنیMل اسMتیک

اسید اکسید میکند.

33

تعیین ویتامینها

اMیین ویتامینهMرای تعMروبی بMورهای میکMبیوسنسجملMMMه اسMMMید B-12 (E.coli L15)از و توسMMعه Enterobacter agylomeransآسMMکوربیک) )

ویتMامین سMریع تشMخیص اسMت.برای B-6یافتMه فعMالیت اسMاس بMر بیوسنسMور سیسMتم یMک

Saccharomyces)تنفسی یک مخمر تثبیت شده)uvarum هMود دارد.کMیژن وجMترود اکسMر روی الکMب

ویتMMامین سMMاده و سMMریع در B-6تشMMخیص حMد در دریMایی امکانپMذیر ng/mlمحصMوالت را

ساخته است.

34

کاربردهای بیوسنسورهای میکروبی در محیط

هMروبی در زمینMور میکMاربرد بیوسنسMترین کMبیشسنسMMورهای مMMزیت اسMMت. BODمحیطی

بMا تمMاس در میکروارگانیسMمها بMاالی واکنش الکترودهMا بMوده کMه نسMبت اکسMیژن مصMرف شMده

احتیMاج بMه BOD را انMدازه گMیری میکننMد. سMنجش بMر 5 شMده انجMام آنالیزهMای در کMه دارد روز

دقیقMه زمMان الزم اسMت. 15اسMاس بیوسنسMور انتخMاب میکروبهMا از مهمMترین معیارهMا در یکی از وسMیعی دامنMه بتواننMد بایMد آنهMا کMه اینسMت بایMد کنند.بیوسنسMورها رامصMرف سوبسMتراها جملMه از محیطی نامسMاعد عوامMل بMه نسMبت

.سمیت و شوری مقاوم باشند

35

Biosnsors for Biosnsors for detection of:detection of:

L. monocytogen

es

36

Ye et al. (1997) described a piezoelectric biosensor for detection of Salmonella typhimurium. The device consists of a quartz crystal wafer sandwiched between two metal electrodes. These electrodes provide a means of connecting the device to an external oscillator circuit that drives the quartz crystal at its resonant frequency. A change in mass on the surface of the electrode thus changes the resonant frequency of the quartz crystal microbalance (QCM) device (Following Fig.)(1).

Salmonella

37

38

The antibody against Salmonella was immobilised onto the gold electrode-coated quartz crystal surface through a polyethylenimine–glutaraldehyde technique. The Salmonella cells reacted specifically and bound to the crystal surface resulting in an increase in mass that was directly related to the concentration in the solution.

The biosensor responded to concentrations of S.typhimurium in the range of 5.3*105 to 1.2*109 CFUml-1 in 25 min (1).

Salmonella

39

Another biosensor technique for Salmonella detection developed by Seo et al. (1999) was a direct method, in which Salmonella binding to specific antibodies attached to a waveguide surface were detected in minutes by measuring interferometrically the alteration in phase velocity of a guided optical wave (1).

Salmonella

40

Magnetoelastic biosensor for the detection of Salmonella typhimurium in food products

41

Existing biosensor technologies such as surface acoustic wave devices, optical and thickness-shear mode resonators offer sensitivity, but are limited by their need for direct physical contacts. For example quartz crystal microbalance (QCM) needs direct physical contacts (i.e. electrical wiring) to obtain sensor response which prohibit their usage in the sealed containers. Magnetoelastic Magnetoelastic sensorssensors don’t need direct electrical contacts for the actuation and data acquisition; this makes magnetoelastic sensors suitable for the remote detection of pathogens. The current investigation mainly focuses on the development of a biosensor that addresses these limitations through remote and real-time sensing of pathogens in food products. The Langmuir Blodgett (LB) technique was used for antibody immobilization on the magnetoelastic sensors(2).

Salmonella(2)

42

Fig. Schematic drawing illustrating the wireless nature of the magnetoelastic biosensors and the basic principle for detecting bacterial cells. The fundamental resonant frequency of the biosensor is f0 without antigen binding, which shifts (decreases) to fanalyte due to the increased mass of antigen binding to antibody immobilized on the sensor surface(2).

Salmonella

43

Bacterial binding measurements

After the preparation of biosensors, they were exposed to increasing concentrations (5 * 101 cfu/ml–5 * 108 cfu/ml) of S. typhimurium spiked in different media (distilled water, fat-free milk and apple juice) at a flow rate of 100 µl/min. A peristaltic pump (Ismatec Reglo Digital peristaltic pump) was used to control the flow rate of the media containing the pathogens. The resonance frequency of the sensors was measured using an HP network analyzer 8751A with S-parameter test set both before and after the binding of bacterial cells to the immobilized antibody on the sensor. About of 801 points were recorded over the frequency range with an 11.31 s sweep time. A standard open circuit calibration was used to minimize experimental errors in the test set up. A personal computer was used to acquire data at 2 min intervals. Each data point (Fig. 3) represents the mean values obtained from five independent sensors, subjected to study under identical conditions(2).

44

45

When the test temperature and humidity are constant, the resonant frequency change of the magnetoelastic sensor depends only on the mass change (Δm) on its surface. If the mass increase is small compared to the mass of the sensor the shift in the resonant frequency is given by:

where f is the initial resonance frequency, M is the initial mass, Δm is the mass change, and Δf is the shift in the resonant frequency of the sensor. Equation shows that the resonance frequency shifts linearly, decreasing with increasing mass on the sensor surface. Hence binding of the target organism onto the biosensor surface causes a mass increase with a corresponding decrease in fundamental resonance frequency(2).

46

Sensor surface bacterial densities of 0.105, 0.075 and 0.105 cells/µm2 were observed on the samples which were exposed to S. typhimurium suspensions in water, fat-free milk, and apple juice respectively at the highest bacterial concentrations(2).

47

The Escherichia coli O157:H7 DNA detection on a gold nanoparticle-enhanced piezoelectric biosensor

E. Coli (2)

48

E. coli O157:H7 is most well known to both microbiologists and general public. This organism was first recognized in 1982, and has the ability to cause life threatening complications hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans(6). Illness due to E. coli O157:H7 infection is often misdiagnosed and generally needs invasive and expensive medical tests before it is correctly diagnosed. Primary sources for exposure to E. coli O157:H7 are consumption of ground beef, sprouts lettuce, salami, unpasteurized milk, and juice contaminated by pathogens. Since the loss caused by E. coli O157:H7 is enormous in terms of medical cost and product recall, it is extremely urgent to develop some rapid and sensitive methods to detect the bacteria in food or water supplies(6).

49

As to the selectivity, DNA is an ideal target for specific detection of pathogenic bacteria. The DNA probe, which is an oligonucleotide sequence immobilized on a fixed support and able to hybridize the complementary strand present in solution, is a powerful molecular tool for monitoring and detecting specific microorganisms in the environment or in food. It is well known that QCM is a very sensitive mass-measuring sensor, and the crystal resonance frequency decreases with a mass increase on the Au electrode surface.

50

Nanoparticles are a new class of materials, which has been adopted for improving the detection limit and sensitivity of the DNA biosensors. In order to form a more effective and sensitive system, we adopted the gold nanoparticles in the construction of E. coli O157: H7 DNA biosensor for the goal of signal amplification, because (i) colloidal Au has a good biocompatibility; (ii) gold nanoparticle has a larger surface area, and helps to immobilize more biofunctional molecules onto the surface of the sensor; (iii) avidin-conjugated Au nanoparticle could be used as “mass enhancer” to amplify the frequency change depending on its relatively large mass compared to DNA target.

51

The whole detection course of this QCM DNA sensor mainly included two parts: sensor fabrication and detection. During the process of sensor fabrication, how to immobilize more ssDNA probes was pivotal for the following bacteria DNA detection, so two important routes for DNA probes immobilization were introduced to this part, which were:• Self-assembled monolayers (SAMs).• Au-thiol binding.A stepwise decrease of Δf was observed in each fabrication step(6).

52

53

E. coli

Detection of E.coliE.coli in foods

One of the many applications of surface plasmon resonance )SPR( technology is the detection of E. coli O157:H7. Surface plasmon resonance is a quantum optical-electrical phenomenon based on the fact that energy carried by photons of light can be transferred to electrons in a metal. The wavelength of light at which coupling occurs is characteristic of the particular metal and the environment of the metal surface illuminated. This transfer can be observed by measuring the amount of light reflected by the metal surface )1, in, 2(.

54

The following Fig. shows how a change in the chemical environment 100 nm above the thin metal layer results in a shift in the wavelength of light, which is absorbed rather than reflected. The most common practical implementation of SPR is to use a metal-coated optical prism, but other practical implementations have been demonstrated including metal-coated diffraction gratings, optical fibres and planar waveguides. The SPR biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with different antisera or other molecular species (Fratamico et al., 1998). The lower detection limit of the BIAcore system (a commercial example of an SPR system) is approximately 10 pg of analyte mm-

2.

55

E. coli

56

L. L. monocytogenemonocytogeness L. monocytogenes causes listeriosislisteriosis, and has been detected in a

variety of environments, including foods. Listeriosis occurs around the world, with an annual incidence of approximately 77 cases per million of the normal population. Its incidence tends to be higher in pregnant women, the elderly, and people with weakened or suppressed immune systems. The traditional culture – based technique for the detection of L. monocytogenes requires 5 ~10 days for complete identification, and is a fairly labor intensive labor intensive process. Several alternative detection methods have been developed for the rapid and sensitive detection of L. monocytogenes , including ELISA, PCR, DNA microarray, and biosensorbiosensor methods(4).

57

SPR biosensor technology is one of the most promising detection methods currently in use for rapid pathogenic identification. It is sensitive to changes in the thickness or refractive indices of biomaterials at the interface between a thin gold film and an ambient medium, & is thus capable of characterizing biomolecular interactions in real time without the need for labeling.

58

As SPR sensors are capable of detecting changes in a refractive index of several hundred nanometers over the sensor surface, bacterial cell particles specifically bound to an antibody can induce a large SPR angle shift, as compared with biomolecules covering an identical surface area(4). The direct detection of microbial cells is often difficult, owing principally to their large size. In order to assay whole L.monocytogenes cells with an SPR biosensor, it is first necessary to find a high-affinity antibody [monoclonal anti- L.monocytogenes mouse antibody] for the cell and to optimize the detection surface.

In this study, a specific monoclonal antibody against L.monocytogenes was screened using an SPR biosensor. Monoclonal antibodies were bound to protein L, after which the L.monocytogenes cells were subjected to an affinity assay. Protein L was immoblized on a carboxymethyl dextran surface via an amine coupling method, and utilized repeatedly by regeneration. The monoclonal antibody, ‘A 18’, was selected and employed for the high sensitivity detection of L.monocytogenes. specific antibodies were screened via direct binding to cells using an SPR biosensor. It was determined that the screened antibody evidenced a high degree of sensitivity for the direct detection of L.monocytogenes.

59

In a study that was done in year 2007 by Joung Hyou- Arm et al., they selected a specific monoclonal antibody (mouse IgG1) against L. monocytogenes , and also attempted to detect it directly. The direct detection of microbial cells is often difficult, owing to principally large size. In order to assay whole L. monocytogenes cells with a SPR biosensor, it is first necessary to find a high- affinity antibody for the cell & to optimize the detection surface (4).

60

References1. Maria N. Velasco-Garcia; Toby Mottram (2003). Biosensor Technology

addressing Agricultural Problems. Biosystems Engineering , 84 (1), 1–12

2. Guntupalli, R., Lakshmanan, Ramji S., Johnson, Michael L., Hu, J., Huang, Tung-Shi., Barbaree, James M., Vodyanoy, Vitaly J., Chin, Bryan A. (2007). Magnetoelastic biosensor for the detection of Salmonella typhimurium in food products. Sens. & Instrumen. Food Qual. 1:3–10 DOI 10.1007/s11694-006-9003-8

3. SKLADAL, P., SYMERSKA, Y., POHANKA, M., SAFAR, B., MACELA, A. (2005). ELECTROCHEMICAL IMMUNOSENSOR FOR DETECTION OF FRANCISELLA TULARENSIS. Detection Technologies, Implementation Strategies and Commercial Opportunities, 221–232.

4. Joung, H., et al. (2007). Screening of a Specific Monoclonal Antibody against and Detection of Listeria monocytogenes Whole Cells Using a Surface Plasmon Resonance Biosensor. Biotechnology and Bioprocess Engineering, 12 : 80-85.

5. Nayak, M., Kotian, A., Marathe, S., Chakravortty, D. (2009). Detection of microorganisms using biosensors—A smarter way towards detection techniques. Biosensors and Bioelectronics 25, 661–667.

6. LiJiang, W., QingShan, W., ChunSheng, W., ZhaoYing, H., Jian, J. & Ping, W. (2008). The Escherichia coli O157:H7 DNA detection on a gold nanoparticle-enhanced piezoelectric biosensor. Chinese Science Bulletin, vol. 53 | no. 8 | 1175-1184.

7. D’Souza, S.F.(2001). Microbial biosensors. Biosensors & Bioelectronics 16, 337–353.

61

Any Questions???

62

The EndThe End Thanks Thanks