Seminar 1

Presented By : Laleh Mehryar (871101007)

Dear Professor : Dr. Rezazadeh

Department of Food Science, Urmia University 1

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) 2
 The ability to determine the occurrence of food
contamination due to foodborne pathogens at
every stage of food production, processing, and
distribution is crucial to improving the safety of
our food supply.
There are more than 250 known food borne
diseases caused by bacterial and viral infections in
the United States.
Annually, these foodborne diseases result in an
estimated 76 million illnesses, 325,000
hospitalizations, 5,000 deaths, and 6 billions
dollars in unneeded expenditure. Bacterial
contamination accounts for 91% of total
foodborne diseases.
Salmonella sp., Escherichia coli, Listeria
monocytogenes, Staphylococcus aureus,
Campylobacter jejuni, Campylobacter coli
and Bacillus cereus were found to be main source
of bacterial contaminations in our food supply.

3
5
6
⦿ 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).

7
Advantage
s• 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.

8
 ‫یکیژولویب رصانع عاونا‬

‫ه دش صل ا خ ی ا ه می زنآ ‪1.‬‬ ‫⦿‬

‫ریغ و هدنز‬ ‫⦿ ل م ا ک ی ا هل ولس ‪2 .‬‬

‫هدنز‬

‫‪9‬‬
 ‫ه د ش صال خ ی ا ه میزنآ‬

‫⦿ و تیالعف تللعب اهروسنلسویب تخالس رد ه د ش صلالخ ی ا ه ملیزنآ‬

‫دن ری گی م ر ا رق ه د افت س ا در و م ل و ا دت م ر و ط ب الب یص اص ت خ ا ی ا ه زیل انآ ‪.‬‬
‫اهروسنسویبهن ی مز رد ا ه ن آ یاهدربراک ی رادیاپان و ی ن ارگ تلعبهک ن و ر د ه د ش‬
‫هتخانش ی ا ه ملیزنآ ‪ 90%‬زا شلیب‪ .‬دوش یلم د و د ح م ن اونعبل م ا ک ی ا ه ل ولس زا‬
‫هدافتس ا بیت رتنیا هبدنتس ه ی لولس ن یرتهبفلت خم ی تعنص یاهدنیارفرد ی لولس ل خ ا د ی ا ه‬
‫ب م یزاس ص الخ ت میقنارگ و ینلوط دنیارفعنام هک دشابی م ویتانرتآل ی م س ت ابیکرت اب‬ ‫میزنآ عن‬
‫و هد ن ا م ی قاب ل ولس ل خ ا د رد ا ه میزنآ هد ش میزنآ‬
‫دنه دیمن ش نکاو ن یگنس ی اهزلف هل م ج زا )‪.(7‬‬

‫‪10‬‬
 ‫یاهلولس‬
‫لماک‬
‫⦿ ه دنز ری غ م ه و ه دنز مرف ر د مل ه لل م اک ی ا هل وللس‬
‫ت ی م ه ا هدنز یاهلولس ‪.‬دنریگیم رارق هدافتس ا د ر و م‬
‫دنراد اهروسنسویب تخا لس رد یا هل ظ ح ل م ل لباق ار یفلت خ م ‪.‬‬
‫ک لین اگرا ی ا هبیک رت ه دنز ی ا هب و ر کی م ر جن م یز ا و ه ری غ‬
‫ای یز ا و ه تر وص ب ‪ .‬د ن کی م زی ل وب ات م دیس کا ی د ‪،‬‬
‫ک اینومآ هللمج زا ی تلولصحم دلیلوتهب ل دب م زا ه د افتسا اب هک‬
‫د وشی م ‪ ...‬و ا ه دی س ا ‪ ،‬ن ب رک ین ا م ز ه دنز‬
‫ا ر ت س ب وس ه م‬ ‫ی ا ه ل و ل س ‪ .‬د ن و شی م ر وتین و م ف ل ت خ م ی ا ه‬
‫ه ب ذ ج تلیفر ظ هل ک دلن وشی م ه د افتلس ا ت یالعفص لخاش‬
‫ن اونع بملس یناگراورکیم طلسوت ی د ر ا و م رد ‪ .‬دوش هتفرگ رلظن رد‬
‫ی لسفنتی لکیلوباتم‬
‫ی کیژولویبن ژیسکا زاین هبساحم هل م ج زا ‪.‬‬

‫‪11‬‬
 ‫ی ا هتی د و د حم‬
‫ل م ا ک یاهلولس‬

‫⦿ ذوفن ل م ا ک یاهلوللس زا هدافتلسا تلیدودحم نلیرتمهم ‪1.‬‬

‫هب رلجنم هلک اهلوللس هراوید زا تلولصحم و ارتسبوس یاهروسنس الب‬
‫هلسیاقم رد خلساپ ن ا م ز ن د ش یلنلوط‬
‫دوشیم ی میزنآ ‪.‬‬

‫⦿ ندوب نیئاپ ل م ا ک یاهلولس یاهتیدودحم زا رگ ید یک ی ‪2.‬‬

‫ییاهروسنسویب الب هلسیاقم رد الهنآ یصالصتخا درکلم ع اس اسا هک ه د ش‬
‫هدافتسا ه د ش ص ال خ ی ا ه میزنآ زا تس ا رگید طلسوت هتلساوخان ه د ش‬
‫زلیالتاک یاهشنکاو تللع هب‬
‫دش اب ی م ل ولس ر د د و ج و م ی ا ه می زنآ)‪.(7‬‬

‫‪12‬‬
 ‫هدافتسا للکشم نلیا رب هلبلغ یاهشور زا یلکی‬ ‫‪.1‬‬
‫ی ا هل ولس و‬ ‫هک تل ل سا رل لی ذپ ذ وفن‬ ‫ی ا هش ور زا‬
‫ی ا هل ل ح) یللی ای میش ‪ ( ،‬د ا م جن ا) یلل کی زیف نیرت جی‬ ‫( کین اگ ر ا‬
‫ار ‪.‬دوشیم هدافتس ا (نی یاپاپ ای موزوزیل )یمیزنآ‬
‫نئولوت هللم ج زا کلیناگرا ی اه ل ل ح هدافتلسا ش و ر ییایمیش رامیت نی‪،‬‬
‫ا ‪.‬دشابیم لوناتوب و لوناتا ‪،‬مرفورلک ی ز ا س ا د ج هلیلسوب یلکچوک یا هذفنم‬
‫داجیا ثلعاب عیرس ذوفن ثلعاب هلک دوشیم لوللس حطلس زا الهدیپیل ییاشغ‬
‫ک چ و ک یاهذفن م نی ا زا تلوص حم ای ارتسبوس یریگولج یلولس ن و ر د‬
‫ی ا ه ملیزنآ جورخ زا و ه د ش ل ولس گر م ثل ع اب دلنی ا رف‬
‫نلی ا م ا جن ا دلن چ ر ه‪ .‬د وشی م ورد ی ا ه میزنآ زا علبنم کلی‬
‫ناونعب الما دوشیم‬
‫ه د ا س ی ا ه د ر ک ل م ع یارب‪.‬دنکیم لللمع یلولس‬
‫اهروسنسویب‬
‫‪.‬‬ ‫دنریگیم رارق هدافتسا د ر و م‬

‫‪13‬‬
‫ت سا هد ش ی سررب اهشنکاو ن یا ش ه ا ک ی ارب ی یاهشور ‪. 2.‬‬
‫زا ی د ا د ع ت ن دش جر ا خ ث ع اب ل ول ل س یری ذپ ذ وفن‬
‫ل م ع نلیا هلک ه د ش ملک یللوکلوم نز و الب الهروتکافوک ل ول س کی‬
‫ه چ رگ ‪ .‬د ه دی م ش ه اک ار هت س ا و خ ان ی ا هشنک ا و ار زوتکل‬
‫یلول س ل خ ا د زادیزوتکلگاتب ل م ا ش ر م خ م ل م ا ک رد لول س ن ا م ه‬
‫و لوناتا هب ‪ co2‬هکیالح رد دنکیم لیدبت‬
‫زوتکالگ و زکولگ هلب طلقف ار زوتکلریذپذوفن طلیارش رگید یکی ‪.‬دنکیم‬
‫لیدبت اهروتکافوک نتفر تس د زا تلعب یاهشور زا هدافتسا اهمیزنآ ندرک‬
‫العفریغ یاهشور زا هدافتسا ه د ر م یاهلوللس زا هلکینامز هدوب‬
‫ش ل ه اک ر د ر ل گ ی د‬ ‫یلکیزیف ر د هتس ا و خ ان ی ا هشنک ا و‬
‫ش ور ‪ .‬د وش هتس اوخان یکیلوبات م ی ا ه ه ا ر ندرک هکول ب هدنز‬
‫یاهلولس‬
‫ای‬
‫ت سا ال قتنا متسیس ‪.‬‬

‫‪14‬‬
 ‫د ا و م ثبیتت‬
‫ی کیژ ول ویب‬
‫دن وی‬ ‫یی ای میش ی ا ه ش ور‬ ‫⦿‬
‫پ‬ ‫یسنال ووک‬

‫ت ل اصتا‬
‫یض ر ع‬

‫یب ذج‬ ‫ی کی زیف ی ا ه ش ور‬ ‫⦿‬

‫یزادنا هلت‬
‫‪15‬‬
 ‫دن ویپ‬
‫یسن ل ا و وک‬
‫⦿ میزنآ ثبیتت یارب جیار کینکت کی یسنال ووک دنویپ‬
‫لولس ثبیلتت یارب هلک تلسا اله یداب یلتنآ و ا ه نی ا یس اس ا‬
‫ت ل کش م زا ی ک ی ‪ .‬تس ی ن یب س ان م ش ور ی ا ه ه و ر گ‬
‫ض ر ع م رد اهلولس هلک تلسنیا کلینکت شنک ا و ت خس طی ارش و‬
‫ی وق ه دن ه د ش ن ک ا و ل م اع یاهلولس هلب ندیلسر بیلسآ ثلعاب‬
‫هلک هلتفرگ رارق یلول س ن و ر د یاهمیزن آ روطنیمه و ه د ش‬
‫هدنز‬
‫دنکیم ادیپش ه ا ک‪.‬‬

‫‪16‬‬
 ‫یکیزیف یاهشور‬

‫نتخادنا م ا د هلب و ب ذ ج یاهشور‬

‫ه د افت سا ه دنز ی ا هل ول لس زا ه ل کیتقو‬
‫ل و م ع م هار‪.‬دنشابیم دیفم دللنوشیم لدبم ح ط س‬
‫یکیدزن رد اهلولس یرادهگن زیل ای د اش غ لث م‬
‫یی ا ه ا ش غ زا ه د افتس ا ظ ا ح ل زا دیاب اشغ‬
‫یلک تال ح رد ‪.‬دشابیم هدوب رادیاپ یکیناکم و‬
‫یللیایمیش ذف ان م هز ا دنا و‬ ‫نآ ‪μm 15-10‬‬
‫ت م ا خض‬
‫د ش اب‪.‬‬ ‫‪μm 0.1-1‬‬
‫نآ‬

‫‪17‬‬
 ‫یزادنا هلت‬
‫‪ ‬یزتنس یاهرمیلپ‬

‫لکال لینیو یلپ و ناتروا یلپ س ا س ا رب یاهلژوردیه ‪ -‬دیمآ لیرکآ یلپ بیسآ ال متحا تابیکرت نیا‬
‫یاهتیدودحم نیرتمهم زا یکی‪.‬دشابیم‬
‫ت سا هدنز ی اهلولس هبن دیسر ‪.‬‬

‫‪ ‬یعیبط یاهرمیلپ‬

‫اهرمیلپ‬
‫‪-‬‬ ‫ن انیگاراک ‪-‬ت انیژآل ل م ا ش اهلولس ن تخادنا م ا د هبی اربهدافتسا د ر و م ی ارب‬
‫ن یا هک دشابیم ‪ ...‬ن ازوتیک ‪-‬ن یئاپب و ذ هطقن ابزراگآ‬
‫ت‬‫دنتسه دیفم رایسبهدنز ی اهلولس ت ثبی‪.‬‬

‫‪18‬‬
 ‫رد یبورکیم یاهروسنسویب یاهدربراک‬
‫اذغ‬

‫یکیتیالنآ یاهرازبا یارب الضاقت رلیخا یاهاللس رد ⦿ نویساتنامرف و ا ذ غ‬

‫زی النآ یارب یصاص تخا و عیرس ن د ا د ناشن یارب اهزیالنآ ‪.‬تلسا‬
‫هلتفای شلیازفا‬
‫اهیگدوآل ‪ ،‬یی ا ذ غ یاهیندوزفا ‪ ،‬ی ذ غ م یاهرتماراپ و ‪،‬‬
‫یرادهگن ن ا م ز ت د م نییعت و یلبورکیم ش ر ا م ش‬
‫ت سا زایندروم ی یایوبت ایصوصخ رگید ‪.‬‬

‫‪19‬‬
 ‫ریش تیفیک لرتنک‬

‫⦿ تسا یل م ه م رلت م ار اپ کلیریش تلیفیک ن درک لرتنک‬

‫الی نویسادیلسنار ه ات وک بر چ ‪ off- flavour‬تلوصحموریش رد‬
‫ی ا ه دیسا ن دش د ازآ ت لل عب ی ریش‬
‫‪ (C4-‬ری اس ف ل خ رب‪ .‬د وشی م د ا جیا‬ ‫ر ل ی ج نز )‬
‫‪C12‬‬
‫میزنآ یاراد نایتوکین رتکابورترآ ا ه مسیناگراورکیم‬
‫هب تبلسن هلک هدوب نویسادیلسکا تلاب عون زا یلیاه‬
‫دنکیم ل م ع ی صاصتخا ریجنز هاتوک برچ ی ا ه د یس ا روسنسویب ک لی ‪.‬‬
‫م ی خ ض مل‬ ‫ن ا ر ا ک م ه و ت لیمشا شرازگ ربانبزا ه ک‬
‫ی ف ی ژ ول ون ک ت س اس ا رب ی ب و ر کی م ت انیژآل میسلک رد‬
‫ه د ش تثبیلت ن ایتوکین رلتکابورنرآ هد ش هدافتسا ن ژیلسکا دورتکال حطلس‬
‫یور رب ن ییعتی اربهتس ویپان متسیس رد روس نس ن یا ‪ .‬تس ا ر ا رق‬
‫ه د افت س ا د ر و م ر ی ش ر د د ا ز آ ب ر چ دیس ا ن ا ز ی م ی ا ر ب‬
‫یر ا میت شیپ چی ه ش ور نی ا ر د‪ .‬تس ا هت فرگ‬
‫رد ار هجیتن روسنلس و هلتفرگن ماج نا ا له هلنومن ‪3‬‬
‫‪31‬‬
 ‫ا ه هنیمآدیسا نییعت‬

‫⦿ نییعت یارب یلبورکیم یاهروسنلسویب زا یدادعت‬

‫نیزوریت هلللمج زا اهدیللساونیمآ کیماتولگ و)‪phenologens‬‬
‫‪ (Aeromonas‬نافوتپیرت) ‪( Ps.fluorescens‬‬
‫دیس) ‪ (B.subtilis‬ی ا رب نین لآ لی نف نی ی عت‪ .‬در ا د د و ج و‬
‫ا هک تس ا ز این در و م نین لآ لینف ری م خت دن ی ا رف‬
‫ل ر ت نک‬
‫یاهلولس س السا رب یلبورکیم روسنلسویب کی ت انیژلآ میسلک ر د‬
‫ه دش ت ی ثبت س ی ر ا گل و و س وئت و رپ لینف نلییعت روظن م هلب‬
‫نژیلسکا دورتکال یور رب د و ج و م زانیمآ د نینآل لینف‪.‬تلسا ه د ش‬
‫هلیهت نلینآل دیسا کیتلسا لینف هلب ار نلینآل لینف لوللس رد‬
‫دنکیم دیسکا‪.‬‬

‫‪21‬‬
 ‫اهنیماتیو نییعت‬

‫⦿ زا اهنیماتیو نلییعت یارب یلبورکیم یاهروسنلسویب‬

‫و‬ ‫دی س ا‬ ‫‪(B-12‬‬ ‫‪(E.coli L15‬‬ ‫هلل لل مج‬
‫‪ B-‬کیبروکللسآ کی)‪ ( Enterobacteragylomerans‬هعسوت‬
‫نلیماتیو عیرلس صلیخشت یارب ‪.‬تلسا هلتفای کی یلسفنت تلیالعف سالسا ‪6‬‬
‫رب‬ ‫متسیس‬ ‫‪ Saccharomyces‬رب روس نلسویب‬
‫ه د ش بیتثت رلمخ م عیرس ص یخشت هک ‪.‬دراد ) )‪uvarum‬‬
‫د و ج و نژیسکا دورتکال یور د ح ر د یی ایر د ت ل وص ح م ر د‬
‫نی م اتی و ه د اس و ‪B-6‬‬
‫‪ ng/ml‬ت سا هتخاس ریذپناکما ار ‪.‬‬

‫‪22‬‬
 ‫یبورکیم یاهروسنسویب یاهدربراک‬
‫طیحم رد‬

‫نل ی ر ت ش ی ب‬ ‫⦿ هنی مز ر د یلب ور کی م ر وسنلس ویب د ربر اک‬

‫یاهروسنلس تلیزم ‪.‬تلسا یلطیحم هدوب ‪ BOD‬شنکاو‬
‫اهدورتکال اب سا م ت رد اهمسیناگراورکیم یالب یریگ هزادنا‬
‫ار ه د ش فرصم نژیلسکا تبلسن هک رد هک دراد زور ‪5‬‬
‫شجنس ‪.‬دننکیم ‪ BOD‬هب جایتحا‬
‫روسنسویبس ا لسا ربهد ش ماجنا ی اهزیالنآ ا هر ای ع م نی ‪15‬‬
‫رت م ه م زا یک ی ‪ .‬تس ا مز ل ن ا مز هق یق د دنناوتبدلیابا لهنآ هلک‬
‫ت لسنیا ا لهبورکیم ب اختنا رد فرصمار اهارتسبوللس زا‬
‫ی عیللسو هللنماد دعاسمان ل لماوع هلب ت بلسن دلیاب‬
‫اهروسنلسویبدننک‬
‫‪.‬‬
‫دنشاب مو ا ق م ی روش و ت یمس هل م ج زا ی طیحم ‪.‬‬

‫‪23‬‬
Biosnsors for detection
of:

L. monocytogenes

24
Salmonella

⦿ 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).

25
26
Salmonella

⦿ 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).

27
Salmonella

⦿ 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).

28
Magnetoelastic biosensor for the detection of
Salmonella typhimurium in food products

29
Salmonella(2
)
⦿ Existing biosensor technologies such as surface
wave devices, optical and thickness-shear mode
acoustic
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
sensors 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).

30
Salmonella

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).
31
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).

32
33
⦿ 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).

34
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).

46
E. Coli (2)

The Escherichia coli O157:H7 DNA detection on a

gold nanoparticle-enhanced piezoelectric
biosensor

36
 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).

37
⦿ 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.

38
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.

39
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).

51
41
E.
coli

D etection of E .coli
in foods
⦿ One of the many applications of surface plasmon
resonance (SPR) technology is the detection of E.
coli O 157: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).
42
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.

43
E.
coli

44
L . monocytogenes

⦿ L. monocytogenes causes listeriosis, and has been detected in a

variety of environments, including foods. Listeriosis occurs around
the world, with an annual incidence of approximately 7 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
process. Several alternative detection methods have been
developed for the rapid and sensitive detection of L.
monocytogenes , including ELISA, PCR, DNA microarray, and
biosensor methods(4).

45
 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.

57
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.

47
⦿ 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).

48
References
□ Maria N. Velasco-Garcia; Toby Mottram (2003). Biosensor Technology
addressing Agricultural Problems. Biosystems Engineering , 84 (1), 1–12
e 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
0 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.
o 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.
d 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.
i 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.
B D’Souza, S.F.(2001). Microbial biosensors. Biosensors & Bioelectronics 16, 337–
353.

49
Any
Questions???

50
The End
Thanks

51