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Enzyme Based Biosensors

Prepared by 1.Pritam Singh 2.Raja Chaudhary 3.Munish Kumar #.!urpreet Singh &.Jitendra Kumar *.'sho( Kumar -.Rohit !upta 8.Shubham Rathore 9.Manish Joshi 1 .!opa" Singh Rana 11.Ra$i %ada$ 12.'ni"(umar )iddi 13. +idhan So,

Table of contents
Introduction Why enzyme based biosensors? Working principle of enzyme based biosensor Components of biosensor - Basic parameters of biosensor - Main components of biosensor Types of enzyme based biosensors E amples of enzyme based biosensors !arameters of biosensor performance "pplications of biosensor "d#antages and disad#antages Challenges $uture prospects Conclusion %eferences

$ather of the Biosensor

!rofessor &eland C Clark 'nr ()(*-+,,-

Introduction

"n enzyme based biosensor is a self- contained Integrated de#ice that is capable of pro#iding specific .uantitati#e or semi- .uantitati#e analytical information using an enzyme/ a biological recognition element 0hich is in direct spatial contact 0ith a transducer element1

In shortbiosensor Biological response %esponse 2an analytical de#ice3 Electrical

Why enzyme based biosensors?


Enzymes ha#e a high selecti#ity for their substrates1 Enzymes can operate at moderate potentials because dri#ing force or potential that is needed to achie#e the enzymatic bio-catalysis is often #ery close to that of the substrate of the enzyme1 Impro#ement in enzyme stability 0hen enzymes are immobilized on transducer surface1

Working principle of biosensor


Working principle of a biosensor in#ol#ed in three elements 0hich are follo0ing $irst biological recognition element 0hich highly specific to0ards the biological material analytes produce1 'na"yte4- It is a chemical constituent or substance that is the sub5ect of an analytical analysis1 6econd transducer detects and transduces signal from biological target receptor molecule to electrical signal 0hich is due to reaction occur1 Third after transduction signal from biological to electrical/ 0here its amplification is necessary takes place and read out in detector1 "fter processing the #alues are displayed for monitoring and controlling the system1

Components of biosensor
+asi. parameters o/ biosensor
The analyte 2What 0e 0ant to detect3 6ample handling 27o0 to deli#er the analyte to the sensiti#e region3 8etection9recognition 27o0 do 0e specifically recognize the analyte3 6ignal 27o0 do 0e kno0 there 0as a detection3

Main .omponents o/ biosensor


- The biocatalyst 2a3 0hich con#erts substrate to product1 - This reaction is determined by the transducer 2b3 0hich con#erts it to - The output from the transducer is amplified by amplifier 2c3 if re.uired1 - !rocessed by processor 2d3 - 8isplayed on display 2e3
an electrical signal1

Types of enzyme based biosensors


1. 0"e.tro.hemi.a" biosensor
Chemical reaction !roduction or consumption of ions or electrons Electrical properties change changes are sensed out and used as measuring parameter There are three basic types of electro chemical biosensor1 . 'mperometri. biosensor Measures4 Electric current .Based on o idase enzymes that generate hydrogen pero ide and consume o ygen1
"nalyte

"nalyte : ; ygen : 0ater


; idase

7ydrogen pero ide : o idized analyte

$ormation of hydrogen pero ide can be detected by the help of !telectrode1 E ample4 <lucose biosensor Measures- <lucose content <lucose mediator : surrounding o ygen 2<;83
Catalase

7ydrogen pero ide : <lucose o idase

7ydrogen pero ide


<;8

; ygen : Water

<lucose

<luconic acid : + electrons : + protons

<lucose content can be detected by !t-electrode1

Potentiometri. biosensor Measures- =oltage Change in distribution of charge is detected using ion-selecti#e electrode/ such as p7-meter1

These biosensors are intended primarily for monitoring le#els of carbon dio ide/ ammonia and other gases dissol#ed in blood > other li.uids using enzyme as biological recognition element1

2. Condu.tometri. biosensor
Measures- conducti#ity change in the solution1 Conducti#ity changes due to the production or consumption of ions1

3. Ca"orimetri. biosensor
Measures- %esistance difference If the enzyme catalyzed reaction is e othermic/ t0o thermistors may be used to measure the difference in resistance bet0een reactant and products and hence the analyte concentration1

#. 1pti.a" biosensor
Co"orimetri. /or .o"or Measures4 Change in light adsorption1 Photometri. /or "ight intensity !hoton output for a luminescent or fluorescent process can be detected 0ith photomultipliers tube or photodiode system1

&. 0"e.tro.hemi.a" )2' hybridi3ation biosensor


$ormation of 8?" recognition layer1 "ctual hybridization e#ent1 Transformation of hybridization e#ent into an electrical signal1 This type of biosensor is moti#ated by the application to clinical diagnosis and genome mutation detection1 These sensor are a#ailable in form of electrodes/ chips and crystals1

E amples of enzyme based biosensors

So"4ge"4immobi"i3ed urease Condu.tometri. biosensor


@sed for hea#y metal ions determination in li.uid samples1 Works on the enzyme inhibition principles1 @rease enzyme is used 0hich hydrolyze the urea1 Constru.tion @rease is immobilized by cross linking 0ith bo#ine serum albumin from biological sensiti#e membrane1 <old electrode is used as transducer1 5or(ing prin.ip"e $irst/ the electrode dips in the urea solution to standardized the condition 2to check the #ariable range of urea re.uired to proper functioning31 7ydrolysis of urea on the electrode surface takes place according to /irst order (ineti.s bet0een (-(-mM concentration of urea solution1

By using absorption spectroscopy techni.ue/ 0e can calculate the remaining concentration of urea in solution1 )ete.tion o/ meta" ion !uts the electrode in the li.uid solution 0here metal ions immobilizes the enzymes present on the electrode1 ?o0 keeps this electrode in urea solution 0here response of biosensor for #arying concentration of metal ions 2,1(-(,mM3 is e#aluated by measuring the urease acti#ity1 7ydrolysis of urea 0ill change the impedance of the o#erall circuit and hence #oltage 0ill #ary according to impedance1 This output #oltage is ac.uired for further analysis1

Per/orman.e /a.tors 6torage time4 A-B days @sed for C-- times 0hen used to measure urea and only once for hea#y metal ions1 "mount of inhibition4 CdDCuD!b 6ensiti#ity4 - (mM in spectrophotometric method1 - -mM in electrical method1 'd$antages Easy production &o0 cost Ease operation 7igh sensiti#ity

Cho"estero" dete.ting biosensor based on .ho"estero" o6idase en3yme4


$or determination of cholesterol in blood1 Cholesterol o idase used as bio element1 Constru.tion In cholesterol biosensor based on direct electron transfer of cholesterol o idase on Multi-0all Carbon ?anotubes 2MW?Ts3/ cholesterol o idase is immobilized onto MW?Ts modified screen printed electrode #ia co#alently bonding bet0een the carbonyl group of carbon nanotubes and amino group of enzyme by using (-ethyl-E-2E-dimethyl-aminopropyl3 carbodiimide and ?hydro ysuccinimide as a coupling agent1
Cholesterol o idase

Cholesterol : o ygen pero ide


Enzyme

Cholest-C-en-E-one : hydrogen

7ydrogen pero ide

; ygen : + proton : + electron

)ire.t e"e.tron trans/er o/ M527s8Cho"estero" 16idase8SP0 e"e.trode

?o redo peak 0as obser#ed at screen printed electrode 26!E3 and MW?Ts-C;;796!E but the current increases in MW?Ts96!E due to its high conducti#ity of Carbon ?ano Tubes 2C?Ts31

"fter Ch; immobilized on MW?Ts/ a pair of re#ersible redo peaks 0ere clearly obser#ed 0hich indicated that Ch; immobilized on C?Ts and C?Ts play an important role in facilitating the electron transfer e change bet0een the acti#e center of Ch;

'mperometri. response o/ M527s8Ch168SP0 to .ho"estero"

The amperometric current response of MW?Ts9Ch; 96!E to successi#e addition of cholesterol concentration 0as performed in ,1( M !B6 p7 B1, at -,1C =1 "ccording to the calibration cur#e of the current response of this biosensor to cholesterol concentration/ the current increases 0ith increasing cholesterol concentration1

Enzyme logic biosensor for security sur#eillance

Chara.teristi.s94 Works on biocatalytic cascade1 8etect the presence of nitro aromatic e plosi#e and organophosphate ner#e agent 1 Enzyme used 4 ?itroreductase 2?%d3 7orseradish pero idase27%!3 "cetyl cholinesterase 2"ChE3 Choline o idase 2Ch; 3 $orm 7+;+ in presence of threat1 %esult sho0ed as FGE69?;H1

(A) Biocatalytic cascade used to perform NOR logic operation in connection to trinitrotoluene (TNT) and paraoxon (PAX) inputs. (B) the e ui!alent logic system" and (#) the corresponding truth ta$le %ith assessment dra%n from the com$inations of the input signals .

!arameters of biosensor performance


Sensiti$ity4-=alue of the electrode response per substrate concentration1 :inearity94 &inearity of the sensor should be high for the detection of high substrate concentration1 8etecti#e > .uantitati#e determination limits1 Se"e.ti$ity94 6ystem should pro#ide a high degree of selecti#ity for the analyte to b measured1 Response time94 Time necessary for ha#ing )-I of the response1 5arm up time94 The time it takes to get ready for ne t detection1 Pre.ision94 The accuracy of information pro#ided after detection1 Cost per measurement94 The amount of money being spent per measurement1 %eproducibility 6ize / 0eight and price of the de#ice. 6tability &ifetime

"pplications of biosensors
8etection of e plosi#es and ner#e agents1 8rug disco#ery and e#aluation of biological acti#ity of ne0 compounds1 To monitor the freshness of food in food industry1 <lucose monitoring in diabetes patients1 8etermination of pesticides and ri#er 0ater contamination1 $or continuous monitoring of compounds such as methanol/ acetonitrile/ phenolics in process streams/ effluents > ground 0ater1

');'27'!0S
6imple and effecti#e prototype1

)<S');'27'! 0S
8enaturation of biological part of the biosensor due to temperature rise or p7 change1 !ure form of reagents and buffer is re.uired1 7omogeneity in sample mi ture is re.uired1 <as bubble formation1

%egular monitoring of pesticides contamination1 Considerably small size compared to an a#erage sim card1 Elimination of radio label and lo0 sample re.uirements compared to

Cha""enges
Impro#ement of protein immobilization and long term stability of biochemical/ for storage/ calibration/ reproducibility/ and drift1J Coupling of molecular recognition to signal transduction at the molecular le#el1 Biocompatibility and antifouling coatings1 J
0==0C7S 1= 7>0 PR0S02C0 1= 7>0 S02S1R 12 7>0 =?2C7<12 1= 7>0 +<1:1!<C': S%S70M +0<2! M12<71R0). ' 751 =1:) PR1+:0 M 0==0C7 1= +<1:1!<C': 02;<R12M027 12 7>0 =?2C7<12 1= 7>0 S02S1R.

8ecreasing of sensor response times1


;#ercoming mass transport limitations1 KK Minimizing diffusion paths1 KK @sing forces like electrostatics to speed mass transport of analytes1 Increasing of rates for binding reactions 0ithout increasing off rates1 In#enting ne0 transduction schemes that are inherently fast1

Modeling that bridges fluid continuum and molecular forces1

Miniaturization to micro and ?ano fluidic systems1


!otential to automate #ery comple procedures1 Compatibility 0ith small sample #olumes1 !otential for packing many de#ices in small spaces - parallel processing. &ittle 0aste1 !ossible integration 0ith pumping/ detection and processing components1 %eproducibility of function1 !otential for mass fabrication1 !otentially lo0 inherent cost1

=uture prospe.ts

Con."usion
$rom all these studies/ 0e conclude that biosensors are cheap/ small and portable de#ices1 They are capable of being used by semiskilled operators1

%eferences

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