Enzyme Based Biosensors

8/13/2019 Enzyme Based Biosensors

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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

• Eamples 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 isin direct spatial contact 0ith a transducer element1

• In short-

biosensor

Biological response Electrical%esponse

2an analytical de#ice3





Working principle of biosensor Working principle of a biosensor in#ol#ed in three elements

0hich are follo0ing-

• $irst biological recognition element 0hich highly specificto0ards 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 0hichis due to reaction occur1

• Third after transduction signal from biological to electrical/0here its amplification is necessary takes place and read outin detector1 "fter processing the #alues are displayed formonitoring 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 thesensiti#e region3

•

8etection9recognition 27o0 do 0e specifically recognizethe 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 an electrical signal1- The output from the transducer is amplified by amplifier 2c3 if re.uired1- !rocessed by processor 2d3- 8isplayed on display 2e3



Types of enzyme based biosensors1. 0"etrohemia" 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 oidase enzymes that generate hydrogen peroide andconsume oygen1

"nalyte

"nalyte : ;ygen : 0ater 7ydrogen peroide : oidized analyte

;idase



$ormation of hydrogen peroide can be detected by the help of !t-electrode1

Eample4 <lucose biosensor

Measures- <lucose content

<lucose mediator : surrounding oygen 7ydrogen peroide :

<lucose oidase2<;83

Catalase

7ydrogen peroide ;ygen : Water

<;8

<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#eelectrode/ such as p7-meter1



These biosensors are intended primarily for monitoringle#els of carbon dioide/ ammonia and other gasesdissol#ed in blood > other li.uids using enzyme asbiological recognition element1

2. Condutometri 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 eothermic/ t0othermistors may be used to measure the difference inresistance bet0een reactant and products and hence the

analyte concentration1





Eamples of enzyme based biosensors

• So"4ge"4immobi"ied urease Condutometri 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

Constrution

@rease is immobilized by cross linking 0ith bo#ine serum albumin frombiological sensiti#e membrane1

<old electrode is used as transducer1

5or(ing prinip"e

$irst/ the electrode dips in the urea solution to standardized the

condition 2to check the #ariable range of urea re.uired to properfunctioning31

7ydrolysis of urea on the electrode surface takes place according to/irst order (inetis bet0een (-(mM concentration of urea solution1



By using absorption spectroscopy techni.ue/ 0e cancalculate the remaining concentration of urea insolution1

)etetion 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 ofmetal ions 2,1(-(,mM3 is e#aluated by measuring theurease acti#ity1

7ydrolysis of urea 0ill change the impedance of theo#erall circuit and hence #oltage 0ill #ary according toimpedance1

This output #oltage is ac.uired for further analysis1



Per/ormane /ators

6torage time4 A- days

@sed for - 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" deteting biosensor based onho"estero" o6idase enyme4

$or determination of cholesterol in blood1

Cholesterol oidase used as bio element1

Constrution

In cholesterol biosensor based on direct electron transfer ofcholesterol oidase on Multi-0all Carbon anotubes 2MWTs3/

cholesterol oidase is immobilized onto MWTs modified screenprinted electrode #ia co#alently bonding bet0een the carbonylgroup of carbon nanotubes and amino group of enzyme by using(-ethyl--2-dimethyl-aminopropyl3 carbodiimide and -hydroysuccinimide as a coupling agent1

Cholesterol oidase

Cholesterol : oygen Cholest--en--one : hydrogenperoide

Enzyme

7ydrogen peroide ;ygen : + proton : + electron

i " / / h " "



)iret e"etron trans/er o/ M57sCho"estero"6idaseSP0 e"etrode

o redo peak 0as obser#ed atscreen printed electrode 26!E3 andMWTs-C;;796!E but the currentincreases in MWTs96!E due to itshigh conducti#ity of Carbon ano

Tubes 2CTs31

"fter Ch; immobilized on MWTs/a pair of re#ersible redo peaks 0ereclearly obser#ed 0hich indicated thatCh; immobilized on CTs and CTsplay an important role in facilitating

the electron transfer echangebet0een the acti#e center of Ch;

' i / M57 Ch SP0



'mperometri response o/ M57sCh6SP0to ho"estero"

The amperometric current response of MWTs9Ch;96!E tosuccessi#e addition of cholesterol concentration 0as performed in ,1(M !B6 p7 1, at -,1 =1 "ccording to the calibration cur#e of the current response of this

biosensor to cholesterol concentration/ the current increases 0ithincreasin cholesterol concentration1



•Enzyme logic biosensor for securitysur#eillance

Ch t i ti



Charateristis4

Works on biocatalytic cascade1

8etect the presence of nitro aromatic eplosi#e and

organophosphate ner#e agent 1

Enzyme used 4 itroreductase 2%d3

7orseradish peroidase27%!3

"cetyl cholinesterase 2"ChE3

Choline oidase 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 eui!alent logic system" and (#) the corresponding truth ta$le %ithassessment dra%n from the com$inations of the in ut si nals.

f bi f



!arameters of biosensor performance• Sensiti$ity4-=alue of the electrode response per substrate concentration1

• :inearity4 &inearity of the sensor should be high for the detection of highsubstrate concentration1

- 8etecti#e > .uantitati#e determination limits1

• Se"eti$ity4 6ystem should pro#ide a high degree of selecti#ity for theanalyte to b measured1

• Response time4 Time necessary for ha#ing ) of the response1

• 5arm up time4 The time it takes to get ready for net detection1

• Preision4 The accuracy of information pro#ided after detection1

• Cost per measurement4 The amount of money being spent permeasurement1

• %eproducibility

• 6ize / 0eight and price of the de#ice.

• 6tability

• &ifetime



"pplications of biosensors• 8etection of eplosi#es and ner#e agents1

•

8rug disco#ery and e#aluation of biological acti#ity ofne0 compounds1

• To monitor the freshness of food in food industry1

• <lucose monitoring in diabetes patients1

• 8etermination of pesticides and ri#er 0atercontamination1

• $or continuous monitoring of compounds such as

methanol/ acetonitrile/ phenolics in process streams/effluents > ground 0ater1



');'7'!0S

)<S');'7'!

0S6imple and effecti#eprototype1

8enaturation of biologicalpart of the biosensor due totemperature rise or p7

change1%egular monitoring ofpesticides contamination1

!ure form of reagents andbuffer is re.uired1

Considerably small sizecompared to an a#eragesim card1

7omogeneity in samplemiture is re.uired1

Elimination of radio labeland lo0 samplere.uirements compared to

<as bubble formation1

Ch ""



Cha""enges• Impro#ement of protein immobilization and long term stability of

biochemical/ for storage/ calibration/ reproducibility/ and drift1

• Coupling of molecular recognition to signal transduction at themolecular le#el1

• Biocompatibility and antifouling coatings1

• 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

0==0C7S = 7>0

PR0S0C0 = 7>0S0SR 7>0

=?C7< = 7>0+<:!<C':

S%S70M +0<!M<7R0).

' 75=:)

PR+:0M

0==0C7 =+<:!<C':

0;<RM07 7>0 =?C7< =

7>0 S0SR.

• Modeling that bridges fluid continuum and molecular forces



• 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 prospets

C " i



Con"usion• $rom all these studies/ 0e conclude that biosensors are

cheap/ small and portable de#ices1

• They are capable of being used by semiskilledoperators1

% f



%eferences



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Enzyme Based Biosensors