CHOLESTEROL BIO-SENSOR

• NIHA Agarwalla• NAUSHAD Rahman• KARTHIKA Gogulakrishnan• Sun Chenxi

MT-5009: ANALYZING HI-TECHNOLOGY OPPORTUNITIES

For information on other technologies, please see Jeff Funk’s slide share account (http://www.slideshare.net/Funk98/presentations) or his

book with Chris Magee: Exponential Change: What drives it? What does it tell us about the future?

http://www.amazon.com/Exponential-Change-drives-about-future-

ebook/dp/B00HPSAYEM/ref=sr_1_1?ie=UTF8&qid=1398325920&sr=8-1&keywords=exponential+change

CONTENT

• What is Biosensor?

• Why Cholesterol Biosensor?

• Conventional Techniques V/s Biosensor

• Current Trend of CB

• Basic Parameters of CB

• Different Materials of Biosensor

• Commercial Biosensor

• Future CB Biosensor

• Market Analysis

BIOSENSOR

• Biosensor is a compact analytical devices or sensor thatintegrates a biological element with a physiochemicaltransducer to produce an electronic signal proportional to asingle analyte which is then conveyed to a detector.

• Biosensor = Analyte + biorecognation element + transducer .

• A biosensor is an analytical device that detects the level ofglucose, cholesterol, urea or any other chemicals in our bodyby using the blood ,urine ,saliva or skin as a sample.

Niha

Niha

WORKFLOW OF BIOSENSOR

Transducer

Electrochemical Optical Piezoelectric pH Change

Matrix

Electrode Array chip Metal Surface

Immobilization of enzyme

Entrapment Covalent Crosslinking Adsorption

Bio receptor /Biomolecules

Enzymes Antibodies Reagent Other molecules

Analyte

Blood Urea Saliva Skin

Niha

CONTD..

DetectorNiha

WORKING PRINCIPLE OF BIOSENSOR

Niha

CONTD…

Niha

EXPLANATION

• Analyte:- It is a sample of Blood ,Urine, Saliva ,Skin

• Analyte is reacted with bio receptor.

• Bio receptors are enzymes, antigens.

• E.g.:- For Glucose detection in blood an enzyme called glucoseoxidase is used.

• For Cholesterol detection in blood an enzyme calledcholesterol oxidase or cholesterol esterase is used.

• Matrix is a solid support which is used to holds the bioreceptor.

• Examples of matrixes are electrodes, array chip or any metalsurface.

Niha

EXPLANATION CONTD…

• The only necessary property for matrix that it should not erode orget affected by pH, temperature or outside environment.

• It is difficult to attach bio receptor to the matrix

• So different immobilization techniques are used for the attachment

• Immobilization techniques are entrapment, adsorption ,crosslinking, covalent bonding etc.

• The bio receptor comes in contact with the analyte and generatesdifferent kinds of signal.

• Signal can be either movement of electron, change in color ,masschange etc. which is detected by transducer and covert this biosignals to electrical signals.

• The electrical signal are amplified and is displayed in a monitor.

Niha

Food Analysis Study of biomolecules and their interaction Drug Development Crime detection Medical diagnosis (both clinical and laboratory use) Environmental field monitoring Quality control Industrial Process Control Detection systems for biological warfare agents Manufacturing of pharmaceuticals and replacement organs

APPLICATION OF BIOSENSOR

Niha

BIOSENSOR FOR MEDICAL

• Biosensor are used for applications ranging from screening fordisease to allow early intervention, through to themanagement of chronic disease and the monitoring of healthand wellbeing.

• Biosensors are an essential tool in the detection andmonitoring of a wide range of medical conditions from cancerto Parkinson’s disease.

Niha

CHOLESTEROL BIOSENSOR

• Cardiovascular diseases and cardiac arrest are number one cause ofdeath globally. One of the most important reasons ishypercholesterolemia i.e. increased concentration of cholesterol inblood.

• An estimated 17.3 million people died from CVDs in 2008,representing 30% of all global deaths .

• The number of people who die from CVDs, mainly from heartdisease and stroke, will increase to reach 23.3. million by 2030.CVDs are projected to remain the single leading cause of death .

• Estimation of cholesterol level in blood hence is the most importantand challenging task for medical industry.

• Development and Improvement of existing Cholesterol biosensorhas got a worldwide attention.

Niha

CONVENTIONAL TECHNIQUES TO DETERMINE CHOLESTEROL

1.Lieberman-Burchard Test

Lieberman–Burchard is a reagent used in a colorimetric test todetect cholesterol, which gives a deep green color.

This test uses acetic anhydride and sulfuric acid as reagents.

Niha

ADVANTAGE OF BIOSENSOR OVER L-B TEST

L-B Test Biosensor

Requires lot of reagents Do not require any regent

Time for detection is more than 20 min. Its response time is as less as 5 sec.

It uses acetic acid and sulfuric acid as reagent which can cause severe burns so require specific care.

It do not use any such reagent so handling is easy.

For L-B test sample should be extracted from plasma and this extraction step constitutes a cumbersome extra step in the assay

Sample can be taken from any part of body. It simple blood.

Requires pretreatment of sample to avoid optical interference because of hemoglobin in blood

No pretreatment of sample is required.

Expensive and can only be used by trained people Less expensive and can be used by any people.

Niha

CONTD..

L-B Test device :Huge apparatus Bio-sensor : Small and portable .

Niha

NON BIOSENSOR TECHNIQUES TO DETERMINE CHOLESTEROL

2 .High-Performance liquid Chromatography(HPLC)

HPLC used to separate the components in a mixture, toidentify each component, and to quantify each component

HPLC uses mass transfer process involving adsorption technique to separate different components.

Niha

ADVANTAGE OF BIOSENSOR OVER L-B TEST

HPLC Biosensor

Irreversibly adsorbed compounds notdetected

Do not require any regent

Requires pretreatment of sample to avoid optical interference because of hemoglobin in blood.

No pretreatment of sample is required.

Complex setup and can only be used by trained people

Easy to handler it

Bulky and Costly Smaller and Cheaper

NIha

CONTD….

HPLC : Bulk Setup Bio-sensor : Small setup

NIha

HISTORY OF CHOLESTEROL BIOSENSOR

• First cholesterol biosensor was developed in 1993.

Components Used

Matrix Ppy/Pt covered with

polycarbonate membrane

Bio receptor Cholesterol Oxidase

Immobilization technique Entrapment

Transducer Amperometric

Niha

PUBLICATIONS TREND IN CB

0

5

10

15

20

25

30

1993 1995 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013

No of Publication

No of Publication

Niha

VARIOUS TYPES OF TRANSDUCER USED IN CHOLESTEROL BIOSENSOR

Electrochemical

Potentiometric

Amperometric

Cyclic Voltammetry

Optical

Fluorescence

Absorption

Reflection

Niha

ELECTROCHEMICAL BASED CB.

• Electrochemical biosensors are normally based on enzymaticcatalysis of a reaction that produces or consumes electronsand generates signal, which are captured by transducer.

• The signal is proportional to concentration of the analysedsubstance.

• Electrochemical are considered to be the most importantcholesterol biosensor.

• Electrochemical sensor may be divided into conductometric,potentiometric, and amperometric biosensors dependingupon the electrochemical property to be measured bydetector system.

Naushad

OPTICAL BASED CB

• An optical fiber-based biosensor is a biosensor that employsan optical fiber, as a platform for the biological recognitionelement, and as a conduit for excitation light and/or theresultant signal.

• The optical measurement method is critical to the sensitivityand detection limit of the sensor. Optical transducers usedifferent types of measurement such as fluorescence,absorbance ,and chemiluminescence.

Naushad

ADVANTAGES OF ELECTROCHEMICAL OVER OPTICAL

Electrochemical Optical

The color of the sample will not interfere with the redox reaction in electrochemical method and hence do not cause anychange in the electrical signal.

Color of the sample interfere with the wavelengths when using optical method which result in inaccurate data.

The life time of the reagents used during reaction donot decrease.

The life time of the reagents can be short under incident light.

Its response time and sensitivity is very high. Because of the diffusion of analytes, it may cause slow response time.

No specific reagent is required for electrochemical biosensor

Fiber Optic Biosensor only works for specific reagent.

Low cost and low power requirement High cost and high power requirement

Naushad

ELECTROCHEMICAL V/S OPTICAL

0.00E+00

5.00E-01

1.00E+00

1.50E+00

2.00E+00

2.50E+00

3.00E+00

3.50E+00

4.00E+00

4.50E+00

5.00E+00

2001 2002 2003 2004 2005 2006 2007 2008

De

tec

tio

n L

ev

el

Year

Optical Biosensor

Electrochemical

Acrylamine glass

beads

+ ChOx, ChEt, HRP+

Alkylamine glass

beads+ Chox,

Chet, HRP+ Cross-

linking via

Glutaraldehyde

PANI/ITO+

ChOx+

Covalent via

EDC/NHS

TEOS (sol–gel)ITO+

ChOx, ChEt+

Entrapment

Ferrocene

monocarboxylica

cid-PPy/Pt/Pt+

ChOx+

PPy/Pt+ ChOx+

Entrapment FeMC

(physisorbed)-

P(NMPY)-PTS/ITO+

ChOx+ Physical

adso

Naushad

𝜇𝑔/𝑚

𝑙

1. Linearity2. Sensitivity3. Response time4. Reusability5. Shelf life6. Detection Limit

Basic Parameters to Measure Efficiency of CB

Naushad

SENSITIVITY

0

0.5

1

1.5

2

2.5

1994 1996 1998 2000 2002 2004 2006 2008

Se

nsi

tivity

(n

A M

-1)

Year

Improvment In Sensitivity In CB

Sensitivity

1

2

3 4

5

6

7

8

9

Naushad

CONTD….

Material No

Tetramethoxy silane sol–gel/poly(1,2-diaminobenzene)/Pt + ChOx+ Entrapment followed by treatment

with Glutaraldehyde vapors+ Ampero. at 0.6 V vs. Ag/AgCl1

PPy/Pt/Pt+ ChOx+ Entrapment+ Ampero. at 0.5 V vs. Ag/AgCl2

PPy-p(HEMA)-TEGDA/Pt+ ChOx+ Entrapment+ Ampero. at 0.7 V vs. Ag/AgCl 3

Ferrocene monocarboxylicacid-PPy/Pt/Pt+ ChOx+ Entrapment+ Ampero. at 0.375 V vs. Ag/AgCl 4

PPy/ITO+ ChOx, ChEt+ Entrapment+ Ampero. at 0.5 V vs. Pt wire 5(a) BSA/dilauroylphosphatidylcholine (DLPC)/rhodium–graphite; (b) Agarose gel-Riboflavin-

DLPC/rhodium–graphite + (a) RfP450scc; (b) Cytochrome P450scc+ (a) Cross-linking via Glutaraldehyde;

(b)+ Electro. (CV) vs. Ag/AgCl

6

3-aminopropyl-modified controlled-pore glass (APCEG)/rotating disk + Chox, ChEt, HRP+ Cross-linking via

Glutaraldehyde+ Ampero. at −0.15 V vs. Ag/AgCl) withTBC 7Streptavidin/biotin/thioctic acid (SAM)/Au nanowires + ChOx, ChEt+ Covalent via Biotin-avidin+

Voltammetric (SWV) vs. Ag/AgCl 8

FeMC (physisorbed)-P(NMPY)-PTS/ITO+ ChOx+ Physical adsorption+ Electro. (CV) vs. Ag/AgCl 9

Naushad

EXPLANATION OF GRAPH

• Sensitivity of an sensor indicates the capacity of the sensor torespond truly to the change in the output, corresponding tothe change in the input.

• Its depends on various factor like electrode , Enzymes andImmobilization technique.

Naushad Rahman

RESPONSE TIME

0

0.5

1

1.5

2

2.5

3

3.5

1996 1998 2000 2002 2004 2006 2008

Re

spo

nse

Tim

e (

sec

)

Year

Response time in sec

Response time in sec

1

2

3

4

5 6

7

8

9

12

11

1013

14

15

Naushad

TABLE

1 PPy/Pt covered with polycarbonate membrane +ChOx + Entrapment+Ampero. at 0.7 V vs. SCE

8PPy/Pt+ ChOx+ Entrapment+ Ampero. at 0.7 V vs. Ag/AgCl

2 2-aminoethanethiolate/Au +ChOx, ChEt for TC, ChOx for FC +Cross-linking via Glutaraldehyde +Amperometric at 0 V vs. SCE with thioninas electron mediator

9

ITO glass+ Cytochrome P450SCC (5 g in 40 LB)+ LB films+ Electro. (CV) vs. Ag/AgCl

3

Hexadecyl mercaptan (SAM)/Au+ Molecular imprints+ Template+ Electro. (CV) vs. Ag/AgCl

10 BSA/dilauroylphosphatidylcholine (DLPC)/rhodium–graphite; (b) Agarose gel-Riboflavin-DLPC/rhodium–graphite + (a) RfP450scc; (b) Cytochrome P450scc+ (a) Cross-linking via Glutaraldehyde; (b)+ Electro. (CV) vs. Ag/AgCl

4 TEOS derived solgel+ ChOx, HRP+ Entrapment+ Ampero. at 0.75 V vs. Ag/AgCl

11 Sol–gel/CNT-Pt/graphite electrode+ ChOx+ Entrapment+ Electro. (CV) and Ampero. −0.2 V vs.

5 Poly(ethylene imine)(PEI)/poly(styrene sulfonate)/ITO + ChOx+ LBL deposition of PEI and ChOx + Ampero. at 0.6 V vs. Ag/AgCl

12

1-hexadecanethiol (SAM)/Au+ Molecular imprinted layer+ Templete + Voltammetry (CV) vs. Ag/AgCl

6

PPy- doecylbenzene sulfonate/ITO+ ChOx+ Physical adsorption+ Electro. (CV) vs. Ag/AgCl

13 MWCN/Screen Printed Carbon Electrode+ Chox, Chet, HRP, K4 Fe(CN)6+ Physical adsorption+ Ampero. at 0.3 V vs. Ag/AgCl

7 (a) Octadecylsilica (ODS)/TEOS sol–gel for cholesterol in aqueous micelle solution; (b) ODS/HECMC/PVA gel for cholesterol in hydrophobic organic solvent + ChOx+ Entrapment+ Optical oxygen transducer

14

Multilayer of Pt-MWCNT-CHIT and poly(sodium-p -styrenesulfonate) onto Au + ChOx+ Cross-linking via Glutaraldehyde+ Ampero. at 0.1 V vs. SCE

15 ZnO/Au+ ChOx+ Physical adsorption+ Electro. (CV) vs. Ag/AgCl

Naushad Rahman

EXPLANATION OF GRAPH

• Response Time:- Time taken by a sensor to detect thecholesterol in a sample.

• Lower the response time higher is the efficiency of thebiosensor.

NIha

REUSABILITY

0

20

40

60

80

100

120

140

160

180

200

1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

Re

usa

bility

(Tim

es)

Year

Reusability

RE

6

1

2

3

4

5

TABLE

Component Component

1

PANI/Pt+ ChOx+ Electro. doping+ Ampero. at 0.6 V vs. SCE and 1% tritonX-100 5

Poly(dialyldimethylammonium chloride) (PDDA)/MWCNTs/Au + ChOx covered by o-PDD+ LBL deposition of PDDA and ChOx+ Ampero. at 0.7 V vs. SCE

2 Acrylamine glass beads+ ChOx, ChEt, HRP+ Covalent via diazotization+ Spectro. at 520 nm using APZ+ phenol 6

PANI/ITO+ ChOx+ Covalent via EDC/NHS+ Spectro. at 500 nm using o-dianisidine

3 Silisic sol–gel/PB/GC+ ChOx+ Entrapment+ Electro. (CV) at −0.05 V vs. Ag/AgCl

4 Polyacrylonitrile fiber+ ChOx+ Cross-linking via Glutaraldehyde+ Spectro. at 520 nm using APZ+ phenol

Naushad Rahman

EXPLANATION OF GRAPH

• Reusability means number times sensor can be use beforelosing its precision level .

• It depends upon the electrode quality .

• As sensor electrode will erode by interfacing element andreduce its surface area to volume ration will decrease.

Naushad Rahman

SHELF LIFE

Naushad

0

20

40

60

80

100

120

2000 2001 2002 2003 2004 2005 2006 2007 2008

Sh

elf L

ife

( D

ay

s)

Year

Improvment in Shelf Life

1

2

34 5

6

7

TABLE

1 Tributylmethyl phosphoniumchloride polymer membrane/pyrolitic graphite electrode + ChOx, HRP+ Entrapment+ Ampero. at −0.28 V vs. Ag/AgCl

4

PPy/ITO+ ChOx, ChEt+ Entrapment+ Ampero. at 0.5 V vs. Pt wire

2 Acrylamine glass beads+ ChOx, ChEt, HRP+ Covalent via diazotization+ Spectro. at 520 nm using APZ+ phenol

5Sol–gel/CNT-Pt/graphite electrode+ ChOx+ Entrapment+ Electro. (CV) and Ampero. −0.2 V vs.

3 Silisic sol–gel/PB/GC+ ChOx+ Entrapment+ Electro. (CV) at −0.05 V vs. Ag/AgCl

6 BSA/TEOS/ITO+ ChOx, HRP+ Covalent+ Ampero. at 0.75 V vs. Ag/AgCl

7 BSA/Polycarbonate/Oxygen electrode+ ChOx, ChEt+ Cross-linking via Glutaraldehyde+ Polarographic at −0.7 V vs. Ag/AgCl

Naushad Rahman

EXPLANATION OF GRAPH

• Shelf Life:- Duration till which one can use electrode to detectcholesterol.

• Higher the shelf life higher is the efficiency of the biosensor.

Naushad

DETECTION LIMIT

Naushad

0

10

20

30

40

50

60

70

1992 1994 1996 1998 2000 2002 2004 2006 2008

De

tec

tio

n L

imit (

mg

\d

l)

Year

Detection Limit

DL

21

3

4

5

6 7

8

9

1310

11

12

TABLE

Naushad Rahman

Component Component

1 PPy/Pt covered with polycarbonate membrane +ChOx + Entrapment+Ampero. at 0.7 V vs. SCE

7 Poly(o-phenylenediamine)/PPy/Pt/Pt+ ChOx+ Entrapment in PPy+ Ampero. at 0.5 V vs. Ag/AgCl

2 HRP-hydroxymethyl ferrocene-carbon paste electrode +ChOx ChEt+In solution +Chronoamperometry at 0 V vs. SCE

8 Acrylamine glass beads+ ChOx ChEt HRP+ Covalent via diazotization+ Spectro. at 520 nm using APZ+ phenol

3 Laponite clay nanoparticls-poly((12-pyrrol-1-yldodecy)triethylammonium tetrafluoroborate)/Ptdisk electrode + ChOx ChEt for TC ChOx for FC +Entrapment +Ampero. at 0.53 V vs. Ag/AgCl

9

PPy/Pt+ ChOx+ Entrapment+ Ampero. at 0.7 V vs. Ag/AgCl

4 Glassy carbon+ ChOx+ In solution + Electro. at 0 V vs. SCE using thionin as mediator

10 ITO glass+ Cytochrome P450SCC (5 g in 40 LB)+ LB films+ Electro. (CV) vs. Ag/AgCl

5 Tetramethoxy silane sol–gel/poly(12-diaminobenzene)/Pt + ChOx+ Entrapment followed by treatment with Glutaraldehyde vapors+ Ampero. at 0.6 V vs. Ag/AgCl

11Silica sol–gel- chitosan (CHIT)-MWCNT/PB/GC + ChOx+ Entrapment+ Ampero. at −0.05 V vs. Ag/AgCl

6Hexadecyl mercaptan (SAM)/Au+ Molecular imprints+ Template+ Electro. (CV) vs. Ag/AgCl

12 CHIT/NiNPs/histidine/MWCNT/GC electrode + ChOx+ Cross-linking via Glutaraldehyde+ Ampero. −0.2 V vs. SCE

13 TEOS (sol–gel)ITO+ ChOx ChEt+ Entrapment+ Spectro. at 500 nm using 4-APP + phenol

EXPLANATION OF GRAPH

• DL is the lowest quantity of a substance that can bedistinguished from the absence of that substance within astated confidence limit.

• Lower the DL greater is the selectivity of the CB.

Naushd

DIFFERENT MATERIALS FOR AMPEROMETRIC BIOSENSORS

Metallic Materials Thermoplastic Polymeric Material

Thermosetting Polymeric Material

Applications flow through reusable sensors

Moderate temperature with no strong solvents, used as working electrode

Used in combination with polar solvents, used as working electrode

Advantages lower electronic noise low detection limits, high sensitivities, lowerapplied potential, reduction of background, efficient electron transfer

Disadvantages Moderate performance

commercial production of pure and defect-free polymers is difficultand costly

Examples Gold, Ion, Silver Carbon, conductive polymer, sol-gel polymer, functional polymer

Chenxi

METAL

Chitosan-gold

High electron transfer rate (Electron transfer rate constant wasestimated to be 15.6 s−1)

Enhanced stability

Moderate bioactivity

magnetic Fe3O4/chitosan

Fast response to H2O2 and excellent linear relationships

Excellent bioactivity

Long-time stability and good reproducibility

Chenxi

CARBON NANOTUBES

• Accuracy, efficiency and detection limit of a biosensor can beincreased with the use of carbon nanotubes immobilized onbare electrodes

• High electron transfer, high surface area, minimization of thesurface fouling, high stability, excellent adsorptive andbiocompatibility.

Chenxi

SOL-GEL POLYMERIC MATERIALS

Sol-gel materials provide a versatile way for immobilization dueto the presence of inorganic M–O or M–OH–M bridges forminga continuous network containing a liquid phase which can thenbe dried out to form a solid, porous polymeric matrix

Excellent sensitivity

Good reproducibility

Remarkable stability

Rapid response

Chenxi

CONDUCTIVE POLYMERIC MATERIALS

Superior reusability

Excellent thermal stability

Large surface to volume ratio

High conductivity, sensitivity

Good biocompatibility

Wide linear range

Graphene/chitosan

Chenxi

FUNCTIONAL POLYMERIC MATERIAL

These are polymeric materials which possess differentfunctional groups, for example, thiols, amines, carboxylicacids and others

Low detection limit, wide detection range

Fast response

Good stability.

Anti-interference ability

Chenxi

COMPARISON OF SELECTED MATERIALS

Chenxi

INVASIVE CB

• Invasive biosensors are the one in which blood sample isrequired and is obtained from the finger tip by pricking.

• These sample is placed in the test strip and the test strip isinserted into the biosensor device and amount of cholesterolis detected.

NIHA

NON INVASIVE BIOSENSOR.

• Non Invasive biosensor are the one in which no pricking isrequired it uses skin cholesterol to measure the cholesterollevel in human body.

• Skin contains over 11% of the body cholesterol and ages inparallel with vascular connective tissue. As arterial wallsaccumulate cholesterol, so do the skin tissues.

• A high skin cholesterol level is a reliable predictor of highercholesterol accumulation in the arteries and, accordingly, canbe used in combination with other risk factors to assess risk ofcoronary artery disease.

NIHA

CONTD..

• The simple test is conducted by placing a drop of digitonin, which binds selectively to thecholesterol in the skin, on the palm of the hand. This liquid also contains an enzyme linked tothe digitonin by a copolymer. After a one-minute incubation period, the area is blotted dry toremove any unbound digitonin solution. A second drop of liquid is then added, containing asubstrate for the horseradish peroxidase enzyme. When combined, a blue color changeoccurs in direct proportion to the amount of digitonin that is bound to skin cholesterol. Aftertwo minutes, a hand-held spectrophotometer (color reader) is placed over the drop tomeasure the precise blue color, which indicates the skin cholesterol value.

PREVU* Skin Cholesterol Test NIha

INVASIVE V/S NON INVASIVE

Invasive Non Invasive

It requires sample so finger pricking is done which is very painful

No pricking is required so painless cholesterol determination

It require patient preparation such as fasting before cholesterol determination

No prior patient preparation is required.

More sensitive and more accurate Less Sensitive and less accurate

Latest cholesterol biosensor can detect total cholesterol, LDL,HDL and triglycerides separately.

It can detect only total cholesterol

It is the present. It can be the future as high research is done in the non invasive biosensor.

NIha

Commercial Cholesterol biosensor

Electrochemical techniques are simple, relatively cheap, and rapid as compared to other methods; but also have a potential for further improvement.Other methods.colorimetric, high performance liquid chromatography (HPLC) using gel electrophoretic chip, capillary electrophoresis, spectrophotometricand fluorometric

1st generation of Commercial cholesterol biosensors use cholesterol esterase or cholesterol oxidase .They depend on the presence of oxygen for the enzyme –catalysed reaction and the production and measurement of hydrogen peroxide.

•2nd Generation use electron mediators which help to transfer electrons from the enzyme to electrode surface .Ferro/ferricyanide, ferrocene, conducting organic salts and redox dyes were used as mediators.

Karthika

http://pubs.acs.org/action/showImage?doi=10.1021%2Fcr068123a&iName=master.img-005.jpg&type=master

Commercial Cholesterol biosensor

3rd generation of cholesterol biosensors: Use direct electron transfer between enzymes and electrodes in the absence of mediator. The distance between immobilised enzyme and electrode should be as small as possible. Such biosensors are more sensitive.

A wide range of cholesterol biosensors are available in market today using a range of matrix ,immobilisation and transducer technologies.

Karthika

COMMERCIAL CB

• Cholesterol Bio sensors used in Clinical analysis are usuallymore accurate than home kits.

• Cholesterol biosensors used in Labs devices like Autoanalyzerwhich is capable of doing dozen of analyses simultaneously bya single machine from a small amount of serum.

Karthika

COMMERCIAL CHOLESTROL BIOSENSOR

cholesterol oxidase for assay of total and free cholesterol in serum by continuous-flow analysis.

Desktop version

http://en.wikipedia.org/wiki/AutoAnalyzer

Hitachi 7070

http://japancare.trustpass.alibaba.com/product/

124017974-

103336093/Auto_Analyzer_HITACHI_7070_91

1_.html

Evolution of Commercial biosensors

Standalone Huge devices

PortableHome

kits

Karthika

DESKTOP VERSION OF CB

Karthika

CONTD..

Karthika

HOME KITS

Detects as little as 200 nM (80 ng/ml) cholesterol

Helps accurately measure cholesterol content of 0.01 µl ofhuman serum

Detects both free cholesterol and cholesteryl esters.

High sensitivity and excellent linearity of the assay at lowlevels of cholesterol (0–0.015 µg⁄ml).

Karthika

AMPLEX® RED ASSAYS DETECTION LIMIT

Different product use different reagent (polyethylene glycol cholesteryl ether

, Cholesterol oxidase , horseradish peroxidase ) and electrode combinations.

Karthika

high sensitivity and

excellent linearity of

the assay at low

levels of cholesterol

(0–0.015 µg⁄ml)

𝜇𝑔/𝑚𝐿

Flu

ore

sce

nc

e

COMMON TECHNOLOGY IN COMMERCIAL CB

Most common technique in Home Kits

Cholesterol Kit uses cholesterol oxidase to produce hydrogenperoxide, which is then detected by the reagent in thepresence of horseradish peroxidase (HRP).

Enzyme coupled technology

Measure by reflectance of light(photometric).

An electrochemical reaction which generates an electricalcurrent proportional to the amount of Cholesterol-Electrochemical biosensors.

Karthika

CONTD..

Karthika

FEATURES ON WHICH SUCCESS OF CBDEPENDS

• Be stable under normal storage conditions and show good stabilityover a large number of assays (i.e. much greater than 100)

• The reaction should be as independent of physical parameters aspH and temperature

• The response should be accurate, precise, reproducible and linearover the useful analytical range. It should also be free fromelectrical noise.

• Should be cheap , small, portable• Capable of being used by unskilled person.• Screen Printing played an important role in biosensors

commercialization.• Variation of enzymes and electrode materials had improved

performance in sensitivity storage and shelf life.

Karthika

COMMERCIAL CB CONTD..

Karthika

CONTD..

Karthika

CONTD…

Karthika

CONTD..

Karthika

FUTURE CHOLESTEROL BIOSENSOR

Non invasive Biosensors and Wearable Biosensors are thefuture of CB.

Wearable Biosensor

• These are the devices that will monitor continuously thephysiological signals.

• They rely on wireless sensor ,the data are recorded and isused to monitor patients health condition.

• They are very helpful to athletes, handicapped, old aged andto professional people.

NIha

WEARABLE BIOSENSOR

• Examples of wearable biosensor:

Shoes BiosensorShirt BiosensorRing Biosensor

NIha

WEARABLE BIOSENSOR

• Ring sensor Can measure the heart rate and

oxygen saturation rate.

• Shirt Sensor can measure body temperature, heart

rate and respiration rate.

• Their main applications are :-

Chronic Surveillance of abnormal heart failure

In cardio-vascular dieses to measure hyper tension.

Wireless Monitoring for people in hazardous

operations.

NIha

WEARABLE BIOSENSOR

Advantage:-

Continuous Monitoring

Easy to use

Reduce Hospitalization Fee

Disadvantage:-

Initial Cost is high

Limited number of physiological parameter is

measured

Niha

EFFECT OF TECHNOLOGY IN DEVELOPMENT OF CB

Two most important technology that will play a significant rolefor the development of CB is

Nanotechnology

Microsystem Technology.

Various kind of nanomaterial are applied to cholesterolbiosensor such as gold nanoparticles, carbon nanotubes,Nanowires, Graphene and Quantum dots.

Niha

WHY NANO TECHNOLOGY??

• These materials are generally used because of their uniquephysical, chemical, mechanical, magnetic and opticalproperties,

• They markedly enhance the sensitivity and specificity ofdetection.

• They have great potential in the detection of DNA, RNA,proteins, glucose ,pesticides and other small molecules fromclinical samples, food industrial samples, as well asenvironmental monitoring.

Niha

MULTI ANALYTE DETECTION

They also help in Multi analyte Detection.

• Development of sensors capable of determining several analyte simultaneously can represent an interesting tool in clinical industry.

• This will help to reduce the cost and will also lessen the diagnostic time.

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MINIATURIZATION

They also led to Miniaturization of biosensor.

• Miniaturization allows the handling of low-volume samples, areduction in reagent consumption and waste generation, andincreases sample throughput .

• Miniaturization can benefit Biosensor by making itinexpensive and easy-to-handle analytical devices.

• Nanowire are smaller than the red blood cell whose diameteris 6.8 micro meter.

• Use of Nanowire Biosensor can help to develop implantablebiosensor too.

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EFFECT OF NANOPARTICLE ON SENSITIVITY

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Sensitivity(mV)

Sensitivity(mV)

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EFFECT OF NANOPARTICLE ON DETECTION LIMIT

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1

1.2

1.4

Detection Limit(microM)

Detection Limit(microM)

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EFFECT OF MWCNT ON DETECTION LIMIT

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Detection Limit(micromol)

Detection Limit(micromol)

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EFFECT OF GRAPHENE ON DL AND SENSITIVITY

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1.5

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3

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M1

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TOTAL MARKET OF BIOSENSOR

• The biosensor market is dominated by only a few products

• For medical diagnostics, approximately 90% of biosensors are glucose monitors, blood gas monitors, and electrolyte or metabolite analyzers

• Half of all biosensors produced worldwide are glucose monitors Sales are projected at $1.28 billion in the US in 2012

• The majority of the remaining market includes biosensors directed at environmental control, fermentation monitoring, alcohol testing, and food control

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GROWTH IN THE USAGE OF BIOSENSOR

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MARKET ANALYSIS OF BIOSENSOR

• According to a new market report published by Transparency Market Research “Biosensors Market - Global Industry Analysis, Size, Share, Growth, Trends and Forecast, 2012 -2018,” in 2011, the global biosensors market was valued at USD 9.9 billion and it is expected to grow at a CAGR of 9.6% from 2012 to 2018 to reach a market of USD 18.9 billion by 2018.

• In 2011, the biosensors market was valued to be USD 9,973.5 million and is expected to grow at a CAGR of 9.6% from 2012 to 2018.

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FURTHER ANALYSIS

• The development of new biosensors and devices based onthis technology is a highly capital intensive exercise.

• Although miniaturization allows for economies of scale to beachieved in the actual manufacturing of biosensors, hugecapital investment is required for research and development.

• Though the U.S. remains the largest market in the world,Asian countries namely India and China are witnessing fastgrowth and are predicted to emerge as dominating markets inthe near future.

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FURTHER TREND

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Thank You