biosensor construction and molecular ...dna biosensor (ii-1) vasif hasirci metu turkey 40 biomat 1st...

Vasif Hasirci METU Turkey1

1st ExperTissues School and 12thCIRMIB Biomaterials School, 12 July 2005, IschiaBioMat

VASIF HASIRCI

BioMatMiddle East Technical University

Ankara - TURKEY

BIOSENSOR CONSTRUCTION ANDMOLECULAR RECOGNITION INVOLVED IN

THE PROCESS



BIOSENSORSCHEMICAL-BIOLOGICAL HYBRIDs

Biosensors fall in the middle of a continuum of sensors characterized byanalytical assays that use chemical and biological processes. Increasingly,analytical procedures are benefiting from the sensitivity of biologicalconstituents such as enzymes and antibodies

Chemical sensor (Bio) chemical sensor Bioanalytical assays

pH O2 Enzyme electrode Immunosensors ELISA

electrode electrode Microbe electrode Enzyme assays

Toxicity assays



BIOSENSOR

BiologicalAnalyte Detection TransducerSolution Element

Signal

SignalProcessor

Readout



CONFORMATION OF BASIC MACROMOLECULES

Protein Beta-sheet

DNA-helix

Amylose

Protein Alpha-helix



STRUCTURAL UNITS

• Sugars



• Nucleotides

STRUCTURAL UNITS



DNA DOUBLE HELIX



THE A, B AND Z CONFORMATIONS



A AND B FORMS OF DNA

C2’-endo5883.3710DNA-B(humidity 66%)

C3’-endo16702.5511DNA-A(humidity 75%)

SugarConfiguration

Dihedral Anglebtw baseplanes

Angle btwBase Planesand helix axis

Translation perResidue

Residues perTurn



PEPTIDE BOND AND PLANARITY



PRIMARY, SECONDARY TERTIARY AND QUATERNARYSTRUCTURES

•

cmgm.stanford.edu/biochem201/ Slides/Protein%2...



ALPHA HELIX END VIEW

cmgm.stanford.edu/biochem201/ Slides/Protein%2

Alpha helixhas a 185symmetry,with 3.6 peptidesper turn,residue repeatdistance is 1.50 A.



BETA SHEET

Beta sheethas a 21 symmetry,with 2 peptides per turn,residue repeat distance is3.25 A.



PROTEIN QUATERNARY STRUCTURE

Quaternary structure of Protein Kinase C Interacting Protein lectures.molgen.mpg.de/.../ Levels/quaternary.gif



PROTEIN QUATERNARY STRUCTURE (CONTD)

http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%202/protein%20types.jpg



GLUCOSEAMINOGLYCANS



STARCH AND CELLULOSE

cellulose

Starch (amylopectin)



BIOLOGICAL ELEMENT

Biocatalytic(Enzymes, organelles, whole cell or organisms)

• Catalytic transformation• Specific inhibition of enzyme activity by analyte• Modification of enzyme activity by analyte that acts as modulator

or cofactor • General inhibition of cellular respiration• Promoter recognition by specific analyte followed by gene

expression, enzyme synthesis, and catalytic activity

Bioaffinity(Antibodies, non-catalytic proteins of non-immune originand nucleic acids)

Compound- or class-specific affinity



TRANSDUCER

Transducers are the components which convert the chemicalor electrical response to a signal such as a digital display thatcan be visualized

Electrochemical Optical Potentiometric Absorbance Amperometric Fluorescence Conductimetric Luminescence

Optical-electronic Piezoelectric Devices Light-addressable Surface acoustic wave potentiometric sensor Bulk acoustic wave Surface plasmon resonance

ThermalMagnetic



PERFORMANCE FACTORS

Selectivity

Linear working range

Reproducibility

Response time

Lifetime



Glucose oxidase subunitwith FAD (red)

GOD holoenzyme

GLUCOSE OXIDASE



INDUCED FIT



IMMUNOASSAYS (2)

Label Types

Particlesmetal and dye solsradionuclidesenzymessubstrates and cofactorselectrochemically active compounds



IMMUNOASSAYS

1) Competitive, solid phase immunoassay in whichantibody is immobilized on a solid phase and the antigen islabelled. In the test there is competition between thelabelled and unlabelled antigen (i.e. the analyte). For theavailable antibody bimding sites. After the binding reactionthe amount of label associated with the solid phase will beinversely related to the concentration of the analyte.

2) In the displacement immunoassay in which antibody isagain immobilized on a solid phase and the antigen islabelled. At the start of the assay all of the available bindingsites on the immobilized antibodies are occupied by thelabelled antigen. On the addition of the unlabelled antigenthere is displacement of labelled antigen, and this will bedependent of the amount of the analyte added.



A COMPETITION ASSAY BASED THEOPHYLLINE SENSOR

Analyte: Theophylline, a drug, acts as the antigen.

Assay System: Theophylline conjugated to Alkaline phosphatase

Antibody specific to theophylline is immobilized on animmunoaffinity membrane. The assay is based on competitionbetween the sample drug and the drug-ALP conjugate for a fixednumber of antibody binding sites. After a proper reaction time themembrane is exposed to p-aminophenyl phosphate. Thesubsequent formation of p-aminophenol occurs to an extent thatdepends upon the amount of conjugate present on the membrane.The greater the concentration of the drug in the sample the smallerthe quantity of conjugate that will be able to bind to the membraneand therefore lower product formation. The potential of the systmedepends on the amount of product.



ANTIBODY



ANTIGEN-ANTIBODY CONJUGATION

The Fab portion of the antibody has specificity forbinding an epitope of an antigen. The Fc portiondirects the biological activity of the antibody.



ANTIBODY SPECIFICITY



ANTIBODY SPECIFICITY II



ANTIBODY SPECIFICITY III

Model of putative dsRNA antibody interaction

ALP (III)



ALKALINE PHOSPHATASE

ALP (I)

The immobilized enzymecan be coupled to ahydrogen peroxideelectrode for biosensorconstruction

ALP (II)



BOD BIOSENSOR

Oxygenelectrode

Enzyme immobilized membrane



BIENZYME SYSTEM



DNA BIOSENSOR (I-1)

Single strands of DNA recombine via hybridization to double stranded DNA (double helixstructure). The authors demonstrated that hybridization of DNA on a gold coated cantileverproduces bending of the cantilever. monet.physik.unibas.ch/ nose/biosensor/hybrid.jpg



Schematic drawing of the experiment. A. Two cantilevers covered by single stranded DNA.B. Bending of the first cantilever by hybridization. C. Bending of the second cantilever byhybridization

DNA BIOSENSOR (I-2)



Nanoscaled interdigitated electrode arrays are made withdeep UV lithography. Electrode widths and spacings from500 nm down to 250 nm are produced on large activeareas (0.5 mm x 1 mm). An impedimetric DNA- orimmunosensor measures the binding of a target moleculeto selective probes by measuring changes in the electricproperties in the vicinity of an electrode. For instance, whenantibodies bind to antigens, the change in electricproperties results in a change of impedance, enabling themeasurement of a direct electrical signal. Advantages overoptical detection methods are that no labeling or enzymaticreactions are needed. The direct electrical read-out alsoeliminates the need for optical components.

DNA BIOSENSOR (II-1)



DNA BIOSENSOR (II-2)



A DUAL ENZYME BIOSENSOR FOR PESTICIDE DETECTION

• Acetylcholinesterase (AChE) and Choline oxidase (ChO) were co-immobilized on poly(2-hydroxyethyl methacrylate) (pHEMA)membranes to construct a biosensor for the detection of anti-cholinesterase compounds.

• pHEMA membranes were prepared with the addition of SnCl4 toachieve the desired porosity.

• Immobilization of the enzymes was done by surface attachmentvia epichlorohydrin (Epi) and Cibacron Blue F36A (CB) activation.

• Enzyme immobilized membrane was used in the detection of anti-cholinesterase activity of aldicarb (AS), carbofuran (CF) andcarbaryl (CL), as well as two mixtures, (AS + CF) and (AS + CL).



IMPORTANCE OF PESTICIDE BIOSENSORS

PESTICIDE

USAGE

CONTAMINATION OF •DRINKING WATERS•GROUNDWATERS•PLANTS

FOODCHAIN

HUMAN

WILDLIFE

RAPID AND ON-SITE DETERMINATION



THE PESTICIDES

• Carbaryl (CL)• 1-naphthyl N-methylcarbamate

• Carbofuran (CF)• 2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate

• Aldicarb (AS)• 2-methyl-2-(methylthio) propionaldehyde o-(methylcarbamoyl)

oxime

ACHASE INHIBITION

Carbamates.Act on PAS

Active site and the PAS of AChE.Numbers refer to positions in Torpedo Californica AChE

AS: Anionic substrate binding sitePAS: peripheral anionic siteES: Esteratic siteAACS: Active site selective aromaticcation binding site



ALDICARB

CH3 O CH3-S-C-CH=N-O-C-NH-CH3

CH3

Aldicarb has a very high mammalian toxicity (oral LD50 values in rats:0.84 mg\kg body weight)

Its degradation products, aldicarb sulfoxide and aldicarb sulfone,are also very toxic (oral LD50 values in rats: LD50: 0.49-1.13 and 20-25 mg\kg of body weight)

Aldicarb is a Restricted Use Pesticide (RUP) in the United States asdetermined by the US Environmental Protection Agency

It is very effective against cotton white fly (Bemisia tabaci)



• Acetylcholinesterase (AChE) is avital enzyme of nervous system

• Carbamate and organophosphorus insecticides show theireffect by inhibiting AChE activity by carbamylation andphosphorylation of the serine residue in the active site of theenzyme

• AChE catalyzes the hydrolysis ofthe neurotransmitter acetylcholine(ACh)

• It is present in a wide range ofanimals, including mammals,birds, fish, reptiles and insects

ACETYLCHOLINESTERASE



ABOUT THE BIOSENSOR

Enzymes : Acetylcholinesterase and Choline oxidase

Detection method : Based on O2 measurementSubstrate : AcetylcholineAnalyte : Aldicarb, Carbaryl, CarbofuranImmobilization method : Surface immobilizationImmobilization matrix : Poly(2-hydroxyethylmethacrylate)

(pHEMA) membrane



ENZYMATIC REACTIONS

AChEAcetylcholine + H2O Choline + Acetic Acid

ChOCholine + 2O2 + H2O Betaine + 2H2O2



PREPARATION OF THE MEMBRANES

2-Hydroxyethylmethacrylate (HEMA)

a,a’-Azo-isobutyronitrile (AIBN)

Ethylene glycol dimethacrylate (EGDMA)

Monomer

Initiator

Aqueous solution

Crosslinker

Electrolyte solution (SnCl4)



CHARACTERIZATION OF THE MEMBRANES

Water ContentPermeability

PorosityMechanical Properties



POROSITY OF THE MEMBRANES

SEM Micrographs for theMembrane Prepared in 0.3 MNaCl

SEM Micrographs for theMembrane Prepared in 0.6 MNaCl



IMMOBILIZATION OF ENZYMES bySURFACE IMMOBILIZATION

EpichlorohydrinCibacron Blue

Cl CH2 CH CH2

O

N

N N

X

R Cl



OXYGEN ELECTRODE UNIT



EFFECT OF TEMPERATURE



STORAGE STABILITY



EFFECT OF SUBSTRATE CONCENTRATION ONPESTICIDE DETECTION



REUSABILITY

In the absence ( ) and presence (D) of aldicarb, (100 ppm, inc. 5min)



RESIDUAL ACTIVITY vs INCUBATION TIME &CARBARYL CONCENTRATION

0

20

40

60

80

100

120

0 5 10 15 20 25 30 35

Incubation Time (min)

Resid

ual A

ctivity (

%)

1 ppb

10 ppb

20 ppb

50 ppb



CALIBRATION CURVE FOR CARBARYL DETECTION WITH CB-EPI IMMOBILIZED BI-ENZYME SYSTEM (Incubation time 5 min).

y = -0.0126x + 1.9791

R2 = 0.9733

1

1.2

1.4

1.6

1.8

2

0 5 10 15 20 25

Carbaryl Concentration (ppb)

Lo

g(R

esid

ua

l A

ctivity)



RESIDUAL ACTIVITY vs INCUBATION TIME &CARBOFURAN CONCENTRATION

0

20

40

60

80

100

120

0 5 10 15 20 25 30 35

Incubation Time (min)

Re

sid

ua

l A

ctivity (

%)

1 ppb

10 ppb

20 ppb

50 ppb



CALIBRATION CURVE FOR CARBOFURAN DETECTION WITHCB-EPI IMMOBILIZED BI-ENZYME SYSTEM (Incubation time 5 min)

y = -0.0288x + 1.9648

R2 = 0.9887

1

1.2

1.4

1.6

1.8

2

0 5 10 15 20 25

Carbofuran Concentration (ppb)

Lo

g(R

esid

ua

l A

ctivity)



PROPERTIES

•For both Carbofuran and Carbaryl there is a logarithmic relationbetween the residual activity and pesticide concentration unlikealdicarb where a linear relation was observed (Kok et al, 2002).

•Carbaryl and Carbofuran (0-20 ppb) had a shorter workingrange compared to aldicarb (0-250 ppb).

•Although bi-enzyme system was found to be able to detect all threepesticides, its sensitivity towards carbofuran was highest.

•Minimum detection limit is 0.4 ppb for carbofuran and 1.9 ppb forcarbaryl. These values indicate that the sensitivity towards aldicarbwas significantly lower in comparison to carbaryl and carbofuran(11.7 ppb).



EXPERIMENTAL AND THEORETICAL INHIBITION VALUES FORTHE DETECTION OF BINARY PESTICIDE MIXTURES

WITH ACHE-CHO BIOSENSOR

AS-10 ppb AS-50 ppb

Experimental

(E)

Theoretical

(T)

(E/T)*100 Experimental

(E)

Theoretical

(T)

(E/T)*100

AS+CL CL -1 ppb 18.5 23.9 77.4 28.3 38.7 73.1

CL-10 ppb 31.2 38.8 80.4 39.6 53.6 73.9

CL-20 ppb 54.4 58.1 93.6 61.2 72.9 83.9

AS-CF CF -1 ppb 21.2 30.0 70.7 32.6 44.8 72.8

CF-10 ppb 57.6 65.9 87.4 62.4 80.7 77.3

CF-20 ppb 81.2 86.1 94.3 88.3 100.9 87.5



PESTICIDE MIXTURE RESULTS

• The total degree of the inhibition by Aldicarb+Carbaryl andAldicarb+Carbofuran is more close to the theoretical valueswhen low aldicarb concentrations were used (10 ppb).

• Aldicarb caused lower inhibition than the other two pesticidesat the same concentrations.



BY CREATION OF PHYSICALLY ANDCHEMICALLY DISTINCT REGIONS ONSURFACES

HOW NANOTECHNOLOGY COULD BEINCORPORATED INTO BIOSENSORS?



CREATION OF PHYSICALLY SEPARATED REGIONS ON

ELECTRODE SURFACES



PRODUCTION OF MICROPATTERNED TEMPLATES

Þ Micropatterned Si template with parallel grooves (ridge width 27mm, groove width 2 and 12 mm alternating and groove depth 20 m

m) was prepared by photolithography



PHOTOLITHOGRAPHY

Ü Spin coated Photoresist

ÜPhotomask with defined pattern

Uniform UV exposure

Ü Silicon Wafer

Immersion intodeveloper solution

Positive Photoresist Negative Photoresist



CREATION OF CHEMICALLY DISTINCT REGIONS ON

ELECTRODE SURFACES



PATTERNING ON 2D

http://www.cheme.cornell.edu/~saltzman/Projects/Tissue_Engineering/Polymers_for_cell_assembly/polymers_for_cell_assembly.html

QUALITATIVE DETECTION:COOMASSIE BLUE STAINING



DNA SENSOR

SAM deposition of DNA probeby single drops onto thephotopatterned samples.Detects complementaryoligonucleotide sequences.

thiooligonucleotides

LANGMUIR BLODGETT FILMS (1)

LANGMUIR BLODGETT FILMS (2)

Phospholipids are thereforeDeposited on ionicallyconductive polyacrylamidegiving a structure capableof electrochemical responseGOD and monoamine oxidase

Were incorporated into monolayersof a variety ofcationic amphiphilic polyelectrolytesbased on PEI or PVN

SMART HYDROGEL

Three components of the novel hydrogel composite matrix-the crosslinked hydrogel,the positively charged electroactive ppy component and the negatively charged(net) oxidase enzyme

biosensor construction and molecular ...dna biosensor (ii-1) vasif hasirci metu turkey 40 biomat 1st...

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