Biosensores

Biosensors and the Nanoscale
Processing of Biomolecules
Valtencir Zucolotto
Laboratrio de Nanomedicina e Nanotoxicologia
Instituto de Fsica de So Carlos - USP
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BRASONN Nov/2011
Prof. Dr. Valtencir Zucolotto

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Biosensors
Basic Concepts in Biosensing
Classification
Immobilization Mechanisms
Detection Techniques
Applications of Biosensors
Final Remarks
Analytical Parameters

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Biosensors
World Analytical Market is 12 bi/year
30% of that amount is related to Health Care
Biosensors: 0.1% of HC market with na annual
increase rate of 60%
OBS: Around 6% of occidental people suffer from
diabetics and may benefit with the development of
specific biosensors.
Source: http://www.lsbu.ac.uk/biology/enztech/biosensors.html

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Biosensors
CONTOUR Blood Glucose Meter
http://www.bayercarediabetes.com/us/prodserv/products/contour/index.asp
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Components of a Biosensor
Analyte
Immobilized
biological material
Signal
Processing
Output
Transductor
Reference
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Classification of Biosensors
Recognition Mechanism
Bioreceptor
Antigen
Antibodies
DNA
Enzymes
Interaction
Piezo/gravimetric
Electrochemical
Impedantiometric
Optical (Luminescence,
absorption, SPR)
Signal
Transducer

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Immobilization of the biological species on the

transducer
Y Y Y
YY Y Y Y
Transducer

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Biomolecules Immobilization
. Entrapment
. Covalent Binding
. Cross-Linking
. Electrostatic Interactions, H bonds
. Specific Inteactions: Avidin-Biotin
. Incorporation in Lipidic Membranes

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Biomolecules Immobilization
Immobilization
Mechanism
Biomolecule
Orientation
Packing
Efficiency
Accessibility
Mobility
Biological
Activity

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Protein Immobilization via Layered Thin

Films

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Immobilization via Thin Films
The Electrostatic Layer-by-Layer Technique

The Langmuir-Blodgett Technique
Self Assembled Monolayers (SAMs)

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1. The Langmuir Blodgett Technique
-Fabrication of ultrathin (organized) films;

-Amphiphilic molecules in a volatile solvent
spread on a water sub-phase with further
compression by mobile barriers (not a
spontaneous process);

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-After compression, the molecules can undergo phase transition
forming a dense monolayer (Langmuir film);
-Requires sophisticated, costly equipment (Langmuir Trough).
Surface Pressure
Mobile Barriers
Area per Molecule
KSV Instruments Application Note # 107

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Monolayers may be transferred to a solid platform upon immersion
and withdrawal of a solid substrate (Langmuir-Blodgett LB film).
The films can be molecularly thin as

in the case of phospholipds films.
Highly control over film thickness.
KSV Instruments Application Note # 107

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Useful for incorporation of proteins in the lipidic films.
Substrate

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2. Self-Assembled Monolayers
Monolayers spontaneously adsorbed on
a solid surface
Silanization: Adsorption of silanes on silica-based

substrates
Thiolation: Adsorption of thiol-based molecules on
specific (metallic) surfaces (ex: gold)

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Advantages
Experimental simplicity and organization at the molecular level;

Higly stable and dense monolayers;
Appropriate environment for biological applications: cell growth
and tissue engineering;
Versatility: Various functionalized molecules can be used to
functionalize the surface.

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2.1 Silanization
Substrates:
Glass, Quartz, Silicon.
Self-organization is driven by
polysilanes formation, which
are linked to silanol groups ay
the surface

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2.2 Functionalized Thiols
Cysteine
Substrates:
Au, Pt, Cu, Ag.
3-Mercaptopropionic acid

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Applications in Biosensing: SAMs as linkers for

Biomolecules Immobilization (protein, enzyme,
DNA, antibody, peptide, etc.)
YY Y YY
Transducer

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2. Electrostatic Self-Assembly
Iler (1966): Assembly of Colloidal particles
bearing opposite charges
Sagiv (1983): Chemically Adsorbed MultiLayers:

Required Bifunctionalization

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Bilayer.
Monolayer
Activated Monolayer
Ativaction
O
O
Adsorption
O
Si
Si
Si
Si
OH
OH
Adsorption
Si
Si
O
Si
Si
O
Solid Substrate

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Dechers Approach (1992): Polymeric Layers
Assembled via electrostatic Attraction
Build up of organized multilayer

thin films based upon the
electrostatic interaction btween
polycationic and polyanionic
species

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Simplicity and versatility

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Polyelectrolytes
PAH
H
( C
PDAC
CH 2 ) n
PEI
H
N
H2
C
H2
C
CH 2
n
N+
NH3 Cl
H3C
ClCH3
( CH )
PSS
SO3-Na+
PVS
n
Dendrimers
SO3-Na +

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Experimental Parameters that Influence
Film Properties
-Solution Concentration;
-Ionic Strength;
-Immersion time;
-Drying
-Solution pH.

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Applications in Biosensing

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Adsorption Mechanisms
1) Films from highly charged polyelectrolytes:

Adsorption via electrostatic interactios; Molecules in
an extended configuration: Very thin layers;
2) Films from partially charged polyelectrolytes:

Amount of charges controlled by pH; Highly control
over layer thickness (pH-dependent)

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3) Films assembled via secondary interactions:

Adsorption mainly driven by H-bond, Van der waals,
etc
4) Films assembled via specific

Biotin/Avidin
functionalized
Antigens/antibodies systems, etc.
interactions:
molecules,

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Transduction Methods

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Analyte
Immobilized
biological material
Signal
Processing
Output
Transductor
Reference
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Quartz Crystal Microbalance (QCM)
Monitoring the amount of adsorbed material in real time upon changes in the
oscilation frequency of a quartz crystal
Correlation between adsorbed mass and frequency given by Sauerbry
equation:
Espcies
no antgena
Antgeno
Anticorpo
Eletrodo (Au)
Protena
Substrato de cristal Piezeltrico
2f 2
0
f
A q q
m
filme
Eletrodo (Au)
q = Mdulo de cisalhamento do quartzo,

f0 = Freq. natural oscilao quartzo,
q = Densidade do cristal
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Enzyme: Alcohol dehydrogenase (ADH) from yeast
(E.C. 1.1.1.1), molecular weight of 141-151 kDa
0
1500
1250
F (Hz)
750
m (ng)
1000
IDE/(PAMAM/ADH)n
Side View
500
250
0
Number of bilayers
10
Deposition
139.5 ng per bilayer
52.1 ng attributed to ADH.
A.C. Perinotto et al., Thin Solid Films, 2008

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Optical Detection
Colorimetry;
Photoluminescence (PL);
SPRs

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Surface Plamon Resonance (SPR)
Based on the total internal reflection of ligth and resonance of the plasmon
band;
Variations in the local refractive index at the surface of a metallic thin
film deposited on a dielectric;

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Determination of Ka and Kd;
Determination of the adsorbed mass;
(mg / m ) d f
2
n f nbuffer
dn dc
nf = film refracive index,

df = film thickness
dn/dc ~ 0.2 cm3/g; nf ~ 1,57

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Electrochemical Methods
Monitoring of the redox reactions occurring between immobilized
species and the analyte
Amp
Volt
Cottrell Equation:
Contra-Eletrodo
1
nFAD 2 C
id (t )
1
(t ) 2
Eletrodo
Referncia
Eletrodo de
Trabalho

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35
30
20
15
I /A
I / nA
25
-120.0n
10
1,2 nA
Intervalo linear
50 s
-150.0n
2400
t/s
0
0.0
0.1
0.2
0.3
0.4
0.5
-1
Concentrao de glicose / mmol L
Crespilho, et al., Electrochemistry Communications, 2006

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Field Effect Transistors (FETs) Platforms
Detection of analytes using the Field Effect Transistor (FETs)

Architecture
Conversion: Biological Response
Electrical properties
(Conductance ) Variation

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Impedanciometric Detection
Variation in the dielectrical properties of a matrix containing the
immobilized biological molecules deposited between capacitor plates.
Eletrodos Metlicos
Material Biolgico

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Impedanciometric Detection
Combination of capacitors: Interdigitated electrodes

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Capacitance
Frequency
Antgeno
Anticorpo

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Application of biomoleculescontaining films in biosensing

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AuNPs / Enzyme complexes

Detection of Glucose
ITO-(PVS/PAMAM-Au@CoHCF@GOx)3
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AuNPs / Enzyme complexes

Amp
Volt
Contra-Eletrodo
Glucose Detection
Eletrodo
Referncia
35
30
Eletrodo de
Trabalho
20
15
I /A
I / nA
25
-120.0n
10
1,2 nA
Intervalo linear
Linear Response: 10-100 mol L-1

Sensibility: 115,11 nA mmol L-1
LOD = 5,5 mol L-1
50 s
-150.0n
2400
t/s
0
0.0
0.1
0.2
0.3
0.4
0.5
-1
Concentrao de glicose / mmol L
Crespilho, et al., Electrochemistry Communications, 2006

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Nanowires/Enzyme Complexes
alumina
Deposio de ouro
Contato
eltrico
2
Eletrodo
3
Eletrodeposio nanofios
4
5
Remoo
Alumina
Nanofios
orientados
6

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Nanowires/Enzyme Complexes
Glucose Detection
Detection limit: 22 M
(at 0.0 V).
Apparent MichaelisMenten constant : 2 mM
Crespilho, et al., J. Phys. Chem., 2009

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AgNPs / Enzyme complexes

Urea Detection
ITO
PAni
Urease
PVA-AgNp
ITO/PAni/Urease
Urea injection (mmol L-1)

2.0
1.0
5.0 1.0
1.5
ITO/PAni/PVA-Ag/Urease
NH4+
2 A
NH4+
e-
PAni+ + NH4+
diffusion
NH4+
NH4+
NH4+
50
Time / s
100
PAniNH4+

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Phthalocyanine/Enzyme Complexes
Tyrosinase
Mixed Langmuir Film
LB FIlm
ITO
ITO Electrode covered with LB Immobilized
Tyrosinase
J. Mater Chem., 2011, 21, 4995

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Phthalocyanine/Enzyme Complexes
J. Mater Chem., 2011, 21, 4995

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FET Devices
Electrochemical Ion Sensitive FETs containig CNTs
and Penicilinase for Penicilin Detection
Siqueira Jr. et al, Biosens. Bioelectron, 2009
PAMAM
dendrimer
SWNT-COOH
Penicillinase
Fernandes, EG et al, J. Phys. Chem C, 2010

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FET Devices
Ta2O5
SiO2
p-Si
Al
EIS Architecture
6-bilayer PAMAM/SWNT LbL film

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FETs Devices
Sensitivity of 55 mV/pH (Ta2O5: 56-58 mV/pH)
Stability and fast response

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Catechol Biosensors
-Immobilization of Cl-catechol 1,2 dioxygenase (CCD) and
Polyamidoamine G4 Dendrimer (PAMAM)
0.30
-2
ABS at 260 nm
10 M Catechol
0.020
0.25
0.20
0.15
0.10
-11
0.05
10
M Catechol
0.00
0
0.015
20
40
60
80
100
Time (S)
0.010
-11
10
M Catechol
0.005
Optical Detection
0.000
0
10
20
30
40
Zucolotto et al., Biosens. and Bioelectron., 21, 1320, 2006.

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Catechol Biosensors
100 Hz
-7
2x10
-7
C (F)
1x10
-8
8x10
Mili-Q Water
- 10
10 M Catechol
-8
10 M
-6
10 M
-4
10 M
-2
10 M
Electrical Detection
-8
4x10
0
Bare Electrode
PAMAM/PVS
PAMAM/CCD
Sensing Units
Zucolotto et al., Biosensors and Bioelectronics, 21, 1320, 2006.

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Ethanol Biosensors
Enzyme: Alcohol dehydrogenase (ADH) from yeast
(E.C. 1.1.1.1), molecular weight of 141-151 kDa
0
1500
1250
F (Hz)
750
m (ng)
1000
500
250
0
10
Number of bilayers
Deposition
139.5 ng per bilayer
52.1 ng attributed to ADH.
A.C. Perinotto et al., Thin Solid Films, 2008

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Pasteurellosis Detection
Pasteurellosis: Zoonosis caused by Pasteurella multocida
Strategy
-Incorporation of Pasteurella multocida membrane protein into
liposomes.
-Proteoliposome immobilization in LbL films.
Substrate
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Pasteurellosis Detection
100 Hz
PC2 (23.1%)
IgG
IgG
IgG+ = IgG anti-P. multocida

IgG- = IgG anti-sheep erythrocytes
PC1 (73.8%)
Zucolotto et al., Analytical Chemistry 79, 2163, 2007.

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A biosensor for Thyroid hormone

Immobilization of TRB1 TH nuclear receptor for Thyroid
hormone detection
Substrato de vidro
Trilhas de ouro
Monocamadas
de tiol
Eletrodo
interdigitado
Monocamadas
de silano

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A biosensor for Thyroid hormone
[C] = 50,0 nM at 100 Hz

A
Bendo et al., Submitted.

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Final Remarks

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Industrial Applications of Biosensors

Biomolecule
Analyte
Transducer
LOD
Aplication
26 M
2.4 M
Soft Drinks
Honey
Glucose oxidase Glucose

Fructose
Frutose
dehydrogenase
Amperometric
Amperometric
Alcohol
dehydrogenase
Ethanol
Amperometric
Glycerol
dehydrogenase
Glycerol
Absorbance
8 mg/L
Wine
Cholesterol
oxidase
Sulfite oxidase
Cholesterol
Fluorescence
70 M
Butter
Sulfite
Amperometric
4 ppm
Water
Monoamino
oxidase
Histamine
Amperometric
Acetyl
cholinesterase
Pesticides
Termometric
0.2 ng/ml
Fruits,
Vegetables,
Carboxy
peptidase
Penicillin G
SPR
5.2 g/kg
Milk
Wine, Beer
Fish, Meat,
Wine, Beer
Sensors and Actuators B 102 (2004) 179194

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Analytical Parameters
Response Signal
Sensitivity (SEN)
Increase in the response signal per a specific amount of analyte
[C]
Limit of Detection (LOD)
Lowest amount of analyte a biosensor can properly recognize
LOD = 3Sb/SEN
(Sb = standard deviation for blank measurements*,

SEN = Sensitivity)
*When blank analysis gives
non-zero deviation
Limit of Quantification (LOQ)

What is lowest amount of analyte a biosensor can quantify?
LOD = 10Sb/SEN
Thevenot et al., Pure Appl. Chem., Vol. 71, 12, 2333, 1999.
Long et al., Analytical Chemistry, 55, 7, 1983

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Important Parameters
Specificity, Selectivity
How specific a biosensor can be?
Response time
Typically 90% of steady-state response
Reproducibility
Measure of the scatter in consecutive measurements
Stability
Lifetime
Storage vs operation conditions
Reusability
Are the biosensors reusable or disposable?
Long et al., Analytical Chemistry, 55, 7, 1983

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Sources
An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to SelfAssembly, Abraham Ulman, Academic Press (April
28, 1991).
Biomaterials Science, 2nd Edition : An Introduction to Materials in
Medicine by BD. Ratner, A.S. Hoffman, F. J. Schoen, J. E. Lemons
Academic Press (7/29/2004).
Biosensors: Theory and Applications, by Donald G. Buerk (Author)
CRC; 1st edition (August 24, 1995) ISBN-10: 0877629757, ISBN-13:
978-0877629757.
Long et al., Analytical Chemistry, 55, 7, 1983.

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Muito Obrigado
Valtencir Zucolotto
zuco@ifsc.usp.br
www.nanomedicina.com.br
www.lnn.ifsc.usp.br
www.twitter.com/nanomedicina
Instituto de Fsica de So Carlos - USP

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Biosensors and the Nanoscale

Prof. Dr. Valtencir Zucolotto

Prof. Dr. Valtencir Zucolotto

Prof. Dr. Valtencir Zucolotto

CONTOUR Blood Glucose Meter

Prof. Dr. Valtencir Zucolotto

Immobilization of the biological species on the

Prof. Dr. Valtencir Zucolotto

Prof. Dr. Valtencir Zucolotto

Prof. Dr. Valtencir Zucolotto

Protein Immobilization via Layered Thin

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

The Electrostatic Layer-by-Layer Technique

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

-Fabrication of ultrathin (organized) films;

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Area per Molecule

KSV Instruments Application Note # 107

Immobilization via Thin Films

The films can be molecularly thin as

KSV Instruments Application Note # 107

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Silanization: Adsorption of silanes on silica-based

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Experimental simplicity and organization at the molecular level;

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

2.2 Functionalized Thiols

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Applications in Biosensing: SAMs as linkers for

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Sagiv (1983): Chemically Adsorbed MultiLayers:

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Build up of organized multilayer

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

1) Films from highly charged polyelectrolytes:

2) Films from partially charged polyelectrolytes:

Prof. Dr. Valtencir Zucolotto

Immobilization via Thin Films

3) Films assembled via secondary interactions:

4) Films assembled via specific