BIOELECTROCHEMISTRY
ELECTROCHEMICAL ANALYSIS OF NUCLEIC ACIDS, PROTEINS AND
POLYSACCHARIDES IN BIOMEDICINE
Iveta Třísková
Outline
 Introduction to electrochemical methods
 Electrochemistry of nucleic acids and their components
 Electrochemistry of proteins
 Electrochemistry of polysaccharides
 Biosensors
 Nanoelectrochemistry
Electron transfer
Electrolysis
Battery
Redox reactions
Electron transfer system
Introduction to electrochemical
methods
Polarography
•1922 – Jaroslav Heyrovský
•Electrolysis of the electroactive compound in the supporting
electrolyte
•The potential is insert between working (Hg) and reference
electrode (Ag/AgCl/3M KCl)
•Polarographic wave
•Electrode polarization
Polarographic (voltammetric) currents
Charging current
• Important for electrode double layer charging
• Non-faradayic character
Migration current
• Associated with transport of electroactive compound to the electrode
surface
• Eliminated with addition of big amount of supporting electrolyte
Diffusion current
• For reactions when the rate determinig step rds is diffusion
• 1934 – Dionýz Ilkovič
x
c
zFAD
dt
dn
zFId
cmkzFDI 6/13/22/1
D
Illkovič equation
Cyclic voltammetry and Linear sweep
voltammetry
• Electrolysis of electroactive compounds in the supporting electrolyte
• Three electrode set
•dE/dt (scan rate)
• Voltammetric signals have a peak shape
• The study of redox reactions – mechanism of electrode reaction and its
reversibility
Cyclic voltammetry
•Reversible processes: Randles – Ševčík equation
2/10
ox
2/12/35
p vcADn1069,2I
when Ip is peak current(A); n number of electrones; A effective area of electrode(cm2);
D is diffusion coeffient (cm2/s); cox is concentration(mol/cm3) a v is scan rate (V/s).
•Irreversible processes: Delahay equation
2/1v0
oxc2/1DA2/1
ann51099,2pI
kde α is charge transfer coefficient; na is number of electrones in rate determinig
step(rds)
9
Elimination voltammetric procedure
• Elimination voltammetric procedure (EVP) – developed parralel
with elimination polarography (EP), but compared to EP, EVP is
easier, faster and it is possible to applicate it at solid electrodes
• EVP – mathematic procedure eliminating/conserving some of
partial voltammetric currents (diffusion, charging, kinetic) from
measured LSV or CV curves
• Elimination function as linear combination of total currents
measured at different scan rates
10
2nd condition
vWEYI jjj
k
j
jII
1
I = Id + Ik + Ic + ...…
1st condition
0
ν)E(YI kk
1
ν)E(YI cc
21
ν)E(YI ddId …diffusion current
Ik …kinetic current
Ic …charging current
xx
νconst.νY(E)I
-1.45 -1.35 -1.25
25
50
100
200
300
400
500
600
800
1000
f (I) = - 11,657 I1/2 + 17,485 I - 5,8284 I2
Ik + Ic = 0 Id ≠ 0EVP E4
The two basic conditions of EVP
d
ref
c
ref
k
ref
vrefv I
v
v
I
v
v
I
v
v
I
2110
Adsorptive transfer stripping
voltammetry (AdTSV)
DNA or RNA
adsorption
PA buffer
Rinsing in MILI Q
water
pH 1.8, 5.8 or 12.8
ta = 120 s
Voltammetric analysis in
PA buffer; pH 5.8
11
Differential pulse voltammetry
doba kapky
I1
I2
Potential,V
time, t Potential, VI2-I1,A
ΔI = I2 – I1
Square-wave voltammetry
•Ramaley and Krause
PotentialE
Ei
0
Forward
sample
ΔEs
ΔEp
tp
Backward
sample
Time
ΔI = If - Ib
Itotal
Iforward
Ibackward
14
Equipments
• Electrochemical analyzers:
 AUTOLAB PGSTAT 20 (Eco Chemie, Utrecht, The Netherland)
 μAUTOLAB TYPE III (Metrohm, Switzerland)
• GPES Manager 4.9
• Hanging Mercury Drop Electrode (HMDE)
• Polymer pencil graphite electrode(pPeGE)
Electrodes
• Hanging Mercury Drop Electrode (HMDE)
•Graphite electrodes
Basal plane pyrolytic graphite
electrodes
Polymer pencil graphite electrode
Pyrolytic graphite electrodes
Edge plane pyrolytic graphite
electrodes
Inner structure of EPPG a BPPG electrodes
Carbon fibre electrodes
Glassy carbon electrodes• Graphite electrodes
Carbon paste electrodes
Boron dopped diamond
electrodes
• Screen printed electrodes
Nucleic acids
Nucleic acids and their components
N
NH2
O
R
NNH
N
O
O
R
CH3
N
N
N
NH2
R
N
N
N
N
O
R
NH
NH2
3’5’
purine bases
thymine (T)
pyrimidine bases
cytosine (C) adenine (A) guanine (G)
DNA double helix
base
pairs
sugar-
phosphate
backbone
major
groove
minor
grooveBasepairs
C•G
T•A
A
B C
D
O
O
P-O
O
O
baseCH2
2-deoxyribose
phosphate
nucleotide
minor groove
major groove
1953: James Watson, Francis Crick, Rosalind Franklin,
Maurice Wilkins: DNA double helix
1962: Nobel prize (JW, FC, MW)
Explanation of the basic principles of preservation,
transmission and expression of genetic information
20
Hairpins
LOOP
STEM
A
G A
C G
G C
5' 3'
Tm of DNA heptamer = 76 °C
(0.1 M NaCl)
The shortest and thermodynamically the most stable hairpin DNA
heptamer d(GCGAAGC) – replication origins of phage ФX 174
and herpes simplex virus, promotor regions of heat – shock genes
of bacteria E. coli and rRNA genes
20
21
Hairpins
• Hairpins are linked with triplet repeats expansion associated
with many neurodegenerative diseases (fragile chromosome
syndrom, Huntington disease, Friedriech’s ataxia)
Analysis of hairpins:
d(GCGAAGC): UV, Tm (Hirao 1989, Yoshizawa 1994, 1997), NMR (Hirao
1994, Yoshizawa 1997, Padrta 2002, Sychrovský 2002),
Ramanova spektroskopie (Chraibi 2000),
electrophoresis (Hirao 1989, Yoshizawa 1994, 1997), CD
(Hirao 1989), X – ray analysis (Sunami 2004), molecular
dynamics (Nakamura 1999, Padrta 2002),
electrochemistry (Trnková 2004)
21
22
G - quadruplexes
• Stabilized by G-quartets (four molecules of guanine bound by
Hoogsteen hydrogen bonds)
• Especially formed in presence of Na+ a K+ ions
• Structural polymorphism
N
N
N
N
N
O
H
H
H
R
N
N
N N
N
O
H
H
H
R N
N
N
N
N
O
H
H
H
R
N
N
NN
N
O
H H
H
R
M+
N
N
N
N
N
O
H
H
H
R
N
N
N N
N
O
H
H
H
R N
N
N
N
N
O
H
H
H
R
N
N
NN
N
O
H H
H
R
M+
22
a) Four - stranded tetraplex
b) Double - stranded intra-molecular tetraplex
c) Single - stranded intra-molecular tetraplex
23
I - motifs
• Hemiprotonized C – C+ pair as the basic structural unit
• Formed at acidic or neutral pH
• Diabetes mellitus and triplet repeats expansion linked with many
neurodegenerative diseases
• Structural polymorphism
N
+
N
N
O
H
H H
R
N
N
N
O
HH
R
C+
C
N
+
N
N
O
H
H H
R
N
N
N
O
HH
R
C+
C
23
a) Double-stranded structure
b) Four-stranded structure
c) Four-stranded structure with labeled ends
More than 55 years ago, Emil Paleček: DNA polarography (1960)
Oscillopolarography
• Mercury electrode: redox processes of A,C a G bases
• Carbon electrodes: oxidation of purine and pyrimidine bases
• Copper electrode: oxidation of sugar moieties in NA
Singhal, P.; Kuhr, W. G.: Anal. Chem. 1997, 69, 3552-3557; Anal. Chem. 1997, 69,
4828-4832.
Nucleic acids are electroactive
Electrochemistry of NA and ODN
Paleček, E., Bartošík, M.:
Electrochemistry of Nucleic
Acids. Chem. Rew., 2012
d(GCGAAGC)
DNA
DNA vs. RNA
-1
-0,8
-0,6
-0,4
-0,2
0
-1500 -1400 -1300 -1200 -1100 -1000
I/µA
E/mV
-0,05
0,05
0,15
-500 -400 -300 -200 -100 0
I/µA
E/mV
A B
A + C
G signály
DNA
RNA
redukce
oxidace
28
DNA heptamers with different sequence
in molecule center
-0,05
0
0,05
0,1
0,15
-350 -250 -150 -50
E /mV
I/μA
-0,8
-0,6
-0,4
-0,2
0
0,2
0,4
-350 -300 -250 -200 -150 -100 -50
E /mV
f(I)
LSV
GI
GII
EVLS
A
B
-0,05
0
0,05
0,1
0,15
-350 -250 -150 -50
E /mV
I/μA
-0,8
-0,6
-0,4
-0,2
0
0,2
0,4
-350 -300 -250 -200 -150 -100 -50
E /mV
f(I)
-0,05
0
0,05
0,1
0,15
-350 -250 -150 -50
E /mV
I/μA
-0,8
-0,6
-0,4
-0,2
0
0,2
0,4
-350 -300 -250 -200 -150 -100 -50
E /mV
f(I)
LSV
GI
GII
EVLS
A
B EVP
AAA CCC GAA GGG
LSV
EVP
29
The pH effect for d(GCGAAGC)
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0,08
-500 -400 -300 -200 -100 0
I/µA
E/mV
-1
-0,9
-0,8
-0,7
-0,6
-0,5
-0,4
-0,3
-0,2
-0,1
0
-1600 -1400 -1200 -1000
I/µA
E/mV
A B
A + C
GI
GII4,2
5,12
5,5
5,6
5,99
6,1
30
The concentration effect for (GCGAGC)
-1
-0,9
-0,8
-0,7
-0,6
-0,5
-0,4
-0,3
-0,2
-0,1
0
-1600 -1500 -1400 -1300 -1200 -1100 -1000
E/mV
I/µA
-0,05
-0,03
-0,01
0,01
0,03
0,05
0,07
0,09
0,11
-500 -400 -300 -200 -100 0
E/mV
I/µA
-0,4
-0,3
-0,2
-0,1
0
0,1
0,2
0,3
0,4
0,5
-400 -350 -300 -250 -200 -150 -100 -50
E/mV
f(I)
-1,5
-1
-0,5
0
0,5
1
-1500 -1450 -1400 -1350 -1300 -1250 -1200
E/mV
f(I)
LSV
D
B
A
A+C
C
GI
GII
EVLSEVLS
LSV
-1
-0,9
-0,8
-0,7
-0,6
-0,5
-0,4
-0,3
-0,2
-0,1
0
-1600 -1500 -1400 -1300 -1200 -1100 -1000
E/mV
I/µA
-0,05
-0,03
-0,01
0,01
0,03
0,05
0,07
0,09
0,11
-500 -400 -300 -200 -100 0
E/mV
I/µA
-0,4
-0,3
-0,2
-0,1
0
0,1
0,2
0,3
0,4
0,5
-400 -350 -300 -250 -200 -150 -100 -50
E/mV
f(I)
-1,5
-1
-0,5
0
0,5
1
-1500 -1450 -1400 -1350 -1300 -1250 -1200
E/mV
f(I)
LSV
D
B
A
A+C
C
GI
GII
EVLSEVLS
LSV
EVP EVP
45 nmol·l-1
90 nmol·l-1
135 nmol·l-1
180 nmol·l-1
225 nmol·l-1
315 nmol·l-1
405 nmol·l-1
495 nmol·l-1
675 nmol·l-1
855 nmol·l-1
0
0.02
0.04
0.06
0.08
0.1
0 2 4 6 8 10
Ip
c
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
-350 -250 -150
f(I)
E/mV
ELIMINATION
E1
E6
E5
ADSORPTION
Electrochemistry of purine and purine
derivatives
• The typical electroactive compounds
• 1962 - Smith a Elving – the first study of electrochemical reduction of
purine and adenine by using polarography and coulometry on mercury
dropped electrode (DME).
• The two-step 2e- reduction
32
N
1
2
6
N
3
5
4 8
N
7
N
H
9
+2H+
+ 2e-
N
N
N
N
H
H
H
H
+2H+
+ 2e-
N
N
N
N
H
H
H
H
H
H
H
purin 1,6 -dihydropurin
1,2,3,6 -tetrahydropurin
N
1
2
6
N
3
5
4 8
N
7
N
H
9
+2H+
+ 2e-
N
N
N
N
H
H
H
H
+2H+
+ 2e-
N
N
N
N
H
H
H
H
H
H
H
purin 1,6 -dihydropurin
1,2,3,6 -tetrahydropurin
Electrochemical reduction of adenine
2,3 – dihydropurin (IV)
N
N
NH2
N
N
H
+2H+
+ 2e-
N
N
NH2
N
N
H
H
H +2H+
+ 2e-
N
N
H
NH2
N
N
H
H
H
H
H
-NH3 N
N
H
N
N
H
H
H
+2H+
+ 2e-
N
N
H
N
N
H
H
H
H
H
H
H2ON
N
N
N
H
H
H
H
H
OH
H
6-aminopurin (I)
1,6 – dihydro – 6 – aminopurin
(II)
1,2,3,6 – tetrahydro – 6 – aminopurin
(III)
1,2,3,6 – tetrahydropurin (V)(VI)
2,3 – dihydropurin (IV)
N
N
NH2
N
N
H
+2H+
+ 2e-
N
N
NH2
N
N
H
H
H +2H+
+ 2e-
N
N
H
NH2
N
N
H
H
H
H
H
-NH3 N
N
H
N
N
H
H
H
+2H+
+ 2e-
N
N
H
N
N
H
H
H
H
H
H
H2ON
N
N
N
H
H
H
H
H
OH
H
6-aminopurin (I)
1,6 – dihydro – 6 – aminopurin
(II)
1,2,3,6 – tetrahydro – 6 – aminopurin
(III)
1,2,3,6 – tetrahydropurin (V)(VI)
•Smith and Elving (1962)
•6 e- reduction process is accompanied
by deamination process (e.g.
coulometry)
•Polarographic reduction is finished in
point III (formation of 1,2,3,6-tetrahydro-
6-aminopurine)
•Problem of adenine reduction at pH > 6
Electrochemical reduction of guanine
guanine 7, 8 - dihydrogenguanine
d(GCGAAGC)
Cathodic part
Anodic part
N
NH
NH2
O
+H+
-H+
N
+
NH
NH2
O
H +2e-
-2e-
NH
NH
NH2
O
+H+
NH
NH
NH2
O
H
-NH3N
NHO
+H+
-H+
N
+
NHO
H
+e-
-e-
N
NHO
H
+H+
+e-
1/2
N
NHO
H
H
N
NH O
H
H
N
NHO
H
H
H
(I) (II) (III)
(IV)(V)
(IX)
(VI)
(VII) (VIII)
N
NH
NH2
O
+H+
-H+
N
+
NH
NH2
O
H +2e-
-2e-
NH
NH
NH2
O
+H+
NH
NH
NH2
O
H
-NH3N
NHO
+H+
-H+
N
+
NHO
H
+e-
-e-
N
NHO
H
+H+
+e-
1/2
N
NHO
H
H
N
NH O
H
H
N
NHO
H
H
H
(I) (II) (III)
(IV)(V)
(IX)
(VI)
(VII) (VIII)
Electrohemical reduction of cytosine
•Elving (1972)
•Reduction is initialized by fast protonation of
cytosine(I) in N-3 position to electroactive
form(II). The two-electron reduction of N-3=C-4
follows and karbanion (III) is formed.
•The reduction in polarography and volatmmetry
is finished by 4-amino-
3,4-dihydrogenpyrimidine-2-on (IV) formation
•The other intermediates is possible to obtain by
using electrolysis or coulometry
-2.00
-1.80
-1.60
-1.40
-1.20
-1.00
-0.80
-0.60
-0.40
-0.20
0.00
-1500 -1400 -1300 -1200 -1100 -1000
I/A
E /mV
pH 1.55
pH 1.84
pH 2.15
pH 2.72
pH 3.22
pH 4.24
pH 4.85
pH 5.61
pH 6.14
pH 7.2
Electrochemical oxidation of adenine
N
N
N
N
H
NH2
+H2O
-2H+
- 2e-
N
N
H
N
N
H
NH2
O
+H2O -2H+
- 2e-
N
N
H
NH
N
H
NH2
O O
N
N
H
N
N
NH2
O O
-2H+
- 2eadenin
2 – oxyadenin
2,8 – dioxyadenindiimin
N
N
N
N
H
NH2
+H2O
-2H+
- 2e-
N
N
H
N
N
H
NH2
O
+H2O -2H+
- 2e-
N
N
H
NH
N
H
NH2
O O
N
N
H
N
N
NH2
O O
-2H+
- 2eadenin
2 – oxyadenin
2,8 – dioxyadenindiimin
0
10
20
30
40
50
60
700 800 900 1000 1100 1200 1300
I/µA
E/mV
3,14 3,29
3,67 4,16
4,55 5,10
5,35 5,80
6,80 7,54
•Dryhurst, Compton
•6e- and 6 H+ electrode process
37(V.Sharma, F. Jelen and L. Trnkova: Sensors, 2015, 15(1), 1564-1600)
38
The complex formation and its oxidation can be described by the following scheme:
1. Cu(II) + e- → Cu(I) (at a deposition potential of 0.15 V)
2. Cu(I) + purine → [Cu(I)-purine] (in the reaction layer on PeGE surface)
3. [Cu(I)-purine] → [Cu(I)-purine]ads (adsorption of the complex)
4. [Cu(I)-purine]ads – e- → [Cu(II)-purine]ads (oxidative stripping, peak OxCom)
5. [Cu(II)-purine]ads – e- → purineox + Cu(II) (oxidative stripping, peak Ox)
Figure 6: Concentration dependence of 1-mXan in the solution with constant concentration
of Cu(II) ions, cCu = 20 µM , reference scan rate 400 mV/s; phosphate-acetatebuffer pH 5.1
0
20
40
60
80
100
120
140
160
180
200
0 200 400 600 800 1000 1200 1400
I[µA]
E[mV]
Formation of the complex Cu(I)-1-mXan
0 µM
0.5 µM
1 µM
2 µM
3 µM
5 µM
7.5 µM
10 µM
15 µM
20 µM
40 µM
0
40
80
120
160
0 10 20 30 40
I[µA]
c[μM]
Electrochemical oxidation of guanine
NH
NNH2
O
N
N
H
+H2O
-2H+
- 2e-
NH
NNH2
O
NH
N
H
O
-2H+
- 2e-
2H+
+ 2e-
NH
NNH2
O
N
N O
guanin 8 – oxyguanin
diimin
NH
NNH2
O
N
N
H
+H2O
-2H+
- 2e-
NH
NNH2
O
NH
N
H
O
-2H+
- 2e-
2H+
+ 2e-
NH
NNH2
O
N
N O
guanin 8 – oxyguanin
diimin
• Dryhurst
•4e- and 4 H+ electrode process
0
10
20
30
40
50
60
70
500 700 900 1100 1300
I/µA
E/mV
G pH 3.12 G pH 3.3 G pH 3.71
G pH 4.12 G pH 4.64 G pH 5.06
G pH 5.35 G pH 5.7 G pH 6.81
G pH 7.54
Before 1990…
Year
1962-1966 ssDNA and dsDNA resolution
1967 DNA damage detection
1967
Interaction of DNA with low
molecular weigth ligands
1978 Application of solid electrodes
1981-1983
DNA labeling with electroactive
substance
1986 DNA-modified electrodes
…after 1990
• The big expansion in electrochemistry of NAs due to the considerable progress in
genomics (Human Genome Project)
• The synthesis of DNA probes – electrochemical detection of hybridization
• Later, approaches targeted to improvement of sensitivity and reproducibility of
analysis:
 ELISA analogy (A)
 Molecular beacon (B)
 Nanotechnology (C) – inorganic NPs, carbon nanotubes, grafen
• Detection of oncogenes, tumor suppressor genes, mononucleotide polymorphism,
repetitive sequence, viral and bacterial NAs, genetically modified organisms
Bartošík M., Paleček E., Vojtěšek B.: Klin Onkol, 2014, 27 (Suppl 1), S53-S60
Electrochemistry of NA in oncology
• Interaction of DNA with antitumor drugs
• 2000 – Brabec – electrochemical biosensor based on carbon electrodes – the monitoring
of guanine oxidation signal decrease due to platinum derivatives establishing
•Antitumor drugs yield electrochemical signal too
Brabec V.: Electrochim Acta, 2000, 45, 2929-2932
Horakova P., Tesnohlidkova L., Havran L. et al.: Anal Chem, 2010, 82, 2969-2976
Monitoring of selective cisplatination of probe oligonucleotides in the presence of competitor
plasmid DNA using magnetoseparation
and AdTS CV. (A) Separation of the cisplatinated ODN probes using magnetic beads. The
recovered ODNs were analyzed by AdTS CV. (B) Sections of AdTS CVs obtained for the ODNs
noG (black) or G25 (red) treated with cisplatin (rb ) 0.1) in the mixture with plasmid DNA. (C)
Dependence of the current value measured at the anodic part of the AdTS CV at -1.3 V on the
concentration of cisplatin used for modification of the ODN probes in the presence of plasmid
DNA: noG (black); G25 (red).
DNA methylation
•Epigenetic modification playing important role in gene expression
•Changed methylation patterns of DNA associated with carcinogenesis
•Cytosine methylation – 1) reaction with NaHSO3(cytosine is deaminated to uracil,
methylcytosine is not changed), after that amplification of DNA and m-DNA by PCR (uracil
is amplificated as thymin and methylcytosine as cytosine) and finally electrochemical
detection by using suitable redox labels
2) After reaction with NaHSO3 electrochemical reduction of DNA at Hg and solid amalgam
electrodes (uracil is unreducible at Hg electrode, but methylcytosine is reducible at Hg
electrode→after reaction with NaHSO3 m - DNA yields higher signal than DNA)
Bartošík M., Fojta M., Paleček E.: Electrochim Acta, 2012, 78, 75-81
Bartošík M., Fojta M., Paleček E.: Electrochim Acta, 2012, 78, 75-81
• Guanine methylation – at first electrochemical reduction at Hg electrode,
later boron – doped diamond electrodes and carbon electrodes
DNA methylation
miRNA
Why to study miRNA?
• Gene expression regulation
• Regulation of processes during tumor
grow
• Present in all human tissues and fluids
– easily accessible
• Other miRNA between healthy and
diseased individuals
• Oncomarker?
• miRNA as a drug
Cancer, cardiovascular diseases,
neurodegenerative diseases
•DNA probes and enzymatic or NPs labels for signal amplification
•The method using miRNA labeling by using electroactive complex on the base of
hexavalent osmium
Electrochemical detection of miRNA
Proteins
Primary structure
The order of AA in polypeptide
chain
Secondary structure
The geometrical arrangement
of polypeptide chain
Tertiary structure
The spatial arrangement of
polypeptide chain
-helix
β – folded sheet
Quaternary structure
Subunits in protein agglomerates
forming one functional protein
Proteins are electroactive
• The first biomacromolecules investigated by using electrochemical methods
• Herles and Vančura – polarographic study of human body fluids (blood serum
and urine). „Presodium wave“ – cathodic wave occuring at potentials more
positive (300 mV) that cathodic reduction of sodium ions. Preliminarily this
wave was assigned to proteins
• Heyrovský and Babička – albumin in presence of ammonium ions produces in
dc polarography so called „presodium wave“ (H peak), caused by catalytic
hydrogen evolution reaction
• „Presodium wave“ not suitable for analytical purposes
Polarographic catalytic waves of human serum. (2) the
“presodium” catalytic wave in 0.1 M ammonia/ammonium
chloride
Brdička‘s catalytic reaction (BCR)
•1933 – Brdička‘s catalytic reaction – polarographic double – wave of proteins
containing Cys residues in buffered solutions of cobalt (Brdička solution – ammonium
buffer NH4OH + NH4Cl and cobalt complex [Co(NH3)6]Cl3)
•Originally designed for detection sulphur rich substances, such as organic compounds
(2- mercaptopropionic acid, 2-diethylaminoethanethiol hydrochloridum), amino acids
(cysteine, cystine) and proteins (albumin)
•Application of Brdička‘s catalytic reaction in clinical medicine and pharmacology (cancer
diagnostic)
•Mechanism of electrode process of Brdička catalytic reaction is not known in details,
but it is propossed that complex Co(II) with –NH2 and –SH moieties plays a key role
Polarographic catalytic waves of human
serum. (4) the catalytic double-wave in
Brdička solution
What is catalytic hydrogen evolution reaction
(CHER)?
2PH(surf) + 2e- → 2P(surf)
+ H2(g)
P(surf)
+ BH(aq)↔ PH(surf) + B-(aq)
PH a P-: protonized/deprotonized form of AA residues in protein molecule
BH is acid component of buffer; B- is its conjugated basis
• The reaction shows, that catalyst is protein immobilized on the electrode surface
• Basic AA - Cys, Lys, Arg a His residues of protein molecule – catalyze the hydrogen
evolution on the Hg electrode
•Electrochemical phenomenon caused by a catalyst, in which presence the hydrogen
evaluates at the cathode polarized to more positive potentials than in the catalyst
absence
•The hydrogen evolution is produced by cathodic catalytic current. The current intensity
is depend on the catalyst concentration and the kinetic catalyst efficiency
H peak
• In recent years – The „presodium wave“ (J. Heyrovsky) in combination with CPSA
(chronopotentiometric stripping analysis) at stationary and chemically modified Hg
electrodes (amalgam electrode included) – suitable tool for proteins analysis
• Catalytic signal - H peak (discovered due to catalytic hydrogen evolution reaction –
CHER; named according J. Heyrovsky) – is sensitive to structural and conformational
changes of proteins
• H peak (by proteins) used to monitoring of denaturation, agreggation, interaction with
low molecular weight ligands or DNA, structural changes as the result of mutation and
redox state
CPSA – chronopotentiometric
stripping analysis
)(Ef
dt
dE
Electrochemical oxidation of proteins
• Free aminoacids (Cys, His, Met, Tyr a Trp) are oxidized at carbon electrodes
• Proteins are oxidized at carbon electrodes (CPSA method)
• Tyr a Trp residues in proteins yield oxidation signals at carbon electrodes → the study
of DNA-protein interaction, the resolution of fosforylated and unfosforylated forms,
membrane Na-K pump, determination of insuline and α-synuklein (important protein
in the Parkinson‘s disease)
OH
NH2
O
OH -2e, -2H+
NH2
O
OH
O
Tyrosine
-2e, -2H+
N
H
NH2
OH
O
N NH2
OH
O
Tryptophan
e
Tyr
Trp
Bartošík M., Paleček E., Vojtěšek B.: Klin Onkol, 2014, 27 (Suppl 1), S53-S60
Proteins are electroactive
•External protein labeling (sensitive detection of specific proteins in the mixture of other
molecules)
Immunoassays (ELISA)
•Nanotechnology – nanoparticles, nanotubes
•Aptamers
Bartošík M., Paleček E., Vojtěšek B.: Klin
Onkol, 2014, 27 (Suppl 1), S53-S60
Bartošík M., Paleček E., Vojtěšek B.: Klin
Onkol, 2014, 27 (Suppl 1), S53-S60
Polysaccharides
Polysaccharides
• Naturally occuring polysaccharides (PSs) and oligosaccharides (OLSs) are free or fixed on
proteins or lipides
•Structural flexibility – ideal indetificators of intermolecular or intercells interactions
•Most mammalian proteins occur in the form of glycoproteins
•Protein glykosylation in the human health and diaseases (cancer)
Are polysaccharides electroactive?
• Since 2009 PSs and OLSs considered as electrochemical inactive compounds
•In 2009 – some sulphated PSs catalyze hydrogen evolution reaction and give CPS signals
at Hg electrodes
•PSs and OLSs are easy modifiable with Os(VI)L complexes (with nitrogen ligands);
electroactive aducts
•Lectine biosensors for glycane detection(sugar residues of glycoproteins and
glycolipides); electrochemical impedance spectroscopy (EIS) biosensors
Easy and quick detection of oncomarkers and other proteins important in
biomedicine
Bartošík M., Paleček E., Vojtěšek B.: Klin Onkol, 2014, 27 (Suppl 1), S53-S60
Biosensors
What is biosensor?
• Analytical instrument containing senstivite bioreceptor, which is the part of
physicochemical tranducer or it is in the close proximity with physicochemical
transducer
Bioreceptor TransducerAnalyte Electronic signal
Biosensor
•Bioreceptors
Biocatalytic (enzyme, organela, cell, tissue, organ, organism) – analyte is
conversed during chemical reaction
 Bioaffinity (lectine, antibody, NA, receptor) – analyte is specifically
bound in bioaffinity complex
•Physicochemical transducers – electrochemical, optical, piezoelectric and
acoustic, calorimetric
From the history
•The beginning of the 20th century – the conception of redox potentials and the
fisrt pH measurement
•1922 – Jaroslav Heyrovský – discovery of polarography
•1935 – Müller and Bamberger – measurement of O2 concentration in biological
fluids at Hg electrode
•1938 – Petering and Daniels – measurement of O2 consumption with living
oragnisms at Hg electrode
•40s of 20th century – cathodic reduction of O2 at noble metals (Au, Pt) – bare
electrodes lost their lifetime in the biological material
•1956 – Leland C. Clark Jr. – the first membrane electrode permeable for gases
→ the birth of biosensors
•1962 – Clark and Lyons – enzyme electrode (experiment with glukose oxidase
immobilized on the oxygen electrode surface by the dialysis membrane)
•60s of the 20th century – ion-selective electrodes (ISE)
•70s of the 20th century – progress in the field of enzyme electrodes
•1975 – the first commercial biosensor for glukose (Yellow Springs Instrument
Company)
•The end of 70s – the beginning of the immunosensors research
•Biosensors emerge from the scientific laboratories into the real world!
Discovery of biosensor
•Clark (1962) – amperometric sensor for glucose with glucose oxidase and oxygen
electrode
Glucose + O2 → Gluconic acid + H2O2
GOD
Oxygen electrode (1956)
Working electrode: Pt cathode
O2 + H2O + 2e- → H2O2 + 2OHH2O2
+ 2e- → 2OHReference
electrode: Ag/AgCl electrode
The electrodes are separated from the
measured solution with semipermeable
membrane enabling passage of gasis
Requirements for biosensors
•Sensitivity
•Calibration
•Linearity
•Limit of detection
•Noise
•Background signal
•Hysteresis
•Long time stability
•Selectivity
•Response rate
•Response time
•Convection rate
•Temperature dependence
•Lifetime of the biosensor
•Biocompatibility
Measurement conditions
• Direct contact with a sample – biosensor in the monitored medium (river,
tissue, bloodstream)
•Closed vessel– biosensor in the vessel equipped with the water coat (due to
tempering) and magnetic stirrer
•Flow system – biosensor in the flowing cell
Types of biosensors
•Optical biosensors
•Piezoelectric biosensors
•Calorimetric biosensors
•Electrochemical biosensors
•Enzyme biosensors
Electrochemical biosensors
1) Amperometric and voltammetric biosensors
Electrochemical biosensors
2) Potentiometric biosensors
 ISE with enzyme surface
enzyme
jz/iz
jiji
0
akaln
nF
RT
EE Nicolsky – Eisenman equation
Electrochemical biosensors
3) Conductometric/impedimetric biosensors
Nanoelectrochemistry
Elektrochemické metody využívající modifikace povrhu
elektrod nanočásticemi
Voltametrické metody (klasické elektrody i mikroelektrody)
Potenciometrie – příprava ISE, senzorického pole a elektronického jazyka
Imobilizace na povrchu elektrod se provádí buď fyzikální adsorpcí nebo pomocí chemického
navázání, kdy modifikované nanočástice reagují s povrchem elektrody, na jejímž povrchu je
navázaná vhodná látka
Schéma imobilizace nanočástic zlata na povrch modifikované zlaté
elektrody
Stanovení dusičnanů, měďnatých iontů, pesticidů a
herbicidů ve vodách
Biosenzory ve farmacii a lékařství
Farmacie – stanovení léčiv
Lékařství – analýza biologických vzorků
(hemoglobin, cytochrom c, glukosa, peroxid vodíku)
DNA diagnostika
Gold nanoparticles
•Attractive electronic, optical, thermal and catalytic properties
•Potential applications in the fields of physics, chemistry, biology, medicine and material
science and their interdisciplinary fields
•The unique physical and chemical properties of nanostructured materials provide
excellent prospects for interfacting biological recognition events with electronic signal
transduction and for designing a new generation of biosensors.
•Especially AuNPs represent excellent biocompatibility and display unique structural,
electronic, magnetic, optical and catalytic properties – attractive material for biosensor,
chemisensor and electrocatalyst
•The use of AuNPs for amperometric or voltammetric electrochemical nanobiosensors
AuNPs quantitative analysis following its
production and application
•Very important subject in bioelectrochemistry –
construction of biochemical sensors
•Direct electrochemistry of proteins (very important is
establishment of satisfactory electrical communication
between the active site of the enzyme and the electrode
surface)
•Modification of electrode surfaces with the AuNPs will
provide a microenvironment similar to that redox-proteins
in native systems and gives the protein molecules more
freedom in orientation, thereby reducing the insulating
effect of the protein shell through the conducting tunnels
of AuNPs
•1996 – Natan and co-workers - electrochemistry of horse
heart cytochrome c at SnO2 electrodes modified with 12
nm AuNPS
Gold nanoparticles-based electrochemical sensor and
bioelectrochemical sensor
Direct electrochemistry of redox-protein on AuNPs
Possible ways of coupling an
enzymatic and an electrochemical
reactions:
a) mediated electron exchange and
b) direct, mediatorless, electron
transfer.
Gold nanoparticles-based electrochemical sensor and
bioelectrochemical sensor
Direct electrochemistry of redox-protein on AuNPs
•The nanoparticle/protein conjugates can be assembled on the electrode via self-assembly
technology – the AuNPs could be immobilized on the self-assembled monolayer and
complete DET
•DET of hemoglobin on the citrated-capped AuNPs assembled on a cysteamine
modified gold substrate
•Investigation of electrocatalytic activity of NPs/hemoglobin electrode towards
H2O2 reduction
•DET of glucose oxidase and HRP on AuNPs immobilized cysteinamine modified
gold electrode
•The AuNPs modifed carbon paste electrodes have provided a good microenvironment for
completing the DET of different redox-proteins
• DET between immoblized myoglobin and colloidal gold modified carbon paste
electrode
•Xanthin oxidase biosensor, based on a carbon paste electrode modified with
electrodeposited AuNPs for the amperometric determination of hypoxanthine
•The polymer-nanoparticles composites possess the interesting electrical, optical and
magnetic properties superior to those of the parent polymer and nanoparticles
•The nanocomposite composed of AuNPs and biopolymer such as chitosan as excellent
matrix for completing the DET of some redox protein
•Biocomposite made of chitosan hydrogel, GOD and AuNPs for glucose biosensor
Gold nanoparticles in DNA immobilization
•AuNPS can strongly adsorb DNA
•DNA can also be immobilized onto AuNPs through special functional groups such as
thiols and others, which can interact strongly with AuNPs
•DNA oligonucleotides that contain several adenosyl phosphothiolate residues at their
ends have been used to interact with the metal surface of NPs
Schematic of the methods used for conjugating oligonucleotides to gold nanoparticles. a) Thiol-modified and b)
disulfidemodified oligonucleotides spontaneously bind to gold nanoparticle surfaces. Asymmetric disulfide
modification adds an additional mercaptoalcohol ligand to the Au surface, but the density of oligonucleotides
formed on the nanoparticle surface is the same as for thiol-terminal oligonucleotides. c) Di and d) trisulfide
modified conjugates. e) Oligothiol – nanoparticle conjugates. Although four thiol connections are shown, any
number are possible via sequential addition of a commercial dithiane phosphoramidite during solid-phase
oligonucleotide synthesis. f) Oligonucleotide conjugates from NanoprobesQ phosphine-modified nanoparticles.
Adapted from Nanotechnology, 2003, 14, R63.
Gold nanoparticles in DNA immobilization
• Monomaleimido gold clusters have been coupled with thiolated DNA oligomers to
synthesize probes for homogenous nucleic acid analyses and ensure a 1:1 DNA/AuNP
connection with interest for sensitivity improvements
Schematics of A) Formation of particle-linked DNA network structure due to the interconnection
between magnetic beads in the case where AuNPs modified with more than one DNA strands
are used; B) The previous network is not created by using the 1:1 Au-DNA connection; C) The
reaction of maleimido-Au67 with thiol-oligonulceotide that make possible the 1:1 Au-DNA
connection. Adaped from Langmuir , 2005, 21, 9625
Gold nanoparticles-based electrochemical sensor and
bioelectrochemical sensor
Gold nanoparticles for genosensors
• The development of electrical DNA hybridization biosensors has attracted considerable
research efforts
•The AuNPs modified electrochemical sensing interfaces offer elegant ways for interacting
DNA recognition events with electrochemical signal transduction, and for amplyfying the
resulting electrical response
•AuNPs –based electrochemical device will provide new opportunity for gene diagnostics
•Merkoci and co-workers reviewed recent important achievements on the electrochemical
sensing of DNA using AuNPs
Schematic procedure of the different strategies used for the
integration of AuNPs into DNA sensing systems: (A) previous
dissolving of AuNPs by using HBr/Br2 mixture followed by
Au(III) ions detection, (B) direct detection of AuNPs
anchored onto the surface of the genosensor, (C)
conductometric detection, (D) enhancement with silver or
gold followed by detection, (E) AuNPs as carriers of other
AuNPs, (F) AuNPs as carriers of other electroactive labels.
Reprinted with permission from Ref. [85], A. Merkoc¸i, 19
(2007) 743. Copyright Wiley-VCH (2007).
Gold nanoparticles-based electrochemical sensor and
bioelectrochemical sensor
Gold nanoparticles for genosensors
•The use of colloidal gold tags for electronic detection of DNA hybridization – capturing
the AuNPs to the hybridized target, followed by highly sensitive anodic stripping
electrochemical measurement of the metal tracer
•Due to the toxicity of HBr/Br2 solution the novel AuNPs –based protocol was reported –
a novel AuNPs – based protocol for detection of DNA hybridization based on
magnetically trigged direct electrochemical detection of gold quantum dot tracers
•Enhancement by precipitation of silver or gold onto the AuNPs for amplifying signals
and lowering detection limits
•Analyzing sequence-specific DNA using AuNPs marked DNA probes and subsequent
signal amplification step by silver enhancement (electrostatic adsorption of target ODNs
onto the sensing surface of the GCE and its hybridization to the AuNPs-labeled ODNs
DNA probe)
•Another signal amplification strategy is to attach electroactive ferrocenylhexanethiol
molecules or electrogenerated chemiluminiscence indicator to the AuNPs labels
•The AuNPs/streptavidin conjugates covered with 6-ferrocenylhexanethiol
were attached onto a biotinylated DNA detection probe of a sandwich DNA
complex
•DNA ultrasensitive electrochemical detection by using AuNPs will play an important role
on the development of specific and sensitive assays for clinical diagnosis, detection of
pathogenic microorganisms
Gold nanoparticles-based electrochemical sensor and
bioelectrochemical sensor
Gold nanoparticles for immunosensors
•Immunosensors are important analytical tools based on the detection of the binding
event between antibody and antigen
•Electrochemical immunosensors are attractive tools and have received considerable
attention becuase they are easy, economy, robust and achieve excellent detection limits
with small analyte volumes
•Several strategies have been proposed to develop electrochemical immunosensors with
high sensitivity using AuNPs
•DNA –free ultrasensitive electrochemical immunosensors have received considerable
interests because of their simplify, rapidness and high sensitivity
(a) Schematic representation of the
preparation of an immunosensing layer. (b)
Schemiatic view of electrochemical detection
of mouse IgG or prostate specific antigen.
Reprinted with permission from Ref. [139], H.
Yang, J. Am. Chem. Soc. 128 (2006) 16022
Gold nanoparticles as enhancing platform for
electrocatalysis and electrochemical sensor
•AuNPs have been studied extensively for the design and fabrication of electrocatalysts
and using as an enhancing component of catalytic activity or selectivity
Scheme illustrating (a) the formation of Ru-AuNPs in
aqueous medium due to electrostatic interactions
between Ru(bpy)3
2+ and citrate-capped AuNPs and
(b) the immobilization of Ru-AuNPs on a sulfhydrylderivated
ITO electrode surface.
Platinum nanoparticles
•Platinum nanoparticles have been an intensive research subject for the design of
electrodes
•Platinum films modified microelectrodes were shown to be excellent amperometric
sensor for H2O2 in a wide range of concentration
•Pt nanoparticles and single walled carbon nanotubes were combined to modify a glassy
carbon electrode to improve their electroactivity for H2O2 (glucose sensor)
•Pt nanoparticles were used in combination with multi-walled carbon nanotubes
(MWCNTs) for fabricating sensitivity-enhanced electrochemical DNA biosensor
Carbon nanotubes (CNTs)
•CNTs are built from sp2 carbon units – a seamless structure with hexagonal honeycomb
lattices
•Carbon nanotubes with one hundered time the tensile strength of steel, thermal
conductivity, electrical conductivity similar to copper, but with the ability to carry much
higher currents, they seem to be very interesting material
•Since their discovery in 1991, CNTs have generated great interest for applications based
on their field emission and electronic transport properties, their hiugh mechanical
strength and chemical properties
•CNTs appliccation in the field of emission devices, nanoscale transistors, tips for
scanning microscopy or components for composite materials
•Two groups of CNTs: A) multi-wall carbon nanotubes (MWCNTs) – concentric and
closed graphite tubules with multiple layers of graphite sheet
B) single-wall carbon nanotubes (SWCNTs) – single graphite sheet
rolled seamlessly, defining a cylinder of 1-2 nm diameter
Schematic representation of Single Walled Carbon Nanotube
(SWCNT) and Multi Walled Carbon Nanotube (MWCNT)
•CNTs exhibits strong electrocatalytic activity for a wide range of compounds, such as
neurotransmitters NADH, hydrogen peroxide, ascorbic, cytochrome c, hydrazines,
hydrogen suphide, amino acids, glucose and DNA
•Various types of CNT modified electrodes were prepared including physical adsorption
of CNT onto electrode surface, like glassy carbon and composite paste electrodes
•CNT was incorporated into an epoxy polymer, forming an epoxy composite hybrid
material as a new electrode with improved electrochemical sensing properties (CNTEC
– carbon-nanotube epoxy composite)
•SWCNT-glassy carbon modified electrode for the highly sensitive and selective
detection of dopac in the presence of 5-hydroxytryptamin
•SWCNT film modified glassy carbon electrodes towards the reduction/oxidation of
cytochrome c
•MWCNT biosensor was obtained by means of GOx immobilization through physical
entrapment inside and epoxy resin matrix and its performance was examined for
glucose determination
•MWCNTEC modified with bacterial cells for application as a microbial sensor
•CNTPE (carbon nanotubes paste electrode) for determination of dopamine, ascorbic
acid, dopac, uric acid and hydrogen peroxide
Carbon nanotubes