MASARYKOVA
UNIVERZITA
P Ř Í R O D O V Ě D E C K Á FAKULTA
Nanočástice pro detekci
acetylcholinesterasy
Diplomová práce
ONDŘEJ ŠABACKÝ
Vedoucí práce: doc. RNDr. Petr Skládal, CSc.
Ústav biochemie
Specializace: Analytická biochemie
Brno 2021
Bibliografický záznam
Autor:
Název práce:
Studijní program:
Specializace:
Vedoucí práce:
Rok:
Počet stran:
Klíčová slova:
Ondřej Šabacký
Přírodovědecká fakulta
Masarykova univerzita
Ústav biochemie
Nanočástice pro detekci acetylcholinesterasy
Biochemie
Analytická biochemie
doc. RNDr. Petr Skládal, CSc.
2021
66
acetylcholinesterasa, inhibitory acetylcholinesterasy,
elektrochemie, stříbrné nanočástice, ITO
Bibliographic record
Author:
Title of Thesis:
Degree Programme:
Specialization:
Supervisor:
Year:
Number of Pages:
Keywords:
Ondfej Sabacky
Faculty of Science
Masaryk University
Department of Biochemistry
Nanoparticles for detection of acetylcholinesterase
Biochemistry
Analytical biochemistry
doc. RNDr. Petr Skladal, CSc.
2021
66
acetylcholinesterase, acetylcholinesterase inhibitors,
electrochemistry, silver nanoparticles, ITO
Abstrakt
Tato diplomová práce se zabývá problematikou stanovení inhibitorů
acetylcholinesterasy (AChE) pomocí biosenzorů s AChE imobilizovanou na jejich
povrchu. Jako modelový inhibitor byl použit eserin (physostigmin).
V praktické části byly nejdříve stanoveny amperometrické odezvy na ITO
(cínem dopovaný oxid inditý) páscích a platinových elektrodách s imobilizovanou
AChE s použitím substrátu acetylthiocholinu (ATCh). Dále byly připraveny stříbrné
nanočástice stabilizované citrátem sodným a draselným (citrate-capped AgNPs) a
jejich velikost stanovena pomocí mikroskopie atomárních sil (AFM). Tyto nanočástice
byly využity pro stanovení inhibice AChE vyvolané agregační reakcí. Průběh agregace
těchto nanočástic byl také ověřen pomocí Ramanovy spektroskopie.
Nakonec byly u platinových elektrod s imobilizovanou AChE pomocí cyklické
voltametrie (CV) stanoveny rozdíly v odezvách na různé koncentrace inhibitoru
eserinu.
Abstract
This diploma thesis deals with the determination of acetylcholinesterase (AChE)
inhibitors using biosensors with surface-immobilized AChE. Eserine (physostigmine)
was used as a model inhibitor.
In the practical part, amperometric responses for ITO (indium tin oxide) strips
and platinum electrodes with surface-immobilized AChE were first determined using
acetylthiocholine substrate (ATCh). Furthermore, silver nanoparticles stabilized with
sodium and potassium citrate (citrate-capped AgNPs) were prepared, and their size
was characterized using atomic force microscopy (AFM). These nanoparticles were
further used for the determination of AChE inhibition triggered by aggregation reaction.
The course of aggregation of these nanoparticles was also verified by Raman
spectroscopy.
Finally, for platinum electrodes with surface-immobilized AChE, differences in
responses to different concentrations of eserine inhibitor were determined by cyclic
voltammetry (CV).
MUNI
SCI
ummovA u N L V E uz I I A
P í l R U D O V E D E C krt F A K U L T A
KOT L A.Ě S K A í , S i l 37 BRNO
I C : 0Ů5AS 53J
Z A D Á N Í
DIPLOMOVÉ PRÁCE
Akademický rok; 2020/2021
U s Lav; Ustav biochemie
Studení: Bc. Ondřej Sabacky
Program; Biochemie
Specializace: Analytická biochemie
Ředitel ůslavu PFF MU Vám ve smyslu Studijního a zkušebního řádu MU určuje diplomovou práci s názvem:
Název práce: Nanoíástice pro detekci acetylcholinesterase
Název p race an gl leky: Nan oparticles for detection of acelylchol I nesterase
J azyk záverečné p race: ang I liti na
Oficiální zadání:
Klasické stanovení aktivity acelyle holi neste rasy (AChE) využívá hydrolytlcké štěpení aeetylcholinu a redox n I reakci
vzniklého thloehollnu s Elmanovým činidlem za vzniku žlutého zbarvení; fotometrické méření přitom nepatří mezi
riejcltllvéjší. Mnohem nižěí aktivity lze přitom stanovil s využitím nanoiástic stříbra (Ag NP)_ Thlocholln může sloužit
k jej leh tvorbé ii následné agregaci, což lze výhodne sledoval. C íle m práce bude po rovnat Jedn otl lve stratég le spoje n í
AChE s Ag N P delekeí, vybral 1 až 2 systémy a ly pak porovnal v kombinovaném měření na bázi průhledné ITO
elektrody Bude tak možné paralelně sledoval elektrické změny (Impedance, vollametrle) čl optickou odezvu [změna
fluorescence rl barvy}. Optimalizovaný systém bude vyzkoušen pro stanovení inhibitorů AChE fInsekticidy, léílva) a
nebo spPažen s modelovým Imunosensorem využívajícím AChE jako značku.
Literatura
Xia N. Nanomaterlals-Based Optical Techniques for the Detection of Acetylcholinesterase and Pesticides. Sensors
2015,15, 499-514.
LI Z. Unmodified silver nanopartlcles for rapid analysis of the organophosphorus pesticide, diplerex, often found in
different waters. Sensors and Actuators B 2014.193, 205-211.
Liu R. Application of gold-silver nanocluster based fluorescent sensors for determination of acetylcholinesterase activity
and Its Inhibitor. Materials Research Express. 2018, 5.065027.
Kumar ND. Acetylcholinesterase [AChE)-medlated Immobilization of silver nanopartlcles for the detection of organophosphorus
pesticides RSC Advances 2016, 6, 64769-64777.
Vedoucí práce: doc. PiNDr. Petr Skládal. CSc.
Datum zadáni práce: 15.10.2019
V B m é dne: 13. 5. 2021
Zadání bylo schváleno prostřednictvím IS MU.
Be. Ondřej Šabacký, 3. 2021
doc. RNDr. Petr Skládal, CSc, 14.4.2021
RNDr. Jílka Kaíparovská, Ph.D., 11. 5 2021
Acknowledgments
I would like to express my gratitude to doc. RNDr. Petr Skládal, CSc. for his
help, patience, willingness, and valuable advice he provided me with during the
creation of this work. I would also like to express my appreciation to Ing. Jakub Máčala
and Mgr. Matěj Pastucha for their help with practical experiments.
Declaration
Prohlašuji, že jsem svoji práci vypracoval samostatně pod vedením vedoucího práce
s využitím informačních zdrojů, které jsou v práci citovány.
Brno May 31, 2021
Ondřej Šabacký
Table of Contents
GLOSSARY 12
1 INTRODUCTION 13
2 ACETYLCHOLINESTERASE 14
2.1 Determination of the activity of AChE 18
2.2 Inhibition of AChE 19
2.3 Inhibitors of AChE 20
2.3.1 Reversible AChE inhibitors 20
2.3.2 Irreversible AChE inhibitors (organophosphorus compounds) 22
3 SILVER NANOPARTICLES (AGNPS) 24
4 INDIUM TIN OXIDE (ITO) 25
5 BIOSENSORS 26
5.1 Definition of the biosensor 26
5.2 History of biosensors 26
5.3 Physical-chemical converter 27
5.4 Basic concepts 28
5.5 Enzyme immobilization 29
6 ELECTROCHEMICAL METHODS 30
6.1 Amperometry 31
6.2 Cyclic voltammetry 31
7 AIMS AND OBJECTIVES 33
8 MATERIALS AND METHODS 34
8.1 Apparatus 34
8.2 Reagents 36
8.3 Methodology 37
8.3.1 Preparation of phosphate-buffered saline (PBS) 37
8.3.2 Synthesis of citrate-capped AgNPs 37
8.3.3 Immobilization of AChE on ITO-coated PET foil 37
8.3.4 Immobilization of AChE on platinum electrodes (AC1 .W2.RS) 38
8.3.5 Amperometric measurements 38
8
8.3.6 Cyclic voltammetry measurements 40
8.3.7 Spectrophotometric measurements 41
8.3.8 AFM analysis of citrate-capped AgNPs 41
8.3.9 Raman spectroscopy analysis of citrate-capped AgNPs 41
9 RESULTS AND DISCUSSION 42
9.1 Results of amperometric measurements with platinum electrodes 42
9.2 Results of amperometric measurements with ITO strips 47
9.3 CV measurements with platinum electrodes 52
9.4 Spectrophotometric measurements of citrate-capped AgNPs 54
9.5 AFM 57
9.6 Raman spectroscopy 60
10 CONCLUSION 62
11 BIBLIOGRAPHY 63
9
List of Figures and Tables
Figure 1 - Native AChE from Torpedo californica. [2] 14
Figure 2 - AChE from Electrophorus electricus (tetramer). [4] 15
Figure 3 - ACh E at the synaptic cleft. [6] 17
Figure 4 - Mechanism of AChE-catalyzed hydrolysis of ACh. [1] 18
Figure 5 - General structure of biologically active carbamates. [1] 22
Figure 6 - General structure of OPs. [1 ] 23
Figure 7 - Structural formulas of selected nerve agents and DDVP. [11] 24
Figure 8 - Schematic illustration of the novel analysis of organophosphorus pesticides
(OPs) based on the inhibition of an enzyme and the use of the aggregation
process of citrate-capped AgNPs. [16] 25
Figure 9 - Cyclic voltammetry. [26] 32
Figure 10 - USB Potentiostat EmStat 34
Figure 11 - Amperometric sensor AC1 .W2.RS 34
Figure 12 - Spectrophotometer NanoPhotometer® NP 80 35
Figure 13 - Diagram of measuring apparatus 39
Figure 14 - Diagram of measuring apparatus 40
Figure 15 - Amperometric response to ATCh 43
Figure 16 - Amperometric response to ATCh 44
Figure 17 - Amperometric response to ATCh 45
Figure 18 - Amperometric responses to ATCh all in one graph 46
Figure 19 - Amperometric responses ATCh on an electrode without immobilized
enzyme (control) 46
Figure 20 - Connection of the flow cell using crocodile clips 47
Figure 21 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with
surface-immobilized AChE (1 nkat pi1
). Potential 600 mV 48
Figure 22 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with
surface-immobilized AChE (1 nkat pi1
). Potential 700 mV 48
Figure 23 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with
surface-immobilized AChE (1 nkat pi1
). Potential 800 mV 49
Figure 24 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with
surface-immobilized AChE (1 nkat pi1
). Potential 1000 mV 49
10
Figure 25 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with
surface-immobilized AChE (0.1 nkat uM). Potential 800 mV 50
Figure 26 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with
surface-immobilized AChE (0.01 nkat pi"1
). Potential 800 mV 51
Figure 27 - CV response to ATCh/AgNOß mixture 52
Figure 28 - CV response to ATCh/AgN03 mixture (various inhibitor concentrations).
53
Figure 29 - Current at 0.8 V (top half-wave) against eserine concentrations 54
Figure 30 - Citrate-capped AgNPs prepared using sodium citrate 55
Figure 31 - Citrate-capped AgNPs prepared using potassium citrate 55
Figure 32 - Recorded spectra for solutions prepared using AgNPs prepared with
sodium citrate 56
Figure 33 - Recorded spectra for solutions prepared using AgNPs prepared with
potassium citrate 57
Figure 34 - AFM images of citrate-capped AgNPs (sodium citrate) 58
Figure 35 - AFM images of citrate-capped AgNPs (sodium citrate) 58
Figure 36 - AFM images of citrate-capped AgNPs (potassium citrate) 59
Figure 37 - AFM images of aggregated AgNPs (potassium citrate - left, sodium citrate
-right) 59
Figure 38 - S E R S of fresh and aggregated AgNPs 60
Table I - Responses to additions of ATCh (AChE 0.01 nkat pi1
) 43
Table II - Responses to additions of ATCh (AChE 0.1 nkat pi1
) 44
Table III - Responses to additions of ATCh (AChE 1 nkat pi1
) 45
Table IV - Responses to additions of ATCh (AChE 1 nkat pi1
) 50
Table V - Responses to additions of ATCh 51
Table VI - Intensity at 1590 cm 1
60
11
Glossary
4-MBA 4-Mercaptobenzoic acid
ACh Acetylcholine
AChE Acetylcholinesterase
AD Alzheimer's disease
AFM Atomic force microscopy
AgNP(s) Silver nanoparticles
APTES (3-Aminopropyl)triethoxysilane
ATCh Acetylthiocholine (chloride)
CV Cyclic voltammetry
EA Ethanolamine
ITO Indium tin oxide (coated PET)
OPs Organophosphorus compounds
PBS Phosphate-buffered saline
SERS Surface-enhanced Raman spectroscopy
12
1 Introduction
Determination of the activity and inhibition of acetylcholinesterase is a powerful
tool in medical diagnostics of several neurodegenerative diseases, as well as pesticide
and nerve agent intoxication. Although Ellman's method has been widely used to
determine acetylcholinesterase activity, it has limits, especially for field use.
With the increasing use of pesticides based on acetylcholinesterase inhibitors,
the need arises for a simple and compact device capable of detection thereof.
Transparent ITO electrodes (ITO-coated pet foil) or the corundum ceramic-based
platinum electrodes combined with immobilized acetylcholinesterase could serve well
for this purpose.
Furthermore, attention has recently turned to silver nanoparticles for their
electrochemical and surface plasmon resonance properties. Therefore, silver
nanoparticles could act as signal enhancers for the electrochemical biosensors with
surface-immobilized acetylcholinesterase. In addition, silver nanoparticles can also be
used for the spectrophotometrical determination of acetylcholinesterase inhibition.
This thesis deals with the preparation of a biosensor capable of detecting
acetylcholinesterase inhibition while using silver nanoparticles as response signal
enhancers.
13
2 Acetylcholinesterase
Acetylcholinesterase (AChE, EC 3.1.1.7) is an enzyme of the hydrolases class
found in various sorts of electric impulse conducting tissue: nerve and muscle, central
and peripheral tissues, motor and sensory fibers, and cholinergic and noncholinergic
fibers. The activity of AChE is higher in motor neurons than in sensory neurons. [1]
Figure 1 - Native AChE from Torpedo californica. [2]
This cholinergic enzyme instantly degrades or hydrolyzes acetylcholine (ACh),
a neurotransmitter present in many living organisms, including humans, to acetic acid
and choline (Equation 1). The primary purpose of AChE is to end neuronal
transmission and signaling between synapses to prevent ACh from spreading and the
activation of nearby receptors. AChE is inhibited by organophosphates and
carbamates, which are an essential component of pesticides and nerve agents. [3]
14
acetylcholine water acetate choline
Equation 1 - AChE-catalyzed hydrolysis of ACh.
AChE is a monomer that often forms a disulfide bond dimer. Thanks to Van der
Waals forces, two dimers can be further joined into tetramers. The tetramers group
and bind themselves to what is described as "tails" composed of three chains. From a
chemical and immunological point of view, the structure of these tails resembles the
structure of collagen, and thus they can be disintegrated by collagenases. With another
disulfide bond, the tetramer dimers associate with each tail. [3]
Figure 2 - AChE from Electrophorus electricus (tetramer). [4]
The active site of AChE consists of two subsites: the anionic and esteratic
subsites. The anionic subsite serves as the binding site for the positively charged
quaternary amine of ACh. The esteratic subsite is the place where ACh becomes
hydrolyzed to acetate and choline. [5]
15
The main role of AChE is the termination of neural transmission. However,
AChE is also crucial for neural development. AChE is intricately involved in the
embryological development of the nervous system and is expressed by developing
neurons and during periods of axonal growth (a time in which enzymatic activity does
not seem to be most important). Transient AChE activity was locally present in the
dorsal root ganglions in the peripheral nervous system of chicks. These discoveries
suggest that AChE, in addition to its primary enzymatic function, contributes to
morphogenesis during fetal development. [3]
AChE is known to be distributed in nervous tissue such as the brainstem,
cerebellum, peripheral, and autonomic nervous systems. Skeletal muscles also
contain AChE with distribution patterns seemingly related to the type of muscle (fast
versus slow-twitch) and their specific function. [3]
Less commonly known is the presence and function of AChE on red blood cells.
Blood group antigens are found on the outer lipid bilayer of red blood cells, which
serves for convenient antibody recognition. Similarly, AChE is present on red blood
cell membranes as well. [3]
The neurotransmitter ACh is released when a neural signal propagates and
excites or activates a cellular membrane. The ACh receptor then undergoes a
conformational change, and the membrane releases calcium ions. These calcium ions
play a role in exciting the fibers of nerves and muscles by triggering an additional
change in phospholipids. Essentially, the downstream effect of a signal initiated with
ACh results in amplification and propagation of the cellular signaling. [3]
16
i
{
{
Acetylcholine (AChiis made
from choline ar>d acetyl CoA
In the synapse ACh is rapidly
broken down by the enzyme
acetylcholinesterase (AChE)
Choline s transported back
into the axon terminal and
used to make more ACh
Acetylcholinesterase (AChE)
Figure 3 - AChE at the synaptic cleft. [6]
The interaction of AChE with the substrate ACh results in its breakdown,
hydrolysis, inactivation, and subsequent control of the amount of ACh at the synapse.
AChE is a serine hydrolase that creates a tetrahedral intermediate through acid-base
reactions with a catalytic triad (serine 200, histidine 440, glutamate 327). Histidine
allows for the transference of a proton between the serine and ACh oxygen molecules,
thereby removing choline to form a new acylated serine. The regeneration of free AChE
begins when the acylated serine is deacylated. Aspartate stabilizes the protonated
histidine, leading to acetic acid and a new, free enzyme release. The interaction
between amino acid residues (tyrosine, phenylalanine, tryptophan) that make up a
peripheral anionic site influences the conformational binding of ACh to that site. [3]
17
Figure 4 - Mechanism of AChE-catalyzed hydrolysis of ACh. [1]
2.1 Determination of the activity of AChE
Knowledge of the current state of cholinesterase activity in the body is crucial
for the early diagnosis of organophosphate intoxication and monitoring the
effectiveness of administered therapy, especially AChE reactivators. Erythrocyte AChE
is most commonly used for this assay, as it is readily available, and its degree of
inhibition corresponds very well to the severity of poisoning. [7]
Many methodologies have been developed to determine cholinesterase activity.
The most commonly used methods are electrometric, titration, colorimetric,
determination of pH change using an indicator, spectrophotometric, fluorimetric,
radiometric, polarographic, and enzyme. However, the methods mentioned above
cannot be introduced into routine practice for many reasons, such as sample pretreatment,
long measurement times, or insufficient substrate specificity of the
enzyme. [7]
18
A susceptible and suitable method for everyday use was described by Ellman.
This colorimetric method is now used to evaluate the health status of people who
generally come into contact with organophosphorus inhibitors (industrial and
agricultural workers). [7]
The principle of this method is based on the hydrolysis of acetylthiocholine. After
enzymatic hydrolysis, acetic acid and thiocholine are released. Thiocholine containing
the SH group in its molecule is detected by 5,5'-dithiobis-2-nitrobenzoic acid (DTNB)
by releasing the yellowish 5-mercapto-2-nitrobenzoate anion (TNB) after the reaction
with thiocholine. This anion is then detected spectrophotometrically at a wavelength of
412 nm. [7]
Although the Ellman's method is fast, cheap, and straightforward, it also has its
disadvantages. Interference with hemoglobin is very crucial in measuring blood
cholinesterase activities. The absorption maximum of the color indicator TNB" is 412
nm. However, the hemoglobin present in the blood also absorbs radiation at this
wavelength. If we want to rule out such an error, a blood sample has to be significantly
diluted. Another problem is the reaction of Ellman's reagent (DTNB) with slow-reacting
sulfhydryl groups in the solution, which can affect the measurement results. [7]
These shortcomings have led to many modifications to the original Ellman
method. For example, to reduce interference with hemoglobin, two-beam
spectrophotometers or other chromogenic disulfides have been used to measure
different wavelengths. Another way to improve the determination of AChE in the blood
is to use selective BChE (butyrylcholinesterase) inhibitors such as either quinidine or
phenothiazine derivatives. [7]
2.2 Inhibition of AChE
Inhibition of hydrolysis of ACh leads to serious health risks. Inhibition or
alteration of AChE activity may occur for several reasons. Reduced levels of the
neurotransmitter ACh in the body lead to neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, or Huntington's disease. Another reason for
reduced enzyme activity may be a disorder of proteosynthesis or an inherited defect,
e.g., familial idiopathic acholinesterasemia. AChE may also be inhibited by inactivation.
The inhibitor (pesticide, nerve agent) binds to the hydroxyl group of serine at the active
19
site of AChE, thereby preventing the breakdown of ACh to acetate and choline in the
synaptic cleft. As a result of inhibition, excess ACh accumulates in the tissues, and
signal transmission is interrupted, resulting in muscle weakness, muscle paralysis,
seizures in the C N S , impaired neuromuscular disc function, and, in severe cases,
respiratory problems, fibrillation, and death. [8]
2.3 Inhibitors of AChE
AChE inhibitors inhibit the ability of cholinesterase to decompose ACh, thereby
increasing the concentration of neurotransmitter and duration of action. AChE
inhibitors are usually divided into two groups based on their mode of action: irreversible
and reversible. Reversible inhibitors, both competitive and non-competitive, find
application in therapy. Irreversible AChE inhibitors, on the other hand, have toxic
effects. [1 ]
2.3.1 Reversible AChE inhibitors
Reversible AChE inhibitors play an essential role in the pharmacological
manipulation of enzyme activity. These inhibitors include compounds with different
functional groups (carbamate, quaternary or tertiary ammonium group) and have been
used to diagnose and treat various diseases such as myasthenia gravis, AD,
postoperative ileus, bladder distension, glaucoma, as well as antidotes for
anticholinergic overdose. [1]
Alzheimer's disease treatment using reversible AChE inhibitors
Alzheimer's disease (AD) is the most common cause of dementia and accounts
for up to 80 % of all dementia diagnoses. [9] AD is a progressive neurological disorder
characterized by loss of memory and other intellectual abilities severe enough to
interfere with daily life. The disease is associated with the loss of cholinergic neurons
in the brain and decreased ACh levels. Inhibition of cerebral AChE is a primary
therapeutic target in AD treatment strategies. There is no cure for AD, and the
reversible AChE inhibitors used in therapy treat the symptoms associated with
memory, thinking, language, judgment, and other thought consequences. Various
20
physiological processes related to AD damage or cell destruction that produce and use
ACh, thereby reducing the amount of ACh available for message delivery to other cells.
Cholinesterase inhibitor drugs that inhibit AChE activity maintain ACh levels by
reducing the rate of AChE degradation. Consequently, they increase cholinergic
neurotransmission in areas of the forebrain and compensate for the loss of functional
brain cells. However, no drug indicates delaying or stopping the progression of the
disease. Drugs that are currently approved by regulatory agencies, such as the US
Food and Drug Administration (FDA) and the European Medicines Agency (EMA), to
treat cognitive manifestations of AD and improve patients' quality of life are donepezil,
rivastigmine, and galantamine, such as are reversible inhibitors of AChE and
memantine as NMDA receptor antagonists. Tacrine was the first AChE inhibitor
approved for AD treatment in 1993, but its use was discontinued due to a high
incidence of adverse events, including hepatotoxicity. [1]
Carbamates
Carbamates are organic compounds derived from carbamic acid (NH2COOH).
Carbamate compounds find use in human medicine as therapeutic drugs (AD,
myasthenia gravis, glaucoma, Lewy bodies, Parkinson's disease). In addition, these
reversible AChE inhibitors have been applied as pesticides and parasiticides in
veterinary medicine and prophylaxis of organophosphorus compounds (OPs)
poisoning. [1]
Carbamates, unlike organophosphorus pesticides, have a relatively quick
decarbamylation step which means that substantial recovery of the enzyme can occur
in a finite period of time. [5] Figure 5 shows a biologically active carbamate structure,
where X can be oxygen or sulfur (thiocarbamate), Ri and R2 are usually organic or
alkyl substituents, but R1 or R2 may also be hydrogen, and R3 is primarily an organic
substituent or sometimes a metal. [1]
21
Figure 5 - General structure of biologically active carbamates. [1]
Carbamates have a similar toxicological presentation to OP poisonings with a
duration of toxicity that is typically less than 24 hours. Common agents resulting in
toxic exposure are aldicarb, carbofuran, carbaryl, ethinenocarb, fenobucarb, oxamyl,
methomyl, pirimicarb, propoxur, and trimethacarb. [10]
2.3.2 Irreversible AChE inhibitors (organophosphorus compounds)
Organophosphorus compounds (OPs) are either oxones or thiones derived from
phosphoric, phosphonic, phosphinic, or phosphoramidic acid (Figure 6). Ri and R2 are
aryl or alkyl groups bonded to the phosphorus atom either directly (forming
phosphinates) or through an oxygen or sulfur atom (forming phosphates or
phosphothioates). R1 might sometimes be directly bonded to the phosphorus atom,
and R2 is bonded to an oxygen or sulfur atom (forming phosphonates or
thiophosphonates). In phosphoramidates, at least one of these groups is -NH2 (un-,
mono- or bi-substituted), and the atom double-bonded with phosphorus is either
oxygen or sulfur. Also binding to the phosphorus atom through oxygen or sulfur atom
is the - X group, which may be halogen, aliphatic, aromatic, or heterocyclic groups.
This leaving group is released from the phosphorus atom when the OP is hydrolyzed
by phosphotriesterases or upon interaction with protein targets. In medicine and
agriculture, the word "organophosphates" refers to a group of insecticides and nerve
agents that inhibit AChE. [1]
22
0 ( S )
Figure 6 - General structure of OPs. [1]
OP pesticides such as diazinon, dichlorvos (DDVP), parathion, and paraoxon
are widely used in modern agriculture to protect crops from pests and insects. [11 ] [12]
However, the OP-induced contamination of agricultural products, environments, and
even foods has raised increasing public concerns. It has been reported that OPs can
cause neurotoxic poisoning to both animals and humans because of their strong ability
to irreversibly block the activity of AChE in cholinergic synapses and neuromuscular
junctions at very low concentrations, resulting in the accumulation of ACh throughout
the body and, thus, exerting a malignant influence on human health with severe
consequences. Therefore, reliable and sensitive analytical methods for detecting OPs
in trace levels have become increasingly important. Many sensitive analytical methods,
including colorimetric assays, fluorescence methods, photoelectrochemical
approaches, photonic sensing platforms, and liquid crystal sensing platforms, have
been established to detect OPs. Many of these OP assay methods rely on the
mechanism of AChE-catalyzed hydrolysis of the artificial substrate acetylthiocholine
(ATCh) due to its high sensitivity and rapid response. Although these reported methods
showed satisfactory results for OP analysis, it is still attractive to achieve higher
sensitivity. [13] Biosensor techniques based on the inhibition of AChE activity have
lately gained considerable attention due to benefits, such as simplicity, rapidity,
reliability, and low-cost instrumentation. Different AChE-based amperometric,
potentiometric, or optical biosensors have been reported. In amperometric AChEbased
biosensors, enzyme inhibition is monitored by the change in the oxidation
current of thiocholine at a specific potential. Nanomaterials have also been employed
to make electrochemical biosensors with improved analytical performance. [14] In
recent years, surface-enhanced Raman spectroscopy (SERS) has been considered a
tool for OP detection. [15]
23
Besides being used as pesticides, OPs are also used as potent nerve agents,
namely, sarin, soman, tabun, cyclosarin, and VX.
Fx ,0
X
H,C O
Sarin
CH3
CH3
X
H,C 0
Cyclosarin
Fx ,P CH,
H3 C
Soman
,CH3
^ C H 3
H,CNECN
O
X
CH3CO N '
H3 C
Tabun
CH,
CH,
,p
X
H3 C S- -N
C H 2 C H ;
C H 2 C H 3
Russian VX
CH3 CON O H3 C
H3 C S " ^ _ N
CH,
-CH,
H,C
VX
H 3 C - O x O
^ x
H3C-O O A CI
CI
DDVP
Figure 7 - Structural formulas of selected nerve agents and DDVP. [11]
3 Silver nanoparticles (AgNPs)
The practical use of noble metal nanoparticles (NPs) for biological and
biomedical applications is widely explored due to their strong surface plasmon
resonance (SPR). The metallic NPs possess unique physical, chemical, and biological
properties due to their nanometer-size range and shape. The unique optical properties
of gold and silver NPs result from their strong absorption and scattering of light. Among
other NPs, AgNPs have been utilized in surface-enhanced Raman scattering (SERS),
plasmonic absorbance, catalysis, antimicrobials, and biosensors. AgNPs are often
used combined with biologically active molecules that can pass on the response to
toxic chemical agents. Several studies have reported the rapid and direct detection of
AChE inhibitors in aqueous solutions by sensitive and straightforward colorimetric
24
AChE-based assays using NPs. AuNPs and AgNPs are widely used for OP pesticide
detection in environmental and biological systems due to their distinct color properties.
Therefore, AgNPs have been receiving significant interest in recent years in the field
of novel biosensors and as a very efficient support matrix for enzyme immobilization.
Enzyme immobilization on NPs can lower protein unfolding, which increases the
enzyme stability (and activity) and biosensor performance. [12] Enzyme-catalyzed
deposition of AgNPs can also serve to enhance the response signal output. [13]
AgNPs can be prepared using a citric acid salt as a stabilizing agent. Such
AgNPs (citrate-capped) can be used in the spectrophotometric determination of AChE
inhibition. A synthetic substrate, acetylthiocholine (ATCh), is hydrolyzed to thiocholine
(TCh), which induces the aggregation of AgNPs, leading to a color change (Figure 8).
[16]
Active Inactive Dispersed
Figure 8 - Schematic illustration of the novel analysis of organophosphorus pesticides (OPs) based
on the inhibition of an enzyme and the use of the aggregation process of citrate-capped AgNPs. [16]
4 Indium Tin Oxide (ITO)
The first transparent conductive thin film of oxide was prepared from cadmium
oxide, and it is not used anymore due to its toxicity. Later on, a thin film of tin dioxide
was prepared in 1937 and a thin film of indium oxide in 1954. [17]
Indium tin oxide (ITO) is a composition of indium oxide and tin dioxide in a ratio
of 9:1. It is one of the most widely used transparent and, at the same time, conductive
25
thin films based on metal oxides. Transparent conductive oxide thin films have, as the
name suggests, good electrical conductivity and low light absorption, but also good
substrate adhesion, low capacitive current, and stable electrochemical and physical
properties. These properties make them ideal for the production of biosensors and
electrodes and electrodes in photovoltaic devices. [18]
ITO thin films are prepared by various techniques, such as electron beam
evaporation, radiofrequency magnetron sputtering, chemical deposition of vapors, and
the most commonly used chemical deposition from solution due to its simplicity and
low cost. [17][19]
5 Biosensors
5.1 Definition of the biosensor
For the first time, the biosensor was defined by G. Rechnitz as an analytical
instrument consisting of an immobilized biorecognition component and a
physicochemical transducer. [20] The biorecognition part recognizes the analyte by
two basic principles - biocatalytic (tissue, cell, enzyme), where the analyte is a
substrate of the enzyme reaction, and bioaffinity (antibody, receptor, nucleic acid,
lectin), where the analyte is bound in an affinity complex. The physical-chemical
transducer of which the bio-cognition part is a part or is in close contact with it provides
a final electronic analytical signal proportional to the concentration of the analyte or
group of substances. [21]
IUPAC (International Union of Pure and Applied Chemistry) defines a biosensor
as: "A device that utilizes specific biochemical interactions mediated by isolated
enzymes, immunosystems, tissues, organelles, or whole cells to detect chemical
compounds, usually by electrical, thermal, or optical signals." [22]
5.2 History of biosensors
At the beginning of the journey to biosensors was Leland C. Clark Jr. and his
discovery of the oxygen sensor. Clark separated the working platinum electrode from
26
the sample with a gas-permeable membrane and moved the reference electrode
behind the membrane, creating the sensor itself. [23]
Thanks to this discovery, there was a massive development of biosensors with
immobilized enzyme on the membrane. If there is dissolved oxygen on the side of the
reactants or products, the sensor makes it possible to measure the loss or increase of
oxygen, which is directly proportional to the concentration of analyte = substrate. [23]
The first commercially successful biosensor was a blood glucose biosensor.
Yellow Spring Instruments introduced a commercially available enzyme electrode for
the determination of blood glucose in 1975. In 1987, the introduction of GlucoPen
ExacTech became an essential milestone for people with diabetes. [21]
We currently encounter many biosensors used in clinical diagnostics, the
military, agriculture, food, sports, and other industries. [21]
5.3 Physical-chemical converter
A physical-chemical transducer is a system that converts changes arising from
specific biochemical reactions into a measurable electrical signal suitable for further
evaluation. Depending on the type of biocatalyst, the correct type of transducer must
be selected.
In principle, the converters can be divided into the following groups:
• Optical
• Electrochemical
• Piezoelectric and acoustic
• Electromagnetic
• Calorimetric
The most often encountered are electrochemical converters, mainly due to low
production and acquisition costs, simple construction, and high sensitivity. [21]
27
5.4 Basic concepts
Sensitivity - a stable change in the output signal caused by a change in the analyte
concentration. Ideally, the sensitivity should be constant; in practice, recalibration is
necessary.
Calibration - measuring the response using standard solutions of known
concentration.
Linearity - ideally in the entire work area. In practice, this is a narrower interval within
the work area.
Detection limit - this is the smallest possible detectable analyte concentration.
Noise - most often electromagnetic, can be prevented by shielding the sensor and
wires. It can also be caused by turbulence in a stirred arrangement.
Background signal - generated in the absence of analyte, usually read automatically.
Hysteresis - is caused by previous measurements on the current signal. It manifests
itself by a change in the shape of the calibration curves, on which concave or convex
motion can be observed.
Long-term stability - depends on changes in the sensitivity of the biosensor over
time. The decrease in sensitivity may be due to oxidation of the surface or deposition
of layers of molecules on the surface of the biosensor.
Selectivity - ideally, the signal should change only after the addition of the test
substance. In practice, it is necessary to eliminate substances causing interference
(e.g., by a selective barrier) or to determine their share in the total signal by another
sensor at the same time.
28
Response rate - depends on the physical properties, especially the rate of diffusion
from the environment to the surface of the biosensor and then to diffusion within the
biosensor system.
Response time - the time required to reach a specific constant signal size.
The convection rate - is determined by the supply of substances from the
environment. It can be accelerated by agitation or flow of the medium, but excessive
speed, which could lead to turbulent phenomena, must be avoided.
Temperature dependence - affects the ongoing reaction and diffusion. The solution
is to work in an isothermal environment or monitor temperature.
Service life is given by the lifespan of the biorecognition part. A lower temperature is
usually suitable for long-term storage. Appropriate conditions vary for each biosensor.
Biocompatibility is essential when working in living tissues - treatment against blood
clotting and disinfection. [21]
5.5 Enzyme immobilization
It is necessary to properly attach the biorecognition component to the transducer
to ensure the correct functioning of biosensors. This process is called immobilization,
and five basic methods are distinguished:
Adsorption
This fast and straightforward method uses adsorbent materials such as
graphite, alumina, cellulose, or silica gel. Enzymes are weakly bound to the material,
which is a disadvantage. This method is suitable for short-term use. The material is
sensitive to changes in temperature and pH.
29
Encapsulation
The biorecognition component is located behind the inert membrane, where it
is in close contact with the transducer. This method is reliable and flexible, stable to
changes in temperature and pH. It is used in the glucose biosensor.
Capture
The biorecognition component is mixed with the monomer solution and then
polymerized into a gel. The biological sample is then retained in the gel.
Polyacrylamide is usually used for capture. The problem is the slow diffusion of the
substrate and the associated longer measurement time.
Cross-linking
Glutaraldehyde is usually used to capture the biorecognition component on a
solid surface. The advantage is the possibility of stabilizing the adsorbed enzyme.
However, enzyme damage and reduced substrate diffusion may occur. In addition, the
method does not show good resistance to mechanical influences.
Covalent bond
A covalent bond is formed between the functional group and the transducer or
membrane. The enzyme is attached tightly. The reaction conditions must be mild. [14]
6 Electrochemical methods
Electrochemistry deals with processes in systems containing charged particles.
For example, electrochemical methods are often used to measure the catalytic activity
of enzymes. It is prevalent mainly due to the simple construction of the electrochemical
system, low acquisition costs, and high sensitivity. The electrochemical measuring
system must contain at least a working and a reference electrode. [21 ] Today, modern
technology allows the construction of microscopic systems, for example, by screen
30
printing. There are several electrochemical methods. This thesis further deals with
amperometry and cyclic voltammetry (CV).
6.1 Amperometry
Amperometry works on the principle of monitoring the current that arises during
the oxidation or reduction of an electroactive substance. The current is usually
measured at a constant voltage at the working electrode. The current generated during
a reaction over a given period indicates a charge that is proportional to the
concentration of analytes converted on the electrodes. The measurement is most often
performed in a two-electrode arrangement, where the voltage on the working electrode
is set against the auxiliary electrode. Another possibility is to measure in a threeelectrode
arrangement, in which there is also a reference electrode. The voltage of the
working electrode is set against the reference electrode. The benefit of the threeelectrode
arrangement lies in the elimination of the effect of the magnitude of the
current in the solution on the voltage at the working electrode. Thanks to this, it is
possible to achieve a much higher current value using a three-electrode system
compared to a two-electrode arrangement. Therefore, the three-electrode system is
more versatile, but a potentiostat is required for its operation. [21]
6.2 Cyclic voltammetry
CV is an electrochemical technique that measures the current that develops in
an electrochemical cell under conditions where voltage is more than that predicted by
the Nernst equation. CV is performed by cycling the potential of the working electrode
and measuring the resulting current. [24]
The basic principle of CV is step changes of potential in time, from maximum to
minimum value (forward scan) and backward (reverse scan). The breakpoint is called
Es. A typical voltammetric curve of a reversible redox process can be described from
an electrochemical point of view as follows: by gradually changing the potential from
maximum to minimum, a reduction occurs until the maximum cathodic current is
reached (at that moment, the further current increase limits the transport of substance
to the surface of the electrode) at the potential of Epc. As the rate of loss of oxidized
31
form of the substance increases, its amount around the electrode decreases, and once
there is too little of the substance, the current gradually decreases, while the potential
decreases to the point Es. At that moment, the direction of potential growth changes,
and the reverse process gradually begins (oxidation of the accumulated reduced form).
There is a gradual increase in the reduced form of the analyte, and the maximum
anodic current is reached. After exceeding the Epa potential to which this maximum
corresponds, there is not enough of the reduced form of the substance around the
electrode that could be oxidized, and the current gradually decreases until the system
returns to its initial state at maximum potential (Figure 9). In the field of biosensors, this
method is of great importance, especially in the study of new electrochemical systems.
[21][25]
current
Figure 9 - Cyclic voltammetry. [26]
32
7 Aims and Objectives
1. Immobilization of AChE on ITO strips and platinum sensors.
2. Preparation of citrate-capped AgNPs.
3. Determination of the size and physical properties of prepared nanoparticles.
4. Amperometric determination of response to ATCh substrate on prepared sensors
with surface-immobilized AChE.
5. Determination of AChE inhibition using CV and spectrophotometrical methods
combined with AgNPs.
33
8 Materials and methods
8.1 Apparatus
USB potentiostat EmStat
• Manufacturer: Palmsens BV, The Netherlands
• Measurable current range: 1 nA - 100 mA
• Associated with PSTrace software
•
Figure 10- USB Potentiostat EmStat.
Amperometric sensor AC1.W2.RS
• Manufacturer: BVT Technologies, a.s., Czechia
• Dimensions: 25.4 x 7.26 x 0.63 mm
• Working electrode: platinum (Pt)
• Base: corundum ceramic
Figure 11 - Amperometric sensor AC1 .W2.RS.
Spectrophotometer NanoPhotometer® NP 80
• Manufacturer: Implen GmbH, Germany
34
• Wavelength range: 200 nm - 900 nm
• Sample: cuvette, drop
Figure 12 - Spectrophotometer NanoPhotometer® NP 80.
Atomic force microscope (AFM) Dimension FastScan Bio
• Manufacturer: Bruker Co., USA
• Probe: Scanasyst-Air, tip height: 2.5-8 pm
• Image processing software: NanoScope Analysis
Raman Spectrometer AvaRaman
• Manufacturer: Avantes, The Netherlands
• Laser probe: 532 nm
• processing software: Avasoft 8
Other devices:
• Analytical balance Scaltec S B C 22, Germany
• Electromagnetic stirrer Color squid, IKA, Germany
• Peristaltic pump Miniplus 3, Gilson, USA
• pH meter inoLab pH 720 equipped with SenTix 21 pH electrode, Germany
35
8.2 Reagents
• Acetone, 58.08 g moľ1
, supplied from: Penta, Czech Republic
• AChE (Acetylcholinesterase from Electrophorus electricus (electric eel), EC
3.1.1.7), lyophilized powder, 200-1 000 units/mg protein, manufacturer: SigmaAldrich,
USA
• AgN03 (Silver nitrate), 169.87 g moľ1
, manufacturer: Penta, Czechia
• APTES ((3-Aminopropyl)triethoxysilane), 221.37 g moľ1
, manufacturer: SigmaAldrich,
USA
• ATCh (Acetylthiocholine chloride), 197.73 g moľ1
, manufacturer: Sigma-Aldrich,
USA
• Cystamine dihydrochloride, 225.20 g moľ1
, manufacturer: Sigma-Aldrich, USA
• Ethanolamine, 61.08 g moľ1
, manufacturer: Sigma-Aldrich, USA
• Glutaraldehyde, 50% water solution, 100.12 g moľ1
, manufacturer: SigmaAldrich
(Fluka), USA
• ITO (Indium tin oxide) coated PET, 100 O/sq, manufacturer: Sigma-Aldrich,
USA
• 4-MBA (4-Mercaptobenzoicacid), 154.19 g moľ1
, manufacturer: Sigma-Aldrich,
USA
• NaBhU (Sodium borohydride), 37.83 g moľ1
, manufacturer: Ubichem, UK
• NaCI (Sodium chloride), 58.44 g moľ1
, manufacturer: Penta, Czechia
• NaH2P04 • 2H2O (Sodium phosphate monobasic dihydrate), 156.01 g moľ1
,
manufacturer: Penta, Czechia
• PBS (50mM phosphate, 150mM NaCI, pH = 7,4)
• Eserine (Physostigmine), 387.4 g moľ1
, provided by Petr Skladal
• Potassium citrate, 306.40 g moľ1
, manufacturer: Chemapol, Czechia
• Sodium citrate, 258.06 g moľ1
, Penta, Czechia
36
8.3 Methodology
8.3.1 Preparation of phosphate-buffered saline (PBS)
The PBS was prepared with 50 mM NahtePCM and 150 mM NaCI, weighed
quantities were dissolved in an appropriate volume of distilled water, and then the pH
was adjusted to a final value of 7.4 with 1 mM NaOH solution.
8.3.2 Synthesis of citrate-capped AgNPs
The citrate-capped AgNPs were prepared according to Z. Li et al. First, 250 pi
of 100 mM AgN03 solution was added to 100 ml of distilled water in a beaker in a
cold-water bath (~5 °C) while stirring vigorously. Then, 250 pi of 10OmM sodium citrate
solution and 6 ml of 5 mM sodium borohydride (NaBFU) solution were sequentially
added to the solution. NaBFU reduces A g +
ions to Ag° atomic silver. NaBFU in the
presence of sodium citrate also forms AgNPs, with the citrate salt acting as the
stabilizing agent. The instant color change from colorless to light yellow indicated the
creation of dispersed, colloidal AgNPs. The AgNP mixture was stirred vigorously for
another 30 minutes. The resulting product was then stored in the refrigerator at 5 °C
for further use. [16]
Another batch of colloidal citrate-capped AgNPs was prepared using a slightly
modified procedure when the 100 mM sodium citrate solution was substituted with 100
mM potassium citrate solution. Similar results were obtained with potassium citrate
solution.
8.3.3 Immobilization of AChE on ITO-coated PET foil
AChE was immobilized on 3 x 1 cm ITO-coated PET foil strips. The strips were
cut to desired dimensions using scissors. The first step was to wash the strips in
acetone for at least 10 minutes. The strips were then silanized by immersing in 2%
APTES solution (0.2 ml of APTES, 9.8 ml of acetone) for 1 hour. After the incubation
in APTES solution, the strips were then washed with acetone and dried. In the next
step, the strips were incubated in 3% glutaraldehyde solution in PBS for 1 hour. After
37
this step, the strips were washed with distilled water, dried and 70 pi of AChE solution
of the desired activity (1 nkat p i 1
, 0.1 nkat p i 1
, and 0.01 nkat pi1
) were then applied
on each strip. The strips placed in a sealed Petri dish to avoid evaporation of AChE
solution were stored in a refrigerator overnight.
On the other day, the AChE solution was washed off with distilled water, and
the strips were incubated in a 1 % solution of aminoethanol in PBS for 30 minutes. Then
the strips were washed with distilled water, dried, and stored dry in a sealed Petri dish
in the refrigerator.
8.3.4 Immobilization of AChE on platinum electrodes (AC1.W2.RS)
AChE was immobilized on AC1.W2.RS corundum ceramic-based platinum
electrodes. The first step was to wash the electrodes in acetone for at least 10 minutes.
After drying, 2 pi of 10 mg ml"1
cystamine distilled water solution was added onto each
working electrode for 1 hour. After the incubation solution, the electrodes were washed
with distilled water and dried. In the next step, 2 pi of 3% glutaraldehyde solution in
PBS was added onto each working electrode for 1 hour. After this step, the electrodes
were washed with distilled water, dried, and 2 pi of AChE solution of the desired activity
(1 nkat p i 1
, 0.1 nkat p i 1
, and 0.01 nkat pi1
) were then applied on each electrode. The
electrodes placed in a sealed Petri dish to avoid evaporation of AChE solution were
stored in a refrigerator overnight.
The next day, the AChE solution was washed off with distilled water, and the
electrodes were washed with distilled water, dried, and stored dry in a sealed Petri dish
in the refrigerator.
8.3.5 Amperometric measurements
Amperometric measurements were carried out in mixed as well as flow
arrangements at positive potentials ranging from 300 mV to 800 mV.
For amperometric measurements in the mixed arrangement, the AC1.W2.RS
electrodes with surface-immobilized AChE were immersed in a beaker containing 10
ml of PBS and connected to the EmStat potentiostat. The content of the beaker was
stirred at a constant speed of 300 rpm using an electromagnetic stirrer. The
38
chronoamperometry method was selected in the PSTrace software, and the potential
was set to 300 mV. After the background signal stabilization, four volumes of 10 pi, 20
pi, 20 pi, and 50 pi of 100 mM acetylthiocholine (ATCh) solution were sequentially
added to the stirred PBS. It was essential to wait for the signal stabilization between
each addition of the ATCh solution. These steps were repeated for each sensor with
different AChE activities used for the immobilization (1 nkat p i 1
, 0.1 nkat p i 1
, and
0.01 nkat pi1
).
Figure 13 - Diagram of measuring apparatus.
1 - the electromagnetic stirrer, 2 - beaker with a working solution, 3 - electrochemical sensor
AC1 .W2.RS, 4 - potentiostat EmStat, 5 - a desktop computer with PSTrace software.
For amperometric measurements in the flow arrangement, the ITO strips with
surface-immobilized AChE were placed in a flow cell connected to a peristaltic pump.
The cell contained electrodes that were in contact with the ITO strip. The cell was
connected to the potentiostat using crocodile clips. The chronoamperometry method
was selected in the PSTrace software, and the potential was set from 600 mV to
1000 mV.
First, PBS was pumped into the cell at 2.5 rpm (approx 60 pi min1
). Then, after
the background signal stabilization, 1 mM ATCh solution was pumped into the cell at
2.5 rpm. It was again necessary to wait for the signal stabilization. Then, PBS was
pumped into the cell at 2.5 rpm again. These steps were repeated for both potentials
and each ITO strip with different AChE activity (1 nkat p i 1
, 0.1 nkat p i 1
, and
0.01 nkat pi1
).
39
Figure 14 - Diagram of measuring apparatus.
1 - flow cell with ITO strip, 2 - beaker with a working solution, 3 - peristaltic pump, 4 - waste beaker,
5 - potentiostat, 6 - a desktop computer with PSTrace software
All measurements were performed at room temperature of 23 °C.
8.3.6 Cyclic voltammetry measurements
The cyclic voltammetry measurements were performed as follows: 1 pi of 1 mM
ATCh (in PBS) and 1 pi of 1 mM AgN03 (in 5 mM phosphate) solutions were applied
on the working electrode of AC1 .W2.RS sensor with surface-immobilized AChE (1 nkat
pi1
). The mixture was incubated on the surface of the electrode for 5 minutes and then
washed off with distilled water. Then the sensor was immersed in a beaker with PBS,
and the CV measurement was performed using a potentiostat. The potential was set
to 1 Vwith steps of 0.1 V.
The measurements using the inhibitor of AChE eserine were performed in the
same manner, but before incubating the ATCh/AgN03 mixture, 1 pi of eserine aqueous
solution (1 pg m l 1
, 5 pg m l 1
, 10 pg m l 1
, 20 pg m l 1
, 25 pg ml1
) was applied to the
working electrode and incubated for 5 minutes. After that, the inhibitor was washed off
with distilled water. Followed the incubation of 1 pi of 1 mM ATCh (in PBS) and 1 pi of
1 mM AgN03 (in 5 mM phosphate) solutions. The mixture was left on the surface of the
electrode for 5 minutes and then washed off with distilled water. Then the sensor was
immersed in a beaker with PBS, and the CV measurement was performed using a
potentiostat. The potential was set to 1 V with steps of 0.1 V. All measurements were
performed at room temperature of 23 °C.
40
8.3.7 Spectrophotometric measurements
All spectrophotometric were performed in a cuvette with 10 mm pathlength using
NanoPhotometer® NP 80. Absorption spectra were measured for citrate-capped
AgNPs, a mixture of citrate-capped AgNPs with 1 mM ATCh (final concentration in the
cuvette), a mixture of citrate-capped AgNPs with 1 mM ATCh and AChE (1 nkat pi1
),
and a mixture of citrate-capped AgNPs with 1 mM ATCh and AChE (1 nkat pi1
) and the
inhibitor eserine (final concentration in the cuvette was 1 pg pi1
).
In each measurement, the total volume of the solution in the cuvette was 1 ml.
The mixture of 10 pi of ATCh and 10 pi of AChE was preincubated for 5 minutes before
adding 980 pi of citrate-capped AgNPs. The same time of preincubation was applied
for the mixture of 10 pi of ATCh, 10 pi of AChE, and 10 pi of eserine (final concentration
in the cuvette was 10 pg pi1
), which was then topped with 970 pi of citrate-capped
AgNPs.
Wavescan mode was selected on the device, and the respective absorption
spectra were measured.
8.3.8 AFM analysis of citrate-capped AgNPs
The AFM analysis was used to determine the size of prepared citrate-capped
AgNPs and citrate-capped AgNPs aggregated by thiocholine (TCh).
For the analysis, the citrate-capped AgNPs were diluted 100 times with PBS.
Then, the AgNPs were immobilized on AFM mica sheets using 10pM polylysine.
Finally, the analysis itself was performed using the AFM Dimension FastScan Bio with
Scanasyst-Air probes by Mgr. Matěj Pastucha.
The obtained images were processed using NanoScope Analysis software.
8.3.9 Raman spectroscopy analysis of citrate-capped AgNPs
The detection of traces of substances by surface-sensitive techniques such as
surface-enhanced Raman spectroscopy (SERS) explores the interaction of adsorbed
molecules on plasmonic surfaces to improve the limit of detection of analytes. [15]
SERS is ultrasensitive vibrational spectroscopy that uses metal nanoparticles for
41
electromagnetic enhancement called localized surface plasmon resonance (LSPR) to
enhance the Raman intensity of molecules. S E R S is capable of recognizing various
chemical substances in the mixture without the need to separate them. [27]
Surface-enhanced Raman scattering was another method used to determine
the difference between fresh citrate-capped AgNPs and citrate-capped AgNPs
aggregated by TCh. For this method, 10 pi of AgNP solution was first mixed with 4mercaptobenzoic
acid (1.2 mg ml1
), and the mixed drop was allowed to dry on a slide
with an aluminum foil layer. All Raman spectroscopy measurements were performed
by Ing. Jakub Máčala.
9 Results and discussion
9.1 Results of amperometric measurements with platinum electrodes
To determine the amperometric response on AC1 .W2.RS platinum sensors with
surface-immobilized AChE of different activities, the sensors were immersed in a
beaker with stirred (300 rpm) PBS solution (see 8.3.5), and the chronoamperometry
method was selected in PSTrace software. The working potential was set to 300 mV.
Four volumes of 10 pi, 20 pi, 20 pi and 50 pi of 100mM ATCh solution were
added sequentially into the beaker with PBS. These steps were repeated for all three
activities of AChE (see 8.3.5; Figures 15-18). It was necessary to wait for a signal
stabilization between each addition.
Each addition is marked with an arrow in graphs. In addition, a control
measurement with an empty electrode was performed to determine a response to
ATCh itself, as it might become slowly hydrolyzed spontaneously (Figure 19).
The current values in the following tables resemble the difference between two
stabilized signals, i.e., they do not resemble the difference between each stabilized
signal and the baseline signal. The total current value is a difference between the
stabilized baseline signal and the highest stabilized signal.
42
15 n
12 H 1 1 — • — I — I — • — I 1 1 — • — I — I — • — I 1 1 — I —
0 200 400 600 800
t[s]
Figure 15 - Amperometric response to ATCh.
Pt-electrode with AChE (0.01 nkat ur1
).
Table I - Responses to additions of ATCh (AChE 0.01 nkat ur1
).
Time Addition of 100 mM ATCh Final concentration I I (total)
[s] [pi] [mM] [nA] [nA]
240 10 0.1 0
500 20 0.3 0.214
1.021
600 20 0.5 0.163
780 50 1 0.644
The missing response signal to the first addition of ATCh was most likely due to
a combination of low concentration of ATCh and low activity of the immobilized
enzyme.
43
18 n
12 -I . . . 1 . . • 1 . . . 1 -
0 200 400 600
t[s]
Figure 16 - Amperometric response to ATCh.
Pt-electrode with AChE (0.1 nkat ur1
).
Table II - Responses to additions of ATCh (AChE 0.1 nkat ul1
).
Time
[s]
Addition of 100 mM ATCh
[Ml]
Final concentration
[mM]
I
[nA]
220 10 0.1 0.75
340 20 0.3 0.848
440 20 0.5 0.846
3.854
600 50 1 1.41
44
Table III - Responses to additions of ATCh (AChE 1 nkat u.11
).
Time Addition of 100 mM ATCh Final concentration I I (total)
[s] [Ml] [mM] [nA] [nA]
950
10 0.1 2.538
1950
20 0.3 13.605
2700 20 0.5 -
16.143
2900 50
As Figure 17 shows, no response signals were recorded for the last two
additions of 20 ul and 50 ul of ATCh. That might be due to the fact that AChE is inhibited
at high substrate concentrations. [28] In addition, the high concentrations of thiols the
produced thiocholine - might degrade the silver reference electrode surface.
45
0.01
nkat
1 nkat
t[s]
Figure 18 - Amperometric responses to ATCh all in one graph.
It is evident from Figure 18 that the response on the sensor with
surface-immobilized AChE with an activity of 1 nkat p i 1
was the highest.
16 i
t[s]
Figure 19 - Amperometric responses ATCh on an electrode without immobilized enzyme (control).
46
On the other hand, the total response to ATCh additions on an empty electrode
was 1.1 nA, which is similar to the response on the sensor with surface-immobilized
AChE with an activity of 0.01 nkat pi1
.
Based on the obtained results, it was decided that all the other oncoming
measurements with platinum electrodes were going to be performed using AChE with
an activity of 1 nkat p i 1
and 1 mM ATCh. AC1.W2.RS platinum sensors have proven
to be suitable for AChE immobilization and further measurements with ATCh.
9.2 Results of amperometric measurements with ITO strips
For the measurements of the amperometric response on ITO strips with surfaceimmobilized
AChE, the strips were placed into a flow cell connected to the potentiostat,
and the chronoamperometry method was selected in PSTrace software.
First, PBS was pumped into the cell, and after the signal stabilization, 1mM
ATCh was pumped into the cell. Then, after the stabilization of the signal, PBS was
pumped into the cell again. These steps were repeated for all measurements
(see 8.3.5).
ATCh addition is marked with an arrow in each graph. In addition, a control
measurement with an empty ITO was performed to determine a response to ATCh
itself.
The current values in the following tables resemble the difference between the
baseline signal of PBS and stabilized response signal to ATCh addition.
Figure 20 - Connection of the flow cell using crocodile clips.
47
130.0 n
l[nA]
128.0 - I — • — • — • — I — • — • — • — I — • — • — • — I — • — • — • — I — • -
0 200 400 600 800
t[s]
Figure 21 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with surfaceimmobilized
AChE (1 nkat ur1
). Potential 600 mV.
131.0
130.5 •
130.0
l[nA]
200 400 600 800 1000
t[s]
Figure 22 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with surfaceimmobilized
AChE (1 nkat ur1
). Potential 700 mV.
48
l[nA]
132.0 n
131.5
131.0
130.5
130.0
129.5
129.0
200 400 600 800 1000
t [s]
Figure 23 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with
surface-immobilized AChE (1 nkat ur1
). Potential 800 mV.
l[nA]
400 600 800 1000 1200
t[s]
Figure 24 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with
surface-immobilized AChE (1 nkat uľ1
). Potential 1000 mV.
49
Table IV - Responses to additions of ATCh (AChE 1 nkat ul"1
).
E [mV] 600 700 800 1000
l[nA] 0.778 0.836 1.596 3.129
Based on the obtained results (Table IV), it is evident that the amperometric
signal response was stronger at a higher potential. However, higher potentials can
cause an unwanted interference of other compounds present in the system.
For the ITO strips with surface-immobilized AChE with an activity of 0.1 nkat pi"1
and 0.01 nkat p i 1
, it was only managed to record response signals at 800 mV.
400 600 800 1000 1200 1400
t[s]
Figure 25 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with
surface-immobilized AChE (0.1 nkat ul"1
). Potential 800 mV.
50
10.0 n
l[nA]
400 600 800 1000 1200 1400
t[s]
Figure 26 - Amperometric response to an addition of 1 mM ATCh on an ITO strip with
surface-immobilized AChE (0.01 nkat ul"1
). Potential 800 mV.
Table V - Responses to additions of ATCh.
AChE 0.1 nkat uM AChE 0.01 nkat uM
E [mV] 600 800 600 800
l[nA] 0.778 0.836 1.596 3.129
On an empty ITO strip, no signal response was recorded. Although ITO strips
with surface-immobilized AChE showed some amperometric response, the same
results could not be achieved again when trying to replicate the experiments. Many
ITO strips with the same activity of immobilized AChE showed no amperometric
response. It seems that the bond between AChE and the silanized surface of ITO strips
is very weak, and the enzyme is flushed from the surface by the addition of PBS into
the flow cell.
51
For these reasons and lack of time, other planned ITO experiments had to be
discontinued. So instead, attention was turned to platinum electrodes.
9.3 CV measurements with platinum electrodes
CV was another electrochemical method used to determine the response
signals to ATCh on AChE immobilized platinum electrodes. It was also used to
determine the change in signal response when inhibitor eserine was incubated on the
surface of the sensor prior to the addition of ATCh.
All measurements were performed according to the above-described method
(see 8.3.6). For the inhibition analysis, it was essential to wash off the inhibitor before
applying the ATCh/AgN03 mixture to the working electrode.
The first experiment aimed to determine whether there was a difference in
response to ATCh/AgN03 mixture on an empty sensor, a sensor with inhibited AChE
(eserine 10 pg ml1
), and a difference in preincubation times of the ATCh/AgN03
mixture (5 and 10 minutes) on sensors with uninhibited AChE. It was expected that the
AgNPs generated in the solution would adhere to the surface of the electrode and
provide an anodic signal on CVs.
Eserine
I [MA]
-4
0 0.2 0.4 0.6 0.8
E[V]
Figure 27 - CV response to ATCh/AgN03 mixture.
52
It can be seen in Figure 27 that the responses on the empty sensor and the
sensor with inhibited AChE are very similar. Thus, it can be said that the inhibition took
place on the surface of the electrode. As for the difference in incubation time, the signal
response after 5 minutes of incubation was higher. Hence, all the other incubations
were carried out for 5 minutes.
The second CV experiment was set to determine whether there is a difference
in signal responses for various concentrations of eserine. The selected concentrations
of eserine were 1 pg m l 1
, 5 pg m l 1
, 10 pg m l 1
, 20 pg m l 1
, and 25 pg ml1
. A drop of
inhibitor was first incubated on the surface of the electrode for 5 minutes, then washed
off. Then the ATCh/AgN03 mixture was incubated on the surface for another 5 minutes
before being washed off.
-4 H 1 1 1 1 1 1 1 1 1 1
0 0.2 0.4 0.6 0.8 1
E[V]
Figure 28 - CV response to ATCh/AgN03 mixture (various inhibitor concentrations).
It is evident from Figure 28 that with increasing concentration of inhibitor, the
signal response decreased. Thus, there is a dependence between the concentration
of inhibitor and voltammetric response intensity. For comparison, the curve for 5minute
incubation without eserine was added to the graph to demonstrate that even at
1 pg m l 1
of eserine, some inhibition took place.
53
8
7 • •
6 •
5 •
I at 0.8 V
(uA)
4 •
3 •
2 •
1 •
0
0 10 20 30
Concentration of eserine (ug/ml)
Figure 29 - Current at 0.8 V (top half-wave) against eserine concentrations.
Figure 29 shows the decreasing trend of current with increasing inhibitor
concentration. For this graph, the current values at 0.8 V of the upper half-wave
were used.
CV combined with platinum electrodes with surface-immobilized AChE seems
to be a promising method for the determination of the inhibitors of AChE.
9.4 Spectrophotometric measurements of citrate-capped AgNPs
Citrate-capped AgNPs were used for spectrophotometric determination of the
inhibition of AChE. AgNPs prepared with both sodium and potassium citrate were used
for this experiment. This experiment was based on the work of Z. Li et al. [16]
AChE hydrolyzes ATCh to acetic acid and thiocholine, which induces
aggregation of AgNPs, leading to color change from yellow to red-brown (Figures 30
and 31). The absorption maximum of AgNPs lies around 400 nm.
The analyzed solutions were prepared according to the above-described
method (see 8.3.7).
54
Figure 30 - Citrate-capped AgNPs prepared using sodium citrate.
AgNPs, b - AgNPs + ATCh, c - AgNPs + AChE, d - AgNPs + AChE + ATCh,
e - AgNPs + AChE + ATCh + eserine
Figure 31 - Citrate-capped AgNPs prepared using potassium citrate.
AgNPs, b - AgNPs + ATCh, c - AgNPs + AChE, d - AgNPs + AChE + ATCh,
e - AgNPs + AChE + ATCh + eserine
55
UV-VIS spectra were recorded for the solutions in the pictures above. Ideally,
the mixture of AgNPs and ATCh should not undergo any color change. However, due
to the fast self-dissociation of ATCh, it was hard to avoid oxidation of the mixture. On
the other hand, the color change from yellow to red-brown caused by the TCh-induced
aggregation of AgNPs is evident in both pictures. There was also a slight color change
in the inhibited samples. Nevertheless, again the self-dissociation of ATCh made it
hard to distinguish between the sample containing just AgNPs and ATCh and the
inhibited sample.
Figure 32 - Recorded spectra for solutions prepared using AgNPs prepared with sodium citrate.
56
3.0 n
AgNPs
AgNPs+ATCh
AgNPs+AChE
AgNPs+AChE+ATCh
AgNPs+AChE+ATCh+inh.
260 360 460 560
Wavelength (nm)
660 760
Figure 33 - Recorded spectra for solutions prepared using AgNPs prepared with potassium citrate.
In both cases, there is an absorption maximum peak at about 400 nm for AgNPs
and AgNPs with AChE. Also, a broad absorption band at about 520 nM can be
observed in Figures 32 and 33, indicating the color change from yellow to red-brown
caused by TCh.
Ideally, the maximum peaks at 400 nm should be similar for all the mixtures
except the one containing the inhibitor and the one where the AChE-mediated
hydrolysis of ATCh caused the color change from yellow to red-brown.
Although promising, this method would require further investigation on how to
avoid the self-dissociation of ATCh and the oxidation of AgNPs associated with that.
9.5 AFM
The AFM analysis was used to determine the size of prepared AgNPs and the
size of AgNPs aggregated by TCh. For this purpose, the same solutions of pure AgNPs
and AgNPs aggregated by TCh as in the spectrophotometric analysis were used. The
samples were prepared according to the above-described method (see 8.3.8).
57
The size of analyzed fresh nanoparticles varied from 2 to 20 nm. The size of
aggregated AgNPs was around 60 nm. Figures 34-37 show the AFM images of
prepared AgNPs and aggregates thereof.
Figure 34 - A F M images of citrate-capped AgNPs (sodium citrate).
Height Sensor Peak Force Error 400.0 nm
Figure 35 - A F M images of citrate-capped AgNPs (sodium citrate).
58
Height Sensor 400.0 nm Peak Force Error 400.0 nm
Figure 36 - A F M images of citrate-capped AgNPs (potassium citrate).
Peak Force Error 400.0 nm Peak Force Error 400.0 nm
Figure 37 - A F M images of aggregated AgNPs (potassium citrate - left, sodium citrate - right).
Thanks to the AFM imaging, it was possible to determine the size of prepared
AgNPs and to confirm the formation of AgNP aggregates induced by TCh. It was
affirmed that in the absence of thiols from TCh, the AgNPs were smaller and spread
evenly, whereas, in the presence of thiols from TCh, they became larger, aggregated,
and less evenly spread.[12] Furthermore, as starting material, the potassium
citrate-based AgNPs seemed to be more homogeneous, which is an advantage.
Further AFM analyses would have been targeted to compare TCh-aggregated
AgNPs and samples where the hydrolysis of ATCh was inhibited.
59
9.6 Raman spectroscopy
Raman spectroscopy was also used to determine the difference between fresh
and aggregated AgNPs. For this method, 4-MBA was used as a reporter molecule to
functionalize the AgNPs for SERS.
3000 AgNPs_Na
AgNPs_K
Na_aggregated
Kaggregated
2000 3000
Raman shift [cm1
]
Figure 38 - S E R S of fresh and aggregated AgNPs.
The recorded S E R S spectra (Figure 38) show decreased intensity for AgNPs
aggregated by TCh (green and red curves).
Table VI - Intensity at 1590 c m 1
.
I [cnts] at 1590 cnv1
Fresh AgNPs 2057 (Na) 2331 (K)
Aggregated AgNPs 894 (Na) 596 (K)
60
The peak intensity values at 1590 c m 1
were 2057 cnts (prepared with sodium
citrate) and 2331 cnts (prepared with potassium citrate) for the fresh AgNPs and 894
cnts (Na) and 596 cnts (K) for the aggregated AgNPs. Thus, the difference in peak
intensity indicates a size difference between the fresh and aggregated AgNPs.
Thanks to the ability to distinguish between the fresh and aggregated AgNPs,
Raman spectroscopy could serve well for the indication of AChE inhibition. When
analyzing a mixture of AChE, ATCh, and citrate-capped AgNPs, there should be an
observable difference in intensity when an inhibitor blocking the AChE-mediated
hydrolysis of ATCh is present in the mixture - no or fewer AgNP aggregates should be
present in the mixture. On the other hand, when the inhibitor is not present in the
mixture, TCh should induce the formation of AgNP aggregates.
Using S E R S for this purpose also requires further investigation.
61
10 Conclusion
Amperometric and CV measurements, with sensors with surface-immobilized
AChE and ATCh as a model substrate, were performed in the practical part of this
thesis. Unfortunately, some of the experiments could not be replicated. Hence, the
results remain unclear. The measurements were carried out in a stirred arrangement
and a flow cell. It was managed to record differences in response signals for AChE
inhibition caused by various concentrations of inhibitor eserine.
Further, citrate-capped AgNPs were prepared. These AgNPs were used in
spectrophotometric determination of AgNP aggregation caused by TCh and the
inhibition of AChE caused by eserine.
The size of prepared citrate-capped AgNPs and the aggregates thereof was
determined using AFM analysis. In addition, surface-enhanced Raman Spectroscopy
was also used to determine the difference in physical properties of fresh and
aggregated AgNPs.
Creating a robust electrochemical system for detecting AChE inhibitors using
AgNPs as signal enhancers is likely possible given more research time.
62
11 Bibliography
[1] M. B. Colovič, D. Z. Krstič, T. D. Lazarevič-Pašti, A. M. Bondžič, and V. M. Vasič,
"Acetylcholinesterase inhibitors: pharmacology and toxicology," Curr Neuropharmacol,
vol. 11, no. 3, pp. 315-335, May 2013, doi: 10.2174/1570159X11311030006.
[2] "NATIVE ACETYLCHOLINESTERASE ( E C . 3.1.1.7) FROM TORPEDO
CALIFORNIA." May 2021. Accessed: May 15, 2021. [Online]. Available:
https ://www.rcsb.org/structu re/2AC E
[3] A. Trang and P. B. Khandhar, "Physiology, Acetylcholinesterase.," in StatPearls,
Treasure Island (FL): StatPearls Publishing, 2021.
[4] "ELECTROPHORUS ELECTRICUS ACETYLCHOLINESTERASE." Accessed:
May 15, 2021. [Online]. Available: https://www.rcsb.org/structure/1C2B
[5] M. G. Lionetto, R. Caricato, A. Calisi, M. E. Giordano, and T. Schettino,
"Acetylcholinesterase as a biomarker in environmental and occupational medicine:
new insights and future perspectives," Biomed Res Int, vol. 2013, pp. 321213-321213,
Jul. 2013, doi: 10.1155/2013/321213.
[6] E. Fajemisin, O. Bamidele, S. Ogunsola, and A. Aiyenuro, "The Organ
Distribution, Characterization and Modification of Acetylcholinesterase Activity in Adult
African Grasshopper: Zonocerus sp Linn.," Asian Journal of Research in Biochemistry,
pp. 1-9, Nov. 2019, doi: 10.9734/ajrb/2019/v5i430097.
[7] J. Ž. Karasová, K. Kuča, D. Jun, and J. Bajgar, "UŽITÍ ELLMANOVY METODY
PRO STANOVENÍ AKTIVIT CHOLIN- ESTERAS PŘI IN VIVO HODNOCENÍ ÚČINKŮ
REAKTIVÁTORŮ," Chem. Listy, vol. 104, no. 1, pp. 46-50, Feb. 2010.
[8] K. Vrchovecká, "Nanočástice kovů pro zvýšení signálu biosensorů [online].,"
Bachelor's thesis, Masarykova univerzita, Brno, 2015. Accessed: May 07, 2021.
[Online]. Available: https://is.muni.cz/th/i52pd/
63
[9] J. Weller and A. Budson, "Current understanding of Alzheimer's disease
diagnosis and treatment," FWOORes, vol. 7, p. F1000 Faculty Rev-1161, Jul. 2018,
doi: 10.12688/Í1 OOOresearch.14506.1.
[10] A. Vale and M. Lotti, "Organophosphorus and carbamate insecticide
poisoning.," Handb Clin Neurol, vol. 131, pp. 149-168, Nov. 2015, doi: 10.1016/B978-
0-444-62627-1.00010-X.
[11] J. Bajgar, "Laboratory examination in nerve agent intoxication.," Acta Medica
(Hradec Kralove), vol. 56, no. 3, pp. 89-96, Nov. 2013, doi:
10.14712/18059694.2014.15.
[12] D. Nanda Kumar, S. A. Alex, N. Chandrasekaran, and A. Mukherjee,
"Acetylcholinesterase (AChE)-mediated immobilization of silver nanoparticles for the
detection of organophosphorus pesticides," RSC Adv., vol. 6, no. 69, pp. 64769-
64777, 2016, doi: 10.1039/C6RA13185A.
[13] Z. Liu, X. Xia, G. Zhou, L. Ge, and F. Li, "Acetylcholinesterase-catalyzed silver
deposition for ultrasensitive electrochemical biosensing of organophosphorus
pesticides," Analyst, vol. 145, no. 6, pp. 2339-2344, Mar. 2020, doi:
10.1039/C9AN02546D.
[14] N. Chauhan and C. S. Pundir, "An amperometric acetylcholinesterase sensor
based on Fe304 nanoparticle/multi-walled carbon nanotube-modified ITO-coated
glass plate for the detection of pesticides," Electrochimica Acta, vol. 67, pp. 79-86,
Apr. 2012, doi: 10.1016/j.electacta.2012.02.012.
[15] J. M. Dias Soares and H. P. de Oliveira, "Silver-based surface enhanced Raman
spectroscopy devices for detection of organophosphorus pesticides traces.,"
Biotechnol Prog, vol. 35, no. 4, p. e2809, Jul. 2019, doi: 10.1002/btpr.2809.
64
[16] Z. Li, Y. Wang, Y. Ni, and S. Kokot, "Unmodified silver nanoparticles for rapid
analysis of the organophosphorus pesticide, dipterex, often found in different waters,"
Sensors and Actuators B: Chemical, vol. 193, pp. 205-211, Mar. 2014, doi:
https://doi.Org/10.1016/j.snb.2013.11.096.
[17] E. B. Aydin and M. K. Sezgintürk, "Indium tin oxide (ITO): A promising material
in biosensing technology," TrAC Trends in Analytical Chemistry, vol. 97, pp. 309-315,
Dec. 2017, doi: 10.1016/j.trac.2017.09.021.
[18] T. Hlaváčová, "Fotoelektrochemické enzymové biosensory," Diplomová práce,
Masarykova univerzita, Brno, 2020. Accessed: May 14, 2021. [Online]. Available:
https ://is. mu n i .cz/th/oifv8/
[19] S. Elmas, §. Korkmaz, and S. Pat, "Optical characterization of deposited ITO
thin films on glass and PET substrates," Applied Surface Science, vol. 276, pp. 641-
645, Jul. 2013, doi: 10.1016/j.apsusc.2013.03.146.
[20] G. A. Rechnitz, "Biosensors into the 1990s," Electroanalysis, vol. 3, no. 2, pp.
73-76, 1991, doi: 10.1002/elan.1140030202.
[21] P. Skládal, "Biosensory." Masarykova Univerzita, 2002.
[22] D. R. Thévenot, K. Toth, R. A. Durst, and G. S. Wilson, "Electrochemical
biosensors: recommended definitions and classificationl International Union of Pure
and Applied Chemistry: Physical Chemistry Division, Commission 1.7 (Biophysical
Chemistry); Analytical Chemistry Division, Commission V.5 (Electroanalytical
Chemistry). 1," Biosensors and Bioelectronics, vol. 16, no. 1, pp. 121-131, Jan. 2001,
doi: 10.1016/S0956-5663(01 )00115-4.
[23] M. Kronďák, "Clarkovo čidlo pro měření koncentrace rozpuštěného kyslíku,"
Vesmír, vol. 89, no. 631, Oct. 2010. Accessed: Apr. 19, 2019. [Online]. Available:
https://vesmir.cz/cz/casopis/archiv-casopisu/2010/cislo-10/clarkovo-cidlo-pro-mereni-
koncentrace-rozpusteneho-kysliku.html
65
[24] A. Quiroga, "Cyclic Voltammetry," Aug. 2020, Accessed: Apr. 25, 2021. [Online].
Available: https://chem.libretexts.Org/@go/page/311
[25] Skoog, D. A., Holler, F. J . , & Crouch, S. R. (2007). "Principles of instrumental
analysis." Belmont, CA: Thomson Brooks/Cole.
[26] "Cyclic voltammetry." Accessed: May 15, 2021. [Online]. Available:
https://d4ceckwy45dem.cloudfront.net/wp-content/uploads/2017/12/voltammogram-
600x505.png
[27] T. Sukmanee, K. Wongravee, S. Ekgasit, C. Thammacharoen, and P.
Pienpinijtham, "Facile and Sensitive Detection of Carbofuran Carbamate Pesticide in
Rice and Soybean Using Coupling Reaction-based Surface-Enhanced Raman
Scattering.," AnalSci, vol. 33, no. 1, pp. 89-94, Jan. 2017, doi: 10.2116/analsci.33.89.
[28] J.-P. Colletier et al., "Structural insights into substrate traffic and inhibition in
acetylcholinesterase," EMBO J, vol. 25, no. 12, pp. 2746-2756, Jun. 2006, doi:
10.1038/sj.emboj.7601175.
66