Available online at www.sciencedirect.
com Current Opinion in
ScienceDirect
Review Article
Electrochemical detection of ATP release in-vitro and
in-vivo
Andreas Hellmann1, Annika Schundner2, Manfred Frick2 and
Christine Kranz1
Abstract
ATP is one of the most widely distributed extracellular signaling
molecules, regulating many physiological and pathological
processes via activation of purinergic receptors. Extracellular
ATP may originate from cell-regulated release but also from
cell membrane injury. To measure ATP release in-vitro or in-
vivo for insight into the mechanisms of transmission requires
its direct, sensitive, non-invasive detection, ideally with high
spatial and temporal resolution. This current opinion article
highlights recent research in ATP detection using electro-
chemical methods and presents examples how in-vitro elec-
trochemical ATP measurements can contribute to insights in
purinergic signaling for pivotal functions like alveolar homeo-
stasis and how ATP acts as major excitatory neurotransmitter
in the central nervous system.
Addresses
1
Institute of Analytical and Bioanalytical Chemistry, Ulm University,
Ulm, Germany
2
Institute of General Physiology, Ulm University, Ulm, Germany
Corresponding author: Kranz, Christine (Christine.kranz@uni-ulm.de)
Current Opinion in Electrochemistry 2023, 39:101282
This review comes from a themed issue on Bioelectrochemistry
(2023)
Edited by Elena Ferapontova and Heinz-Bernhard Kraatz
For complete overview about the section, refer Bioelectrochemistry
(2023)
Available online 30 March 2023
https://doi.org/10.1016/j.coelec.2023.101282
2451-9103/© 2023 Elsevier B.V. All rights reserved.
Keywords
Adenosine triphosphate, Purinergic signaling, Electrochemical bio-
sensors, Aptamers, In-vitro and in-vivo measurements.
Introduction
Adenosine triphosphate (ATP) is one of the most
important extracellular signaling molecules. Extracel-
lular ATP may be released from cells upon cell mem-
brane injury or cell lysis. In addition, a multiplicity of
regulated ATP release mechanisms has been identified.
ATP can be released via exocytosis of ATP containing
vesicles, or through various channels, including connexin
or pannexin hemichannels, maxi-anion channels,
www.sciencedirect.com
Electrochemistry
CALHM1 ion channel and P2X7 receptors [1*e3].
When released into the extracellular space, ATP binds to
purinergic P2 receptors (metabotropic P2Y or ionotropic
P2X receptors) [4]. Almost every cell type possesses
some kind of purinoceptors, and ATP acts as autocrine
and/or paracrine signaling molecule essentially in all
tissues and organs [5,6]. ATP-mediated purinergic
signaling contributes to a wide range of physiological
processes [7e10]. Malfunction, however, has been
associated with the development or progression of many
diseases [11] and targeting purinergic signaling offers
promising therapeutic potential [12]. For example, in
the lung, extracellular ATP and its metabolites, maintain
mucociliary clearance in the airways [13] and alveolar
homeostasis by regulating secretion of pulmonary sur-
factant and alveolar fluid transport [14**e16]. On the
other hand, extracellular ATP also serves as a danger
signal in the lungs [17]. It is linked to pulmonary
inflammation, development of acute lung injury (ALI/
ARDS) and chronic lung diseases [10,18e20]. Clinical
trials are underway for the use of purinergic receptor
antagonists for the treatment of chronic cough [21] and
inhaled P2Y2 agonists for treatment of cystic fibrosis
(CF) [22].
ATP is detected and quantified by various methods
including chromatographic methods [23,24], micro-
scopic methods (colorimetry and fluorescence, FRET-
based measurements) [25,26], and the well-known
luciferin/luciferase-based bioluminescence assay
[27,28]. Given the complexity of purinergic signaling,
the determination of local ATP concentrations is still
challenging. Also, the structural similarity of ATP with
the adenylate homologues, adenosine diphosphate
(ADP), adenosine monophosphate (AMP) and cyclic
(c)-AMP) is challenging and requires highly specific
methods. Specificity can be achieved via separation
techniques (e.g., reversed-phase HPLC) coupled to
e.g., mass spectrometry [24], the luciferin/luciferase-
based bioluminescence assay [28], or genetically enco-
ded recognition schemes [29,30]. Detection methods
requiring a sampling step are usually limited in
displaying real-time dynamics of ATP release. Electro-
chemical methods have some advantages as they can be
used in-situ, particularly, if micro-sized electrochemical
transducers are used. Since neither ATP nor its
Current Opinion in Electrochemistry 2023, 39:101282
2 Bioelectrochemistry (2023)

metabolic products are reduced or oxidized at suitable
potentials (>1 V [31]), selective detection schemes
using bio-recognition elements such as aptamers [32,33]
or enzymes [34,35] are mostly employed. Enzyme-based
biosensors for ATP were pioneered by Scheller and
Pfeiffer [34].
In the following, we highlight electrochemical ATP
measurements suitable for in-vitro and in-vivo measure-
ments, e.g., stimulated chemically and/or mechanically.
Direct electrochemical detection
Fast-scan cyclic voltammetry (FSCV) using carbon-fiber
microelectrodes (CFMEs), which is well-established for
in-vivo measurements of catecholamines like dopamine
[36e38], has also been used to monitor purinergic
transmitters, including ATP and adenosine [39]
(Figure 1a and 1b). In FSCV, the applied voltage is
alternated in a triangular wave fashion with high scan
rates (up to 2000 V/s), enabling high temporal resolution
[40]. Real-time detection of exogenously delivered ATP
to mimic a transient event in brain slices was
demonstrated using amino (NH2)-functionalized
carbon-fiber electrodes [41]. The amino functionaliza-
tion via N-(3- dimethylaminopropyl)-N0 -ethyl-
carbodiimide (EDC) and ethylenediamine (EDA)
enhances electrostatic interaction and adsorption of the
analyte and increases the sensitivity of the measure-
ments. Besides amino functionalization, physical treat-
ment using N2, O2 or Ar plasma resulted in enhanced
adsorption properties and sensitivity of the carbon-fiber
electrodes towards ATP [42,43]. Recently, the modifi-
cation of carbon fiber electrodes with metal nano-
particles (NPs) (e.g., Au NPs or Pt NPs) has been
reported, which resulted in higher sensitivities in ATP
detection using FSCV [44,45*]. Spontaneous and
mechanosensitive adenosine release was investigated
using FSCV in hippocampal (CA1) brain slices [46**].
The authors could show that Pannexin1 channels seem
not to be involved in the spontaneous release of aden-
osine, as in wildtype (WT) and global deletion pannexin
knockout mice (1Panex1KO), there was neither a sig-
nificant difference in frequency nor concentration. In
case of mechanically induced release, lower amounts of

Figure 1

(a) Direct electrochemical oxidation of ATP and (b) FSCV of ATP and adenosine using a triangular and optimized sawhorse waveform (CFE; −0.4 to
1.45 V; scan rate: 400 V/s). Mechanically stimulated adenosine release was detected in rat brain slices and evaluated using an in-slice training set.
Adapted with permission from Ross et al. [39], copyright 2014. (c) Conformational change of an immobilized and redox labeled aptamer due to binding of
ATP, which results in an enhanced electron transfer. (d) Elevated ATP levels were observed with an electrochemical RNA-aptamer sensor upon chemical
stimulation with Ca2+ of astrocytes in cell culture. Adapted with permission from Santos-Cancel et al. [54**], copyright 2019.

Current Opinion in Electrochemistry 2023, 39:101282 www.sciencedirect.com


adenosine were found in Panex1KO, which was similar to
WT mice treated with inhibitor. Despite the advantages
such as sub-second temporal resolution in FSCV, usually
selectivity remains an issue, as this method relies on
direct oxidation at the electrode surface at relatively
high potentials, and thus may be prone to interferences
from other electroactive species present in solution
(e.g., catecholamines, H2O2 etc.).
Aptamer-based biosensors
Aptamersdsingle- or double-stranded oligonucleotides
are used for selective ATP detection due their high
binding affinity and specificity [47e49]. Aptamers are
produced in-vitro by a systematic evolution of ligands by
exponential enrichment (SELEX) process [50]. Immo-
bilized on the electrode surface, aptamers are designed to
fold their flexible chains into well-defined three-dimen-
sional (3D) structures upon binding to their target
molecule as schematically shown in Figure 1c. The
conformational change leads to enhanced electron
transfer based on a redox-active moiety tethered to the
end of the chain. One of the first aptamer-based elec-
trochemical ATP sensor was introduced by Fan and co-
workers using target-induced structural switching of a
ferrocence-tagged anti-ATP aptamer [33]. Via self-
assembly, the 27-base anti-ATP aptamer containing one
thiol moiety at the 30 and one ferrocene moiety at the 50
terminus was immobilized onto gold electrodes. Binding
of ATP leads to a change in distance of the ferrocene
moiety to the electrode surface enabling “electron
transfer (eT) ON” state, whereas in absence of ATP re-
flects the “eT OFF” state. Cyclic voltammetry (CV) and
square wave voltammetry (SWV) were used for ATP
determination. The aptamer sensor is selective towards
ATP in presence of e.g., guanosine triphosphate (GTP)
and cytidine triphosphate (CTP). However, its metabo-
lites were not investigated, despite their presence in
biological matrices. The binding affinity of ATP-aptamers
towards ADP, AMP and adenosine remains an issue [51].
Aptamers can be used as recognition elements in field-
effect-transistors (FET), which provide high sensi-
tivity, low-cost fabrication, miniaturization and the
advantage that only a small amount of sample volume is
required [52]. For example, bivalent split aptamers can
significantly enhance the sensitivity towards ATP by a
simple hybridization of two identical monovalent split
aptamers as demonstrated recently [53]. As shown in
Figure 1d, real-time, selective detection of ATP release
from an astrocyte containing hydrogel-based cell-culture
was achieved using RNA aptamer-modified electrodes
interfaced with the cell culture (Figure 1d top) [54**].
ATP release was investigated in dependence of various
stimuli, including Ca2þ, glutamate, and ionomycin and
could be detected with a temporal resolution of seconds.
The authors also employed the luciferin/luciferase assay
to validate the results. The detection of small molecules
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Electrochemical ATP detection Hellmann et al. 3
in solutions of high ionic strength like buffered solutions
using FET-based sensing is challenging due to shielding
by the electrical double layer, however this issue can be
overcome by using specific stem-loop aptamers as
recently demonstrated [55]. Besides issues concerning
ionic strength of the media, aptamers, and in particular
RNA-based ones, are prone to degradation in biological
samples. In addition, limited temporal resolution and
sensor size restrict further applications, e.g., implanta-
tion in tissue and displaying real-time dynamics of ATP.
Enzyme-based biosensors
Potentiometric ATP detection
Mainly, the ATPases catalyzed reaction of ATP to ADP is
used in potentiometric ATP measurements. Protons
(Hþ) are generated and potentiometrically detected.
For example, ATPases immobilized at the transducer
surface of a FET, generate Hþ by ATP hydrolysis
(Figure 2a), which are recorded as change of charge
distribution at the transducer, leading to a change in
overall device conductance [56]. A potentiometric
sensor for ATP using a semiconductor charge-coupled
device (CCD) for label-free imaging of pH has been
developed using apyrase, which is immobilized via 3-
aminopropyltriethoxysilane (3-APTES) or carboxyethyl
silanetriol (CEST) at the pH sensitive layer; whereby
CEST gave a homogeneous distribution of the enzyme
(Figure 2b and 2c) [57]. In a follow-up, the distribution
of local ATP (ca. 0.5 mM) at a mouse hippocampal slice
upon electrical stimulation could be obtained without
labeling and further processing of the tissue [58*]. In a
collaborative effort, an ATP-sensing FET has been
realized at the tip of a dual carbon nanoelectrode [59*].
Immobilized hexokinase (HEX) produces Hþ in pres-
ence of ATP and glucose, leading to changes in
drainesource currents or to shifts in gate voltage.
Placing the FET at the orifice of a double barrel nano-
pipette enabled the detection of local ATP secretion
from cardiac cell ensembles as well as release of ATP
from single cells upon mechanical and osmotic stress.
There is a limited number of reports describing the
potentiometric detection of ATP, as they suffer from
drifts and are prone to non-specific interferences e.g.,
pH changes. In addition, if apyrase is used as the
recognition element, the sensor is responding to ATP
and ADP [60], thus selectivity remains an issue.
Amperometric detection of purinergic signaling
molecules
Amperometric ATP biosensors capitalize on the enzy-
matically catalyzed conversion of ATP to generate
hydrogen peroxide (H2O2) via a consecutive or
competitive enzymatic assay (Figure 3a); H2O2 is
reduced or oxidized at the electrode with the resulting
Faraday current being proportional to the ATP amount.
While the consecutive approach uses mainly glycerol
kinase (GK) and glycerol-3-phosphate oxidase (G3POx)
Current Opinion in Electrochemistry 2023, 39:101282
4 Bioelectrochemistry (2023)

Figure 2

(a) Hydrolysis of ATP and ADP by the enzyme apyrase (Apy) releasing H+. (b) Imaging of ATP distribution via a pH sensitive CCD-based FET using
apyrase; no signal response was observed in absence of the enzyme. (c) Histogram of the potential difference with increasing ATP concentrations.
Adapted with permission from Endo et al. [57], copyright 2018.

Figure 3

(a) Enzyme-based detection schemes using either a consecutive enzyme biosensor based on glycerol kinase (GK) and glycerol-3-phosphate oxidase
(G3POx) or a competitive assay based on glucose oxidase (GOX) and hexokinase (HEX). In both approaches H2O2 is generated and detected as
electroactive by-product. (b) An enzyme-based purine sensor is realized using a consecutive approach with adenosine deaminase, nucleoside phos-
phorylase and xanthine oxidase. (c) The purine sensor was applied in a study of 133 patients including 76 stroke and 9 stroke mimics revealing elevated
purine levels in venous blood caused by the stroke. Adapted with permission from Dale et al. [67], copyright 2019.

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 Electrochemical ATP detection Hellmann et al. 5

Figure 4

(a) ATP is loaded into the lamellar body (LB) by a vesicular nucleotide transporter (VNUT) leading to fusion activated calcium entry (FACE) and ultimately
secretion of surfactant. (b) Fusion events are directly linked with released ATP (GOX/HEX-based ATP microbiosensor) after chemical stimulation. The
total ATP concentration could be determined based on the volume under the electrode. Adapted with permission from Fois et al. [1*], copyright 2018. (c)
For determination of mechanically stimulated ATP release, the microbiosensor was positioned 30 mm above alveolar cells seeded on a flexible membrane.
(d) ATP release correlated with mechanical stretch. A variety of control experiments confirmed that ATP was released from ATI cells and depend on the
expression of caveolin-1 and Ca2+-entry via piezo1 channel. Adapted with permission from Diem et al. .[14**], copyright 2020.

with glycerol as co-substrate [61], in the competitive
assay, the enzymes hexokinase (HEX) and glucose oxi-
dase (GOX) compete for the substrate glucose in the
presence of ATP [34,62e64]. Due to the presence of
glucose in many biomedical/biological samples, no
additional substrate must be added, but changes of
glucose levels during measurements may influence the
sensor response. For the consecutive assay glycerol must
be artificially added as substrate. More recently, direct
electron transfer of the enzyme to the electrode surface
has been described by coupling cellobiose dehydroge-
nase with HEX [65*]. Co-immobilization of pyruvate
kinase enables recycling of produced ADP by the
phosphoenolpyruvate catalyzed reaction. Signal ampli-
fication up to 220-fold could be achieved, resulting in a
sub-nanomolar limit of detection (LOD) of ATP. A novel
integrated sensing system for ATP and lactate using a
series of catalytically active enzymes (adenylate kinase,
pyruvate kinase, and pyruvate oxidase) and Prussian
blue as electrocatalytic layer for H2O2 reduction was
demonstrated by K. Nishiyama et al. [66**]. Reliable
detection of ATP in blood samples with high recovery
rates as well as good selectivity in the presence of
ascorbate, pyruvate, ADP, and urate was demonstrated.
In the following, some physiologically relevant examples
using either the consecutive or competitive biosensor
are highlighted for in-vitro measurements.
In-situ ATP release measurements in complex matrices
such as brain slices, complex cell assemblies and whole
blood samples for point of care diagnostics that
contribute to the understanding and early diagnosis of
diseases remain a significant challenge due to rapid
breakdown of ATP and possibly extensive sample
preparation. Dale and co-workers developed a so-called
SMARTchip (Figure 3b) for rapid and on-site analysis
of total purine content in blood samples, as depicted in
Figure 3e [67,68]. Elevated purine concentrations were
observed in mice after status epilepticus correlating
with seizure burden and post-seizure neuro-
degeneration in the hippocampus [69*]. This sensing
approach may contribute to the early diagnosis of
neonatal encephalopathy as demonstrated in a clinical
test of 26 infants [70].
ATP plays a significant role in surfactant secretion,
regulation of alveolar fluid homeostasis, and maintenance
of the gasblood barrier [10,15,16]. P2X4 receptors are

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6 Bioelectrochemistry (2023)
expressed in the membrane of lamellar bodies (LBs),
storage organelles for lung surfactant within alveolar type
II (ATII) cells. Upon exocytosis of LBs, the acidic pH
within the LB is neutralized and binding of ATP to P2X4
receptors results in local fusion-activated cation entry
(FACE) (Figure 4a). ATP for P2X4 activation is also
stored in the LB but can only bind to P2X4 receptors
after pH neutralization [1*]. FACE is essential for sur-
factant secretion and activation [16]. A key aspect of this
study is the ability to correlate the initial fusion event
with a jump in extracellular ATP with high temporal and
spatial resolution. Fusion of LBs could be directly linked
to ATP release from individual LBs by combining live
fluorescence imaging with measurements using the ATP
microbiosensor, which was positioned with the help of a
second working electrode (dual-electrode assembly)
close to the cells (Figure 4b). Similar sensors were used
to unravel the effects of mechanical stretch on surfactant
secretion. Mechanical deformation/expansion of the
alveoli acts as a strong stimulus for surfactant secretion
[71,72]. ATI cells, that cover > 95% of the alveolar
surface, act as mechanosensors in the alveoli and stim-
ulate surfactant secretion from ATII cells in a paracrine
way. Diem et al. used a co-culture model of ATI cells and
surfactant secreting ATII cells on an elastic PDMS
substrate to show that mechanical stretch results in se-
lective, strain-induced ATP release from ATI cells. They
positioned an ATP microbiosensor above the cells while
applying mechanical strain in live cell imaging experi-
ments (Figure 4c,d) [14**].
Conclusions
Purines and in particular ATP as secondary messenger
molecule, regulate numerous cellular processes through
purinergic signaling pathways. For in-vitro or even in-vivo
detection of purinergic transmitters, spatial and/or
temporal resolution is a prerequisite. Electrochemical
approaches have several advantages for local measure-
ments, which may contribute to address signaling
pathways and with that regulation of important cellular
functions. Novel electrode designs of nearly any shape
and size as well as modification with bio-recognition
elements such as enzymes and aptamers enable in-situ
measurements down to the single-cell level. However,
necessary advancements such as improved immobiliza-
tion techniques and rational design are required to
ensure improved operational stability. For in-vivo mea-
surements, FSCV with recent advances in surface
modification of carbon fiber electrodes are promising
given the improved selectivity and sensitivity.
Combining such measurements with principal compo-
nent analysis can assist in distinguishing different ATP
analogues. In our opinion, electrochemical detection of
ATP is and will also in futuredalongside well-
established instrumental techniques and biochemical
assaysdcontribute to further insight into purinergic
signaling and in early diagnosis of diseases.
Current Opinion in Electrochemistry 2023, 39:101282
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in
this paper.
Data availability
No data was used for the research described in
the article.
Acknowledgments
The Deutsche Forschungsgemeinschaft (DFG e German Research
Foundation) is acknowledged in the frame of GRK 2203 (Pulmosens).
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