# Aptamers and MIPs as alternative molecular recognition elements for vasopressin and oxytocin sensing: a review

Essohanam Beke, Chantal Gondran, Benoît Chovelon, Eric Peyrin, Michael Holzinger, Corinne Ravelet

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## To cite this version:

Essohanam Beke, Chantal Gondran, Benoît Chovelon, Eric Peyrin, Michael Holzinger, et al. *Aptamers and MIPs as alternative molecular recognition elements for vasopressin and oxytocin sensing: a review*. Biosensors and Bioelectronics, 2025, 278, pp.117306. 10.1016/j.bios.2025.117306. hal-04968306

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Submitted on 26 Feb 2025

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# Aptamers and MIPs as alternative molecular recognition elements for vasopressin and oxytocin sensing: a review

Essohanam Beke<sup>a,b</sup>, Chantal Gondran<sup>a*</sup>, Benoît Chovelon<sup>b,c</sup>, Eric Peyrin<sup>b</sup>, Michael Holzinger<sup>a</sup>, Corinne Ravelet<sup>b*</sup>

<sup>a</sup>University Grenoble Alpes-CNRS, DCM, UMR 5250, Grenoble, France

<sup>b</sup>University Grenoble Alpes, DPM, UMR 5063, Grenoble, France

<sup>c</sup>Biochemistry Department, Grenoble Alpes University Hospital - Biology and Pathology Institute, F-38041 Grenoble, France

## ABSTRACT

Arginine vasopressin (AVP) and oxytocin (OT) are two important hormones that regulate various physiological and behavioral functions, such as blood pressure, water balance, social bonding, and stress response. The detection and quantification of these hormones are of great interest in clinical diagnosis and research. However, the conventional methods based on antibodies or enzymes have some limitations, such as high cost, low stability, and ethical issues. Therefore, alternative molecular recognition elements, such as aptamers and molecularly imprinted polymers (MIPs), have been developed to overcome these drawbacks. Aptamers are short nucleic acid sequences that can bind to specific targets with high affinity and specificity, while MIPs are synthetic polymers with imprinted binding sites mimicking natural receptors. Both aptamers and MIPs have advantages such as low cost, high stability, easy synthesis, and modification. In this review, we summarize the recent advances in the development and application of aptamers and MIPs for the sensing of vasopressin and oxytocin, and compare their performances. We also discuss the challenges and future perspectives of aptamers and MIPs as alternative molecular recognition elements for vasopressin and oxytocin sensing.
# 1. Introduction

Similar in structure, arginine vasopressin (Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH₂) (AVP) and oxytocin (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂) (OT) are nonapeptides hormones that differ only by two aminoacids (in bold). There is a remarkable similarity in the synthesis and secretion of AVP and OT. They are synthesized by the hypothalamus, and secreted by its posterior lobe, the neurohypophysis (Szczepanska-Sadowska et al., 2021). The secretion of OT and AVP is influenced by changes in body fluid osmolality, blood volume, blood pressure, hypoxia, and stress. AVP interacts with three subtypes of receptors: V1aR, V1bR, and V2R whereas OT activates its own OTTR and V1aR receptors. AVP and OT receptors are present in several regions of the brain (cortex, hypothalamus, pons, medulla, and cerebellum) and in the peripheral organs (heart, lungs, carotid bodies, kidneys, adrenal glands, pancreas, gastrointestinal tract, ovaries, uterus, thymus) (Szczepanska-Sadowska et al., 2021).

AVP is subject to osmoregulation related to consumption of salty foods, or more particularly, in the case of certain diseases, medication, or injury. It promotes vasoconstriction at the vascular level, which raises peripheral resistance and blood pressure. It is also known as anti-diuretic hormone (ADH) as it acts on its receptors in tubular cells at the renal level to accelerate water reabsorption (Stockand, 2010). The available evidence suggests that AVP also plays a complex role in the regulation of the respiratory system. As a circulating neurohormone, AVP influences respiratory activity via the circumventricular organs, especially the area postrema, which is situated in the dorsal surface of the medulla oblongata, and the arterial chemoreceptors, especially those located in the carotid bodies (Proczka et al., 2021).

As for OT, previously known as a hormone that facilitates uterine contractions and milk ejection in mammals, it has also been found to act as a neurotransmitter and is considered to mediate social behavior (Kosfeld et al., 2005), such as maternal and couple relationship (Jones et al., 2017). Preclinical research has also demonstrated the importance of AVP biology in social functioning in multiple mammalian species (Domes et al., 2007; Kosfeld et al., 2005).

Because of their involvement in these important physiological processes, a disturbance of their secretion can be related to serious pathologic states such as the syndrome of inappropriate antidiuretic hormone secretion (SIADH) and diabetes insipidus for AVP
(Qureshi et al., 2014), psychiatric disorders such as autism, schizophrenia, mood and anxiety disorders for OT (Sikich et al., 2021; Szczepanska-Sadowska et al., 2021).

OT can be used as a support treatment of autism. Several studies have shown that for people with autism spectrum disorder, a single dose of intranasal OT enhanced the performance of social cognition or motivation (Guastella et al., 2010; Watanabe T et al., 2014), compared to placebo treatments. However, children and adolescents with autism spectrum disorder, showed that 24 weeks of daily intranasal OT treatments did not improve social interaction (Sikich et al., 2021) compared to placebo treatments. The inconsistent results of these various studies may be due to a different formulation or dose of OT, the duration of the treatment, differences in the age of the participants, or the methods of analysis.

As mentioned above, deficiency of AVP leads to central diabetes insipidus, which needs to be distinguished from nephrogenic diabetes insipidus and primary polydipsia. For an accurate diagnosis of central diabetes insipidus, it is necessary to demonstrate that AVP release is impaired resulting in an increase in plasma osmolality (Robertson, 2016). Patients with central diabetes insipidus present clinical signs of dehydration with paradoxically unincreased urinary osmolality. Over the years, several analytical methods were reported to meet the need of the quantification of these hormones in biological fluids. While early on, the primary means of quantifying OT and AVP were bioassays (Fabian et al., 1969; Davies et al., 1983; Stürmer, 1989). Initially, the “pressor” bioassay measures the blood pressure response to intravenous injection of extracts in rats (Dekanski, 1952). By exploring their various physiological activities, it was possible to detect these peptides in plasma at extremely low concentrations (picomolar range) (Czaczkes and Kleeman, 1964; Segar and Moore, 1968a; Fabian et al., 1969; I. C. Robinson and Walker, 1974). These bioassays were, however, complex, time consuming, expensive, and not specific enough due to the cross-reactivity of AVP and OT towards their respective receptors (Leng and Sabatier, 2016). They were therefore not viable enough for routine use, and soon paved the way to immunoassays (radioimmunoassays and ELISAs) (Chard et al., 1970; Robertson et al., 1970; Burd et al., 1985). Those immunoassays rely on a reaction between the hormone in a sample and a distinguished amount of added antibody. Although just as sensitive as bioassays, those techniques are not easy to implement; because of their low molecular weight. Furthermore, AVP and OT are poor immunogens which makes the preparation of high-affinity antisera difficult. Only the competitive format of immunoassays is efficient for such small molecules, but low specificity has to be accepted. Factors such as endogenous antibodies may also interfere with the antibody-peptide interaction leading to over or underestimations of the
hormone concentrations (Emerson and Lai, 2013). Thus, many papers have used an enzyme-
based immunoassay to measure plasma concentrations without extracting the samples. Like
the first radioimmunoassay on unextracted plasma, these assays gave values that were too
high and completely wrong (Leng and Sabatier, 2016). Matrix extraction prior to
immunoassay is almost mandatory. Recently, MacLean et al. demonstrated that some
commercially available immunoassay kits (e.g. Enzo Life Sciences) are likely to interfere
with plasma samples, while others (e.g. Arbor Assays) do not interfere with unextracted
mouse plasma samples (diluted 1:8) (Gnanadesikan et al., 2021) but may interfere with urine
samples. They have conducted epitope mapping of the Arbor Assays antibody and assessed its
cross-reactivity with known, biologically active fragments of oxytocin. They found
considerable cross-reactivity (0.5-52% by-molarity) for three fragments of OT. These results
suggest that immunoassays in urine samples may be particularly susceptible to interference,
even when using common extraction protocols (Gnanadesikan et al., 2022). Nevertheless, the
two methods radioimmunoassays and ELISAs are considered to be the gold standard methods
and are used in numerous studies to understand the role of the two hormones in the human
body. Indeed, for example, commercially available ELISA kit (Enzo Life Sciences, Inc.,
Farmingdale, NY, https://www.enzo.com) can be used for measuring cerebrospinal fluid or
plasma AVP concentrations (Carson et al., 2015), or OT concentrations in culture
supernatants, milk, plasma, and serum from any species, with detection limits a of 2.84 and 15
pg mL⁻¹ (~ 2.62 and ~ 15 pmol L⁻¹), respectively, addressing the need to detect them at near
picomolar concentrations.

AVP can be measured in urine (reference values: 35 - 79 pmol/24h) or in plasma
(reference values: 0.5 - 13 pmol L⁻¹) (“Eurofins Biomnis,” 2024). To address this issue,
several liquid chromatography - mass spectrometry (LC-MS) methods have been developed
(Zhang et al., 2014, 2011; Franke et al., 2019; Takiwaki et al., 2021) which are more sensitive
and specific than bioassays and immunoassays. Integrated microfluidic systems using
aptamers for sample preconcentration and detection by mass spectrometry (Nguyen et al.,
2011; Yang et al., 2016) enabled reliable monitoring of AVP at 1 pmol L⁻¹ in buffer and 10
pmol L⁻¹ in human plasma ultrafiltrates. However, these techniques using mass spectrometry
are not readily available alternatives for general use, mainly because of the installation costs
and the need for qualified personnel.

New sensing methods for AVP and OT are emerging to offer efficient solutions.
Sensors based on new materials using electrochemical detection are an approach that has
recently been explored to detect OT (Mehrotra et al., 2023; Abedeen et al., 2025). To achieve
the goals of specificity and sensitivity, efforts particularly focused alternative molecular
recognition elements to antibodies: aptamers and molecular imprinted polymers (MIPs)
(Naseri et al., 2020). Aptamers are single-stranded DNAs or RNAs produced by the
*Systematic Evolution of Ligands by EXponential enrichment* (SELEX) process (Famulok and
Szostak, 1992; Tuerk and Gold, 1990) that can specifically bind to a variety of targets,
making them interesting candidates for bioanalytical applications (Zhou et al., 2014; Guan
and Zhang, 2020).

MIPs, known as synthetic biomimetic receptors are more stable and capable of binding
target molecules in a similar way to antibodies or enzyme receptors. MIPs are formed by
imprinting template molecules in a polymeric matrix through mainly non-covalent
interactions between the templates and the monomers. The subsequent removal of the
templates generates recognition cavities with the same shape, size, and functionality of the
template molecules. They can be used for the detection of small molecules (Wei et al., 2007;
Korposh et al., 2014; Rajpal et al., 2021), proteins (Bonini et al., 2007; Sun et al., 2019) and
even whole microorganisms (Schirhagl et al., 2012; Gast et al., 2019).

MIPs and aptamers have a number of advantages such like low cost, easy preparation
and handling, high chemical and thermal stability, long shelf-life, and reusability which make
them very interesting and competitive for meeting the requirements of vasopressin and
oxytocin sensing. As exposed in a recent review of Ali and Omer (Ali and Omer, 2022) the
comparison of these two recognition elements, which have similar advantages and different
disadvantageous will help us to understand why their characteristics and manufacture are
linked to the transduction methods used and their applications in complex environments.

In this review, we give a brief overview of these aptamer- and MIP-based sensors for
AVP and OT detection, classifying them by detection technique (divided into optical,
electrochemical and electrical detection) and highlighting their principles and performance.
The use of aptamers in analysis is promising, since by being synthetic, it allows easy
labelling/immobilisation to precise locations and rational sequence engineering through a
multitude of post-SELEX modifications. Other beneficial features are their inherent high
structural flexibility and capacity for conformational reorganization upon target binding
(known as the ligand-induced adjustment mechanism). It is therefore important to understand
the strategies and mechanisms for the use of these recognition elements. The first paragraph
will focus on aptamers and MIPs dedicated to the assay of AVP and OT, their structural
features and signaling mechanisms in biosensor designs.
## 2. Recognition and signaling mechanisms in aptamer and MIP-based biosensors for AVP and OT

### 2.1 Aptamers for AVP and OT detection: selection, structure and signaling mechanism

The first original aptamer specific to AVP was isolated by Williams et al. using an in vitro selection based on the Spiegelmer technology (Williams et al., 1997). Selection is carried out on D-AVP (non-natural and the enantiomer of the target) using a D-enantiomer DNA library. After identifying the D-DNA aptamer for the target D-enantiomer (D-AVP), the L-enantiomer DNA aptamer is synthesized for the detection of the natural L-enantiomer target (L-AVP). The advantage of this elegant and powerful strategy is that mirror-image analogs (referred to as L-nucleic acids) are highly resistant to the nuclease digestion. The authors clearly identified an imperfect hairpin secondary structure where the large, asymmetric internal bulge (20 nucleotides) is essential for the target binding while the apical loop does not participate in the ligand recognition (Fig. 1A).

Another RNA aptamer towards D-AVP using the same mirror image selection strategy has been developed by Klussmann team (Purschke et al., 2006). This aptamer, named NOX-F37, shows dissociation constants of 1.7 nmol L⁻¹, which is three orders of magnitude better than the dissociation constant of the 55-mer DNA aptamer of Williams et al. that binds to AVP with a K_d of 1.2 µmol L⁻¹. Its secondary structure is presented in (Fig. 1F).

This aptamer has been less used than its precursor, with only two publications (Yang et al., 2015, 2013). This is likely due to the fact that this aptamer is in the RNA series. The modified sequence they used is shown in (Fig. 1F).

Very recently, in 2024, Lin's team developed a D-series aptamer against L-AVP (named 66-DNA aptamer) using the capture SELEX method (Yu et al., 2024). This aptamer is based on the natural enantiomer D-deoxyribose and can be produced at a low cost compared to anti-AVP apta-spiegelmers. This is a major advantage for the assay of the natural target L-AVP. The aptamer has been designed with high affinity; the equilibrium dissociation constants K_d =1.4 nmol L⁻¹ (estimated from the reaction rate constants when the aptamer is immobilized) and K_d =543 nmol L⁻¹ (obtained from measurements in bulk solution).

Recently, the only reported aptamer for OT was selected using a non-SELEX-based selection approach (Raptamer Discovery Group) (Rana et al., 2023). Raptamer library beads incorporate proprietary modified nucleotides in the random region; these modified bases provide a more functionally diverse composition to enhance interactions with target
molecules. Following the selection of oxytocin specific Raptamers (Giner's Oxytocin (gOT) aptamers), they can be synthesized with the appropriate modified nucleotides in the sequences. The functionality of the candidate raptamer gOT-1B was validated using gold nanoparticles. Raptamer binds to nanoparticles and the binding induces a conformational change and the AuNPs aggregate. However, it is unfortunate that the sequences of the aptamers are not disclosed in this article. In addition, there is a lack of control experiments with scrambled sequences to establish the aptamer binding and specificity as is now recommended in many publications (Famulok and Mayer, 2014; McKeague et al., 2015; Jauset Rubio et al., 2016; Zara et al., 2021).

Since the first aptasensor created in 2000 by Ellington and colleagues (Jhaveri et al., 2000), many aptasensing strategies have been designed and developed to make aptasensors simpler, applicable to various methods and versatile for all types of targets. In addition to the simple direct approach often used in field effect transistor (FET) and surface plasmon resonance (SPR) methods, there are many alternatives signaling methods based on the specific characteristics of aptamers. One strategy relies on the intrinsic target-induced **conformational switch**, which will be used to generate a measurable signal (Fig. 1A). Although such switching aptamers have often shown a good affinity for the target, this approach has some limitations. Many aptamers have a partially folded structure and the signal produced by this strategy is often low. In addition, in the case of small molecules, the target may interact with the aptamer without a detectable conformational change. The application of this strategy is further limited to few aptamers (Munzar et al., 2019).

In order to mimic sandwich systems using antibodies for protein assays, two aptamer-based pseudo-sandwich systems have been developed for small molecules. Stojanovic and colleagues (Stojanovic et al., 2000) developed a **split-aptamer**-based strategy in which the aptamer sequence was divided into two subunits that form a ternary complex in the presence of the target (as Fig. 1B). To obtain a signal, the two subunits (labelled, for example, with a FRET or fluorescent-quencher pairs) have to be close enough to each other to allow signal transmission, which occurs only in the presence of a target. This strategy has been used for D-AVP by Du and his colleagues by immobilizing a subunit part of D-AVP aptamer (Fig. 1B), the other being added for the sensing platform (Du et al., 2012). Another “pseudo-sandwich” aptamer biosensing concept based on the **kissing complex strategy** (as Fig. 1C) has been described by Toulmé and Peyrin groups (Durand et al., 2016, 2014; Goux et al., 2015). This approach is based on the capacity of RNA aptamers to recognize RNA or DNA hairpin structures through loop-loop interactions. The aptaswitch, an engineered aptamer modified by
a short RNA/DNA sequence is undergoes an unfolded-to-folded state transition upon addition of the target. The resulting apical loop of the target bound aptaswitch then interacts with a hairpin (named aptakiss) through a kissing complex, thus reporting the presence of the target. This new type of aptasensor is able to detect small molecules based on a dual recognition mechanism that includes a signaling partner that contributes to increase the stability of the complex with the target. This strategy was conceived in order to enhance the specificity and the resulting signal gain of the biosensor. To transform an aptamer into a split or kissing complex aptasensor, it is fundamental to know the aptamer–target structure and understand the role of each nucleobases (Chen et al., 2016) and requires a careful design of the two assembling modules (Perrier et al., 2018). This strategy was applied to D-AVP (Chovelon et al., 2018) (Fig. 1C).

Other strategies called **competitive assays** based on the ability of aptamers to bind to competing element, which may be i) complementary sequence (Fig. 1D) as reported by (Lu et al., 2012) ii) DNA intercalators (Fig. 1E) by (Ren et al., 2012) iii) nanomaterials by (Ren et al., 2013) (Fig. 1F) and iv) an analogue of the target itself (Yang et al., 2015) (Fig. 1G). The dissociation caused by the target binding to the aptamer is converted into a measurable signal during target detection (Lau and Li, 2014).
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Sensing strategies for AVP. (A) Conformational switching aptamer. Expected secondary structure of the D-AVP aptamer (from Williams et al., 1997). The red bases correspond to the bulge involved in D-AVP binding. The red circle corresponds to the localization of the tetramethylrhodamine (TMR) fluorophore for the detection of D-AVP using a TMR-labeled aptamer developed by Liu and Zhao (Liu and Zhao, 2016). (B) Split aptamer. The sequence of D-AVP aptamer was split into two fragments in the sensor developed by Du et al. (Du et al., 2012) (C) Kissing complex with the sequences of DNA vasoswitches and aptakiss 3 used in this strategy (Chovelon et al., 2018). (D) Competition strategy with complementary strand combined with a split strategy, splitting the D-AVP aptamer into two subunits (Lu et al., 2012). (E) Competition strategy with intercalator developed by Ren and colleagues (Ren et al., 2012). (F) Nanomaterial-based competition strategy as described by Wang's team (Ren et al., 2013). (G) Competitive binding between standard AVP immobilized on graphene and a mixture of free aptamer bound to AVP present in sample. This results in a change in the graphene conductance. (Yang et al., 2015). In the box on the left: structure of the D-AVP RNA aptamer developed by Klussmann team (Purschke et al., 2006).</figcaption></figure>
## 2.2 MIPs for OT and AVP detection: synthesis and signaling mechanism

There are various approaches to MIP synthesis. The **first strategy** involves the **formation of reversible covalent bonds** between the monomers and the template. However, after MIP synthesis, template removal requires cleavage of the covalent bonds, which can affect the functionality of the cavity (Huang et al., 2011). **The second approach** involves the **preparation of non-covalent interactions** between the monomer and template, such as ionic, hydrophobic or hydrogen-bond interactions. This method for preparing MIP has been the mostly used due to the ease of template removal without the need for formation and subsequent cleavage of covalent bonds, preventing the collapse of the polymeric structure (Martín-Esteban, 2013; Gama and Bottoli, 2017). In addition, the choice of monomer and cross linker is very important to create highly specific cavities designed for the template molecule and to control the morphology of the MIP matrix. Various monomers and cross-linking reagents used in the MIP synthesis of AVP and OT are summarized in **Table 1**.

In 2003, MIP, specific to oxytocin and vasopressin, were prepared by using a synthesized chiral monomer (N-acrylic acid-L-cysteine benzylamide), immobilized on a gold coated quartz crystal by formation of thiol gold bonds (Lin et al., 2003). The MIP was formed with a drop casted mixture of acrylic acid, acrylamide, N-benzylacrylamide, and the templates (oxytocin or vasopressin) by irradiation. The chirality of this monomer improved the interaction with the peptides while the presence of benzylacrylamide confers hydrophobicity to this mixture and reduced the amount of irreversibly entrapped template molecules. In 2019, the same team proposed another original approach for MIP based QCM sensing. They synthesized a chiral amphiphilic acrylate monomer which clearly reduced the need of other cross linkers (Lin et al., 2019). Furthermore, epitopes instead of whole peptides were used as templates and reduced unwanted irreversible embedding.

In 2015, a MIP based SPR sensor for oxytocin detection was developed by Yola and collaborators (Yola et al., 2015). They mixed oxytocin (OXT = OT)-MAGA monomer and ethylene glycol dimethacrylate (EGDMA) to prepare a stock monomer solution (Fig. 2). EGDMA acted as a cross-linker to make specific binding areas of template molecules more stable after they have been eluted. Then, using the spin coating process, an aliquot was dropped onto the SPR chip surface previously modified by allylmercaptane with AIBN (N,N'-azobisisobutyronitrile) as initiator. The polymerization was performed with UV light, after which oxytocin molecules were removed by breaking electrostatic interactions and hydrogen bonding between MAGA monomer and oxytocin in 1 mol L⁻¹ NaCl solution.
In 2018, Kutner's group synthesised some functional monomers that non-covalently bound the oxytocin template through multiple-point interactions. These monomers contained the polymerisable bisbithiophene moiety. The presence of this moiety allowed the arrangement of functional monomer molecules around the oxytocin molecule to be "frozen" by electropolymerisation. The MIP was deposited as a thin film on an Au electrode by potentiodynamic electropolymerisation. The functionalised electrode was then integrated into a commercial microfluidic system, which allowed electrochemical experiments to be carried out with a small volume of solution. (Sharma et al., 2018).

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Schematic representation of preparation of a SPR chip by surface polymerization. The SPR surface was functionalized with Allyl mercaptane (CH₂CHCH₂SH) and N,N'-azobisisobutyronitrile (AIBN as initiator). A monomer MAGA was added to oxytocin (OXT). MAGA-OXT complex with ethylene glycol dimethacrylate (EGDMA), a cross linker monomer, were mixed to prepare stock monomer solution. After the elution of template molecules, EGDMA as cross-linking increases the stability of specific binding-areas of template molecules. The solution was dropped onto the SPR surface by using spin coating method and after polymerization was started by UV light. Reprinted from (Yola et al., 2015). Copyright © 2015 Elsevier B.V. All rights reserved.</figcaption></figure>

<table><caption>**Table 1.** Functional monomers and cross-linkers used in molecular imprinting for AVP and OT.</caption><thead><tr><td>Target</td><td>Functional monomers</td><td>Cross linker</td><td>References</td></tr></thead><tbody><tr><td>AVP and OT</td><td>Benzylacrylamid<br>Acrylic acid<br>Acrylamide</td><td>(N-Acr-L-Cys-NHBn)₂</td><td>(Lin et al., 2003)</td></tr><tr><td>OT</td><td>MAGA</td><td>Ethylene glycol dimethacrylate (EGDMA)</td><td>(Yola et al., 2015)</td></tr><tr><td>OT</td><td>1-methylamide-4-bis(2,2'-bithienyl)methane</td><td>2,4,5,2',4',5'-hexa(thiophen-2-yl)-3,3'-bithiophene</td><td>(Sharma et al., 2018)</td></tr><tr><td>OT</td><td>Acr- His- NHNH- Fmoc</td><td>N,N- ethylene- bis- acrylamide (EBAA)</td><td>(Lin et al., 2019)</td></tr></tbody></table>

Used in biosensors, MIPs are synthetic biomimetic recognition element analogues of natural and biological antibody systems. As a result, they work on a “lock and key” recognition mechanism, without conformational change. Therefore, there are far fewer
possible signaling strategies compared with what is found with aptamers, which is theoretically limited to approaches based on direct assays or competition assays with target analogues (not reported here for AVP and OT).

The use of these presented MIPs and the aptamers for AVP and OT detection are discussed as followed.

### 3. Optical sensing methods

Compared to conventional analytical methods, optical approaches can provide real-time detection of biological and chemical substances in a cost-effective manner. Due to their selectivity, rapidity, and high sensitivity, they gained competitiveness for biotechnology, disease diagnosis and medical applications (Cooper, 2002; Yoo and Lee, 2016; Kaur et al., 2022). For AVP and OT sensing, optical techniques include colorimetric, fluorescence, RAMAN spectroscopy and SPR approaches are summarized in (Table 2). In this section, all the optical analysis methods used for the determination of AVP have used essentially the aptamer selected by Williams et al. (Williams et al., 1997).
<table><caption>**Table 2.** Comparison of optical analysis methods for determination of vasopressin and oxytocin. NC = Not communicated. RSD = Relative Standard Deviation and n = number of samples.</caption><thead><tr><th>Target</th><th>Immobilization strategy</th><th>Biorecognition element</th><th>Signaling mechanism</th><th>Transduction technique</th><th>Analysis time</th><th>Linear range (mol L<sup>-1</sup>)</th><th>Limit of detection (LOD) (mol L<sup>-1</sup>)</th><th>1) Recovery<br>2) Stability<br>3) RSD (n)</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">AVP</td><td>No immobilisation</td><td>Aptamer</td><td>Competitive</td><td>Colorimetry</td><td>10 min</td><td>125-225 x 10<sup>-9</sup></td><td>-</td><td>1-3) NC (n = 3)</td><td>(Ren et al., 2013)</td></tr><tr><td>No immobilisation</td><td>Aptamer</td><td>Competitive</td><td>Colorimetry</td><td></td><td>-</td><td>16 x 10<sup>-6</sup></td><td>1-3) NC (n = 3)</td><td>(Chovelon et al., 2023)</td></tr><tr><td>No immobilisation</td><td>Aptamer</td><td>Competitive</td><td>Fluorescence</td><td>&lt; 15 min</td><td>100 x 10<sup>-9</sup> - 10 x 10<sup>-6</sup></td><td>100 x 10<sup>-9</sup></td><td>1-2) NC 3) &lt;7.0% (n = 3)</td><td>(Ren et al., 2012)</td></tr><tr><td>No immobilisation</td><td>Aptamer</td><td>Split + amplification</td><td>Fluorescence</td><td>10 min</td><td>-</td><td>2 x 10<sup>-9</sup></td><td>1-3) NC (n = 5)</td><td>(Lu et al., 2012)</td></tr><tr><td>No immobilisation</td><td>Aptamer</td><td>Conformational switch</td><td>Fluorescence anisotropy</td><td>10 min</td><td>-</td><td>0.2 x 10<sup>-6</sup></td><td>1-2) NC 3) &lt;3.0% (n = 5)</td><td>(Liu and Zhao, 2016)</td></tr><tr><td>No immobilisation</td><td>Aptamer</td><td>Kissing complex</td><td>Fluorescence anisotropy</td><td>90 min with incubation time</td><td>2.3-150 x 10<sup>-9</sup></td><td>7.9 x 10<sup>-9</sup></td><td>1-3) NC (n = 3)</td><td>(Chovelon et al., 2018)</td></tr><tr><td>Streptavidin/ Biotinylated aptamer</td><td>Aptamer</td><td>Direct</td><td>Chemiluminescence</td><td>10 min (not including the incubation time)</td><td>0.1 x 10<sup>-9</sup> - 1 x 10<sup>-6</sup></td><td>79 x 10<sup>-12</sup></td><td>1-2) NC 3) 2.3% (n = 7)</td><td>(Li et al., 2014)</td></tr><tr><td>Gold nano-tube array/ aptamer</td><td>Aptamer</td><td>Direct with labelled-AVP</td><td>Surface enhanced Raman scattering (SERS)</td><td>-</td><td>-</td><td>11.28 x 10<sup>-12</sup></td><td>1-3) NC (n = 3)</td><td>(Huh and Erickson, 2010)</td></tr><tr><td>Gold nanopillars/ thiolated aptamer</td><td>RNA Aptamer</td><td>Direct with labelled-AVP</td><td>Surface-enhanced Raman spectroscopy (SERS)</td><td>-</td><td>1 x 10<sup>-12</sup> - 1 x 10<sup>-9</sup></td><td>1 x 10<sup>-12</sup></td><td>1-2) NC 3) &lt;5.0% (n = 3)</td><td>(Yang et al., 2013)</td></tr><tr><td>OT</td><td>EDMA/HMAA/MAGA</td><td>MIP</td><td>Direct</td><td>SPR</td><td>&gt; 1h</td><td>0.09-9.22 x 10<sup>-9</sup></td><td>2.98 x 10<sup>-12</sup></td><td>1) 5 cycles (no difference)<br>2) 5°C-30 d; 20°C-12h (no difference)<br>3) NC</td><td>(Yola et al., 2015)</td></tr></tbody></table>
### 3.1 Colorimetric approaches

To our knowledge, only two studies deal with colorimetric AVP detection with aptamers. First, Ren and al. (Ren et al., 2013) developed two (OR and INHIBIT) enantioselective colorimetric logic gates. Their work was based on the ability of the disulfide group to adsorb onto gold surfaces (Nuzzo and Allara, 1983) and on the observation that the AVP could aggregate the citrate-coated gold nanoparticles (GNPs) without the use of salt by acting as a cross-linking agent. Pure GNPs showed a surface plasmon resonance peak at 520 nm in the UV/Vis spectrum, while a new absorption peak near 620 nm appeared with increasing L-AVP concentration (Fig. 3A). The absorption ratio ($A_{620}/A_{520}$, R) of the absorbance of GNPs at 620 nm and at 520 nm was used as an output signal to identify the aggregation and dispersion state of GNPs and could thus be plotted against L-AVP concentration (Fig 3B). That was caused by the coupling of the GNPs plasma mode and dipole-dipole interactions (Ghosh and Pal, 2007). It was hypothesized that AVP's two main functional groups - disulfide and guanidinium groups derived from cystine and arginine residue could induce the aggregation of GNPs (Choi et al., 2007). In their study, the previously described AVP-D-aptamer (named here V-apt) was used as a chiral selector to distinguish between the enantiomers (D and L) of AVP and as a masking agent to prevent GNP aggregate formation through binding interactions between D-AVP and V-apt (Fig. 3C). In the absence of V-apt, both D-AVP and L-AVP enhanced the R value (Fig. 3B). The R value for D-AVP significantly dropped once V-apt was added to the system which remained until high V-apt concentrations (750 nmol L⁻¹). Investigating GNP aggregation induced by AVP at different pH values showed that the positively charged guanidinium group of the arginine residue was essential for AVP-induced aggregation of GNPs. As the same arginine guanidinium group was found to interact specifically with V-AVP (Michaud et al., 2003), once masked by the D-AVP-aptamer interaction, GNP aggregation was inhibited. As a result, the enantiomers of AVP could be distinguished enantioselectively and colorimetrically by virtue of V-apt acting as chiral selector (Fig. 3).
to the interaction of the target with the colour probe as mentioned before. The presence of the
aptamer protects NB from aggregation due to its intercalation into double-stranded DNA
and/or interaction with nucleobases. After addition of the target, the competition between NB
and the target for binding to the aptamer takes place, leading to an increase in the dye salt
aggregation and then in the blue-to-blank colour change of the solution (Fig. 4A NB
competition).

A decrease of absorbance value of NB at 632 nm was observed for D-AVP with a
limit of detection of 16 µmol L⁻¹ whereas L-AVP did not cause any response decrease over
the concentration range, demonstrating the selectivity of the assay (Fig. 4B).

It can be noticed that in other cases, for example with functional oligonucleotides, the process
is reversed, and the presence of the target facilitates the structuring of the recognition element
and thus the intercalation of NB (NB capture).

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** (A) Schematic representation of the DYSACO assay, NB competition is applied to D-AVP. (B). Dose response curve for D-AVP (blue circle) and L-AVP (red circle) using DYSACO assay. Reproduced from (Chovelon et al., 2023). © 2023 Elsevier B.V. All rights reserved.</figcaption></figure>

Although these two colorimetric methods are interesting, their high detection limits (50 nmol L⁻¹ and 16 µmol L⁻¹ respectively) do not allow the detection of AVP in a picomolar concentration range, which is essential for patient monitoring. Furthermore, these methods have not been tested in complex media for vasopressin detection.

Fluorescence detection, which is generally 100 to 1000 times more sensitive than UV-visible
absorption detection, represents a more promising solution to reach the desired concentration
range.
## 3.2 Fluorescence-based approaches

Fluorescence biosensors have many advantages such as fast response, simple instrument, multiple analysis, high sensitivity and good selectivity (Bauch et al., 2014; Gale, 2010; Leopold et al., 2019; Liu et al., 2020; Zhang et al., 2017).

Wang's team proposed a simple and label-free fluorescent technique via fluorescent indicator displacement (FID) named guanidiniophthalocyanine dye (Zn-DIGP) (Ren et al., 2012) like presented in **Fig. 1E**. Splitting the D-AVP aptamer in two subunits has shown that Zn-DIGP is preferentially attached to the longest internal loop of V-AVP (single-stranded DNA) and produces intense fluorescence. Following the addition of D-AVP, a strong interaction between D-AVP and the internal loop of the aptamer forces the release of the Zn-DIGP from the V-AVP with a fluorescence decrease. While L-AVP was unable to cause any change in fluorescence, the reduced fluorescent signal from the direct fluorophore displacement allowed a sensitive and label-free quantitative assessment of D-AVP. A limit of detection of 100 nmol L⁻¹ was obtained in presence of D-AVP. Whether in pure PBS buffer, or in diluted biological fluids (1% urine and 1% serum in PBS buffer after a centrifugation at 8000 rpm for 10 min to remove the precipitates), the D-AVP could still be clearly discriminated from the L-AVP. While more promising compared to colorimetric assays, this strategy was operational for vasopressin detection in the nanomolar range which is unfortunately still not sufficient for clinical samples.

In most fluorescent biosensors, the readout signal for the analytes is the intensity of the fluorescent signal and their performances can be improved using amplification technologies. Aiming to amplify the aptamer-AVP recognition, Lu and his team (Lu et al., 2012) introduced a versatile sensing platform. The technique is based on the splitting of anti-vasopressin aptamer into subunits (1) and (2) (**Fig. 5**) where each is specific to the substrate. The system also consists of the single strand (3) which functions as a blocker to prevent the formation of the (1)/(2) complex and is complementary to a portion of subunit (1). Each of the subunits is extended at its 3' and 5' ends with complementary sequences that can respectively be cut by endonucleases PvuII and HaeIII restriction enzymes in their duplex structure. The subunit (1) of the aptamer was changed at the 3'-end with the fluorophore (short cyanine, Cy5) and which can be inhibited with the quencher-functionalized nucleic acid (3) (Iowa Black RQ, IAbRQ). The second aptamer subunit (2) was also added to the system (**Fig. 5A**).
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** (A) Endonucleases based amplified detection system of vasopressin using the anti-vasopressin aptamer subunits. (B) Comparison of time dependent fluorescence changes upon analyzing AVP, 50 µmol L⁻¹, by: (a) the amplified sensing platform with the aptamer subunits and endonucleases; (b) the non-amplified aptamer subunits system (without endonucleases). (C) Fluorescence spectra of the analysis of different concentrations of vasopressin using the amplified aptamer subunits/endonucleases system. Concentrations of vasopressin correspond to: (a) 0 µmol L⁻¹; (b) 0.002 µmol L⁻¹; (c) 0.005 µmol L⁻¹; (d) 0.01 µmol L⁻¹; (e) 0.05 µmol L⁻¹; (f) 0.1 µmol L⁻¹; (g) 0.5 µmol L⁻¹; (h) 1 µmol L⁻¹; (i) 5 µmol L⁻¹; (j) 10 µmol L⁻¹; (k) 50 µmol L⁻¹; (l) 100 µmol L⁻¹. (D) Calibration curve corresponding to the obtained fluorescence intensity at = 662 nm after assessing various vasopressin concentrations. Reproduced from (Lu et al., 2012) with permission from the Royal Society of Chemistry.</figcaption></figure>

In the presence of the substrate, the fluorescence of the fluorophore was triggered-on with the formation of the (1)/(2)-AVP complex and the separation of the (1)/(3) structure, providing an optical readout for the substrate-analyte. The aptamer-subunits/AVP complex was separated through biocatalytic cleavage of the complex (1)/(2)-substrate in the presence of endonucleases, which has caused the aptamer-substrate complex to separate, the analyte to regenerate, and the fluorophore to be released. The time-dependent changes in fluorescence intensity that result from assessing 50 µmol L⁻¹ vasopressin in the presence of the (1)/(3) complex, second aptamer subunit (2), and endonucleases are shown in **Fig. 5B**, curve a. As the reaction time was extended, the fluorescence intensities have risen, which was compatible with the vasopressin-stimulated amplified detection mechanism. For comparison, **Fig. 5B**, curve b, displays the variations in fluorescence intensity in the system lacking endonucleases
(Fig.1D). When the AVP was recycled in the presence of endonucleases, the fluorescence intensities were approximately three times higher than without the biocatalysts, demonstrating the amplifying role of the endonucleases in the recycling of the AVP. The amplified aptamer system's fluorescence spectra at various vasopressin concentrations are shown in Fig. 5C. As the vasopressin concentration rises from 2 nmol L⁻¹ to 100 µmol L⁻¹, there is a noticeable increase in fluorescence intensity. The amplified aptamer system's detection limit was 2 nmol L⁻¹ in the presence of biocatalysts which is much higher than the picomolar target range. A comparison of the detection limits of this fluorescent system without amplification (1 µmol L⁻¹) with the colorimetric detection limits (16 µmol L⁻¹) discussed above shows that the complementary strand displacement system (Fig. 1D) is less efficient than a DNA intercalator displacement system (Fig. 1E). There is a significant loss of sensitivity which is not counterbalanced by the fluorometric detection. For this type of structure switching assay, amplification is therefore necessary to lower the detection limits.

Another trustworthy method of fluorescence sensing is the fluorescence anisotropy (FA) assay, which relies on the rotational diffusion of the fluorescence label probe upon target binding (Jameson and Ross, 2010; Lea and Simeonov, 2011; Smith and Eremin, 2008). For labelled biomacromolecules of equivalent dimensions, the degree of depolarization depends on both, the internal adaptability of the molecule domains and the localized movement freedom of the bound dye. This is closely conditioned by the nature of the macromolecule, the fluorophore, the labelling position, as well as the nature and length of the linker between the bound dye and the tagged molecule. FA is related to how fluorophores spin throughout their excited state lifetime. Slow rotation of the fluorescent probe arises in a high FA value while rapid rotation of the fluorescent probe typically affords a low FA value in such assays. As a ratiometric fluorescence method, FA assay exhibits excellent reproducibility and is insensitive to sample fluorescence fluctuations and photobleaching. Detection of small molecules using direct aptamer assays with dye labelling on its extremities was done based on the conformational change of nucleic acid aptamers in presence of its target (Perrier et al., 2010).

However, the method cannot be applied to oligonucleotides that are subjected to a small structural change when complexed to the target.

Switching aptamer assays based on the segmental motion changes of the fluorophore tagged aptamer can be employed as an interesting alternative. The nature of the fluorophore is essential in this assay and can be based on a binding induced change of intramolecular strong interactions between TMR and guanine base (Unruh et al., 2005). In 2016, Zhao and Liu (Liu and Zhao, 2016) have constructed several probes by changing the labelling location of TMR
on internal thymine base of the D-AVP aptamer in order to identify the most responsive
probes. The vasopressin-induced aptamer reorganization generates changes in the dye
freedom degree resulting in a modification of the photoinduced energy transfer quenching
between the dye and the adjacent guanine bases, thus affecting TMR FA value. The resulting
variation in both, the local mobility and the excited state lifetime of the dye produces a
decrease in the FA signal in relation with the labelling site and the conformational change of
the aptamer. Among TMR labelled aptamers, the aptamer with a TMR labelled on the 34th T
base (34T-TMR-Apt) (Fig. 1A) showed the best FA response for D-vasopressin. The FA and
fluorescence intensity of 34T-TMR-Apt decreased and increased respectively with increasing
D-AVP. The detection limit of D-vasopressin was around 0.2 mmol L-1, and the dynamic
detection range was from 0.2 mmol L-1 to 200 mmol L-1. The selectivity of the direct FA
assay was also performed on various substances including L-vasopressin, 7-amino-4-
methylcoumarin, bovine serum albumin, immunoglobulin G, arginine, phenylalanine, aspartic
acid, serine, and tyrosine.

To validate the feasibility of the proposed method in a complex sample matrix, D-AVP was spiked in 100-fold diluted human serum and 5% and 10% urine samples. The FA changes became smaller than in the binding buffer solution, with the FA change decreasing by approximately 15% in the 100-fold diluted human serum and 25% and 30% in the 5% and 10% urine samples, respectively. The detection limit of D-AVP was achieved at 0.5 mmol L⁻¹ in the complex sample matrix tested.

Our group has used the kissing complex strategy (Fig. 1C), which relies on a dual
recognition mechanism involving the formation of functional nucleic acid architectures, for
the detection of AVP (Chovelon et al., 2018). The work was initiated with the same aptamer
previously described recognising the non-natural D-enantiomer. From this aptamer three
aptaswitches (vasosw3' A, vasosw3' B, vasosw3' C) were designed and synthesized by
substituting a part of the sequence, having a little interest in the recognition of the target, by a
sequence capable of performing a "kissing complex" (Fig. 1C). Aptakiss 3 was labelled in 5'
with Texas red. An increase of the fluorescence anisotropy was observed during the formation
of the vasoswitch - target - aptakiss ternary complex. Optimisation of the buffer composition
allowed a clear improvement in the sensitivity: the addition of methanol increased the
detection limit from 340 nmol L⁻¹ to 8 nmol L⁻¹. The presence of methanol enhances the Mg²⁺
- oligonucleotide interaction (particularly important in target recognition and kissing complex
formation) probably due to the decrease in the dielectric constant of the buffer. Finally, the
mirror image of this assay was set up: Spiegelmer synthesis of the L-series aptaswitch (L-
vasosw3' A) recognising L-arginine-vasopressin (natural enantiomer) and the L-series
aptakiss (L-aptakiss 3). The non-natural L-series oligonucleotides have good in vivo stability
which facilitates the transposition of the system in complex media. Application in complex
media was performed in 10% of human serum sample overloaded with L-AVP. As expected,
the L-series oligonucleotides were resistant to nuclease degradation.

The kissing complex system implemented for the detection of vasopressin has
improved the detection limits obtained by FA compared to those obtained by Zhao and Liu
(Liu and Zhao, 2017). However, these LODs are still not sufficient to detect vasopressin at
physiological values.

Within the optical transduction methods, the above presented examples can be
classified as techniques in “homogeneous” media where analyte – receptor interactions and
the resulting signal capture occurs in solution (Table 2). Another principle is based on the
immobilization of the receptor unit on a surface to provide a sensing element with defined
spatial resolution which allows precise measurements with reduced volumes at the
heterogeneous surface-solution interface.

Chemiluminescence is a powerful tool for optical transduction since it does not require
irradiation by a light source which can considerably reduce background noise and detection
limits. Li and al. (Li et al., 2014) developed a straightforward chemiluminescence (CL)
method to detect vasopressin. The method used the AVP-aptamer as the recognition element,
N-(aminobutyl)-N-(ethylisoluminol)-functionalized gold colloid (ABEI Au colloid) as a
signal reporter, and a microplate as immobilizing substrate. The aptamer 5'-end was
biotinylated and then attached to a 96-well microplate via the interaction between biotin and
streptavidin (SA) to form the sensitive area. These immobilized receptors were then incubated
with the target (AVP) solution containing ABEI-Au colloids. CL was induced by adding H₂O₂
to the system, but the catalyst is not specified. This led to the formation of a peroxo-dianion of
the luminol unit of ABEI causing the elimination of nitrogen which provokes exited states of
the resulting aniline dicarboxylate (Ohno et al., 1999). A limit of detection of 79 pmol L⁻¹ was
reached, which represents one of the most performant and reliable examples for vasopressin
detection in physiological concentration ranges even when selectivity of the setup was not
investigated.
### 3.3 RAMAN-based approaches

Surface enhanced Raman scattering (SERS) became a powerful spectroscopic tool for biosensing applications (Ilkhani et al., 2016; Moore et al., 2018; Choi et al., 2020). The principle is based on laser induced harmonic oscillation of electrons called surface plasmons in the valence band of metals. This oscillation generated an electromagnetic field which amplifies inelastic scattered photons, or Raman scattering, provoked by (bio)molecular vibrational modes. This effect is further amplified when the metal sizes are clearly below the excitation wavelength. In this case, the electron density is then polarized and the plasmons oscillate in resonance with the light frequency. Metals like copper, silver, or gold are resonant in the visible light range. Even when silver has the strongest plasmonic properties, gold became the most used material due to its biocompatibility, chemical inertness, and well-established processes for nanoshaping. A further advantage of SERS is the resolution of the molecular signature of the (bio)entity allowing the quantification of the analyte of interest in the presence of other, usually interfering, molecules. The signal amplifying effect of SERS can even be modulated via nanopatterning of the surface with defined inter-particle distances since when two particles are in close distance, the harmonic oscillation interferes constructively leading to so-called hotspots.

An original approach for improved SERS sensing of vasopressin was reported by Huh et al. (Huh and Erickson, 2010). Here, alternating gold/silver/gold layers in nanotube shapes were formed using anodized alumina nanoporous membrane. This nanotube array was integrated in a microfluidic setup and modified with the AVP aptamer via thiol grafting. As Raman dye, fluorescein labelled AVP was used which also allowed optical monitoring with a fluorescence microscope. The authors clearly demonstrated that the multi-metal nanotubes with silver as intermittent layer showed the strongest SERS effect compared to pure gold nanotubes and therefore to highest fluorescein band intensities. The aptasensor showed good sensitivity and a linear range in the concentration range of 5 to 30 µU mL⁻¹ (10 to 65 pmol L⁻¹).

The SERS method has also been used by Yang et al. which have proposed an original approach for such hotspot formation *via* nanopatterning a surface with gold-covered silicon nanopillars (Yang et al., 2013). The distance between these pillars is between 50 and 100 nm which is too far for inter-particle coupling of plasmons. After immobilization of a thiol tagged RNA aptamer previously described (Purschke et al., 2006) and during evaporation of the
liquid, the induced capillary effect provokes a bending of these pillars forming small clusters
with inter-particle distances around 10 nm which is now short enough for such a hotspot
effect. Fortunately, with this aptasensor setup, a detection limit in the concentration range of
interest (1-10 pmol L⁻¹) could be reached.

These examples of SERS based biosensing used vasopressin just as biosensors model
systems and emphasized the original nanopatterns and hotspot formations which are possibly
applicable for any biological analytes. Unfortunately, native vasopressin has no strong Raman
signature and had to be modified with FITC in one case and 5-carboxytetramethylrhodamine,
a commonly used Raman probe in other case. This setup is therefore barely suitable for
clinical samples. Any selectivity tests using relevant interferants were therefore not done. This
method could be used to measure AVP in a complex environment, but only with a
competitive format, which would reduce the Raman response and increase the detection
limits.

3.4 SPR based approaches

One of the other interesting approaches for optical biosensors is surface plasmon
resonance (SPR). Surface plasmons (SPs) are the collective oscillation of free electrons in the
conduction band of metals, and only a p-polarized incident wave can excite them (Lakowicz,
2004). In resonance condition, the maximum energy of the incident light is transferred to the
SPs, causing a sharp dip in the reflectivity curve, when momentum matching is satisfied, that
is, when the component of incident light momentum parallel to the prism/metal interface
equals the SPs momentum. Resonance angle (θres) is the angle at which the surface plasmons
are resonant. Due to its appealing sensing capability, compact design, and simple
implementation, SPR technology has opened a new path for numerous significant applications
in the field of sensing (Olaru et al., 2015; Zhou et al., 2019; Wang et al., 2022). Based on this
technique, Yola and al. (Yola et al., 2015) have developed an oxytocin- MIP nanofilm on gold
surface of SPR chip for oxytocin detection. The sensing platform exhibited a high sensitivity
towards oxytocin with a detection limit of 2.98 pmol L⁻¹ and was selective against
vasopressin, isotocin and dynorphin1-8 making it a really viable technique for oxytocin
detection in biological fluids. The molecular imprinted sensor based on surface plasmon
resonance was also applied successfully to milk sample after centrifugation and dilution of
samples for the determination of oxytocin (around 99% recoveries). This application is the
only known example of the use of an optical sensing method for measuring OT.
For the detection of the hormone vasopressin, chemiluminescence and SERS biosensors were found to have the widest linear range and the lowest LODs, making it possible to approach the desired concentration range.

4. Electrochemical and Electrical sensing methods

Among above-described sensing techniques, electrochemical transduction can be considered as most attractive in terms of performance and utility. Commonly, they are classified into amperometric (current measurement at constant potential), voltammetric (current measurement over a potential range), potentiometric (voltage measurement, generally at equilibrium), and impedimetric (impedance measurement) or conductometric (resistance measurement) methods. Besides their high sensitivity, some electrochemical sensing techniques allow label-free detection, eliminating complexity and cost of sample labeling. Their greatest asset, however, is to be suitable for miniaturization enabling the development of portable and wearable devices. Able to monitor body fluids such as sweat (Oh et al., 2018; Costa et al., 2022; Zhu et al., 2022; Gao et al., 2023), interstitial fluid (Kolluru et al., 2019; De la Paz et al., 2021; Custers et al., 2022) and tears (Kim et al., 2017; Sempionatto et al., 2019), electrochemical sensors attract tremendous attention as alternatives to conventional sophisticated analytical techniques in the healthcare industry. In a common configuration, electrochemical sensors comprise 3 electrodes, the reference, counter and working electrode. The sensing element is immobilized on the working electrode. For AVP and OT detection, voltammetry and impedance measurements were essentially employed. Table 3 summarizes electrochemical and electrical techniques used for vasopressin and oxytocin sensing and their performance.
<table><caption>**Table 3.** Comparison of electrochemical and electrical sensing methods for determination of vasopressin and oxytocin. NC = Not communicated. RSD = Relative Standard Deviation and n = number of samples.</caption><thead><tr><th>Target</th><th>Immobilization strategy</th><th>Biorecognition element</th><th>Signaling Mechanism</th><th>Transduction technique</th><th>Time</th><th>Linear range (mol L<sup>-1</sup>)</th><th>Limit of detection (LOD) (mol L<sup>-1</sup>)</th><th>1) Recovery 2) Stability 3) RSD (n)</th><th>References</th></tr></thead><tbody><tr><td rowspan="4">AVP</td><td>Graphene–mesoporous silica–gold NP hybrids (GSGHs)/thiolated aptamer</td><td>Aptamer</td><td>Split strategy</td><td>Differential pulse voltammetry</td><td>20 min</td><td>4.61 x 10<sup>-9</sup> - 52.1 x 10<sup>-6</sup></td><td>4.61 x 10<sup>-9</sup></td><td>1 and 3 NC 2) RT-10 d (-6.6%)</td><td>(Du et al., 2012)</td></tr><tr><td>ITO/PDDA–MB/PSS–G/Aptamer/BSA</td><td>Aptamer</td><td>Direct</td><td>Differential pulse voltammetry</td><td>&gt; 30 min</td><td>0.92-244 x 10<sup>-9</sup></td><td>0.92 x 10<sup>-9</sup></td><td>1-3) NC</td><td>(Qin et al., 2012)</td></tr><tr><td>Carbon nanotubes/NH<sub>2</sub>-aptamer</td><td>Aptamer</td><td>Direct</td><td>Field effect transistor</td><td>Real-time &lt; 60 s</td><td>100 x 10<sup>-12</sup> - 100 x 10<sup>-9</sup></td><td>43 x 10<sup>-12</sup></td><td>1-3) NC (n = 3)</td><td>(He et al., 2013)</td></tr><tr><td>Graphene functionalized with standard AVP</td><td>RNA Aptamer</td><td>Competitive</td><td>Field effect transistor</td><td>&gt; 90 min</td><td>1-500 x 10<sup>-12</sup></td><td>-</td><td>1-3) NC</td><td>(Yang et al., 2015)</td></tr><tr><td rowspan="3">OT</td><td>Graphene/APTES/GA/Aptamer</td><td>Aptamer</td><td>Direct</td><td>Field effect transistor</td><td>&gt; 10 min</td><td>0.92 x 10<sup>-18</sup> - 0.92 x 10<sup>-12</sup></td><td>3.27 x 10<sup>-18</sup></td><td>1-3) NC (n = 3)</td><td>(Selvarajan et al., 2020)</td></tr><tr><td>Graphene/NH<sub>2</sub>-PASE/Aptamer</td><td>Aptamer</td><td>Direct</td><td>Field effect transistor</td><td>8 min</td><td>1-10 x 10<sup>-12</sup></td><td>0.3 x 10<sup>-12</sup></td><td>1) 4% loss 2) NC 3) 3% (n = 3)</td><td>(Yu et al., 2024)</td></tr><tr><td>Electrodeposited MIP</td><td>MIP</td><td>Direct</td><td>Impedance</td><td>60 min for 5 injections</td><td>0.06 x 10<sup>-3</sup> - 1 x 10<sup>-3</sup></td><td>60 x 10<sup>-6</sup></td><td>1) 20% high 2-3) NC (n = 3)</td><td>(Sharma et al., 2018)</td></tr><tr><td></td><td>Streptavidin-coated carbon screen-printed electrodes</td><td>Aptamer</td><td>Conformational switch</td><td>Impedance and Direct electrochemical oxidation</td><td>&lt; 2 min</td><td>1-100 x 10<sup>-12</sup></td><td>1 x 10<sup>-12</sup></td><td>1-3) NC</td><td>(Rana et al., 2023)</td></tr></tbody></table>
## 4.1 Voltammetry based approaches

In 2012, Wang's group developed a label-free differential pulse voltammetry (DPV)-based aptasensing platform in two different articles (Du et al., 2012) and (Qin et al., 2012). The fabrication of the sensor interface was constructed using a negatively charged indium tin oxide (ITO) electrode surface, various layers, and electroactive molecules.

In one case, (Du et al., 2012) used surface enhanced graphene-mesoporous silica-gold NP hybrids (GSGHs), as depicted in (Fig 6. A). The electrochemical probe ferrocene (Fc) was covalently attached on poly(ethyleneimine) (PEI) to form Fc-PEI. Then, Fc-PEI and poly(sodium 4-styrenesulfonate) PSS were immobilized on the negatively charged ITO electrode through layer-by-layer assembly to obtain a solid-state bilayer probe. Another layer of PEI was used as the outermost layer to fix the GSGHs. In the other case, Qin et al. have deposited a graphene multilayer alternated with electroactive methylene blue (MB) on ITO electrodes surfaces. The multilayer was grown by immersing alternatively the ITO electrode in poly (diallyl dimethylammonium) chloride-methylene blue (PDDA-MB) and poly(styrenesulfonate)-graphene (PSS-G) in aqueous solutions. Graphene was expected to provide adsorption sites for methylene blue and facilitate the electron transfer capability of the multilayer film.

Du et al have used the split strategy (Fig. 1B), SH-P1 a thiolated subunit of the native D-AVP aptamer has been immobilized on gold GNPs appended on GSGHs. 6-Mercaptohexanol (MCH) was used for backfilling to obtain a more regular interface. In the other case (Fig. 6B), the formed MB multilayer was covered by the same D-AVP aptamer and then with 1% BSA to reduce nonspecific adsorption. For the detection using the split strategy, a mixture of D-AVP and the complementary aptamer strand was added to the sensing platform. As expected, a complex resulting from the hybridation P2 and SH-P1 bound the D-AVP has led to a decreasing DPV signal of Fc-PEI. With the entire immobilized aptamer, the electron transfers between the MB and the electrode surface were decreased by the complex formation resulting from the interaction between D-AVP and its aptamer leading to a decreased oxidation current peak of the DPV signal.

The prepared split GSGHs-based sensors have a wide linear range from 5 ng mL⁻¹ to 56.5 mg mL⁻¹, (4.61 nmol L⁻¹ - 52.1 µmol L⁻¹) with a detection limit of 5 ng mL⁻¹ (4.61 nmol L⁻¹) whereas a linear relationship between the current ratio and the concentration of target peptide existed from 1 ng mL⁻¹ to 265 ng mL⁻¹ (0.92-244 nmol L⁻¹) for the full format. In addition, both systems are able to distinguish L and D-vasopressin (enantioselectivity), the
sensor showed, in split format, a 5 ng. mL⁻¹ (4.61 nmol L⁻¹) LOD and the total aptamer setup was sensitive enough to detect 1 ng mL⁻¹ (0.92 nmol L⁻¹) of D-vasopressin, which is an interesting performance even when far from the picomolar range of interest. Finally, 15% of human serum was used to confirm the practical applicability of this sensor (Fig 6, B).

A

<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

**Fig. 6.** Schematic representations of the sensing procedure for the analysis of D-AVP based on (A) the sensing platform with silica-gold NP hybrids (GSGHs) as enhanced materials and ferrocene-poly(ethyleneimine) (Fc-PEI) as electrochemical probe. With PSS = poly(sodium 4-styrenesulfonate) and D-VP = (D-AVP) Reprinted from (Du et al., 2012). (B) The electrochemical aptasensor with alternate adsorption of graphene/polyelectrolyte and methylene blue/polyelectrolyte. Poly (diallyl dimethylammonium) chloride-methylene blue = PDDA-MB and poly(styrenesulfonate)-graphene = PSS-G. In the right, the relative responses of the sensor to D-VP (=D AVP) at different concentrations in PBS and in 15% human serum. Reprinted from (Qin et al., 2012). Copyright © 2011 Elsevier B.V. All rights reserved.

## 4.2 Impedance based approaches

Electrochemical impedance spectroscopy (EIS) appears to be a particularly efficient tool for finding the quantitative parameters of electrochemical processes occurring at the electrode interface. Impedance biosensors can be used in two different ways (Dorledo De Faria et al., 2019). The first method involves the passivation of electrodes, i.e. conditions where no electron transfer (no faradaic current) occurs. The second method, which is the most applied (Guan et al., 2004; Prodromidis, 2010; Bahadır and Sezgintürk, 2016; He et al., 2020) uses a redox probe which diffuses to the electrode surface through the molecular architecture
and generates redox currents. This method allows to record tiny current changes and achieve therefore higher sensitivity than non-faradic devices. Combining a microfluidics platform, MIP technology and non-faradic impedance transduction, Sharma and al. successfully developed a miniaturized chemosensor for oxytocin detection in biological samples (Sharma et al., 2018). Under flow-injection analysis conditions, the capacitive behavior of the electrode-solution interface was studied at a constant frequency. Oxytocin samples of different concentrations were consecutively injected in the carrier solution (10 mmol L⁻¹ KF solution) at a constant flow rate. As interaction of oxytocin with molecular cavities in MIP induced a decrease of the capacitance, the differences of the capacitance vs carrier solution could be correlated to oxytocin concentration. The as-designed setup showed a LOD of 60 µmol L⁻¹ and a linear range extended from 0.06 to 1 mmol L⁻¹. The system has also been tested on OT spiked 1000 times diluted commercial serum containing both human, non-human enzymes as well as the non-protein components. Despite the complexity of the matrix, the sensitivity of the sensor remained nearly the same even if the signal obtained was 20% higher than for the KF solution of OT. The MIP film was also selective against carbatocin, a close analog oxytocin and some other biomolecules, smaller than oxytocin. Although the sensitivity is insufficient to determine oxytocin at picomolar level as found in biological samples, the sensor setup is quite promising related to its selectivity.

Very recently, Rana and colleagues were successful with an aptamer-based electrochemical assay. As shown in **Fig. 7**, when OT was administered in saliva, the aptamer on the carbon surface bound specifically to OT and the electroactive tyrosine residue of the peptide, producing a peak current. The authors have shown that the developed aptasensor can detect as little as 1 pg mL⁻¹ (1 pmol L⁻¹) of OT in less than 2 minutes in commercially available pooled saliva samples, making it the most promising point-of-care assay for OT to date.

<figure><img src="image_9.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_10.png" /></figure>
**Fig. 7.** (A) Schematic representation of the electrochemical detection of oxytocin on an aptamer-modified carbon electrode. The proposed oxidation mechanism of oxytocin at the tyrosine moiety, where R and R' represent the remaining amino acid moieties of oxytocin. (B) Current readout signal to test the specificity of the electrochemical sensor. 1 µg mL⁻¹ (1 µmol L⁻¹) of oxytocin and > 100 µg mL⁻¹ (100 µmol L⁻¹) of vasopressin (VP) were spiked separately into apparently healthy (control) saliva samples. Reprinted from (Rana et al., 2023).

### 4.3 Field effect transistor-based approaches

Field effect transistor (FET) biosensors are composed of a conducting channel placed between two electrodes (i.e. the source and drain). The electrical conductivity of the FET changes when biomolecules are added because of the corresponding interactions that alter the channel's carrier distributions (Ghasemi and Salimi, 2023). Due to its potential for downsizing, parallel sensing, quick response times, and easy integration with electronic manufacturing processes, FET based sensing has drawn a lot of attention among other potentiometric approaches (Kaisti, 2017). These benefits undoubtedly led He and al. (He et al., 2013), Yang and al. (Yang et al., 2015) and Selvarajan team's (Selvarajan et al., 2020) to choose this approach while aiming to face the ongoing challenge of accurate detection of vasopressin at picomolar concentrations.

In 2013, He and al (He et al., 2013) have developed an aptamer-based label-free microfluidic biosensor for the detection of AVP. The detection zone consists of the 55 mer-AVP aptamer immobilized on carbon nanotubes which specifically capture the AVP in solution resulting in changes in conductivity across the sensor. The report a detection limit of 43 pmol L⁻¹ and demonstrate high detection specificity for vasopressin in the presence of different interferents (HAS and Lypressin). To demonstrate the applicability of the sensor in complex environments, tests were carried out on sheep serum. AVP concentrations in serum ranging from 100 nmol L⁻¹ to 1 mmol L⁻¹ were detected, that confirms the capacity of the system to achieve detection at concentrations as low as 100 nmol L⁻¹. But the complicated serum composition imposes obstacle for the direct measurement of AVP without pretreatments.

In 2015, Yang and al. (Yang et al., 2015) introduced a microfluidic aptasensor that combines aptamer-based selective analyte enrichment, isocratic elution, and a graphene sensor in a FET setup. **Figure 8** depicts the principle of this device. Vasopressin present in a sample (referred to as the "sample AVP") were selectively collected by an RNA aptamer (Purschke et al., 2006) immobilized on microbead surfaces at 37 °C (Fig. 8A) and were then enriched by continuous sample infusion into the device. Although not mentioned in the
article, we assume that this aptamer is in the L-series. Nonspecifically adsorbed AVP and impurities were removed through buffer washing (Fig. 8A (b)). Enriched vasopressin molecules were released at a high temperature (55 °C) and eluted with solution-borne aptamer molecules ("free aptamer"), causing isocratic elution of vasopressin captured by the free aptamer (Fig. 8A (c)). The eluate (a mixture of the free aptamer and released vasopressin that was initially caught sample AVP) was then incubated at 37°C with graphene that has been previously functionalized with reagent vasopressin ("standard AVP") (Fig. 8A (d)). A competition is created between the graphene bound standard AVP and sample AVP to bind to free aptamer molecules, causing an increase in the charge on the graphene surface (Fig. 1F and 8A (e)). As a result, more sample AVP eluted in a mixture leads in fewer aptamer molecules binding on the graphene surface, resulting in smaller changes in drain current. Steps a, b and c of the above-described principle were staged in an enrichment microchamber and the last ones (d and e) in a sensing chamber of a microdevice. Even if the sensor performed without a prior enrichment step, detection below the nanomolar level was not achieved. Detection of AVP in the low picomolar range, necessary in clinical settings, has been achieved with enriched vasopressin samples.

In 2020, a FET biosensor was formed in an experimental setup using a suspended graphene-based FET (GFET) to enable the quick, accurate, and sensitive detection of AVP (Selvarajan et al., 2020). The ultrahigh sensitivity of graphene provided justification for choosing it over other materials like one-dimensional CNTs (Chaudhary et al., 2016). Regarding the architecture, a suspended GFET (suspended graphene connects the source and drain electrodes) has been preferred to embedded architecture (graphene supported by silicon dioxide substrate) since charge carriers (electrons or ions) in the suspended GFET would have higher mobilities (Bolotin et al., 2008). For the biorecognition, after two steps modification (with 3-amino propyl triethoxysilane and glutaraldehyde), the traditional 5' amine modified D-AVP aptamer were immobilized on the graphene layer. As a result of the positively charged D-AVP hormone binding to the aptamer, negatively charged charge carriers are drawn to gather in the channel area by attractive forces. Consequently, the region of the transducing channel is affected by charge accumulation, resulting in an increase in drain current. The latter could be plotted against vasopressin concentration. The biosensor showed a significant increment in the relative change of current ratio with the increase of ADH concentration ranging from 10 ag mL⁻¹ to 1 pg mL⁻¹ (0.92 amol L⁻¹ -0.92 pmol L⁻¹). The system exhibited a LOD of 3.55 ag mL⁻¹ (3 amol L⁻¹) which is the lowest value reported for AVP detection. On the other hand, the sensor showed no significant change in drain current
when other biomolecules like urease, BSA, glucose oxidase were tested, which indicates a good specificity for D-AVP detection. Human serum spiked with D-AVP (the dilution factor is not mentioned) successfully helped proving the sensor's selectivity as the drain current significantly increased (Fig 8B). However, what's unfortunate about this study is that the researchers didn't test the system for L-AVP and lumped the two targets together. Nonetheless, these results should be easily extrapolated to the natural target. This study offers encouraging outcomes that motivate additional research on in vivo evaluation of vasopressin in the human body.

<figure><img src="image_11.png" /><figcaption>A</figcaption></figure>

**Fig. 8.** (A) Principle of vasopressin detection of the microfluidic aptasensor developed by Yang et al. (a) Enrichment of sample vasopressin on microbead surfaces by aptamer binding. (b) After the buffer washing. (c) Raising the temperature to 55 °C disrupts the aptamer-vasopressin complexes, sample vasopressin is released into a free aptamer solution. (d) Incubation of the mixture of free aptamer and released sample vasopressin with graphene functionalized with standard vasopressin. (e) Competitive binding of the free aptamer to the standard vasopressin on graphene thus changing the graphene conductance. Reprinted from (Yang et al., 2015) Copyright © 2015, IEEE. (B) Specificity of analysis of vasopressin biosensor with vasopressin spiked in PBS buffer, vasopressin spiked in human serum and other biomolecules Reprinted from (Selvarajan et al., 2020) Copyright ©.

<figure><img src="image_12.png" /><figcaption>B</figcaption></figure>

Fortunately, Yu and his colleagues have developed a FET nanosensor using monolayer graphene as a conducting channel. A new DNA aptamer specifically recognizing L-AVP was attached to the graphene surface at an internal site to facilitate direct interactions between aromatic groups in monomeric residues and hydrophobic elements on the graphene surface. The nanosensor was tested over the clinically relevant AVP concentration range of 1 pmol L⁻¹ to 1 nmol L⁻¹ and achieved an LOD of 0.3 pmol L⁻¹ in the lower concentration range (1-10 pmol L⁻¹). These results demonstrate the potential utility of the nanosensor for real-time monitoring of plasma AVP concentrations.

**5. Other sensing methods**
Lin et al have developed a quartz crystal microbalance (QCM) sensor with MIP (Table 1) for the detection of AVP and OT. The sensor with showed great specificities towards the respective templates up to 1 ng mL⁻¹ (0.92 nmol L⁻¹) where nonspecific interactions with interfering peptides could be observed. Even when the authors did not seek to reach the concentration range of interest for AVP, this work contributes to the understanding of recognition kinetics and processes for peptides (Lin et al., 2003). In 2019, The same team-has reached with an improved setup excellent specificity and reproducibility a detection limit of 3 pg mL⁻¹ (2.77 pmol L⁻¹) towards OT (Lin et al., 2019).

## 6. Analytical performances

The LOD and linear ranges shown in Table 2 for optical analysis methods vary widely between sensors reported in solution or immobilised. For AVP, biosensors in solution typically have linear concentration ranges and LODs between µmol.L⁻¹ and nmol.L⁻¹, whereas immobilised techniques have LODs values and concentration ranges between nmol.L⁻¹ and pmol.L⁻¹. The best LOD performance has been achieved by chemiluminescence (Li et al., 2014) if we avoid methods that cannot be used directly on the non-labelled target, such as SERS (Huh and Erickson, 2010; Yang et al., 2013). In Table 3, some electrochemical and electronic approaches are reported to be capable of quantifying concentrations that span up to 52.1 µmol.L⁻¹ (Du et al., 2012) while others report much more conservative ranges as narrow as 500 pmol.L⁻¹ (Yang et al., 2015). Despite the high level of variation, the overall performance of electrochemically and electronic sensors are better than for the optical biosensors, demonstrating their ability to detect at clinically relevant levels of the targeted analyte. The high level of sensitivity was achieved for AVP quantification with field effect transistor with the work of Yu and colleagues (Yu et al., 2024) and Selvarajan et al (Selvarajan et al., 2020), which achieved LODs of 0.3 pmol.L⁻¹ and 3 amol.L⁻¹ respectively.

For OT, LODs of 2.98 and 1 pmol.L⁻¹ respectively were obtained with an optical biosensor consisting of an MIP (Yola et al., 2015) and an electrochemical method using an aptamer (Rana et al., 2023).

From our point of view, it is difficult to find the best biosensor from among all those presented, as indications are often lacking on stability, quantification limits and their use in complex environments. State-of-the-art analytical validation would be an asset for future publications and we could finally compare them with existing reference methods.

## 7. Conclusion and futures perspectives
The development of vasopressin and oxytocin biosensors promises to significantly advance our knowledge about these hormones in neuroendocrinology, social behavior, and mental health. In this review, major advancements in the field of AVP and OT detection are highlighted, including the use of a variety of methods, including optical, electric/electrochemical, and mass-based transduction.

An analysis of all the works reported in this field shows that there is still a long way to go before these methods can be used in routine practice. Regarding aptamer-based methods for vasopressin sensing, only the D-series species has been determined in most cases without focusing on the natural vasopressin (L-AVP) by using the spiegelmer (L-DNA aptamer) originally developed by Williams and co-workers (Williams et al., 1997). We suspect that this is, at least in part, linked to the fact that aptamer synthesis in the L-series is very expensive (around € 2,000-3,000 for a 40 nmoles synthesis scale). In many cases, the configuration of the aptamer or vasopressin is not mentioned. The authors refer to vasopressin, but do not indicate that the aptamer is in the L-series, leaving some doubt as to whether the experiments were carried out on natural vasopressin. This is the case for (Huh and Erickson, 2010; Lu et al., 2012; He et al., 2013; Li et al., 2014; Selvarajan et al., 2020). However, this DNA aptamer is selective and specific against D-AVP, and has been tested by many different teams using a variety of detection methods. Only one team used the RNA aptamer (Purschke et al., 2006) and although it showed a better Kd (1.7 nmol L⁻¹) than its predecessor (0.9 µmol L⁻¹), the LODs did not appear to be lowered, if we refer to Field effect transistor methods. A new D-series aptamer directed against L-AVP has just been described (Yu et al., 2024) but the data are not yet sufficient to assess its relevance. To date, there is only example of MIPs being used for AVP assay. This approach deserves to be developed and pursued. Another approach worth exploring is the preparation of MIP-aptamer hybrids; the dual recognition of MIP-aptamer can synergize the properties of both MIP and aptamer, making it a promising recognition element for diagnostic devices. It was demonstrated that the affinity and specificity of the prepared MIP-aptamer hybrid towards the target were dramatically improved compared to the aptamer or MIP alone (Ali and Omer, 2022).

Optical methods in solution are invaluable because they have detection limits only down to the nanomolar level. Immobilized systems using methods such as SERS or chemiluminescence reduce detection limits and are competitive with electrochemical and electrical sensing methods. Field effect transistors appear to be the method of choice in terms of sensitivity with the best LOD (3.27 amol L⁻¹) obtained in 2020 (Selvarajan et al., 2020), but they are quite complicated to implement.
Only 9 of the 17 publications demonstrated the ability to monitor vasopressin and oxytocin in complex samples. The matrices studied were diluted serum (present in the test sample at a concentration between 1 and 15%), urine (1 to 10%), saliva and even milk. When these methods were applied to complex media, only 4 of them (He et al., 2013; Yola et al., 2015; Selvarajan et al., 2020; Rana et al., 2023) were able to reach the picomolar range found under physiopathological conditions, showing that they can compete with immunoassays which are known to be sensitive to interference (Gnanadesikan et al., 2022) and LC-MS methods which are laborious and time consuming. Other studies lack of precision on the dilution of the complex medium. Impedance detection may be a method to consider for AVP detection, as demonstrated for OT (Rana et al., 2023).

Biosensors with high sensitivity and specificity are needed to address significant and growing healthcare problems. One of the most pressing challenges in clinical practice is the early detection and diagnosis of disease in complex media. At present, in the examples presented for the L-AVP assay, the techniques used for both aptamers and MIPs, although some of them meet the desired detection limits, have not been rigorously and robustly adapted to complex media. There is still a long way to go to develop an accurate, robust, rapid, economical and easy-to-use detection system suitable for point-of-care techniques that can become the standard for diagnostics. To address these difficulties and to enhance the performance for realistic monitoring of these biomarkers, more research and development efforts are still required.

**Funding**

This work was supported by Labex Arcane and CBH-EUR-GS (ANR-17-EURE-0003). We also acknowledge Polynat Carnot Institute (ANR-11-CARN-007-01) and CDP CerCoG.

**CRediT authorship contribution statement**

Essohanam Beke: Writing – original draft, Investigation.

Chantal Gondran: Writing, Review, Supervision.

Benoît Chovelon: Review

Eric Peyrin: Writing, Review, Supervision, Funding acquisition.

Michael Holzinger: Writing, Review, Supervision, Funding acquisition.

Corinne Ravelet: Writing, Review, Supervision, Funding acquisition.

**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.
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