Electrochemical and photoelectrochemical aptasensors
for the detection of environmental pharmaceutical
pollutants

Teodora Lupoi, Yann R. Leroux, Bogdan Feier, Cecilia Cristea, Florence Geneste

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Teodora Lupoi, Yann R. Leroux, Bogdan Feier, Cecilia Cristea, Florence Geneste. Electrochemical and photoelectrochemical aptasensors for the detection of environmental pharmaceutical pollutants. Electrochimica Acta, 2025, 530, pp.146396. 10.1016/j.electacta.2025.146396. hal-05105969

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# Electrochemical and photoelectrochemical aptasensors for the detection of environmental pharmaceutical pollutants

Teodora Lupoi<sup>a,b</sup>, Yann R. Leroux<sup>a</sup>, Bogdan Feier<sup>b</sup>, Cecilia Cristea<sup>b,#</sup> Florence Geneste<sup>a,#</sup>

## ARTICLE INFO

**Keywords:**

pharmaceuticals
aptamers
biosensing
electrochemistry
environment

## ABSTRACT

Pharmaceutical substances are emerging contaminants with the potential to significantly lower the quality of the environment due to their persistence. Evaluating environmental pollution is a critical area of research that can benefit from on-site, affordable, and accessible analytical methods.

This review highlights recent advancements in electrochemical biosensors that use aptamers as recognition elements for pharmaceutical contaminants. The reasoning behind the selection of antibiotics, non-steroidal anti-inflammatory agents, and estrogen hormones is explained, considering factors such as environmental concentrations, toxicity, and selected aptamers. A thorough revision of the aptamers used in the studied literature, their characteristics, and functionalization was done. The coupling of aptamers with an electrochemical and photo-electrochemical transducer was explained, and the mechanisms for signal registration were detailed.

An important focus was the construction of the aptasensors, where the materials and the integration of aptamers strongly influence the cost, complexity, and performance of the biosensor. The main materials used were classified into carbon-based, metallic, and semiconductors, with a justification for the use of covalent and non-covalent aptamer immobilization in each case. The analytical performance was systematically evaluated through parameters like the limit of detection, linearity range, and selectivity. After the global analysis, a conclusion as to whether electrochemical aptasensors can be applied to environmental analysis is reached, with limitations and possible improvements debated.

## 1. Introduction

The presence of pharmaceuticals in the environment started to draw public attention in the late 1990s [1]. With the increase in the world population and quality of life, the consumption of medication grew, which led to higher concentrations in the environment and more frequent contamination [2].

The most significant source is the domestic waste generated after human consumption and excretion, representing the origin of active pharmaceutical ingredients (API) and metabolites. Hospital and industrial wastewater can induce high concentrations of pharmaceuticals that need to be considered despite the high dilution they undergo with municipal wastewater. Poorly regulated veterinary consumption is another massive pollutant, with substances accumulating in the manure that can further reach the soil and water. Landfills and incorrect drug disposal from patients, although controllable sources are contributing to environmental contamination [3].

The pharmaceutical pollutants pass mainly in the soil, surface water, and groundwater. The water bodies become the arch carriers of these substances, affecting the aquatic biota and human health. The recalcitrant nature to degradation of some pharmaceuticals leads to high quantities in wastewater treatment plants (WWTPs) effluents that further deliver the substances in streams, rivers, and lakes [4].

The sources mentioned above are the root of over a hundred different pharmaceuticals in the environment, with members from the class of antibiotics, non-steroidal anti-inflammatory drugs, hormones, beta-blockers, anti-epileptics, and antidepressants [5]. Continuous exposure to low concentrations of steroidal estrogens leads to hormonal disruptions in humans and aquatic life [6]. Antibiotic pollution points towards a selection of antibiotic-resistant bacteria that further determines a lack of treatment options for microbial infections [7]. Other molecules accumulate in the bodies of animals, interfere with the growth rate and photosynthesis of algae, change fish behavior, and induce oxidative stress [8]. Because their environmental levels are not well monitored, it

<footnote><sup>a</sup> Univ Rennes, CNRS, ISCR, UMR 6226, F-35000 Rennes, France</footnote>

<footnote><sup>b</sup> "Iuliu Hatieganu" University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Analytical Chemistry, 400349, Cluj-Napoca, Romania</footnote>

<footnote># Corresponding authors.</footnote>

<footnote>E-mail addresses: cccristea@umfcluj.ro (C. Cristea), Florence.Geneste@univ-rennes1.fr (F. Geneste).</footnote>
is difficult to make a definitive correlation between them and the observed toxic effects.

The concentrations of pharmaceuticals in the environment vary according to the water matrices, from ng/L to µg/L [9]. The legal limitations of pharmaceutical contaminants are not strict, their levels can be set as high as the predicted no-effect concentrations (PNEC), which is the concentration of a substance below which no adverse effects upon an ecosystem are observed.

To correctly estimate the risk, an appropriate analytical method should have the limit of quantification (LOQ) at least at the level of PNEC [10]. Therefore, the performance of the analytical technique is a decisive limiting factor in monitoring pharmaceuticals in environmental waters.

The standard methods that can reach such a low limit of quantification combine the separation properties of chromatographic techniques like gas chromatography (GC) and liquid chromatography (LC) with the high selectivity of a mass spectrometer (MS) detector [11].

Their powerful resolution, selectivity, and sensitivity are unquestionable advantages in environmental screening, nonetheless, they come with a high cost per analysis, no portability, laborious sample treatment, long analysis time, pollutant solvents, and require skilled operators. An ideal analytical method applied to environmental samples should be sensitive, selective, cost-efficient, and allow in situ continuous monitoring. These requirements can be satisfied by miniaturized systems such as electrochemical sensors. These new analytical tools can address significant drawbacks of conventional methods in environmental analysis.

The discrepancy in environmental monitoring between developed and developing countries can be overcome with sensors because of their accessibility. Moreover, the portability and possibility of online monitoring are valuable features, as there is strong intra-day, inter-day, and seasonal variability in the concentration of pharmaceuticals in the environment [12].

The development of the sensors integrates various new materials to improve the analytical parameters. For increased sensitivity, nanomaterials based on metals, metal oxides, and carbon structures are constantly used for their properties: high conductivity, surface-to-volume ratio, long-term chemical stability, enhanced mechanical strength, and easy modification with biologic and biomimetic elements [13]. To augment the selectivity, numerous designs have been proposed, including naturally recognizing components like enzymes, antibodies, and proteins, or synthetic ones like molecularly imprinted polymers (MIP), ligands, and aptamers. Among them, aptamers are increasingly popular in the development of sensing platforms.

Discovered in 1990, aptamers are single-stranded DNA or RNA chains that exhibit ligand-like properties. Their sequence is selected in vitro through Systematic Evolution of Ligands by Exponential Enrichment (SELEX), a procedure that yields aptamers with high affinity for the targeted molecule [14]. The numerous interactions between the nucleotides generate secondary and tertiary structures responsible for their binding properties. Their function is similar to that of the antibodies, but they have additional advantages such as high stability, easy functionalization, chemical synthesis, lower batch-to-batch variation, and the possibility of regeneration [14]. Their accessible derivatization opens the way for many immobilization methods on the electrode surface. Additionally, aptamers have a broad sensing capability, allowing them to detect molecules with different molecular weights, ranging from small molecules and metal ions to proteins and whole cells [15,16].

Numerous aptamers have been selected for pharmaceuticals, some targeting molecules frequently present in the environment and with toxic potential [17,18]. The most representative classes of pharmaceuticals for which aptamers have been selected are the classes of antibiotics, anti-inflammatorysm, and steroidal estrogens. Other drug classes, while still dangerous for the environment, have received less attention when it comes to aptamer selection.

Given the importance of aptasensor development for the monitoring of pollutants, a significant number of studies have been carried out and

summarized in recent general reviews [19–26]. These reviews focus on emerging contaminants, sometimes including pharmaceuticals, but they are not exclusive to them, diluting the discussion with pesticides, heavy metals, biological markers, etc [21,25]. Also, the sensors discussed are often based on many types of transducers (e.g. colorimetric, fluorescent, electrochemical, ECL, PEC, SERS) and lack the depth of electrochemical detection mechanisms and the structured comparative analysis of sensor performance [19,20,22,23]. Finally, some reviews deal with pollutants in food and not specifically in the environment [24,26].

This review offers a novel perspective on recently developed (since 2019) electrochemical and photoelectrochemical (PEC) sensors based on aptamers as recognition elements for pharmaceutical pollutants. It fills existing gaps by exclusively targeting pharmaceutical pollutants in environmental detection, providing a detailed analysis of the used aptamers and electrochemical detection mechanisms, offering a comparative assessment of materials and analytical performance, and maintaining a strong environmental focus rather than biomedical or food safety applications. Firstly, the occurrence and associated risks of pharmaceuticals in the environment will be discussed, highlighting the necessity for effective detection methods. Following this, the aptamers selected for pharmaceuticals will be analyzed in terms of the selection process, affinity, and post-selection functionalization. Furthermore, the review will explain how the sensing mechanism, modifying materials, and immobilization strategies can lead to sensors with different analytical performances despite using the same aptamer sequence. Lastly, it will explore the practical use of aptasensors in environmental monitoring, discussing their applicability alongside potential improvements.

## 2. Pharmaceuticals in the environment

Among the various substances found in the environment at low concentrations, pharmaceuticals and personal care products have priority for monitoring and decontamination [27].

The sources of pharmaceuticals in the environment can be categorized into point sources (wastewater, landfills, septic tanks) and diffuse pollution (sewage sludge, groundwater-surface interface) [28]. The four main factors contributing to elevated levels of pharmaceuticals in water are domestic consumption, hospitals, industrial processes, and aquaculture [27]. A recent global study analyzing over 1000 sampling sites identified the highest pharmaceutical pollution levels near pharmaceutical manufacturing sites, untreated sewage discharge points, and in arid regions with limited water flow [29]. In contrast, the lowest concentrations were found in remote areas, regions with advanced wastewater treatment, or where river dilution was significant.

Both hospitals and pharmaceutical manufacturers are important contamination points, generating higher concentrations of pharmaceuticals in their wastewater compared to municipal wastewater [30]. Efforts are being made to reduce their contribution through regulations that mandate hospitals and industries to implement pretreatment measures, limiting the quantity of pharmaceuticals released in wastewater [31,32]. The veterinary use of drugs induces in the environment antibiotics and antiparasitic substances, with concentrations exceeding 1000 µg/L in wastewater. Since most of the administered doses are unmetabolized, up to 90% are eliminated as active substances in the manure. The resulting manure is used as fertilizer and can undergo leaching or be discharged in wastewater [33]. Solid waste landfills pose a contamination risk due to the incorrect disposal of human and veterinary medications. The resulting leachate is concentrated in contaminants, and if not well-regulated, it can enter the groundwater [34,35].

While in wastewater, pharmaceuticals can undergo inactivation through decontamination processes in WWTPs. This stage is supposed to eliminate most of the organic matter, however, there are pharmaceuticals like diclofenac (DCF) and carbamazepine that can resist this treatment. The unchanged pharmaceuticals are released into surface waters through the effluents of WWTPs. There is high variability in their
removal due to the physicochemical properties such as the octanol-water coefficient (log K<sub>ow</sub>), acid dissociation constant (pK<sub>a</sub>), sorption, and parameters that depend on the decontamination process, disinfectants, hydraulic retention time, and sludge retention time [31,36]. Pharmaceuticals are described as pseudo-persistent contaminants because of their continuous release in the environment and inefficient decontamination.

Data indicating the presence of numerous pharmaceuticals in water bodies comes from every region of the world. The main therapeutic classes of pharmaceutical pollutants detected include antibiotics, non-steroidal anti-inflammatory drugs (NSAIDs), antidepressants, steroidal hormones, lipid-regulating drugs, anticonvulsants, stimulants, and antihypertensives [36].

To assess potential ecological effects, toxicity is studied using laboratory models corresponding to different levels in the ecosystem. For instance, algae are employed for the phototrophic level, Daphnia magna for invertebrates, and fish for vertebrates. Measured environmental concentrations (MEC) combined with the PNEC is the most used method to assess toxicity [37]. However, prioritizing these substances is a complex process where several factors besides toxicity need to be accounted for, such as detection frequency, risk to human health, and bioaccumulation [38].

The class of antibiotics is extensively discussed in the literature, with members frequently detected in high concentrations in the environment globally [39–41]. Antimicrobial resistance (AMR) is a selection process that has accelerated in recent years due to man-made antibiotics. AMR represents the development of resistance factors of pathogens, limiting our ability to treat infections and causing major economic losses [42]. Exposure of bacteria to concentrations of antibiotics lower than the minimum inhibitory concentrations (MIC) but higher than selection concentrations leads to selection mechanisms of strains that become resistant to the agent. This phenomenon is prevalent in the environment, especially in areas with high industrial antibiotic pollution. Municipal pollution with antibiotics suggests there might be a correlation between their levels in the environment and AMR processes, however, this is difficult to track back, requiring orchestrated efforts to monitor antibiotic levels, antibiotic-resistant genes, and antibiotic-resistant bacteria [7]. Moreover, other non-antibiotic pharmaceuticals, such as propranolol and gemfibrozil, can promote AMR in the environment [43]. Besides the spread of AMR, the toxic effects of antibiotics were observed on bacteria, arthropods, and fish model organisms [44,45].

NSAIDs are widely used medications due to their antalgic and anti-inflammatory properties. Reflecting their extensive use, substantial quantities of DCF, ibuprofen (IBU), and naproxen are detected in the environment along with their metabolites, like 4-hydroxy diclofenac, 1-hydroxy ibuprofen, and 3-hydroxy ibuprofen [46]. DCF stands out as the most studied representative due to its high ecotoxicological potential. Unlike IBU and naproxen, which are biodegradable, DCF is resistant to bioremediation applied in WWTPs, and it is more persistent in surface waters [47]. Average concentrations of DCF reaching 0.1 µg/L were detected in each world region, with levels up to 1.55 µg/L (maximum average in Pakistan) [48,49]. The awareness of its toxicity heightened when DCF was linked to a sudden decline in the vulture population of the Gyps genus in Southern Asia, determined by the consumption of animal carcasses coming from cattle treated with anti-inflammatory drugs [50]. Because of its mechanism to inhibit prostaglandins, which are involved in behavior and reproduction regulation, the growth rate in the egg phase and reduced hatchability were reported for several fish species. Its structure favors the enterohepatic circulation, a factor that increases availability and can lead to gill, liver, and kidney damage [51–53]. Other studies emphasized DCF toxicity in green algae and daphnids at environmentally relevant concentrations [54].

Endocrine-disrupting compounds (EDCs) are priority pollutants that have pharmacological activity at low concentrations, a reason that gives them priority for monitoring even if the levels in the environment might seem insignificant. Steroid estrogens can exert disruptive processes in

the human and animal body when present in excess. They are divided into natural steroids like estrone (E1), 17β-estradiol (E2), estriol (E3), and synthetic, 17α-ethinylestradiol (EE2). The PNEC has a low value, 0.1 ng/L for EE2 and 2 ng/L for E2, meaning that low concentrations can induce harmful effects. The main target is the reproductive system, leading to repercussions on fertility, feminization, and permanent sex reversal [55]. A study analyzing the state of water pollution by estrogens in Europe revealed that levels of estrogen concentrations were in the range of 0.1 ng/L to 10 ng/L, exceeding the PNEC [56].

Other pollutants, like antidepressants, are present in the environment as parent compounds and transformation products that retain their pharmacological properties. Venlafaxine, its metabolite, O-desmethyl venlafaxine, fluoxetine, and citalopram are the most frequently detected antidepressants, generating behavioral changes in fish populations [57]. Carbamazepine has a high incidence in surface water due to its persistence after the purification process in WWTPs, and can be a practical indicator of wastewater treatment efficiency [58,59].

The levels of pharmaceuticals in the environment, often expressed as MEC, exhibit variations depending on the analyzed matrix, region, and season. The most analyzed matrix is surface water, followed by WWTP streams, groundwater, and drinking water. Other matrices that reflect veterinary pharmaceutical contamination, such as manure, soil, and sediment, have been less studied [60]. Their concentration in environmental samples depends on the degree of dilution, with higher levels reported in manure (mg/kg) and wastewater coming from farms and hospitals (µg/L) [61]. After the wastewater treatment, several scenarios can unfold. If the molecule is susceptible to remediation, it will not be detected in WWTP effluents. In some cases, a certain percentage can persist, and levels of ng/L can still be detected after treatment. There are also molecules with levels that remain unchanged after treatment, contaminating surface waters, where concentrations of ng/L to µg/L were identified. The persistence of pharmaceuticals in treated wastewater highly depends on the decontamination processes applied. Biological treatment implies the degradation of organic matter by the metabolism of microbial cultures, and it is an economical, safe, and environmentally friendly method. However, certain molecules such as carbamazepine resist this treatment. The most efficient degradation is achieved by advanced oxidation processes (AOP), where highly reactive radicals are formed and attack the organic molecules. AOP can remove pharmaceuticals up to 99%, but it demands a lot of energy and increases the cost of water treatment. Moreover, if the mineralization is incomplete, the degradation products can sometimes be more toxic than the parent compound [58].

In Table 1, values reflecting the mean and maximum concentrations of some pharmaceuticals targeted in this review can be found together with the reported PNEC. In many cases, the mean concentrations are several orders of magnitude above the PNEC, and the maximum concentrations are in the µM range. However, for an analytical method to be considered suitable for pharmaceutical detection, it must achieve an LOQ as low as the PNEC. The method of choice is high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS), which combines the separatory efficiency of the chromatography with a

<caption>**Table 1**</caption>

Mean and maximum concentrations, and PNEC of targeted pharmaceuticals found in European surface waters, as reported by [37].

<table><thead><tr><th>Compound</th><th>Mean concentrations (ng/L)</th><th>Maximum concentrations (ng/L)</th><th>PNEC (ng/L)</th></tr></thead><tbody><tr><td>Diclofenac</td><td>247</td><td>18740</td><td>1</td></tr><tr><td>Ibuprofen</td><td>337</td><td>31323</td><td>10</td></tr><tr><td>Ciprofloxacin</td><td>657</td><td>13567</td><td>100</td></tr><tr><td>Ofloxacin</td><td>540</td><td>8770</td><td>100</td></tr><tr><td>Norfloxacin</td><td>36</td><td>163</td><td>160</td></tr><tr><td>Amoxicillin</td><td>201</td><td>622</td><td>37</td></tr><tr><td>Oxytetracycline</td><td>101</td><td>680</td><td>310</td></tr><tr><td>17β-estradiol</td><td>9</td><td>120</td><td>2</td></tr></tbody></table>
highly specific detector. Gas chromatography (GC) coupled to MS can be
an alternative for volatile compounds or upon derivatization.

In global analyses, pharmaceuticals were detected in all regions,
with suboptimal coverage in Eastern Europe, Africa, and South America,
as reflected in the world map in Figure 1. The highest incidence is
observed in countries with more measurements, emphasizing that lower
incidence does not necessarily indicate lower contamination.

Due to a gap in understanding their distribution, fate, and levels,
there have been limited regulatory measures regarding pharmaceuticals
in the environment. In response to this, the European Council issued
Directive 2008/105/EC, creating a dynamic watch list designed to pri-
oritize the ten most hazardous emerging contaminants [62]. The first
watch list (WL) was established in 2015 and has been updated period-
ically. The pharmaceuticals that were included in the WL are [63–66]:

* 1st WL, 2015: 17β-estradiol, 17α-ethinylestradiol, DCF, erythromycin, clarithromycin, azithromycin

* 2nd WL, 2018: 17β-estradiol, 17α-ethinylestradiol, erythromycin, clarithromycin, azithromycin, amoxicillin, ciprofloxacin

* 3rd WL, 2020: amoxicillin, ciprofloxacin, sulfamethoxazole, trimethoprim, venlafaxine, O-desmethyl venlafaxine, clotrimazole, fluconazole, miconazole

* 4th WL, 2022: sulfamethoxazole, trimethoprim, venlafaxine, O-desmethyl venlafaxine, clotrimazole, fluconazole, miconazole, clindamycin, ofloxacin, metformin

The importance of pharmaceuticals in the environment is proven by
individual studies, water companies, and law institutions. It is no
wonder that a considerable body of literature exists on the development
of new analytical methods that can monitor the levels of pharmaceuti-
cals and have as main challenges to attain the LOQ of LC-MS/MS while
improving the cost, usability, and portability.

3. Aptamers for pharmaceuticals

Aptamers are single-stranded nucleic acid (DNA or RNA) chains that
bind with high affinity to the molecules they were tailored for. The
succession of nucleotides gives the aptamer the primary sequence,
which is specific to the analyte. The generation of the aptamer sequence
is conducted in vitro from a random nucleotide library by a procedure
called SELEX [67]. The first SELEX step is the incubation of the

randomized nucleotide library with the target molecule when nucleo-
tides with affinity towards the target are selected. The incubation tem-
perature and time are controlled depending on the stability of the target.
Separation of the target-bound DNA from the unbound random DNA
library by a physical process (magnetic beads, filtration, solid phase
extraction) follows the selection. The isolated nucleotides are further
amplified by polymerase chain reaction (PCR), and another selection
cycle is launched. This positive selection screens the sequence with the
highest affinity for the target, however, the selectivity is not ensured.
Counter-selection is applied when the selected sequences are incubated
with structural analogs of the target, which can interfere with the
downstream applications of the aptamers. The sequences bind to the
interferants and are then separated, narrowing down the possible
aptameric sequences [68]. SELEX is a well-controlled process and the
long time for positive and counter selection makes it challenging to
obtain aptamers with high affinity and selectivity [67]. The primary
sequence can adopt different three-dimensional conformations because
of the hydrogen bonds between nucleotides, making the 3D form
responsible for the high affinity that the aptamers have for the target
[69].

Also called artificial antibodies, aptamers share the ability to bind
molecules with high affinity. However, certain advantages can be
emphasized. Firstly, their synthesis can be carried out in vitro once the
primary sequence has been established, resulting in lower batch-to-
batch variability. This addresses the reproducibility challenges associ-
ated with antibodies and eliminates contamination from cell culture.
Secondly, they are more stable towards chemical treatment and can
maintain their function in biological samples. They can be functional-
ized easily, which opens new ways for coupling and immobilization.
Moreover, the folding in the 3D structure is reversible, allowing the
regeneration of the aptamer [14].

Aptamers have been developed for a diverse range of targets. Initially, the majority was designed for proteins, followed by small molecules and whole cells. The applications of aptamers are broad, with significant implications in the field of sensors, therapeutics, and drug delivery.

Before using them for downstream assays and applications, the
binding affinity of developed aptamers is determined to assure their
quality. The equilibrium dissociation constant (K_D) will reflect the
strength of interaction between the aptamer and its target by quanti-
fying the changes in the signal or properties before and after the

<figure><img src="image_5.png" /><figcaption>**Figure 1.** The number of pharmaceuticals detected in surface waters, groundwater, tap water, and/or drinking water per country, according to [60]. The study aimed to offer a global comprehension of pharmaceutical pollution by harmonizing sources of literature, databases coming from European projects, and networks such as NORMAN, KNAPPE, FATE-SEES (JRC 2012), and POSEIDON.</figcaption></figure>
aptamer-target complex formation. The K<sub>D</sub> is a characteristic of the aptamer, with lower values indicating a higher affinity for the molecule. The range of the K<sub>D</sub> is from picomolar levels for large molecules to micromolar values for targets with a lower molecular weight. To determine the binding affinity several methods can be employed. The sensitivity, sample volume, cost, speed of analysis, and buffer environment are parameters that ought to be considered thoroughly before choosing the method to determine the K<sub>D</sub>. Ramesh et al. summarized the possible assays that can be employed to estimate the K<sub>D</sub>, classifying them into label-free techniques (UV-VIS spectroscopy, isothermal titration calorimetry, quartz crystal microbalance, surface plasmon resonance, native electrospray ionization-mass spectrometry) and labeled methods (fluorescence intensity, filter-binding assays, ELISA) [70].

Aptamers are selected against pharmaceuticals with the purpose of targeted drug delivery or for the development of sensing platforms. Aptamer-based sensors (aptasensors) that target pharmaceuticals are developed for therapeutic drug monitoring [71], control of food products and pharmaceutical formulations [72] and monitoring environmental contamination [73]. The main pharmaceutical pollutants for which aptamers have been developed are the class of antibiotics, anti-inflammatories, and steroid hormones. The sequences of aptamers that were used to develop electrochemical and PEC sensors in the articles covered by this review are summarized in Table 2, with details concerning the K<sub>D</sub> and the functionalization of the aptamer.

Quoting the article where the SELEX procedure was used for selection is often omitted in the articles studied, and the K<sub>D</sub> is difficult to trace back. In some cases, articles reference as a source for the aptamer sequence another paper where the aptamer was already used as a bio-recognition element for a developed sensor, making the original sequence challenging to find. The absence of the K<sub>D</sub> value is critical, as modifications to the aptamer sequence are often made without considering that truncating the complete SELEX aptamer can alter its binding affinity. The affinity constants in the table are sourced from studies that selected the aptamer sequence by SELEX.

Guan et al. performed in one study the selection of the aptamer sequence by GO-SELEX and the development of an electrochemical sensor for penicillin G (PEN G) detection [162]. GO-SELEX is based on the property of graphene oxide (GO) to adsorb unbound aptamer molecules *via* π stacking. This way, from an oligonucleotide library, only the aptamers not bound to the target will adsorb into the GO and undergo separation, enriching the solution in aptamers that have an affinity for the target [173]. The K<sub>D</sub> of the aptamers was determined by electrochemistry, plotting the decrease in current given by the redox probe after the interaction of the aptamer with increasing concentrations of PEN G. The sequence with the lowest K<sub>D</sub> (101.15 ± 1.94 nM) was chosen for the sensor development. To maintain the maximum affinity of the aptamer while it is immobilized on the surface, tetrahedral DNA nanostructure (TDN) immobilization was used. TDNs offer better control over the spacing of the aptamer while keeping them in an organized vertical position [162].

GO-SELEX was also employed by Bai et al. to obtain an aptamer specific for tetracycline (TC) and used molecular docking simulations to modulate the structure of the best-performing aptamer to increase its affinity constant. Every aptamer sequence was characterized and the K<sub>D</sub> was determined by fluorescence and isothermal calorimetry methods, being a good example of how aptamer modulation should be conducted. Furthermore, the obtained sequences could detect four tetracyclines, discriminating the class of tetracyclines from other antibiotics (penicillin, gentamycin, chloramphenicol, cephalexin). The truncated aptamer was integrated into an electrochemical sensor for TC with good analytical performance, LOD of 0.46 nM [105].

Another study selected the aptamer sequence by SELEX and used the oligonucleotide with the highest affinity (K<sub>D</sub> = 55 nM) to develop a sensor for azlocillin monitoring in wastewater. Unlike the usual immobilization methods, this study immobilized azlocillin at the surface and used the aptamer in the solution, which allowed the detection of the

analyte with an LOD of 1.2 pg/mL [163].

Due to the time-consuming procedure for aptamer discovery, computational studies are making a breakthrough in the field of aptamers. In silico methods can be applied to obtain new aptamer sequences, make predictions regarding aptamer-target interaction, and improve aptamer sequences after SELEX [174]. Using this tool, a study investigated the affinity between four antibiotics (kanamycin, TC, ampicillin, and sulfadimethoxine) with their corresponding aptamers by molecular docking and molecular dynamic simulations. Surprisingly, the highest affinity was obtained between TC and the aptamer specific for kanamycin (KANA). Therefore, the KANA-specific aptamer was integrated into an electrochemical sensor, resulting in an excellent LOD of 2.28 × 10<sup>-18</sup> M for TC. KANA, however, is an interferent to the method, an expected outcome because the aptamer was initially designed for recognizing KANA [175].

Splitting aptamers is a method to decrease the size of the original aptamers by cutting the sequence at specific points. A 33-mer and a 43-mer split aptamers were obtained from the original 76-mer for E2. The two maintained the same affinity and selectivity but manifested lower limits of detection when used in a colorimetric assay [176]. The sequences were integrated into an electrochemical platform and a low LOD of 0.5 pM was obtained in tap water [172].

The most common functionalization for the aptamers was the modification with SH and NH<sub>2</sub>. The two groups provide single-point attachments, ensuring the formation of S-Au and amide covalent bonds with the surface of the electrode. Although the 3' and 5' functionalization is not equivalent, and the modification can significantly influence the functionality of the aptamer [177,178], the 5' end was chosen preferentially for the immobilization on the surface without further investigations.

To facilitate detection, active redox probes like ferrocene (Fc) and methylene blue (MB) were immobilized onto the aptamer structure as markers. Monitoring the change in signal from the label after the aptamer interacts with its target is proof of the presence of the target in the solution.

Surface immobilization reduces the flexibility of the aptamer, and the reach of the molecule to its binding site can be impeded. To minimize the unfavorable effect the immobilization has on the affinity and selectivity of the aptamer, the oligonucleotide chain can be distanced from the surface [179]. The addition of six CH<sub>2</sub> moieties was often introduced between the binding function and the aptamer, aiming to increase the performance of the sensor.

## 4. Electroanalytical methods

The conversion of the change in the conformation of the aptamer after the target binding into a quantifiable signal can be achieved by coupling the platform with a suitable transducer. In the following section, the main electrochemical transducers developed for pharmaceutical pollutants, namely the amperometric, impedimetric, and PEC transducers will be discussed. Additionally, the field of electrochemiluminescent (ECL) sensors is expanding rapidly and the first aptasensors for pharmaceuticals were developed [180,181]. However, because of their few numbers and their different signal output, they were not included in this review. Other electronic devices, such as field-effect transistors (FET) were combined with aptamers for sensing applications. The readers are referred to the work of Wang et al. where the working principles and applications of FET aptasensors are covered [182].

Among all the amperometric methods, the voltammetric approach consists of sweeping the potential over time and registering the current. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV) are techniques of choice that account for 40% of the developed aptasensors studied. In electrochemical impedance spectroscopy (EIS), a potential wave crosses the surface at different frequencies, and the resulting current wave is recorded. PEC sensors are fabricated with photoactive materials that adsorb photons
<caption>**Table 2**</caption>

The analytes targeted in the studied literature with their corresponding aptamer sequence, the selection reference, K<sub>D</sub>, and functionalization of the aptamer post-SELEX.

<table><thead><tr><th>Analyte</th><th>Aptamer sequence 5'→3' [SELEX ref.]</th><th>K<sub>D</sub> (nM)/Method</th><th>5'</th><th>3'</th><th>Ref.</th></tr></thead><tbody><tr><td>Diclofenac</td><td>ATA CCA GCT TAT TCA ATT GCA ACG TGG CGG TCA GTC AGC GGG TGG TGG GTT CGG TCC AGA TAG TAA GTG CAA TCT [74]</td><td>42.7± 15.9 /Spectrophotometry</td><td>NH<sub>2</sub>-C<sub>6</sub></td><td>-</td><td>[75]</td></tr><tr><td rowspan="3">Ibuprofen</td><td rowspan="3">ATA CCA GCT TAT TCA ATT CCA CAG ACC CTT AGC TTT CCT ATT CTG CGC GAC GCT GAG ATA GTA AGT GCA ATC T [83]</td><td rowspan="3">1500</td><td>NH<sub>2</sub>-C<sub>7</sub></td><td>-</td><td>[76]</td></tr><tr><td>NH<sub>2</sub>-C<sub>6</sub></td><td>FAM</td><td>[77]</td></tr><tr><td>NH<sub>2</sub>-C<sub>7</sub></td><td>-</td><td>[78]</td></tr><tr><td rowspan="3">Tetracycline</td><td rowspan="3">TCT AAC GTG AAT GAT AGA CCT GGC TTG GGT GGT GGG CGA CTG ACT GGC GGT GCA ACG TTA ACT TAT TCG ACC ATA</td><td rowspan="3">-</td><td>SH-(CH<sub>2</sub>)<sub>6</sub></td><td>-</td><td>[79]</td></tr><tr><td>NH<sub>2</sub>-</td><td>-</td><td>[80]</td></tr><tr><td>NH<sub>2</sub>-</td><td>-</td><td>[81]</td></tr><tr><td rowspan="2">Oxytetracycline</td><td>TCT CTC CTC GGT GGT GTC CTC TCT</td><td>-</td><td>-</td><td>-</td><td>[82]</td></tr><tr><td>CCC CCG GCA GGC CAC GGC TTG GGT TGG TCC CAC TGC GCG T CGT ACG GAA TTC GCT AGC CCC CCG GCA GGC CAC GGC TTG GGT TGG TCC CAC TGC GCG TGG ATC CGA GCT CCA CGT G [87]</td><td>63.6 /Equilibrium filtration method</td><td>Fc<br>SH-(CH<sub>2</sub>)<sub>6</sub></td><td>(CH<sub>2</sub>)<sub>3</sub>-SH</td><td>[86]<br>[88-90]</td></tr><tr><td rowspan="10">Oxytetracycline</td><td>TTT TTC GTA CGG AAT TCG CTA GCC CCC CGG CAG GCC ACG GCT TGG GTT GGT CCC ACT GCG CGT GGA TCC GAG CTC CAC GTG GCC GTT CAG A AA AAT GAG GGT AAC ATA ACT GTA ATA CAC AAA CGC C GTT TGT GTA TTA CAG TTA TGT TAC CTC CAT TTT TTC GAAC GAG AGA CGG TGG TG ATT ACA CCA CGA CCC CCG GCA GGC CAC GGC TTG GGT TGG TCC CAC TGC GCG TCG TCG ACG GAT CCA TGG CAC ATG GAT TAC TAG CGA ACG CAT GAG CCG GCTGCG GCG CAT GCG T GGA ATT CGC TAG CAC GTT GAC GCT GGT GCC CGG TTG TGG TGC GAG TGT TGT GTG GAT CCG AGC TCC ACG TG [106]</td><td>4.59 ± 0.1/ Fluorescence 1830/ ITC 12.08 ± 2.25/Equilibrium filtration method</td><td>NH<sub>2</sub><br>SH<br>SH-(CH<sub>2</sub>)<sub>6</sub><br>SH<br>SH-SH-SH-(CH<sub>2</sub>)<sub>6</sub><br>NH<sub>2</sub>- (CH<sub>2</sub>)<sub>6</sub><br>-</td><td>-<br>Fc<br>-<br>-<br>-<br>-<br>-</td><td>[91]<br>[92]<br>[73]<br>[93]<br>[94]<br>[95]<br>[96,97]<br>[98]<br>[99]</td></tr><tr><td>CGA CGC ACA GTC GCT GGT GCG TAC CTG GTT GCC GTT GTG T [122]</td><td>4.7/ ELAA</td><td>COOH</td><td>-</td><td>[100]</td></tr><tr><td>GGG GGC ACA CAT GTA GGT GCT GTC CAG GTG TGG TTG TGG T CGT ACG GAA TTC GCT AGC CGA GGC ACA GTC GCT GGT GCC TAC CTG GTT GCC GTT GTG TGG ATC CGA GCT CCA CGT G</td><td>-</td><td>-</td><td>-</td><td>[101]<br>[102]<br>[103]<br>[104]<br>[105]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[106]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[107]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[108-110]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[111]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[112,113]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[114]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[115]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[116,117]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[118]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[119]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[120]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[121]</td></tr><tr><td>-</td><td>-</td><td>-</td><td>-</td><td>[123]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 2 (continued)**</caption><thead><tr><th>Analyte</th><th>Aptamer sequence 5'→3' [SELEX ref.]</th><th>K<sub>D</sub> (nM)/Method</th><th>5'</th><th>3'</th><th>Ref.</th></tr></thead><tbody><tr><td rowspan="5">Enrofloxacin</td><td>CGT ACG GAA TTC GCT AGC CGA GTT GAG CCG GGC GCG GTA CGG GTA CTG GTA TGT GTG GGG ATC CGA GCT CCA CGT G</td><td>-</td><td>-</td><td>-</td><td>[124]</td></tr><tr><td>CGT ACG GAA TTC GCT AGC GGG CGG GGG TGC TGG GGG AAT GGA GTG CTG CGT GCT GCG GGG ATC CGA GCT CCA CGT G</td><td>-</td><td>NH<sub>2</sub></td><td>-</td><td>[125]</td></tr><tr><td>CGT ACG GAA TTC GCT AGC CGA GTT GAG CCG GGC GCG GTA CGG GTA CTG GTA TGT GTG GGG ATC CGA GCT CCA CGT G</td><td>-</td><td>-</td><td>-</td><td>[126]</td></tr><tr><td>CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA</td><td>-</td><td>-</td><td>-</td><td>[127–129]</td></tr><tr><td>(continued)</td><td></td><td></td><td>NH<sub>2</sub>- (CH<sub>2</sub>)<sub>6</sub></td><td>-</td><td>[130]</td></tr><tr><td rowspan="2">Ciprofloxacin</td><td rowspan="2">ATA CCA GCT TAT TCA ATT GCA GGG TAT CTG AGG CTT GAT CTA CTA AAT GTC GTG GGG CAT TGC TAT TGG CGT TGA TAC GTA CAA TCG TAA TCA GTT AG [134]</td><td rowspan="2">0.11±0.006/Affinity elution assay</td><td>SH</td><td>-</td><td>[131]</td></tr><tr><td>SH-(CH<sub>2</sub>)<sub>6</sub></td><td>-</td><td>[132]</td></tr><tr><td rowspan="2">Chloramphenicol</td><td rowspan="2">ACT TCA GTG AGT TGT CCC ACG GTC GGC GAG TCG GTG GTA G</td><td rowspan="2">-</td><td>NH<sub>2</sub></td><td>-</td><td>[133]</td></tr><tr><td>NH<sub>2</sub></td><td>-</td><td>[135]</td></tr><tr><td rowspan="2">Ampicillin</td><td rowspan="2">CAT TCC TTT TAC ACT TCA GTG AGT TGT CCC ACG GTC GGC GAG TCG GTG GTA G</td><td rowspan="2">-</td><td>MB</td><td>MB</td><td>[136]</td></tr><tr><td>SH-(CH<sub>2</sub>)<sub>6</sub></td><td>-</td><td>[137,138]</td></tr><tr><td rowspan="2">Penicillin G</td><td rowspan="2">TTA GTT GGG GTT CAG TTG G [147]</td><td rowspan="2">9.8</td><td>NH<sub>2</sub></td><td>-</td><td>[139–141]</td></tr><tr><td>-</td><td>-</td><td>[142–145]</td></tr><tr><td rowspan="2">Amoxicillin</td><td rowspan="2">TTG ATC GCG GGC GGT TGT ATA GCG G [147]</td><td rowspan="2">13.4</td><td>-</td><td>(CH<sub>2</sub>)<sub>6</sub>- biotin</td><td>[146]</td></tr><tr><td>-</td><td>-</td><td>[148]</td></tr><tr><td rowspan="2">Azlocillin</td><td rowspan="2">TTA GTT GGG GTT CAG TTG G [147]</td><td rowspan="2">9.8</td><td>NH<sub>2</sub>- (CH<sub>2</sub>)<sub>6</sub></td><td>-</td><td>[149]</td></tr><tr><td>-</td><td>-</td><td>[150–152]</td></tr><tr><td rowspan="2">Penicillin G</td><td rowspan="2">TTG ATC GCG GGC GGT TGT ATA GCG G [147]</td><td rowspan="2">13.4</td><td>-</td><td>-</td><td>[153,154]</td></tr><tr><td>-</td><td>-</td><td>[155,156]</td></tr><tr><td rowspan="2">Azlocillin</td><td rowspan="2">CAG GAA GAC AAC TCC GAC TAG AAT TGA TAA TCA AGA ATT CGT CTG GGG GGA ATG TGC G</td><td rowspan="2">55</td><td>NH<sub>2</sub>- (CH<sub>2</sub>)<sub>6</sub></td><td>-</td><td>[157]</td></tr><tr><td>-</td><td>-</td><td>[158]</td></tr><tr><td rowspan="2">Penicillin G</td><td rowspan="2">GGG TCT GAG GAG TGC GCG GTG CCA GTG AGT [159]</td><td rowspan="2">383.4</td><td>SH</td><td>-</td><td>[160]</td></tr><tr><td>/Fluorescence quenching</td><td></td><td></td><td></td></tr><tr><td rowspan="2">Azlocillin</td><td>TTA GTT GGG GTT CAG TTG G [147]</td><td>9.8</td><td>SH</td><td>-</td><td>[161]</td></tr><tr><td>CAC CAG TCA GAC AGC ACG GTG ACT GGA GTG ACG TCG GTA CCT GAG ATC GAG TGA CGT CGG TAC CTG</td><td>105.15</td><td>-</td><td>-</td><td>[162]</td></tr><tr><td rowspan="2">Azlocillin</td><td>CAG GAA GAC AAC TCC GAC TAG AAT TGA TAA TCA AGA ATT CGT CTG GGG GGA ATG TGC G</td><td>55</td><td>-</td><td>-</td><td>[163]</td></tr><tr><td>/Fluorescence binding assay</td><td></td><td></td><td></td><td></td></tr></tbody></table>

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solution can be voltammetric or impedimetric, and their response is highly dependent on the way the electroactive molecules interact with the surface. For the voltammetric sensors, following the oxidation or reduction of the probe, an initial current is registered by CV, DPV, or SWV. The aptasensor is left in contact with the analyte when the aptamer on the surface recognizes the target molecule and changes its spatial conformation. Subsequently, the aptasensor is washed and re-analyzed under the same conditions. In the case of the most common signal-off sensors, the change of conformation of the aptamer has a shielding effect on the redox probe, which can no longer reach the surface and exchange electrons. The immediate consequence is the decrease in current intensity, proportional to the concentration of the analyte, as in Figure 2A. The calibration curve is obtained by plotting the difference in current (ΔI = I<sub>before incubation</sub> - I<sub>after incubation</sub>) depending on the concentration of the analyte. This approach has been used by many recent studies with the [Fe(CN)₆]³⁻/⁴⁻ redox couple. The reasons for the frequent use of this couple are its well-defined electrochemical behavior, stability, and reversibility. Moreover, its electrochemical response is strongly influenced by the state of the surface, allowing rapid data acquisition regarding the functionalization of the surface [184]. It has been used in numerous protocols, such as the aptasensor developed for chloramphenicol (CAP) in Figure 3A, and other studies for DCF [76], TC [88], and oxytetracycline (OTC) [115,120].

Other original studies going beyond the concept of immobilized
aptamers have also been reported. In one of them, an atypical compet-
itive detection was conducted by SWV following the same redox probe,
but the target molecule, azlocillin, was immobilized at the surface of the
electrode by an amide bond while the aptamer was in solution. The
aptasensor was incubated with a constant concentration of aptamer. If
azlocillin is present in the incubation solution, the aptamer will pref-
erentially interact with the free molecule and bind less to the azlocillin
attached to the surface. Because the aptamer is a large insulating
molecule, if it binds to the surface, it will induce a decrease in current.
The more azlocillin present in the environment, the less aptamer binds

to the surface, and less sensor response is registered (Figure 3B) [163].

Another study used [Fe(CN)₆]³⁻/⁴⁻ to detect CAP while exploiting the difference in the binding capacity of the free and bound aptamer for a single-stranded DNA binding protein. In the blank solution, the free aptamer binds to the protein, leading to significant surface passivation. Alternatively, if CAP is present, it binds to the aptamer, and the protein has less adherence to the surface, causing less decrease in current intensity, as shown in Figure 3C [137].

Impedimetric sensors are sensitive and accessible tools used for detecting numerous analytes. EIS can provide information about mass and charge transfer by applying a small sinusoidal potential excitation to the electrochemical system. The electrochemical cell is a combination of resistors, capacitors, or constant phase elements that induce a phase shift between the applied potential and the read current. The main graphical representation of EIS in biosensors is the Nyquist plot, where the real component of the impedance (Z<sub>real</sub>) is plotted on the X-axis and the imaginary part (Z<sub>imag</sub>) on the Y-axis.

All the studies analyzed use the faradaic form of EIS, meaning that impedance is produced when redox reactions occur, the reason for which EIS measurements are conducted in [Fe(CN)₆]³⁻/⁴⁻ solution. The processes taking place at the electrode/electrolyte interface can be simulated into an electric circuit (Randles equivalent circuit) that will fit the Nyquist plot. A typical circuit contains the resistance in solution (Rₛ), double-layer capacitance at the surface of the electrode (C<sub>dl</sub>), charge transfer resistance (R<sub>ct</sub>), and Warburg resistance (Z<sub>w</sub>).

EIS biosensors translate the change in aptamer properties upon
binding into a change in R<sub>ct</sub>. The usual mechanism implies an increase in
the R<sub>ct</sub> after the target recognition because the conformation of the
biomolecule will impede the electron transfer. For instance, for detect-
ing enrofloxacin (ENR), the authors observed a logarithmic increase in
ΔR<sub>ct</sub> with the increment of concentration in a range of 0.01 – 2000 pg/
mL (Figure 3D). Other impedimetric sensors were constructed on the
same principle for DCF [75,77], tetracyclines [92,99,104], fluo-
roquinolones [127], CAP [139], and penicillins [150,185].

<figure><img src="image_6.png" /></figure>

**Figure 3.** Detection strategies using a redox probe in solution for the detection of (A) CAP when a typical decrease in current is determined by the target incubation [138], (B) azlocillin, when the target molecule is immobilized at the surface. Reprinted from Ref. [163]. Copyright (2024), with permission from Elsevier (C) CAP using a competitive assay. Reprinted from Ref. [137]. Copyright (2024), with permission from Elsevier, (D) ENR, generating an increase in R<sub>ct</sub> with the concentration. Reprinted from Ref. [154]. Copyright (2024), with permission from Elsevier.

<figure><img src="image_7.png" /></figure>
Several voltammetric or PEC sensors use the EIS technique as a reliable electrochemical characterization method of surface functionalization. Each modification step is confirmed by following the trend of Rct. For example, nanomaterial modification generates a decrease in Rct compared to the bare electrode due to their high conductivity and active area while the biomolecule attachment leads to an increase in Rct because of the formation of an insulating layer.

## 4.2. Labeled redox probe

Aptamers can be labeled with redox probes, which undergo distinct oxidation or reduction depending on the state of the biomolecule. Typically, the probe is covalently immobilized on the aptamer at the free end. This approach has the advantage of minimizing false positives, as the change in the signal is attributed to the interaction aptamer-analyte rather than the state of the surface.

When the target binds, the aptamer undergoes a conformational rearrangement, adjusting the distance between the redox probe and the electrode surface. In one scenario, this leads to an increase in current intensity, resulting in a signal-on quantification. In the other scenario, the current intensity decreases, inducing a signal-off detection (Figure 2B).

Ferrocene (Fc) was used as a redox probe where two signal-on aptasensors were obtained for TC and OTC [86,111] using a similar sensor design with a gold platform, thiolated aptamer immobilization, and 6-mercapto-hexanol (MCH) blocking. Another study reported a decrease in the Fc signal following the incubation with TC, which demonstrates that both quantifications are possible and dependent on the particularity of the aptamer-analyte interaction [73].

Methylene blue (MB) is another redox-active molecule used to label the aptamers. In a multiplexed method, an aptamer specific for ciprofloxacin (CIP) was double-labeled with MB. Integrated in an ingenious method, the aptamer is hybridized with two DNA complementary strands (CS) immobilized at the surface by Au-S bonds. The presence of the target molecule in the solution breaks the bond aptamer-CS, and the aptamer carries away the active MB. Consequently, the signal generated initially by MB will decrease proportionally with the amount of aptamer that leaves the surface and with the concentration of the analyte. The sensor showed high affinity for the congeners of CIP, levofloxacin (LFX), and ofloxacin (OFX), but no affinity towards other classes of antibiotics [136].

There are reported cases when MB is fixed on the immobilized aptamer by electrostatic interactions between the negatively charged phosphate groups and the MB cations [78,123]. In the presence of the target, the aptamer undergoes conformation change, leading to the release of MB into solution.

Novel redox probes were used for detecting OTC and ENR. For OTC, a composite made of gold nanoparticles (AuNPs), carboxylated multiwalled carbon nanotubes (cMWCNTs), and complementary strands of DNA was marked with thionine, a redox active molecule. The cDNA hybridized with the aptamer on the surface of the gold electrode, and the signal of the thionine could be registered. The signal decreases if OTC is preferentially interacting with the aptamer, detaching the composite and the thionine from the surface [107]. A similar procedure was employed for the detection of ENR, where thionine is labeled on gold triangular nanosheets modified with cDNA. In this study, the oxidation of thionine is catalyzed by the coordinated effect of a nickel metal-organic framework (MOF) and AuNPs, a factor that increases the sensitivity of the method, with an LOD of $5.6 \times 10^{-3}$ pg/mL [130].

## 4.3. Redox probe-free photoelectrochemical

PEC sensing involves a charge transfer between the photo-electroactive material, the analyte, and the electrode upon light exposure. Since the input signal is the light, and the output signal registered is a current, this difference in energy forms could potentially minimize the

background noise, leading to better sensitivity [186]. The key element in the design of a PEC sensor is the engineering of the photoactive material (semiconductor) that amplifies the photocurrent and can immobilize the recognition molecule. The detection mechanism of PEC sensors is represented in Figure 2C and begins with the absorption of light by the semiconductor. Upon excitation, electrons (e⁻) will migrate from the valence band (VB) of the semiconductor to the conduction band (CB), leaving holes (h⁺) carriers in the VB. A separation of charges occurs, photoelectrons move to the working electrode (determining a photocurrent) and the photoholes will oxidize species in the solution that have a lower oxidation potential than the VB energy of the semiconductor. The electrons produced after the analyte oxidation in the solution will create an additional photocurrent. The increase in photocurrent depends on the concentration of the analyte, as illustrated in the chro-noamperogram in Figure 2D.

This sensing method was employed in the detection of DCF upon visible light illumination, using graphene-doped CdS decorated with gold nanoparticles, where the nanomaterials highly increased the photocurrent response [80]. The increase in photocurrent was observed with the increment of OTC concentration when using a ternary Z-scheme heterojunction of Bi-bridged $BiVO_4/g-C_3N_4$ junction [125]. The same procedure was applied by other studies detecting TC [89,96–98,187], OTC [110,125], ampicillin (AMP) [188], and E2 [170].

In other cases, the inverse response was observed. After the incubation with the analyte, a decrease in photocurrent is generated due to the high resistance of the bound aptamer that blocks the electron transfer and the charge separation [90,117]. A higher analyte concentration will form more aptamer-analyte complexes that increase the resistance, resulting in lower electron transfer and photocurrent response. This phenomenon is described in Figure 4A, where the interaction between ENR and its aptamer determined a decrease in photocurrent [132].

Modified aptamers can be integrated into the PEC platforms, like in the case of CAP detection. Horseradish peroxidase (HRP) was immobilized on the aptamer to catalyze the oxidation of 4-chloro-1-naphthol (4-CN) to insoluble benzo-4-chlorohexadienone (4-CD), resulting in a great decrease in photocurrent due to its adsorption on the electrode surface. When the APT-HRP binds to CAP, it leaves the surface, the formation of the precipitate is prevented, and the photocurrent is proportional to the concentration of the analyte [146].

Song et al. fabricated a dual-modal aptasensor combining two types of signal output, electrochemical and PEC. The dual-modal detection has the advantage of enhanced accuracy by cross-verifying the results, reducing the likelihood of false positives or negatives, and providing a more robust and reliable measurement of target analytes. MB intercalated in the complex formed between an immobilized complementary DNA strand (cDNA) and the aptamer. Upon binding to OTC, MB leaves the surface of the electrode at the same time as the aptamer detaches, and a decrease in current and photocurrent is observed, as illustrated in Figure 4B [123].

The rich array of detection methods explored in this chapter shows the exceptional versatility inherent in aptasensors for pharmaceutical pollutant analysis. The adaptable nature of these methods accommodates diverse sensing needs, offering rapid and cost-effective electrochemical platforms alongside the high sensitivity and selectivity provided by sophisticated voltammetric, impedimetric, and PEC setups.

## 5. Fabrication of the aptasensors

The design of electrochemical aptasensors involves numerous modification steps of the starting material. The surface functionalization aims to optimize the performance of the final sensor by providing a suitable platform for the immobilization of the biorecognition element, thereby enhancing selectivity. Once selectivity is ensured, sensitivity can be improved to the extent that a stable electrochemical response is achieved even in the presence of very low analyte concentrations. An
<figure><img src="image_8.png" /><figcaption>**Figure 4.** (A) The detection process of a PEC aptasensor based on APT/Au/Bi<sub>24</sub>O<sub>31</sub>Br<sub>10</sub>/ITO. Illustration of the formation of highly resistant APT-ENR complexes that inhibit electron transfer. Adapted from Ref. [132]. Copyright (2024), with permission from Elsevier. (B) Fabrication and detection mechanism of a dual-modal EC-PEC aptasensor using MB as an enhancer of the signal. Reprinted from Ref. [123]. Copyright (2024), with permission from Elsevier.</figcaption></figure>

illustration of different strategies to immobilize aptamers on various material substrates is shown in Figure 5. Each type of material and how it can be functionalized with aptamers will be discussed in detail.

### 5.1. Carbon materials

Carbon materials are the most widely used materials in electrochemical sensing due to their excellent stability, high conductivity, and

<figure><img src="image_9.png" /><figcaption>**Figure 5.** Illustration of aptamer immobilization methods and materials employed by the analyzed studies.</figcaption></figure>
wide potential window. Carbon nanomaterials (graphene, GO, rGO, GQDs, CNTs) have already been the focus of several reviews, highlighting their importance in aptasensor development due to their high electrical conductivity and large surface area [189–191]. Emphasizing the advantages of graphene in the design of aptasensors, Liu et al. employed this nanomaterial with a double function. Firstly, the aptamer specific for E2 was adsorbed on graphene sheets in solution by π-stacking. Furthermore, the aptamer detaches from graphene when the analyte is present, promoting graphene assembly on the electrode surface and enhancing the electron transfer between the supporting electrode and ferrocene formic acid (FcCOOH) redox probe in solution. An enzyme is further used to cleave the bonds between the aptamer left in the solution and the target, regenerating the analyte, which can bind to other aptamers, inducing an additional release of graphene. With the synergic effect between graphene and the enzyme, an LOD of 50 fM was obtained [192]. In another approach, Chang et al. used laser-scribed graphene (LSG) electrodes as starting materials. From this material, various patterned electrode designs can be obtained. The developed sensor used a split-aptamer that was reconstructed in the presence of E2. The LSG electrodes were first functionalized with poly(β-cyclodextrin), and then adamantane-modified aptamer fragments were immobilized by host-guest interaction [193]. Graphene quantum dots (GQDs), which consist of one or a few graphene layers with a size smaller than 100 nm, were also used as aptasensor materials. In a PEC aptasensor, Wang et al. used GQDs to improve visible light absorption of TiO₂ nanotubes and immobilize by π-stacking a CAP-specific aptamer [143].

Graphene oxide (GO) can also be employed, and its oxygen functionalities can be used to immobilize the aptamer. For instance, alcohol functions in GO can react with (3-aminopropyl) 3-ethoxysilane (APTES), leading to -NH₂ surface groups. Roushani et al. used such a functional surface as anchoring groups for silver nanoparticles modified with CAP-

specific aptamer [140]. The carboxyl functions of GO can also be activated with EDC/NHS and involved in carbodiimide coupling. Es'haghi et al. used this strategy to design a sensor specific to DCF, using an aptamer home-labeled with methylene blue [78]. rGO was often accompanied by AuNPs in the construction of the aptasensors for the detection of TC [104], OTC [120,126], CIP [135], AMP [194], PEN G [161,162], and CAP [137,139].

Carbon nanotubes (CNTs) are another class of nanomaterials frequently employed in the literature for signal amplification and aptamer immobilization [75,79]. For instance, Bedioui et al. used carboxyl-modified MWCNTs deposited on GCE to immobilize a DCF-specific aptamer, as illustrated in Figure 6A. The MWCNTs serve here two purposes, i.e. they improve the substrate conductivity (with reduced Rct values) and allow aptamer immobilization through carbodiimide coupling [75]. Carboxyl-modified MWCNTs were also used in the composition of a PEC aptasensor specific to TC [96].

Although widely used, bare carbon electrodes often require surface modification to enable effective aptamer immobilization, due to their relatively low surface reactivity and potential for nonspecific adsorption [195]. However, these limitations can be mitigated by surface functionalization through aryldiazonium salts reduction, as a simple, robust, and versatile functionalization method. This method has already been used in the construction of aptasensors, allowing the introduction of reactive moieties such as carboxyl groups for carbodiimide coupling [196] or ethynyl groups for click-chemistry aptamer immobilization [197]. As illustrated in Figure 6B, in-situ generation of 4-carboxyphenyl diazonium salt and its subsequent electroreduction leads to carboxyl-modified GCE that were used to develop aptasensors sensitive to tetracyclines [91,115].

A

<figure><img src="image_10.png" /></figure>

B

<figure><img src="image_11.png" /><figcaption>**Figure 6.** (A) Immobilization of aminated aptamers on activated carboxyl functions provided by MWCNTs deposited on GCE [70], and (B) the electrodeposition of diazonium salts on GCE for surface functionalization. Reprinted from Ref. [110]. Copyright (2024), with permission from Elsevier.</figcaption></figure>
## 5.2. Metallic materials

Among various metallic materials, noble metal nanoparticles are the most commonly used in the development of aptasensors. Noble metal nanoparticles possess high surface area, good conductivity, unique optoelectronic properties, and biocompatibility. Gold nanoparticles (AuNPs) are, by far, the most employed for fabricating aptasensors for environmental applications [198]. One advantage of immobilizing AuNPs on the electrode surface is the increase of active surface area. The immediate consequence is a significant signal increase and any subsequent modification will cover more surface. Because the morphology of the AuNPs (shape, size, distribution) strongly influences their properties and the performance of the sensor, Blidar et al. paid particular attention to the well-controlled design of the AuNPs for developing a sensor specific to OTC. The control of the electrochemical deposition parameters, HAuCl₄ solution, and additives results in fern-like, spear-like, cauliflower-like, thistle-like, and pyramidal structures, as illustrated by the scanning electron microscopy (SEM) images in Figure 7 [111]. In other approaches, gold nanovoids and nanocubes were obtained [73,137].

Gold is known for its strong affinity towards thiols. As indicated in Table 1, numerous aptamers undergo functionalization with an SH group, either directly or through a carbon chain spacer. This aptamer modification is highly advantageous, given its ability to bind easily to gold surfaces: the simple immersion of the gold-modified electrode into the aptamer buffer solution overnight is enough to create the covalent Au-S bond. The thiolated aptamers will organize spontaneously at the surface, forming self-assembled monolayers (SAM). The high surface

area of AuNPs will lead to a high number of aptamers on the surface, a factor that will increase the sensitivity of the sensor [199]. One disadvantage of this strategy is that aptamers can bind non-specifically to gold surfaces because of their multiple nitrogen atoms, leading to chaotic organization. To limit this phenomenon, backfilling with another thiol is required to displace adsorbed aptamers and ensure an upright orientation. The main alkanethiol used for backfilling is 6-mercapto-hexanol (MCH), which blocks the free gold sites and has anti-fouling properties [111]. Another drawback of the thiol-based SAM is the restricted control over the orientation of the building molecules that can adopt randomized conformations [200]. Moreover, the Au-S bond, because of the big difference in electronegativity between the two atoms, has a polar character and is more susceptible to breaking under chemical and electrochemical factors. Despite these limitations, this strategy accounts for 20% of the immobilized aptamers in the studied literature, with thiolated aptamers specific for AINS, antibiotics, and steroidal hormones used in all types of detection [80,104,172].

Indirect immobilization of aptamers on gold surfaces can be realized by hybridization. Two single-stranded DNA chains can hybridize due to the hydrogen bonds between purines and pyrimidines, and form double-stranded oligonucleotides [201]. In many cases, a complementary DNA strand (cDNA) of the aptamer is immobilized covalently on the gold surface, followed by the subsequent hybridization with the aptamer. This approach is more than an immobilization strategy as it plays a pivotal role in many detection techniques where, after the interaction with the target molecule, the aptamer-cDNA bond breaks, and the aptamer is released from the surface with changes in the registered signal.

<figure><img src="image_12.png" /><figcaption>**Figure 7.** SEM images of different gold structures obtained by the electroreduction of a HAuCl₄ solution: (A) fern-like, (B) spear-like, (C) cauliflower-like, (D) thistle-like, and (E) tetragonal pyramid structures; (F) the working principle of the aptasensor. Adapted from Ref. [111]. Copyright (2024), with permission from Elsevier.</figcaption></figure>
Variations of the hybridization technique were used, like the design of a ladder-shaped DNA structure for detecting AMP (Figure 8A). At the base of the structure, there are two single-stranded DNA (ssDNA) chains that further create a joint by hybridization with the aptamer. Successive ssDNA molecules are then added to the surface to form the DNA ladder. In the presence of AMP, the aptamer joint is fragmented and the multiple layers of DNA leave the surface, leading to a positive signal [148].

Studies utilizing self-assembled tetrahedral DNA nanostructures (TDN) have given particular emphasis to controlling aptamer immobilization. TDN is a pyramidal 3D nanostructure formed by the complementary pairing of four ssDNA molecules [202]. The pyramid is anchored at the surface coated by AuNPs by three SH groups, offering better stability to the complex, as illustrated in Figure 8B. The aptamer is spaced from the surface, limiting steric hindrance and increasing the availability of the aptamer for target binding. Moreover, the size of the TDN is programmable, allowing control over the distribution of aptamers. Gual et al. employed TDNs for detecting AMP and penicillin [162,194]. For AMP, an LOD of 1 pM was achieved, which is 100 times lower when using TDN compared to the thiol-based aptamer SAM [194].

### 5.3. MOF and COF

Metal-organic frameworks (MOFs) are a special class of porous functional materials constructed from metal clusters and organic ligands, via coordinate bonds, leading to open crystalline frameworks with high porosity. The variety of metals and organic ligands leads to structural diversity and tunability. They can find applications in gas storage, separation, and catalysis [203]. Their sensing applications have been delayed because of their insulating features, but their high surface area and porosity can represent a method to increase electrode surface area [204]. An additional benefit of using MOFs is the possibility of immobilizing DNA chains by non-covalent interactions. The high surface area and tunable pore size of MOFs allow the immobilization of large aptamer quantities, distributed both on the surface and inside the MOFs [205]. Additionally, due to their tunability, MOFs rich in functional groups can be synthesized to immobilize aptamers covalently.

Carbon or metallic nanomaterials can be added to improve the poor conductivity of pristine MOFs. For instance, CoNi-MOF has high hydrophilicity and good electrochemical stability but low conductivity. To boost their conductivity, AuNPs were added to the MOF surface through electrodeposition. In this case, the AuNP coating enhanced the material's conductivity while also providing a platform for aptamer immobilization via thiol functional groups [131]. Aptamers can also be immobilized directly on the CoNi-MOF surface by π-stacking [127]. Both

CoNi-MOF aptasensors manifested excellent LODs of 0.33 fg/mL and 0.2 fg/mL for ENR detection.

Additionally, electrochemically active MOFs were reported as excellent redox mediators for TC detection [100]. In this example, the first component, Mo@MOF-88, which shows a reduction peak at -1.06 V vs Ag/AgCl, was immobilized on the electrode surface with a ssDNA. Subsequently, a second MOF, NH₂-UiO-66, characterized by an oxidation peak at 0.724 V vs Ag/AgCl, underwent modification with a TC-specific aptamer, which then hybridized with the ssDNA on the surface. The two DNA chains were connected to their corresponding MOFs by carbodiimide coupling between the COOH group of the biomolecule and the NH₂-rich MOF, being an example of covalent immobilization on MOFs. During detection, TC detaches the Apt@NH₂-UiO-66 from the surface, leading to a decrease of the oxidation peak at 0.724 V vs Ag/AgCl and an increase of the reduction peak at -1.06 V vs Ag/AgCl, achieving a dual-response strategy, as illustrated in Figure 9 [100].

The detection of TC was carried out using a Ni-MOF, chosen for its peroxidase-like activity. This MOF can reduce H₂O₂ in solution in the presence of an electron donor, 3,3',5,5'-tetramethylbenzidine (TMB), which is oxidized to TMB⁺. The subsequent electroreduction of TMB⁺ back to its reduced form gives a significant voltammetric signal. Immobilizing the TC-specific aptamer via π-stacking enhances the enzymatic activity of the MOF, resulting in an increased current due to the reduction of TMB⁺. When TC is present, it displaces the aptamer from the surface, reducing the amount of TMB⁺ and decreasing the current intensity [101].

Other interesting properties of MOFs are their wide light-absorption wavelength ranges and moderate carrier recombination rates, which make them excellent candidates for constructing PEC sensors. MOF band gap can be tuned by choosing the right metal clusters and organic ligands [206]. For instance, CdS semiconductors were functionalized with Eu-MOF and MIL-68(In)-NH₂, resulting in synergic light-harvesting effects, accelerated charge transfer, and improved photoelectric conversion efficiency of the PEC sensors [96,188].

Covalent organic frameworks (COFs) share numerous properties with MOFs, as their high porosity, but differ in terms of building blocks and connections. Unlike MOFs, COFs consist of lightweight organic elements without the inclusion of metal atoms in their structure. While MOFs rely on coordinate bonds between metal ions and organic ligands, COFs are linked by robust covalent bonds. Although COFs lose the catalytic activity associated with metallic atoms in MOFs, they gain enhanced chemical and physical stability [207]. COFs were used to develop impedimetric sensors for OTC, AMP, and ENR. The employed

<figure><img src="image_13.png" /><figcaption>**Figure 8.** Design of DNA structures based on hybridization: (A) ladder-shaped DNA structure. Reprinted from Ref. [148]. Copyright (2024), with permission from Elsevier, (B) tetrahedral DNA nanostructure. Reprinted from Ref. [194]. Copyright (2024), with permission from Elsevier.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Figure 9.** Ratiometric sensing of TC based on two electrochemically active MOFs. Adapted with permission from [100]Copyright (2023) American Chemical Society.</figcaption></figure>

COFs exhibited an extended π conjugation system, large specific surface area, big pores, and uniform morphology, representing an ideal substrate for aptamer immobilization by non-covalent interactions [116, 154]. Several studies reported the use of COF/MOF hybrids, which can bridge the gap between pristine MOFs and COFs [99,152].

## 5.4. Semiconductors

The significance of photoactive materials in PEC sensing is pivotal, and the design and engineering of these materials are acknowledged as essential steps in creating a high-performance PEC sensor. To ameliorate the performance of the photoactive material, improving visible-light excitation and charge separation are the main objectives [208].

Metal oxides are central semiconductors extensively employed in the field of PEC sensing due to their photocatalytic properties, availability, non-toxicity, high surface area, and surface modification capabilities. Binary oxides such as TiO₂, ZnO, and WO₃ are often included in the design of PEC aptasensors for pharmaceutical pollutants detection [82, 93,98,141]. An important focus is the development of ternary oxides with tunable band gaps. Combining metal cations in mixed oxides can lead to predictable band gaps and better control of their properties. Bi, V, and Fe are the main metals found in the composition of ternary oxides for aptasensor construction, leading to BiVO₄ [187], Bi₂WO₆ [81], FeVO₄ [82], and BiFeO₃ [157]. However, metal oxides undergo fast photogenerated charge recombination and poor utilization of visible light, and are often combined with other materials in photoelectrode fabrication. To enhance their properties, morphological control, heterojunction formation, and sensitization strategies were employed [209, 210].

MOF-derived In₃O₃ [114] and TiO₂ nanotubes [143] were synthesized and used to detect OTC and CAP. The nanotube structure assures a large specific surface area that enhances light absorption, the transfer of photoexcited electrons, and numerous sites for aptamer binding. TiO₂ was combined with GQDs and In₃O₃ with Ag-doped ZnIn₂S₄ to reduce the recombination of photocarriers, maximize efficiency, and increase the intensity of the photocurrent signal.

Dye sensitization is a common way to improve visible-light absorption. In the method used by Wang et al. bismuth oxy-iodide (BiOI) microspheres were sensitized with bipyridine ruthenium (Ru(bpy)₃⁺), exhibiting 13 times the current intensity of BiOI alone under visible light. Glutaraldehyde (GA) was used as a cross-linking agent to connect

the aminated aptamer specific for AMP to the surface, leading to an impressive LOD of 3.3 × 10⁻⁸ nM [156].

Metal oxides were often involved in heterojunction formation. A heterojunction implies the combination of two or more materials with matching band gaps, allowing photoexcited electrons and holes to move from one semiconductor to another. This transfer of charges limits the recombination rate and contributes significantly to the photoconversion [211]. For AMP detection, an aptasensor was developed where p-type BiFeO₃ nanoparticles were coupled with n-type graphite-like nitride (utg-C₃N₄) nanosheets [157]. The heterojunction improved both visible-light utilization, lowering the band gap of BiFeO₃ from 2.20 eV to 2.04 eV, and gave a 7-fold boost in photocurrent. In Figure 10A, the principle of a p-n heterojunction is illustrated. Under the influence of light, electrons will be photoexcited in both materials. Photoelectrons from the CB of utg-C₃N₄ will move to the CB of BiFeO₃, and holes will move from the VB of BiFeO to the VB of utg-C₃N₄. The aptamer was immobilized on the composite by nonspecific adsorption and bound AMP. Upon oxidation of the bound AMP by the photoholes, the photo-current increased, leading to an LOD of 0.33 pM. Other p-n heterojunctions used were: MoS₂/nitrogen-doped graphene hydrogels for CAP and Cu₃Mo₂O₉/BaTiO₃ for OTC detection [77,112].

Z-scheme heterojunctions are more desirable because of their higher charge separation efficiency. The photoelectrons move from the CB of a photocatalyst to the VB of the second photocatalyst, describing a Z-pattern. This results in reduced carrier recombination and enhanced photocurrent. An example of a Z-scheme heterojunction is provided in Figure 10B, where decorating BiOBr nanoflowers with CdTe quantum dots gives a 5-fold and 8-fold increase in photocurrent compared to the individual materials. As a particularity, the aptamer was immobilized by electrostatic interactions. Aptamers are charged negatively because of the phosphate backbone, and they can interact electrostatically with cationic materials present on the surface of the electrode. Cationic chitosan was loaded on the surface of CdTe-BiOBr to immobilize the aptamer specific to TC. The developed PEC sensor manifested an LOD of 9.25 pM for detecting TC and good performance in soil samples [97].

Metal sulfides (CdS, CuS, MoS₂, Bi₂S₃) are the second most used materials in developing PEC aptasensors for pharmaceutical detection. They are characterized by lower band gaps than metal oxides but have the same intrinsic limitations of fast recombination of photocarriers and low conversion efficiencies [209,212]. TC and OTC detection was achieved by integrating Bi₂S₃ into a Z-scheme heterojunction with graphitic
<figure><img src="image_15.png" /><figcaption>**Figure 10.** Schematic representation of charge carriers separation in a (A) p-n heterojunction for AMP detection. Reprinted from Ref. [157]. Copyright (2024), with permission from Elsevier, and (B) Z-scheme heterojunction for TC detection. Reprinted from Ref. [97]. Copyright (2024), with permission from Elsevier.</figcaption></figure>

carbon nitride (g-C₃N₄) [110,213]. Bi₂S₃ has a narrow band gap of 1.3 eV, enabling visible light harvesting and electrical energy production. Graphitic carbon nitride (g-C₃N₄) as a metal-free semiconductor, possesses abundant π conjugation, suitable electronic structure, high chemical and thermal stability, non-toxicity, and enters in the formation of many composites. Bi₂S₃/g-C₃N₄ material presented a two-fold increase in photocurrent intensity compared to pure Bi₂S₃ owing to the heterojunction formation. The aptamer was immobilized at the surface of the material by non-specific absorption and specifically recognized the corresponding tetracycline. The oxidation of the complexed TCs led to an increase in photocurrent, with LODs of 3 pM for TC [213] and 0.6 pM for OTC [110].

MoS₂ is a transition metal chalcogenide with a 2D layered structure and excellent optoelectronic properties. It was used in contact with 3D materials such as nitrogen-doped graphene hydrogels [144] and ZnO [98] for the detection of CAP and TC. In another approach, MoS₂ was combined with CuS and Bi₂WO₆, highly reducing the electron-hole recombination rates and leading to DCF detection with an LOD of 0.03 nM [81].

Doping and the induction of defects are other strategies employed for the increase of charge separation in metal sulfide semiconductors. Se doping of In₂S₃ resulted in a 4-fold increase in photocurrent compared to the pristine material. The Se-defective composite also presented a narrower band gap of 2.19 eV vs 2.49 eV, which translates to better visible-light harvesting properties. The composite was deposited on a GCE with a coating of AuNPs that anchored the thiol-modified APT. CAP detection was achieved using this composite with a detection limit of 17 fm [141]. Combining metal sulfides with carbon and noble metals nanomaterials, as the example of CdS doped with graphene and gold nanoparticles for DCF detection can have several advantages [80]. Because it is a "zero band gap" semiconductor, graphene can transfer photogenerated electrons from a semiconductor, delaying charge recombination and improving photocurrent response. Metallic nanoparticles amplify the output signal because of the SPR effect and are often present in the composition of photoactive composites. The hot electrons generated by the nanoparticles can be transmitted via SPR in the CB of the semiconductor and then to the electrode and generate photocurrent. Moreover, the photoelectrons generated by the semiconductor will be less involved in recombination pathways; they will be scavenged by the nanoparticles and contribute to the increase in photocurrent [208,214]. Graphene-doped CdS combined with AuNPs led to an enhanced photocurrent, which contributed to the sensitivity of the method, resulting in an LOD of 0.78 nM for DCF [80].

Inorganic sensitizers can also be employed, for instance, AgBiS₂ was used to increase visible light absorption of a Zn/Co bimetallic oxide. Integrating nitrogen-doped graphene quantum dots (N-GQD) for their

high carrier transport mobility in the composite, the photoelectrode attained an LOD of 0.25 pM for AMP detection [155].

The heterogeneity of materials employed in PEC sensor construction opens the way for all the immobilization methods mentioned before. An interesting inclination to non-covalent immobilization is evident in PEC sensors. This can be attributed to the fact that PEC sensing mechanism is not mediated by the interaction of a redox probe with the surface and the change in aptamer conformation after analyte binding does not have a central role. Therefore, the aptamer can be randomly adsorbed at the surface as long as it binds to the target molecule. However, questionable reproducibility and stability remain with this immobilization strategy.

In conclusion, numerous materials are used in the fabrication of voltammetric, impedimetric, and PEC aptasensors. The common objective in using these materials is to provide a substrate for aptamer immobilization using both covalent and non-covalent strategies. In Table 3, a summary of the immobilization methods, including advantages and disadvantages, is provided. Additionally, enhanced conductivity, surface area, and stability are obtained. This multifaceted approach contributes to developing sensors with improved performance, specificity, and reproducibility.

## 6. Performance of the developed aptasensors

The performance of aptasensors for pharmaceutical compounds over the last 6 years is summarized in Tables S1 to S6, according to their class. Several parameters must be considered to assess the efficacy of the fabricated sensors and determine their suitability for application to environmental samples. The limit of detection (LOD) is the most relevant factor, and it represents the lowest quantity of analyte that can be determined with sufficient confidence. The modifying materials and the configuration of the aptamer strongly influence the LOD. Ideally, the LOD has to be as low as PNEC, the maximum concentration at which the analyte manifests no toxic effects. The linearity range (LR) is the interval where the registered signal has a linear tendency with the concentration or the logarithm of concentration. A wider range means better qualitative information regarding the concentration of the targets.

Most studies have tested the selectivity against other pharmaceutical pollutants or molecules that are likely to be present in real samples. This parameter is a good indicator of the selectivity of the immobilized aptamer. Two approaches have been used, the most commonly used one evaluates the signal of the aptasensor in solutions of the interferent in concentrations with some order of magnitude higher than the analyte. The second implies registering the signal in a mixture of analyte-interferent in a certain proportion. In Tables S1 to S6, the interferents studied with the second method are emphasized in bold. Considering that a molecule interferes if it gives a signal of ≥ ±20% from the analyte
<table><caption>**Table 3**
Immobilization methods used by studies developing electrochemical aptasensors for pharmaceutical pollutants detection.</caption><thead><tr><th></th><th>Immobilization strategy</th><th>Aptamer functionalization</th><th>Surface type/ functionalization</th><th>Advantages</th><th>Disadvantages</th></tr></thead><tbody><tr><td>Covalent</td><td>Thiol-based SAM</td><td>-SH</td><td>Au, AuNPs, AgNPs</td><td>Simple, compact layer, reagent-less</td><td>Monolayer defaults, time-consuming, nonspecific adsorption</td></tr><tr><td rowspan="4">Non-covalent</td><td>Carbodiimide coupling</td><td>-NH<sub>2</sub>, COOH</td><td>-COOH, OH, NH<sub>2</sub></td><td>Well-ordered distribution, stable, decreased adsorption</td><td>Reagent use, time-consuming, nonspecific</td></tr><tr><td>Hybridization</td><td>-</td><td>Requires the initial immobilization of a cDNA molecule</td><td>Fast, possible regeneration</td><td>Hydrogen bonds can break easily, low stability, false positives, expensive</td></tr><tr><td>Electrostatic interactions</td><td>-</td><td>Positively charged surface (i.e. chitosan)</td><td>Simple, fast, cost-efficient</td><td>The aptamer can easily detach from the surface, low reproducibility</td></tr><tr><td>π-staking, hydrogen bonds, hydrophobic interactions</td><td>-</td><td>MOF, COF, graphene, GQD, semiconductors</td><td>Simple, fast, cost-efficient</td><td>Low stability, no regeneration, low reproducibility, nonspecific adsorption</td></tr></tbody></table>

signal, interfering species are written in red. In parentheses, it is indicated how many times the concentration of the tested interferant is higher than the concentration of the analyte.

The real samples analyzed varied from environmental samples to food samples and biological fluids. Other matrices than environmental samples were included in this review because a sensor developed for an analyte can be applied to media other than those initially studied. The primary criterion for such a cross-application is a LOD aligning with expected levels in the new samples. For instance, a sensor designed for detecting antibiotics in serum samples might find utility in wastewater if its LOD is sufficiently low, and vice versa. However, the new application requires adaptation to the matrix and plotting of a calibration curve in the new sample.

The most frequently analyzed real samples were water matrices such as wastewater, river water, and tap water. The predominant pretreatment method for wastewater involved filtration as the primary step to eliminate suspended solids and microbial contaminants [73]. In other cases, dilution with PBS at ratios of 1:10 or 1:100 was carried out to adjust the ionic strength and maintain assay performance by reducing matrix complexity [158]. Often, samples were spiked with known concentrations of the target pharmaceutical after filtration or dilution. For lake, river, and tap water, the pretreatment was even more straightforward, typically requiring only simple filtration [109,113,126]. In some instances, the water sample was directly aliquoted and analyzed, without additional treatment, when turbidity was low [91,129,170]. Despite the minimal sample preparation, recovery rates in water samples were excellent, highlighting the robustness of the sensor design and its operational efficiency in direct matrices.

For other types of samples, such as milk, more intricate pretreatment procedures were necessary due to the high content of proteins and fats. Common approaches involved protein precipitation using organic solvents and strong acids [92,99,100,108,119], followed by centrifugation to remove the precipitated proteins and lipids. The protocols included filtration after centrifugation, typically through membranes, to eliminate any remaining particulates. In certain cases, a dilution step with PBS or ultrapure water was incorporated either before or after centrifugation to decrease viscosity and mitigate matrix effects [86,99].

In alternative strategies, sensor construction involved modifying the electrode surface with specially designed proteins to prevent fouling caused by milk components [88]. This innovative approach enabled the direct analysis of tetracycline in milk samples by EIS, achieving good recovery rates and offering an effective solution for the direct assessment of highly complex matrices.

Ensuring reproducibility is crucial, given the numerous fabrication steps, variations between sensors can be substantial. Typically, the reproducibility is quantified through the relative standard deviation (RSD), calculated from values obtained after detecting a specific concentration with the analyte using five or more individual sensors.

Repeatability illustrates the number of consecutive measurements a single sensor can endure while maintaining accuracy. As the sensing mechanism relies on a robust affinity between the aptamer and the target, a natural requirement is a regeneration step between each detection to assess repeatability accurately. Lastly, long-term stability is estimated following the preservation of electrodes over a specific duration. For certain PEC sensors, the stability was evaluated by subjecting the electrode to numerous on-off light cycles.

The sum of these factors: LOD, LR, selectivity, reproducibility, and repeatability gives the overall performance of the sensor. For each pharmaceutical class discussed in this chapter, a table will outline the aptasensor construction details, including electrode modification materials and aptamer immobilization strategy. The sensing method, analytical parameters (LOD and LR), and substances tested for interference will be listed, with the disruptive ones highlighted in red. Spiked samples to assess sensor feasibility in practical scenarios will be noted. Additionally, reproducibility will be indicated by the RSD value and stability in conservation days or cycles for PEC sensors. Repeatability
values, when tested, will also be presented.

## 6.1. Non-steroidal anti-inflammatory drugs

Of the numerous anti-inflammatories in current use (DCF, IBU, naproxen, ketoprofen), only DCF and IBU have electrochemical aptasensors developed for their detection (Chapter 3, Table 2). The PNEC for DCF and IBU is 1 ng/L and 10 ng/L, corresponding to $3.3 \times 10^{-3}$ nM and $4.8 \times 10^{-2}$ nM, respectively. Their mean concentrations in European surface waters, according to [37], are two orders of magnitude higher, with 0.8 nM for DCF and 1.6 nM for IBU. Important information regarding the construction of aptasensors for NSAIDs and their analytical performance are presented in Table S1. There has been a greater variety of aptasensors for DCF in recent years, all using the same sequence of aptamer developed by Joeng et al. [74]. Among them, two sensors have a LOD as low as the PNEC. The first sensor had an excellent LOD of $3.4 \times 10^{-6}$ nM, explained by the synergic effect between three nanomaterials: Fe₃O₄, AuNPs, and MWCNTs. It is also the only sensor where the aptamer was immobilized by hybridization, indicating a possible better orientation and accessibility for this aptamer sequence. On the other hand, the reproducibility of the sensor is lower, with an RSD of 5.7%, because of the many steps of surface functionalization [79]. The second sensor has an LOD of $1.6 \times 10^{-4}$ nM and requires a simpler construction protocol, with the aminated aptamer covalently immobilized on MWCNTs functionalized with carboxyl functions. Presenting two LRs, the sensor had an ultrawide domain for DCF detection [75].

Other studies have obtained good LODs, with the possibility of detecting the mean concentration of DCF in surface waters. The selectivity of the sensors was tested against pharmaceutical pollutants (carbamazepine, paracetamol, sulfadimethoxine) that are often detected together in environmental water samples, and molecules present in biological fluids (glucose, uric acid, dopamine, tryptophan). PAR is an interferent for the aptasensor in one study [75]. The authors treated the response of the aptasensor to PAR as a consequence of nonspecific adsorption. Interference from PAR was not observed in the other studies that used the same aptamer [78,80,82], emphasizing that the sensor construction influences selectivity as well.

Only one study tested the repeatability of the sensor, with an RSD of 2.1 % for seven successive measurements for a concentration of 70 nM of DCF. While the repeatability is good, no additional information regarding the intermediate regeneration step is provided by the authors [76]. The stability of the aptasensors at 4°C was satisfactory, with the maximum period tested of 21 days.

An unprecedented LOD of $3.3 \times 10^{-8}$ nM was recently obtained for IBU detection. The authors attributed the efficiency of the aptasensor to the use of nitrogen-doped GQDs and AuNPs, which synergically increased the surface area and conductivity. Moreover, the use of riboflavin, a bulky negative redox probe, can be another reason for the excellent sensitivity due to the electrostatic repulsion between riboflavin and the negatively charged phosphate backbone of the aptamer, which can contribute to the significant DPV response at low concentrations of IBU [84].

## 6.2. Tetracyclines

The class of antibiotics is the most targeted in the literature for aptasensor development among pharmaceutical pollutants. The risk of AMR and associated toxic effects propels tetracyclines, fluoroquinolones, penicillins, and some isolated compounds like CAP and metronidazole (MET) in the top of substances with the most aptasensors developed for.

TC and OTC are antibiotics frequently used as human and veterinary drugs, contaminating food products and environmental waters. The aptasensors developed for TC and OTC are reunited in Table S2.

In surface waters, TC is detected in concentrations ranging between

$4.5 \times 10^{-2}$ nM in Europe and 22.51 nM in the Asian region. The PNEC for resistance selection is 2.25 nM, and the sensors should ideally have an LOD below this value [215].

Four sensors had the LOD in the range of $10^{-6} - 10^{-9}$ nM, [98,99,104, 175], being able to detect ultra-traces of TC and levels way below the PNEC and the usual detected concentrations. Interestingly, the most performant aptasensor [175], with an LOD of $2.28 \times 10^{-9}$ nM used for the detection of TC a KANA-specific aptamer. Based on in silico studies conducted through molecular docking and molecular dynamic simulations, the KANA aptamer has a better affinity for TC. This shows promising results for in silico aptamer development in favor of conventional SELEX methods. The selected aptamer was immobilized by carbodiimide coupling on a GCE modified with carbon nanofibers (CNFs) and MWCNTs, emphasizing the benefits of using carbon nanomaterials to increase the sensitivity of the sensor. Moreover, the study employed an optimization scheme based on central composite design (CCD), which improved the DPV response of the aptasensor. The repeatability proved satisfactory, enabling five consecutive measurements, although no regeneration treatment was demonstrated. Nevertheless, the aptasensor developed exhibited an affinity for KANA, producing a signal equivalent to 25% of that for TC.

Two impedimetric sensors showed excellent LODs [99,104]. The first one [99], with a $2.76 \times 10^{-6}$ nM LOD used a MOF on COF approach, obtaining a composite that improves electrochemical activity, has nitrogen-rich functions, and strong affinity for the aptamer due to the immobilization via π-stacking. The second aptasensor was developed by coating a pencil graphite electrode (PGE) with rGO and AuNPs [104]. The aptamer was immobilized on the AuNPs, forming a SAM. The use of two nanomaterials led to an LOD of $3 \times 10^{-8}$ nM, with good selectivity, and satisfactory reproducibility.

PEC detection proved highly effective for TC, achieving LODs below the PNEC. The lowest LOD of $3.3 \times 10^{-6}$ nM was attained by constructing an aptasensor based on the semiconductor-semiconductor heterojunction between ZnO and MoS₂, and enhanced charge transfer facilitated by AuNPs [98]. The aptamer was adsorbed onto MoS₂ nanosheets, capturing TC and leading to an increased photocurrent attributed to its oxidation. Notably, the sensor fabrication demonstrated remarkable efficiency, requiring only one minute for construction while maintaining good performance.

Besides achieving high sensitivity, a straightforward sensor design is highly valued. A good LOD was achieved through a simple sensor design with an aptasensor formed from the self-assembly of two aptamers specific for TC on a gold screen-printed electrode [94]. Using two aptamers of different sizes, 76 and 40 nucleotides, promotes an optimal orientation of the aptamers and enhances the availability of binding sites for TC. The aptamer-cocktail sensor demonstrated superior recognition affinity toward the target compared to sensors composed of one aptamer, resulting in a 15-fold lower LOD of $1.6 \times 10^{-2}$ nM.

Certain aptasensors underwent regeneration treatment. For instance, a sensor that combined the aptamer with a molecularly imprinted polymer (MIP) was regenerated in a high ionic strength solution [92]. Another strategy used 3 CV scans in 0.1 M KCl to detach TC from its aptamer [73]. Regeneration in 0.5 M H₂SO₄ for 5 minutes and then rinsing with PBS was applied to a platform where the aptamer was immobilized by adsorption, showing the possibility of recovering the initial signal 5 times with the same sensor [99]. A good regeneration is required to estimate the repeatability of a sensor, however, there were reported RSD values for repeatability where no regeneration treatment is indicated [104,175]. Confusion surrounds this parameter, as certain studies examined the stability of the sensor response in a redox probe solution rather than conducting repetitive measurements on the same electrode in an analyte solution [86].

The selectivity for TC was tested against other tetracyclines, antibiotics, and compounds that might be present in real samples. The results were generally good, with vancomycin (VAN) tested and interfering in one study due to its high molecular weight (1449 g/mol) and possible
adsorption to the surface of the electrode [73]. DCF and chlortetracycline (CTC) interfered with the detection of TC while using a platform constructed with a redox-active aptamer [91]. It is interesting to note that DCF and CTC did not interfere in other studies using the same aptamer [94,96,98,187], showing that the sensing mechanism and the functionalization of the aptamer can modulate not only the sensitivity, but also the selectivity of the sensor, as previously seen in the case of DCF sensors.

For OTC, the mean values detected in the environment are around 0.2 nM, and the PNEC is 0.6 nM. The majority of the aptasensors attained an LOD as low as PNEC. Excellent LODs have been obtained while using highly porous materials that enable the immobilization of aptamers with high density [108,116,119]. A ferriporphyrin-based porous organic framework (Fe-PPOF) was synthesized and drop-cast on a gold electrode (AuE) [108]. The composite had a high surface area, stability, and a π conjugation skeleton, making it a suitable substrate for aptamer adsorption. The LOD attained by the aptasensor based on Fe-PPOF was the lowest, $4.03 \times 10^{-6}$ nM. The other sensors that use MOF on COF [119], and COF [116] had good performance, with a LOD of $3.5 \times 10^{-5}$ nM and $1.24 \times 10^{-5}$ nM, respectively. All three sensors mentioned used EIS as a sensing method, emphasizing the good sensitivity obtained with this type of detection.

Numerous PEC sensors were developed for the detection of OTC. One PEC sensor used the sandwich method, using an aptamer immobilized on an electrode coated with rGO and an OTC antibody immobilized on $MnIn_2S_4-WO_3$ doped with Yb and Tm [126]. After the aptamer binds to OTC, the antibody will immobilize on the surface of the electrode, and the photoexcited electrons present in the $MnIn_2S_4-WO_3$ heterojunction will be scavenged by rGO, resulting in accelerated photo-generated charge separation and photocurrent increase. This amplification technique allowed the ultrasensitive detection of OTC with an LOD of $1.3 \times 10^{-5}$ nM. A similar LOD was observed in the case of MOF-derived $In_2O_3$ hollow nanotubes coupled with Ag-doped $ZnIn_2S_4$. Using MOFs as precursors for metal oxides allows better control over their structure and enhanced properties [216]. $In_2O_3$ obtained from MOF had a hollow nanotube aspect, with high porosity and a large surface where aptamers can be immobilized [114]. In heterojunction with $ZnIn_2S_4$ and under the SPR effect induced by doping with Ag, the developed sensor had an LOD of $3.3 \times 10^{-5}$ nM.

The selectivity of the sensors developed for OTC was tested in priority against other tetracyclines. TC was found to interfere in one study where the aptamer was immobilized at the surface of gold nanoparticles [111]. The interference was explained by the authors as a consequence of the structural resemblance and in accordance with the selection studies that showed a certain affinity of the aptamer towards TC. Interestingly, the immobilization of the same aptamer on COF by nonspecific interactions led to a more selective sensor that could discriminate OTC from TC, indicating again an influence of the immobilization method upon the selectivity of the aptamer [116]. Two other studies that used the same shortened aptamer observed interferences from TC, doxycycline (DOX), chlortetracycline (CTC) [120], and TC and CIP [121]. These results emphasize the importance of conserving the aptamer sequence identified in the selection studies. The immobilization of the aptamers and their significant structural changes can influence their affinity and selectivity, requiring the redetermination of the affinity constant and evaluation of the selectivity.

Only one sensor was successfully regenerated by treating the electrode with 2.0 M NaCl following OTC detection [111]. The aim was to disrupt the hydrogen bonds and electrostatic interactions between the aptamer and its target by the high salt concentration, releasing OTC from the electrode. The stability of the aptasensors developed for tetracyclines was tested at 4°C and -20°C for various conservation periods. The average duration of conservation was 14 days at 4°C and 10 days at -20°C, and the sensors kept more than 90% of the initial response for the analyte, showing good stability.

## 6.3. Fluoroquinolones

Electrochemical aptasensors were developed for ENR and CIP, the most common fluoroquinolones detected in the environment, and with specific aptamers developed. The aptasensors are listed in Table S3. ENR can reach wastewater levels of 33.14 nM and 2.7 nM in river water [217]. The LODs of the sensors developed for ENR are very low, with values of at least $8.3 \times 10^{-4}$ nM. There was no general approach used for constructing the sensors or for sensing. The common element is the use of the same aptamer. The ultra-low LODs can be attributed to the high affinity between the aptamer and ENR. Despite the excellent results obtained, the aptamer employed has one of the highest Kd (188 nM) compared to the aptamers used for other antibiotics [218].

CIP has reported values in wastewater of around 3.44 nM and has a PNEC of 0.3 nM. Of the two sensors recently reported, only one attained an LOD as low as PNEC [136]. In this approach, a double-labeled aptamer was immobilized by hybridization with two complementary strands immobilized at a gold surface by SAM formation. The obtained sensor is not selective towards CIP, but it can discriminate between fluoroquinolones and other classes of antibiotics, which makes it a class-selective sensor. The second sensor had an interesting approach, using dendrimers for their 3D structure, flexibility, and high homogeneity to integrate AuNPs [135]. The sensor had a good LOQ but was unsuitable for water samples and had low stability (1 day). Additionally, amino acids generated a signal corresponding to more than 20% of the signal of CIP, indicating a low selectivity of the sensor. However, using dendrimers is an immobilization method with the advantages of high-loading capacity, structural uniformity, high functional group density, and monodispersity that can serve as a linker for biofunctionalization [219].

## 6.4. Penicillins

Penicillins are the most used antibiotics for human use, found in environmental waters with a mean concentration of 0.5 nM and PNEC of approximately 0.1 nM. Table S4 summarizes the analytical characteristics of recent aptasensors developed for the detection of AMP and PEN G. For AMP detection, a remarkable performance was obtained in the case of impedimetric sensors using porous materials as substrates for aptamer immobilization [150,152,154]. The three sensors use a similar sensing mechanism when an increase in Rct is obtained with the increment of AMP concentration. The sensors manifested similar LODs of at least $3.8 \times 10^{-6}$ nM, levels well below the PNEC. Moreover, one aptasensor presented good repeatability by rinsing the electrode in 0.1 M NaOH for 7 minutes between each analysis [152]. The response of AMP was stable for eight regeneration cycles, owing to the good solubility of AMP in basic solutions.

The DNA structures obtained with a ladder-shaped [148] and pyramidal design (Tetrahedral DNA Nanostructures TDN) [194] on a gold substrate presented good performance. The ladder-shaped structure detaches from the surface in the presence of AMP, which allows the redox probe to reach the electrode and generate a high current difference. Using this detection mechanism an LOD of $1 \times 10^{-3}$ nM, a wide linear range, and specificity were obtained. The same LOD was reached while employing the TDN structure. In this case, the optimal orientation of the aptamer and controlled spacing between TDNs facilitated the binding with AMP, which determined a decrease in signal intensity. However, the two structures are difficult to obtain and might influence the reproducibility of the sensor. Unlike the ladder-shaped structure, the TDN-based sensor can be regenerated by rinsing the electrode with deionized water, resulting in a longer electrode lifetime.

The lowest LOD of $3.3 \times 10^{-8}$ nM was attained with a PEC sensor built on BiOI microspheres sensitized with bipyridine ruthenium (Ru (bpy)₃⁺), emphasizing the importance of surface sensitization of semiconductors for improved sensitivity [156].

Amoxicillin (AMX) detection was obtained by EIS using a
semiconductor-semiconductor heterojunction between g-C3N4 and TiO2 [158]. Otherwise a low conductive material, g-C3N4, when used with TiO2, efficiently increased the electron transfer and conductivity of the surface. The LOD obtained is 0.2 nM, which is insufficient to detect the PNEC for AMX, but it can be efficient in wastewater, where concentrations can go up to 2.7 × 10³ nM.

The sensors designed for PEN G detection exhibited robust performance, achieving favorable LODs. In one study, the SELEX process was employed to select an aptamer sequence, which was subsequently incorporated into a tetrahedral DNA nanostructure (TDN) and immobilized onto a gold electrode [162]. In contrast to the TDN developed for AMP detection, this structure did not undergo a regeneration process.

## 6.5. Chloramphenicol and metronidazole

Chloramphenicol (CAP) is an antibiotic widely used in aquaculture, from where it can pass into other water bodies, such as rivers and the sea. CAP concentrations vary considerably according to the water matrix, with levels around 3 × 10⁻³ nM in household wastewater, 0.15 nM in river water, and the highest levels were recorded in seawater, at 46.4 nM [220]. The aptasensors developed for CAP are reported in Table S5. One interesting approach is a sensor where the specificity of the aptamer is combined with that of MIP [139]. The aptamer-CAP complex was immobilized on the AgNPs present on the electrode. Afterward, resorcinol polymerized, entrapping the aptamer with CAP. The extraction step detached CAP from its aptamer, forming a surface with customized pores where the aptamer could bind CAP. Because of the regeneration capacity of the MIP, the sensor could be reused for five consecutive measurements without a significant change in signal, compensating for the laborious construction of the aptasensor. The aptasensor detected CAP by EIS and resulted in a LOD of 3 × 10⁻⁴ nM.

Other voltammetric sensors had excellent LODs, with the advantage of a wider linear range. Because of the high reported concentrations of CAP, a wide linear range allows better estimation of the environmental levels.

PEC detection was well-studied for CAP, with satisfactory LODs. In one technique, the aptamer was immobilized on Fe₃O₄ nanoparticles and hybridized with a ssDNA (S1) [141]. Under the influence of CAP, the aptamer and S1 separated, allowing S1 to hybridize and generate other ssDNA labeled with SiO₂ NPs (DNA-SiO₂), as illustrated in Figure 11A. The DNA-SiO₂ was further hybridized with a hairpin structure DNA

<figure><img src="image_16.png" /><figcaption>**Figure 11.** Illustration of the PEC sensor (A) Target-triggered recycling signal amplification reaction, (B) Construction of the biosensor, (C) Photocurrent response suppression after SiO₂ NPs inhibition. This image was adapted with permission from [141].</figcaption></figure>

immobilized on the In₂S₃ₓSeₓ/Au layer (Figure 11B). Due to the SiO₂ NPs, the PEC signal decreased, obtaining a “signal-off” aptasensor (Figure 11C). The recycling signal amplification reactions and using Se doping of the semiconductor resulted in an LOD of 1.7 × 10⁻⁵ nM. This approach is a good example of integrating the aptamer in the sensing protocol without immobilizing it at the surface of the electrode.

MET is used frequently in veterinary and human medicine, with potential risks of teratogenicity, carcinogenicity, and neurotoxic effects. The sensitivity of the method for detecting nitroimidazole is set at 3 µg/L (17.5 nM). MET detection was achieved by chronoamperometry with a DNAzyme-assisted method. In the absence of MET, a DNA structure with enzymatic properties can catalyze the reduction of H₂O₂ and generate a significant quantity of current. When MET is present in the solution, the catalyzing structure cannot be formed, and a lower current intensity is registered. The chronoamperometric method led to a good LOD of 3 × 10⁻³ nM, high selectivity, and satisfactory reproducibility [165].

## 6.6. Steroidal estrogens

Using concentrated animal feeding operations (CAFOs) in livestock leads to high levels of estrogens in the environment. Among the estrogens, 17β estradiol (E2) has a corresponding aptamer, allowing the development of electrochemical sensors. The levels of E2 vary, with a mean concentration of 0.075 nM, a maximum concentration of 0.7 nM, and a PNEC of 0.01 nM, a value recommended to be attained by the LOD of the sensors. All sensors specific for E2 have LODs inferior to the PNEC, as illustrated in Table S6.

An important aspect considered by studies was the development of cheap, portable, and robust platforms. For instance, a folding paper-based microfluidic cell was obtained by wax printing the microfluidic channels and screen-printing the electrodes [221]. Afterward, the working electrode was modified with nano assemblies of SWCNTs, new methylene blue, and AuNPs, which was the attachment point for the thiolated aptamer. Incubating the electrode with E2 decreased the current intensity of new methylene blue, obtaining a LOD of 1.8 × 10⁻² nM. In another approach, a laser-scribed graphene electrode (LSGE) was obtained and compared with a standard GCE platform [193]. GCE was a better support for the aptamer, with a LOD of 7 × 10⁻⁷, compared to 6.3 × 10⁻⁵ nM obtained with LSGE. Furthermore, the LSGE manifested low selectivity towards E2 due to its highly porous structure of graphene, and no anti-fouling agents were immobilized on the surface to block the active sites.

The affinity between aptamers and graphene was exploited in a protocol where graphene formed a complex with an E2-specific aptamer, preventing its adsorption on an MCH-modified Au electrode [192]. “Naked” graphene represented a switch for the electron transfer when assembled on the MCH-modified electrode. In the presence of E2, the graphene detaches from the aptamer, deposits on the surface of the electrode and enhances the electron transfer and current intensity. The sensor could be regenerated by immersing the electrode into N, N-Dimethyl formamide (DMF). However, this regeneration involves the MCH-modified Au electrode and not the aptamer.

The split-aptamers design was employed in the easy fabrication of a gold-based sensor [172]. The two parts of the aptamer formed dimers in the presence of E2, determining a surface re-arrangement that led to a decrease in electron permeability and a decrease in the current intensity of the redox probe. E2 was detected by DPV, obtaining a LOD of 5 × 10⁻⁴ nM.

The aptasensors designed for pharmaceutical pollutants exhibit remarkable capabilities in reaching concentrations equivalent to those found in environmental matrices and meeting the PNEC values while maintaining high selectivity. These results can be justified by the use of aptamers with high affinity for their targets, which are integrated into the electrochemical platform without affecting their function. The robustness of aptamers allows their versatile immobilization into various substrates, obtaining a high diversity in sensor design, with little
influence on the LOD and the selectivity. Certain aptasensors have good repeatability and are capable of consecutive measurements after the regeneration of the aptamer. The diverse regeneration methods employed by studies that successfully reused the sensor are summarized in Figure 12.

## 7. Conclusions and future trends

In response to the escalating concern over environmental pollution caused by pharmaceuticals, there has been a notable surge in the development of electrochemical and PEC aptasensors dedicated to monitoring these contaminants in recent years. The quantification of pharmaceutical substances from the environment is challenging in terms of achieving a very low sensitivity. The developed aptasensors proved their possible applicability in environmental analysis, attaining ultralow LODs and wide LR. The construction of aptasensors employed a diverse array of materials. What stood out is the frequent combination of these materials, resulting in ingenious hybrid structures that contributed to the overall enhancement of sensor performance. Electrode modification primarily relied on carbon nanomaterials, metallic nanoparticles, porous materials (MOF, COF), and semiconductors. These materials served as substrates for the immobilization of the aptamer.

The diverse range of materials and the flexibility in aptamer functionalization allowed the implementation of various immobilization strategies. Thiol-functionalized aptamers were immobilized on gold electrodes or electrodes coated with gold nanoparticles. Aminated aptamers were linked to the surface through carbodiimide coupling or cross-linking with glutaraldehyde. Non-covalent immobilization involves either hybridization with complementary DNA (cDNA) or non-specific adsorption, facilitated by hydrophobic interactions, electrostatic forces, van der Waals forces, hydrogen bonds, and π-π stacking. Surprisingly, there were no significant differences in the analytical performance between the sensors obtained by covalent and non-covalent immobilization methods.

The aptamer had a crucial role in the detection process, contributing to the exceptional selectivity of the sensors. The sensors demonstrated the ability to discriminate between their target and closely related structures, as well as other pollutants or compounds present in real samples.

Using electrochemistry as a transducer facilitates straightforward and easily interpretable detection readouts. The detection process avoids the need for specialized sample treatment, reagents, solvents, and preconcentration steps. Although the sensor construction may involve some laborious steps, the payoff lies in the simplicity of the subsequent detection process. Additionally, the instrumentation can be readily miniaturized, enhancing portability and enabling on-site monitoring possibilities.

Nonetheless, certain aspects are to be improved in the electrochemical aptasensing of pharmaceutical pollutants. Despite the

theoretically claimed advantage of easy regeneration, which distinguishes aptamers from antibodies, only a few studies have conducted a regeneration treatment. Greater attention should be devoted to this step, considering its potential to reduce costs, extend the sensor lifespan, and enable consecutive measurements. This, in turn, would facilitate online analysis of water samples and the understanding of pharmaceutical substances distribution. Additionally, the $K_D$ should be determined while the aptamer is immobilized on the substrate to ensure it maintains its affinity toward the target.

While the sensor's high specificity is advantageous for single-component detection, it poses a limitation in terms of detecting multiple components simultaneously. Multiplexing lowers the cost and labor of single-analyte tests. An interesting direction in constructing aptasensors involves functionalizing the working electrode with multiple aptamers, each providing distinct signal readouts for a variety of pharmaceuticals.

Furthermore, a certain superficiality can be observed when evaluating the aptamer immobilization. Investigations of the surface density, orientation, and functionality of the aptamer are often omitted. For covalent immobilization, the 5' or 3' terminus functionalization can influence the proper folding and accessibility of the analyte to the binding sites. However, no comparison between the 3' and 5' functionalization has been conducted. In contrast, non-covalent immobilization demonstrates enhanced sensitivity and reproducibility, despite the randomized surface distribution and the potential variability in aptamer orientation. These nuances in aptamer immobilization strategies underscore the need for more comprehensive investigations to optimize sensor performance and reliability.

Despite the significant progress in the development of electrochemical aptasensors for pharmaceutical detection, none have reached the market. This gap between academic research and real-world application is likely due to several challenges. Firstly, aptamers, although highly specific and synthetically accessible, often suffer from stability issues, particularly under important temperature, ionic strength, and pH variations. Additionally, most reported systems are designed for the detection of a single analyte, lacking the multiplexing capabilities needed for environmental monitoring. Furthermore, the absence of standardized manufacturing protocols and regulatory pathways for aptamer-based devices poses a significant barrier to commercialization. Lastly, the regeneration of the aptasensors is often overlooked, another factor that can make them unsuitable for online monitoring of pharmaceutical contaminants. These factors collectively contribute to the current absence of aptasensors for pharmaceuticals on the market, despite promising advances at the proof-of-concept level.

The advancements presented in this work underscore the remarkable potential of aptasensors as robust tools for monitoring pharmaceutical pollutants in diverse environmental matrices. While the field continues to progress, bridging the gap between laboratory success and real-world applicability, the demonstrated capabilities in controlled settings lay a

<figure><img src="image_17.png" /><figcaption>Figure 12. The percentage of the aptasensors that underwent a regeneration step, and the methods employed to achieve consecutive measurements.</figcaption></figure>
strong foundation for future developments. Looking ahead, integrating
aptasensors into practical environmental monitoring scenarios holds
great promise, offering a sensitive, selective, and versatile solution for
addressing the challenges posed by pharmaceutical pollution.

Funding

This work received financial support under the EUR LUMOMAT
project and the Investments for the Future program ANR-18-EURE-0012
Nanosens.

CRediT authorship contribution statement

**Teodora Lupoi:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Yann R. Leroux:** Writing – review & editing, Validation, Supervision, Methodology, Conceptualization. **Bogdan Feier:** Writing – original draft, Methodology, Formal analysis, Conceptualization. **Cecilia Cristea:** Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization. **Florence Geneste:** Writing – review & editing, Supervision, Resources, Funding acquisition, Formal analysis, Conceptualization.

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.

Acknowledgments

Teodora Lupoi thanks the program “Bourses du Gouvernement français France Excellence” for her scholarship.

Supplementary materials

Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.electacta.2025.146396.

Data availability

No data was used for the research described in the article.

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