Electrochemical aptasensor for the selective detection of vancomycin based on nanostructured "in‐lab" printed electrodes

Vol.:(0123456789) Microchimica Acta (2025) 192:107 https://doi.org/10.1007/s00604-025-06952-1 RESEARCH Electrochemical aptasensor fortheselective detection ofvancomycin based onnanostructured “in‑lab” printed electrodes MalekBibani1,2 · MagdolnaCasian1 · BogdanFeier1 · DianaBogdan3 · OanaHosu‑Stancioiu1 · NadiaKtari2 · RafikKalfat2 · CeciliaCristea1 Received: 20 October 2024 / Accepted: 2 January 2025 / Published online: 25 January 2025 © The Author(s) 2025 Abstract Alabel-free, flexible, and disposable aptasensor was designed for the rapid on-site detection of vancomycin (VAN) levels. The electrochemical sensor was based on lab-printed carbon electrodes (C-PE) enriched with cauliflower-shaped gold nanostructures (AuNSs), on which VAN-specific aptamers were immobilized as biorecognition elements and short-chain thiols as blocking agents. The AuNSs, characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM), enhanced the electrochemical properties of the platform and the aptamer immobilization active sites. The developed disposable aptasensor allowed label-free detection of VAN via electrochemical impedance spectroscopy (EIS) across a wide range of concentrations (50–1000 nM), with a limit of detection (LOD) of 1.721 nM. The aptasensor presented good selectivity against some commonly found interferences in human serum and milk and was successfully applied tothe analysis of these samples. Keywords Vancomycin detection· Electrochemical aptasensor· EIS· Gold nanostructure· Cauliflower-shaped gold nanostructures· Antibiotic resistance Introduction Glycopeptide antibiotics have been a key weapon in the fight against bacterial infections for over half a century and are the most frequently used antibiotics for the treatment of severe infections, such as endocarditis, pneumonia, and meningitis [1, 2]. Vancomycin (VAN) is an important antibiotic, used as a first-line treatment for infections caused by antibiotic-resistant Gram-positive pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus sp. [3]. As one of the “emergency” antibiotics, VAN is used in both human and veterinary medicine to treat severe infections, administered intravenously through infusion and orally for local effect. It is characterized by a narrow therapeutic index and is associated with a relatively high occurrence of nephrotoxicity and ototoxicity. If used at sub-therapeutic concentrations, VAN helps the spread of antimicrobial resistance (AMR). Therefore, therapeutic drug monitoring (TDM) for VAN is recommended [4]. Recently, a correlation was observed between VAN sweat concentration and serum concentration [5], thus, a noninvasive TDM could be achieved using wearable sensors. Malek Bibani and Magdolna Casian contributed equally to this work.. Highlights • Fabrication of flexible in-lab printed carbon electrodes for the development of disposable sensors • Vancomycin-specific aptamer characterization study using isothermal calorimetry and surface plasmon resonance techniques • Construction of cauliflower-like gold nanostructures and vancomycin-specific aptamer-based sensing platform • Label-free electrochemical detection of vancomycin in the low nanomolar range • Successful development of a disposable on-site rapid testing device for antibiotic concentration monitoring from real samples. * Bogdan Feier feier.geor[email protected]; [email protected] 1 Department ofAnalytical Chemistry, Faculty ofPharmacy, “Iuliu Hațieganu” University ofMedicine andPharmacy, 4 Pasteur Street, 400349Cluj-Napoca, Romania 2 Laboratoire Matériaux, Traitement Et Analyse, INRAP, BiotechPole Sidi-Thabet, 2020Ariana, Tunisia 3 National Institute forResearch andDevelopment ofIsotopic andMolecular Technologies, 67-103 Donat St., 400293Cluj-Napoca, Romania Microchim Acta (2025) 192:107107 Page 2 of 15 Although the use of VAN in animals is limited in order to impair the spread of AMR [6], its excellent antibacterial activity encourages the use of VAN as a feed additive or veterinary drug to treat bacterial infections, especially cow mastitis. VAN abuse may lead to potential residues in milk or in the environment, thus favoring the AMR [7]. To combat this concern and ensure that milk entering the food supply adheres to regulatory standards, the European Union has set maximum residue limits (MRLs) for antibiotics that are still allowed to be administered to animals, along with the additional requirement of implementing a withdrawal period of antibiotics in animal food products to avoid food safety problems [8]. Traditional antibiotic detection techniques include methods based on chromatography coupled with mass spectrometry [9, 10], microbiological assays [11, 12], and enzymelinked immunosorbent assays [13, 14], which are expensive, time-consuming, and involve laborious pretreatment protocols, long assay time, and lack of portability. Although ELISA kits have been made available for easier public use, there are still concerns that need to be addressed, including enzyme inactivation, cross-reactivity, and long detection times, which are unsuitable for antibiotic monitoring in real-world situations [15]. In the area of animal health management, biosensors have come into the spotlight as versatile and innovative devices, gaining recognition in the global market. Currently, precision livestock farming techniques are applied in sweat and salivary sensing, serodiagnosis, and animal product safety monitoring [16]. Electrochemical sensors offer distinct advantages over conventional methods for the detection of antibiotics, making them a superior choice for certain applications [17]. One key advantage is their remarkable sensitivity, which enables the detection of analytes at low concentrations. The realtime nature of the electrochemical measurements allows rapid and dynamic monitoring, facilitating timely responses insituations where rapid analysis is crucial. In addition, electrochemical sensors often exhibit excellent selectivity, minimizing the interferences from complex samples. Their relative simplicity, portability, wearability, flexibility, and cost-effectiveness make them particularly attractive for onsite and point-of-care applications, allowing the analysis of different types of samples (human serum, sweat, food, and environmental samples). Furthermore, electrochemical sensors can be easily miniaturized (allowing their integration into various automated devices for continuous monitoring) and modified with nanomaterials and (bio)recognition elements (leading to great sensitivity and selectivity). Various gold nanostructures (AuNSs) can be (electro)chemically synthesized, enhancing the catalytic and electrical properties, the active surface for aptamer immobilization through Au–S bonds and the interaction of the aptamer with the target. Overall, all these attributes make electrochemical sensors advantageous tools for efficient and reliable antibiotic detection in diverse settings [18, 19]. Aptamers, often referred to as “chemical antibodies,” are short, single-stranded DNA or RNA sequences that exhibit high affinity and specificity for a specific target molecule. In electrochemical sensors, aptamers play a crucial role because of their unique advantages. One key advantage is their ability to recognize specific targets, including small molecules, proteins, and whole cells, making them versatile for various sensing applications [20, 21]. Aptamers offer a cost-effective and more stable alternative to traditional antibodies, with the added benefit of easier synthesis and functionalization, with few batch-to-batch variations. Their stable and well-defined three-dimensional structures and target-induced conformational changes contribute to their enhanced sensitivity and selectivity in electrochemical sensing, making them ideal candidates for use as biorecognition elements [22]. Moreover, aptamers can be easily immobilized on electrode surfaces, thereby providing a stable and reproducible sensing platform. This immobilization process facilitates the development of robust and reusable electrochemical sensors, contributing to the cost-effectiveness and sustainability of sensing technologies [23]. Overall, the unique properties of aptamers make them valuable components for the design and development of highly efficient electrochemical sensors for a wide array of applications in fields such as medical diagnosis, environmental monitoring, and food safety. This study developed an innovative, sensitive, and disposable aptasensor for on-site VAN detection. The electrochemical sensor was based on an in-lab printed carbon electrode (C-PE) functionalized with gold nanostructures (AuNSs), on which a VAN-specific aptamer was grafted as a biorecognition element. Building on the stability and reproducibility of aptamers, the in-lab printed carbon electrodes (C-PEs) represent an important aspect of this study, significantly enhancing the versatility and cost-effectiveness of the sensing platform. C-PEs reduce dependence on expensive commercial options and provide greater accessibility and customization, allowing researchers to design and tailor them to specific experimental needs with precise control over dimensions, materials, and configurations. The flexible support for C-PEs further facilitates their use in wearable sensors, offering a convenient and affordable starting point. Thus, in-lab C-PEs align with the growing emphasis on sustainable and resource-efficient methodologies. The AuNSs electrosynthesis was optimized and the AuNSs were characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The aptamerVAN affinity was determined using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). Each step in the aptasensor fabrication and optimization process Microchim Acta (2025) 192:107 Page 3 of 15 107 was characterized electrochemically by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). Based on the fact that the binding of VAN to the aptamer induces changes at the electrode surface that alter the electron transfer of the ferro/ferricyanide redox probe, which is then measured using EIS, the quantification of VAN was assessed using the EIS spectra in the presence of 5 mM ferro/ferricyanide as redox probe, obtaining an aptasensor with LOD in the nanomolar range. The developed aptasensor presented good selectivity against some commonly found interferences in serum and milk and was successfully applied for the analysis of these samples. To the best of our knowledge, this is the first electrochemical sensor that combines the advantages of in-lab C-PEs, AuNSs, and aptamers for VAN detection from milk and human serum. VAN is currently the antibiotic of choice in severe cases of cow mastitis and the occurrence of antibiotic residues in animal products can produce significant health hazards to consumers, together with portable potentiostats, this platform could be a great starting point for the fabrication of easy-to-use, disposable, on-site rapid testing devices for the assessment of possible contaminations that could occur in milk and derivates. For therapeutic drug monitoring, given the therapeutic range of this antibiotic (10–20 mg/L or ~ 7–14 µM), our sensor matches the criteria required for safety and efficacy assessment. Moreover, given the recent advances in personalized medicine using wearable noninvasive sensors, along with the correlation found between VAN concentrations in plasma and sweat [16], the flexible design of our sensor could be a starting point for the development of a noninvasive wearable next-generation point-of-care monitoring system that could provide sweat-based real-time VAN treatment monitoring. Materials andmethods Materials All chemicals were of analytical grade and used without further purification. Sulfuric acid (H2SO4), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium chloride (NaCl), magnesium chloride hexahydrate (MgCl2·6H2O), potassium chloride (KCl), sodium carbonate (Na2CO3), sodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O), tetrachloroauric (III) acid (HAuCl4), tris(hydroxymethyl) aminomethane (TRIS), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), potassium hexacyanoferrate (II) trihydrate K4[Fe(CN)6]·3H2O), potassium hexacyanoferrate (III) (K3[Fe(CN)6]), 6-mercapto-1-hexanol (MCH), histidine, lactose (Lac), glucose (GLU), and human serum were purchased from Sigma-Aldrich (Germany). Vancomycin hydrochloride (VAN) was obtained from Linaris Biologische Produkte GmB (Frankenweg, Germany), and gentamicin sulfate (Gen) from Bioworld (Louis Park, MN, USA). The three different types of milk used in this study (low-fat, 1.5%, and 3% fat) were purchased from a local supermarket. The VAN-specific aptamer (Apt) used in this study was previously selected [24] and synthesized by Eurogentec (Belgium) with a customized thiol group at the 5′ end and a ferrocene group at the 3′ end, having the following sequence: 5′-SH-(CH2)6-CGA GGG TAC CGC AAT AGT ACT TAT TGT TCG CCT ATT GTG GGT CGG-Ferrocene-3′. The supporting electrolyte solutions used in this study included a 0.1 M KCl solution and a TRIS buffer (pH 7.4) containing 10 mM TRIS, 100 mM NaCl, 100 mM KCl, and 10 mM MgCl2·6H2O. All solutions were prepared using UltraPure™ DNase/RNase-free distilled water (Thermo Fisher, USA). The lyophilized Apt was resuspended in 10 mM TRIS buffer pH 7.4 to a stock solution of 100 µM, aliquoted, and stored at –20°C. The HAuCl4 and histidine solution mixture was prepared in 0.5M H2SO4. For MCH, a 10mM initial stock was prepared in TRIS buffer containing 20% ethanol, after which further dilutions were performed using TRIS buffer alone. For VAN analysis, fresh concentrations of VAN were prepared in TRIS buffer. For the analysis of real samples, human serum (Sigma-Aldrich) and milk (from a local supermarket) were used without further filtration. Methods andinstruments All electrochemical experiments were performed using an Autolab PGSTAT302N potentiostat (Metrohm Autolab, The Netherlands) equipped with Nova 1.11 software. The heating of the Apt was performed using an Eppendorf Thermomixer® C. The pH measurements were carried out using a micropH meter (Hanna Instruments). Isothermal titration calorimetry (ITC) was performed using an Affinity ITC microcalorimeter (TA Instruments, New Castle, USA) controlled by ITCRun v.3.8.4.24000 and Nano&Affinity ITC Data Collection software. For degassing the solutions, the compatible Degassing Station (TA Instruments, New Castle, USA) was used. The thermodynamic parameters were obtained by fitting the titration curves against the built-in independent sites using NanoAnalyze v.3.12.5 software. The surface plasmon resonance (SPR) experiments were performed on a three-channel Biosensing BI-2500 instrument using the compatible bare gold chips (Biosensing Instrument Inc., Tempe, AZ, USA). To extract the SPR kinetic parameters, the kinetic data were analyzed Microchim Acta (2025) 192:107107 Page 4 of 15 in the framework of the Langmuir isotherm 1:1 binding model using BI-software version 2.4.4 (Biosensing Instrument Inc., Tempe, AZ, USA), which includes the software Scrubber (BioLogic Software Pty Ltd., Campbell, Australia). In this study, in-lab printed electrodes with a carbonbased working surface (C-PE) were used. For the electrode printing process, the carbon conductive ink Electrodag 423SS was purchased from Henkel (Dusseldorf, Germany), and Ag/AgCl ink Electrodag PF-410 was purchased from Acheson (Delaware, USA). An autoclave (Memmert GmbH, Schwabach, Germany) was used for the successive and controlled drying of the conductive ink during the printing process. Morphological and topographical surface characterization of the developed sensor was performed using atomic force microscopy (AFM). The measurements were conducted on a Cypher S microscope (Asylum ResearchOxford Instruments, Santa Barbara, CA) in tapping mode, in air, under ambient conditions, with silicon probes (AC240TS-R3, Olympus, Japan), reflex side aluminum coated, tip radius typical 7 nm, with a spring constant of 2 N/m (0.6–3.5 N/m) and a resonance frequency of 70(± 20) kHz. Data acquisition and image analysis were performed using the integrated Asylum Research software (AR 16.33.234, Asylum Research) written within the Igor Pro software package (Igor Pro 6.38B01, WaveMetrics, Inc., Lake Oswego, OR, USA). Several areas of the sample surface were analyzed, with 512 pixels/ line and with a scan rate of less than 1 Hz. Scanning electron microscopy (SEM) images were obtained using a Hitachi SU8230 SEM (Tokyo, Japan) at 30 kV, 10 µA, and a working distance of 15 mm, using Aztec software from Oxford Analytics. Electrochemical techniques The electrochemical techniques used in this study were cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). CV (the potential was cycled twice between – 0.2 V and + 1.2 V, with a scan rate of 100 mV/s), DPV (the potential was scanned from – 0.2 and + 0.5 V, with a modulation amplitude of 50 mV, modulation time of 40 ms and a scan rate of 10 mV/s), and EIS (the frequency range was between 0.01 and 100,000 Hz, with an amplitude of 0.01 V and DC potential determined by Open Circuit Potentials) measurements were performed in the presence of 5 mM [Fe(CN)6]3–/4– redox probe prepared in 0.1 M KCl solution. The measurements using ferrocene-labeled aptamer were performed in a 0.1 M KCl solution. Aptamer affinity evaluation Isothermal titration calorimetry (ITC) The ITC analyses were conducted according to the following protocol. Prior to each measurement, the Apt solution was subjected to thermal treatment and each solution was degassed for 10 min under vacuum (25 inHg) at 25 °C using the Degassing Station. For the Apt solution, 300 µL of 10 µM Apt in TRIS buffer was prepared, loaded into the ITC sample cell, and titrated with 150 µM VAN solution prepared in the same buffer. The reference cell of the calorimeter was filled with 300 µL nuclease-free water. For titration, 23 titration steps were performed for each injection using 5 µL of VAN solution, except for the first injection, for which 2 µL was used. The other titration parameters were as follows: initial baseline (300 s), injection interval (150 s), stirring rate (100 rpm), and temperature setpoint (25 °C). Surface plasmon resonance (SPR) For SPR analysis, the bare Au chip was initially washed with ethanol, dried under a nitrogen stream, and hydrated with 300 µL TRIS buffer for 1 h at room temperature. The Au chip was then covered with 300 µL of 1 µM Apt solution prepared in TRIS buffer (previously reduced and thermally activated) and left overnight at 4 °C in a water-saturated atmosphere. Subsequently, the surface was rinsed with 300 µL of TRIS buffer and incubated again for 30 min at room temperature with 300 µL of 100 µM MCH solution prepared in the same buffer. After a final wash with 300 µL of TRIS buffer, the modified Au chip was mounted on the SPR prism. The running buffer for the affinity evaluation was TRIS buffer, maintaining a constant flow rate of 60 µL/min. Prior to analysis, the buffer was filtered through a 0.2 µm pore diameter filter and degassed for 1 h under vacuum (25 inHg) at 25 °C using the Degassing Station. After achieving a stable baseline, increasing concentrations (0.05–10 µM) of VAN solutions prepared in TRIS buffer were injected. For each concentration, 300 µL of the solution was injected for 300 s (association phase), followed by 300 s of buffer running through the system before a new injection (dissociation phase and new baseline stabilization). Before the first injection of VAN, 300 µL of TRIS buffer was injected for blank correction. In‑lab electrode printing process The components of the electrochemical cell were printed on a thin flexible polymeric substrate by optimizing the printing procedure described in two previous studies [25, 26]. Briefly, the stencil was placed on top of the polymeric substrate, a layer of conductive ink was applied, and a rubber squeegee was dragged along the stencil to distribute the ink Microchim Acta (2025) 192:107 Page 5 of 15 107 uniformly in the aperture. Ag/AgCl ink was printed first to provide the reference electrode (RE), after which the working (WE) and counter (CE) electrodes were printed using carbon conductive ink. After each printing step, the polymeric substrate was autoclaved for 15 min at 50 °C. To minimize shortcuts on the connections, insulator tape was used to cover the interconnections, after which the resulting C-PEs were left to dry out completely at 50 °C overnight. In addition, serpentine connections were printed using Ag/AgCl conductive ink. Finally, silver wires were attached to the serpentine connection to facilitate the connection between C-PE and the potentiostat. C‑PE conditioning andgold nanostructured platform fabrication The first step in the aptasensor development consisted of the activation of C-PEs with 1 M Na2CO3 solution using an amperometric procedure by applying a constant potential of + 1.2 V (vs. Ag/AgCl) for 600 s. After activation, the C-PEs were modified with gold nanostructures (AuNSs) in the optimal conditions: 10 mM HAuCl4 solution in 0.5 M H2SO4 containing 150 mM histidine by chronopotentiometry (CP)-assisted electrodeposition (− 100 µA current applied for 600s). After modification, the electrode was rinsed three times with 100 µL of nuclease-free water. Aptasensor development The obtained gold nanostructured platform (C-PE/AuNSs) was further used for the immobilization of the VAN-specific aptamer through a gold-thiol covalent bond. Two different immobilization methods were tested: (i) overnight incubation at 4 °C and (ii) multipulse pulse amperometry (MPA). For MPA, the applied potential was switched between + 0.5V and –0.2V (vs. Ag/AgCl) with a 10ms pulse duration for 300s. Prior to use, the possible disulfide bonds formed during Apt storage were reduced with TCEP, by incubating at room temperature for 1h in the dark, a 1:1 (v/v) mixture of 100µM Apt solution with 20mM TCEP, both prepared in TRIS buffer. The reduced aptamer was then diluted with TRIS buffer to a final concentration of 1µM and subjected to thermal treatment by heating to 95 °C for 5 min, followed by cooling to –20°C for 5min. After Apt immobilization, the electrode was rinsed three times with 100 µL of TRIS buffer. To prevent non-specific adsorption on the surface of the electrode, the remaining gold active sites were blocked with 20 µL of 100µM MCH solution prepared in TRIS buffer by incubation for 30min at room temperature. After blocking, the electrode was rinsed three times with 100 µL of TRIS buffer. The electrode surface was consistently covered during aptasensor development with a drop of TRIS buffer to prevent dehydration and spatial degeneration of the Apt. The electrode surface was electrochemically characterized after each modification step by EIS and DPV, using a 5 mM [Fe(CN)6]3−/4− solution. Vancomycin quantification procedure For VAN quantification, the obtained sensing platform (C-PE/AuNSs/Apt/MCH) was incubated with 50 µL of various concentrations of VAN prepared in TRIS buffer for 45 min at room temperature in a water-saturated atmosphere. After incubation, the electrode was rinsed three times with 100 µL of TRIS buffer to eliminate the possibility of nonspecific adsorption. Two different VAN quantification procedures were tested, using the changes before and after VAN incubation in the electrochemical signal of the (i) Apt ferrocene label or (ii) a 5 mM [Fe(CN)6]3−/4− solution. For the first procedure, a 50 µL 0.1M KCl solution was applied on the electrode and the electrochemical signal of the Apt ferrocene label was recorded. The analytical signal was determined based on the anodic current intensity (I) values before and after VAN incubation. For the second quantification procedure, the analytical signal of the redox probe was determined based on the charge-transfer resistance (Rct) and anodic current intensity (I) values before and after target incubation. The EIS and DPV signals were calculated as a percentage of reduction in Rct, respectively increase in I after incubation with VAN, according to the following Eqs. (1) and (2): Interference studies For the treatment of serious MRSA infections, VAN is often combined with a second antibiotic, most often gentamicin. In the aptasensor development process, the potential interferents were established depending on the final applicability on real sample analysis (human serum and milk). Therefore, the selectivity of the developed aptasensor in complex matrices was evaluated against gentamicin, glucose, and lactose. The assessment involved examining the EIS signal responses for individual interferent solutions (1.0 µM) and 1:1 mixture with VAN (1.0 µM each), following the protocol described for the VAN quantification procedure (2.3.5). (1) S incubation = ( Rct MCH −Rct VAN ) × 100 R ct MCH (2) S incubation = | |IMCH −IVAN | |× 100 I MCH Microchim Acta (2025) 192:107107 Page 6 of 15 Real sample analysis To evaluate the performance and applicability of the developed aptasensor for the quantification of VAN in real samples, artificial human serum and milk with different fat contents (0.1%, 1.5%, and 3.5%) were used for analysis. All samples were diluted 10 times with TRIS buffer to ensure the electrolytic composition and ionic strength necessary for proper recognition and binding of the aptamer. The resulting VAN solutions were then incubated for 45 min at room temperature in the same manner as described for VAN quantification. The applicability of the developed sensing method was then evaluated by spiking the diluted samples with a known concentration of VAN (1 µM) and calculating the recovery rates as follows: recovery (%) = Cs/Ct × 100, where Cs is the mean VAN concentration of the spiked sample and Ct is the expected theoretical concentration of the sample after spiking. Results anddiscussion Aptamer affinity studies Since their discovery in the 90s, many aptamers have been reported in the literature for numerous applications. To be used as a biorecognition element in electrochemical sensing, an aptamer must undergo a binding-induced conformational change that produces a significant change in the electron transfer of the redox probe. The selected VAN-specific aptamer was designed to exhibit this property by destabilizing the parent DNA sequence with the removal of four base pairs from its stem (4trunc), leading to a binding-induced conformational change observed via circular dichroism [27]. The 4trunc aptamer with the most stable predicted secondary conformation, characterized by the lowest Gibbs free energy (ΔG = − 7.03 kcal/mol), is shown in Fig.1A. Secondary structure prediction was carried out at 25 °C, with a 0.15 M Na+ and 0.01 M Mg2+ concentration, using the Nupack Web server [28]. The sequence was linked to a thiol functional group via a C6 linker, to enable sufficient spacing for aptamer folding and target binding. When selecting an aptamer from literature, in addition to the characterization presented in the selection paper, it is essential to test whether the aptamer is reliable and works in the designed settings. Therefore, the affinity of the selected Apt sequence, modified with thiol group, was evaluated using two different and complementary affinity evaluation techniques, ITC, and SPR. ITC is a highly effective technique for studying binding interactions, along with the determination of affinity and thermodynamic constants, in a label-free manner. ITC experiments showed that the binding reaction between the aptamer used in this study and its target molecule was exothermic (Fig.1B). The affinity and thermodynamic parameters were obtained by further integrating the resulting heat profiles (Fig.1C): dissociation constant (KD = 13.35 ± 2.74µM), binding enthalpy (ΔH = –40.27 ± 0.73kJ/mol), and entropy (ΔS = –42.92 ± 1.06J/mol·K), stoichiometry of the reaction (n = 1.10 ± 0.05). Another technique intensively applied in the study of aptamer binding kinetics is SPR, a surface-sensitive optical technique that measures the refractive index changes due to analyte binding. The SPR experimental setup consisted of the immobilization of the Apt on the surface of a gold chip, blocking off the remaining gold sites with MCH, followed by a series of injections using increasing concentrations of VAN (0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10µM). The interaction of the immobilized Apt with different concentrations of VAN in solution was monitored, and subsequently processed and analyzed for obtaining both kinetics and equilibrium parameters. The normalized, blank-subtracted sensograms were fit to a 1:1 kinetic binding model (Fig.1D) and a steady-state affinity model (Fig.1E). The rate constants obtained were as follows: kon 3.44 ± 1.38 × 103µM−1 s−1, koff 0.064 ± 0.03 × 103 s−1. According to the expression KD = koff/kon, the dissociation constant was calculated to be 18.31 ± 1.15µM. The obtained KDs through the two different affinity evaluation methods (ITC and SPR) were similar, as for the KD previously reported in the aptamer selection paper (45.5 ± 2.2µM) [24], the difference can be attributed to the different media used for the affinity evaluation (bloodstream vs. TRIS buffer) and the different method applied (calibration-free E-AB sensor vs. ITC/SPR). Platform design andsensing principle The aptamer-based electrochemical sensing platform used in this study was designed starting from an in-labfabricated flexible C-PE functionalized with cauliflowerlike AuNSs, on which thiolated VAN-specific Apt was grafted via Au–S covalent bonds. To prevent nonspecific adsorption, the remaining available Au sites were blocked using a short-chain thiol (MCH). The working principle of the developed aptasensor was based on the selection strategy of the chosen aptamer that presented a key stemloop closing configuration, meaning that the aptamer was selected to specifically respond to the presence of VAN by detaching the complementary part and opening the stem-loop [24, 27]. In the case of our sensing platform, before VAN binding the stem-loop configuration of the aptamer presumably hinders the electron transfer rate, whereas when VAN is present the stem-loop is opened, thus exhibiting a binding-induced change in the electron transfer rate. Moreover, VAN is positively charged at pH 7.4 [29], which could further facilitate the electron transfer rate from the negatively charged redox probe [30]. The Microchim Acta (2025) 192:107 Page 7 of 15 107 schematic illustration of the C-PE printing and aptasensor fabrication process, along with the “signal-on” sensing principle is shown in Fig.2. For a preliminary characterization of the obtained C-PEs, a simple CV voltammogram was recorded in 5 mM [Fe(CN)6]3−/4− in 0.1M KCl. As shown in Fig.S1A, Fig. 1 A Secondary structure prediction of the modified VAN Apt. The analysis was carried out at 25 °C, with a 0.15 M Na+ and 0.01 M Mg+2 concentration, using the Nupack web server [28]. B ITC measurements: ITC data obtained for the exothermic reaction during titration of 10µM Apt (green) and 0µM Apt (black) with 150µM Van. C Integrated heat rates for each injection plotted against the Van Apt mole ratio in the ITC cell; inset: mean thermodynamic and affinity parameter values obtained from three different measurements. D SPR analysis: Sensograms representing the kinetic binding profile of VAN-Apt (representative normalized, blank subtracted SPR sensograms for eight concentrations of VAN (0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10µM); the colored more transparent lines represent the fit of experimental data to a 1:1 kinetic binding model). E Corresponding equilibrium binding curve (fitted to a steady-state affinity model (n = 3)). Experimental conditions: 60 µL/min flow rate, injections of 300s, 100s dissociation time Microchim Acta (2025) 192:107107 Page 8 of 15 the carbon surface did not exhibit clear redox peaks of the [Fe(CN)6]3−/4− redox couple; therefore, to enhance and standardize the electrochemical signal, a simple chronoamperometric surface treatment was applied using a 1M Na2CO3 solution. After pretreatment, the peak potential separation (ΔEp) changed from 0.64 to 0.15V, highlighting a lower barrier to electron transfer and electrochemical reversibility. The size of the in-lab-fabricated C-PEs was 1.5 × 1 cm (length × width), with a geometric surface of the WE of 0.196 cm2. The surface area available for electron transfer to the species in solution was calculated using the Randles–Sevcik Eq.(3): where Ip is the peak current (A), n is the number of transferred electrons, A is the electroactive area (cm2), D is the diffusion coefficient of the oxidized species (cm2 s−1), v is the scan rate (V s−1), and C is the analyte concentration (mol L−1). The CV measurements were performed on the bare C-PEs in 5mM [Fe(CN)6]3−/4− in 0.1M KCl by varying the scan rate from 25 to 300mV s–1 (Fig.S1B). From the slope of the Randles–Sevcik plot (peak currents vs. square root of the scan rate; Fig.S1C), the electroactive area was calculated to be 0.226 cm2. AuNSs electrodeposition andsurface characterization In the field of biosensors, gold nanoplatforms not only allow easy grafting of specific biorecognition elements via Au–S (3) Ip = ( 2.69 ×10 5) ×n 3∕2 ×A×D 1∕2 ×v 1∕2 × C covalent bonds but also enhance interfacial molecular recognition by accelerating molecular diffusion and reducing steric hindrance [31]. By examining the Au electrodeposition process, two essential phases can be distinguished, nucleation and growth, which are both influenced by the reaction conditions [32]. Thus, optimization of the electrodeposition conditions, such as the electrochemical technique, electrodeposition potential, HAuCl4 concentration, and deposition time, influences the regulation of nucleation and directional aggregation. By setting the appropriate conditions, a non-equilibrium system can be created, promoting the formation of AuNSs with distinct architectures. Our approach included the optimization of a previously reported AuNSs formation strategy involving the incorporation of amino acids into a HAuCl4 solution for controlled Au nanostructure formation on the surface of C-PE. The galvanostatic deposition of AuNSs on screen-printed electrodes by combining HAuCl4 with amino acids, such as cysteine [33] and histidine [31], has been previously explored by our group. Those initial conditions served as a starting point for this study and were systematically optimized. Hence, the augmentation of the electrodeposition process for the formation of AuNSs on the sensing surface of C-PE involved the optimization of three key parameters: (i) histidine concentration, (ii) HAuCl4 solution concentration, and (iii) electrodeposition time. Under different experimental conditions, AuNSs with various shapes were obtained via one-step electrochemical deposition. Two different negative currents were applied to the electrode (− 100 µA and − 200 µA) in the presence or absence of His to obtain the three-dimensional Fig. 2 Schematic representation of the aptasensor fabrication process and its working principle Microchim Acta (2025) 192:107 Page 9 of 15 107 structures. The morphologies of the resulting AuNSs were characterized by SEM and AFM (Fig.3). The first goal of the optimization study was to assess whether the presence of His exerted a crucial influence on the shape or size of the AuNSs. The SEM images (Fig.3A–C) revealed captivating AuNSs, with distinct morphological differences based on the protocols employed. As can be observed in Fig.3A, the electrodeposition of Au from a 10 mM HAuCl4 at − 100 µA, in the absence of His resulted in the formation of thistle-like structures. Conversely, the addition of 150 mM of His to the electrodeposition solution resulted in the formation of cauliflower-like structures, as shown in Fig.3B. At more negative current (− 200 µA), crown-like nanostructures formed on the C-PE surface (Fig.3C). The AFM images of the obtained three platforms are presented in Fig.3A’–C’. The surface features identified by AFM corresponded to the surface characteristics observed by SEM. For the thistle-like structures, the height ranged between 0.9 and 1.7 µm, the crown-like structures had a height range of 0.2–1.0 µm, whereas the cauliflower-like nanostructures reached heights of 0.5–1.0 µm. The resulting C-PE/AuNSs platforms were analyzed electrochemically by CV measurements in 5 mM [Fe(CN)6]3−/4− in 0.1M KCl. The parameter values and their influence on the anodic current peak are shown in Fig.4A-C. As shown, using 150mM of histidine in a 10mM HAuCl4 solution and an electrodeposition period of 600s led to the most favorable and reproducible platform, with the highest charge transfer of [Fe(CN)₆]3⁻/4⁻ redox Fig. 3 SEM images of the obtained C-PE/AuNSs platforms: thistlelike (obtained using 10 mM HAuCl4, 0 mM His, CP –100 µA) (A), cauliflower-like (obtained using 10 mM HAuCl4, 150 mM His, CP –100 µA) (B), and crown-like (obtained using 10 mM HAuCl4, 150 mM His, CP –200 µA) (C) structures at different magnifications and the corresponding AFM images (A’, B’, and C’, scan size 10 µm, scale bar 2 µm)