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. 2025 Sep 3;10(36):41868–41877. doi: 10.1021/acsomega.5c06120

Reagentless and Label-Free Electrochemical Aptasensor Using Polyaniline Incorporated with a Battery-Free NFC Potentiostat for One-Step Detection of Cortisol

Supada Khonyoung †, Nuttanan Thanedsed †, Warawut Tiyapongpattana †, Sopon Butcha †, Orawon Chailapakul ‡, Chawin Srisomwat †,*
PMCID: PMC12444599  PMID: 40978363

Abstract

Cortisol serves as a vital biomarker for stress levels, but current evaluation methods are complex and resource-intensive. Traditional detection approaches require elaborate procedures and sophisticated equipment, limiting their accessibility and practical application. There is a pressing need for user-friendly, cost-effective, and portable technologies that can efficiently monitor cortisol levels in real-world settings. We developed a reagentless and label-free electrochemical aptasensor for one-step detection of salivary cortisol utilizing polyaniline (PANI) as an active conductive polymer and integrating it with a battery-free near-field communication (NFC) potentiostat. The PANI-modified screen-printed electrode exhibited excellent conductivity and surface-enhancing properties, facilitating efficient aptamer immobilization and enabling reagentless electrochemical detection of cortisol without the need for additional redox reagents. The battery-free NFC integration enables seamless data transfer to mobile devices, providing real-time analysis capabilities. The proposed sensor achieved a low detection limit of 27 pM within 33 min of analysis time and exhibited high selectivity for cortisol, which makes it suitable for patients with Addison’s disease. Also, the matrices in artificial saliva were not affected by cortisol detection. A performance comparison between commercial EmStat4s and our NFC-based potentiostat in artificial saliva validated the accuracy of the system. This innovative platform represents a significant advancement in point-of-care diagnostics by combining advanced conductive materials with modern electronics. The system’s portability, cost-effectiveness, and user-friendly design make it particularly valuable for noninvasive stress monitoring through salivary analysis, opening new possibilities for accessible bioanalysis and personalized health monitoring.

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1. Introduction

Cortisol, a steroid hormone produced by the adrenal glands, serves as a crucial biomarker for evaluating stress, assessing adrenal function, and monitoring various physiological responses. In particular, salivary cortisol level monitoring offers a noninvasive and convenient approach to monitoring stress and related health issues. Normal levels of salivary cortisol are about 15.5 nmol L–1 in the morning and 3.9 nmol L–1 in the evening. The body regulates cortisol production based on its level in the bloodstream; an abnormal cortisol level in the body leads to Cushing’s syndrome (higher concentration) and Addison’s disease (lower concentration), respectively. , Salivary cortisol levels for patients with Cushing’s syndrome are 23.4 nmol L–1 in the morning and 24.0 nmol L–1 in the late night, whereas for patients with Addison’s disease, it shows 4.1 nmol L–1 level in the morning. Human saliva contains only free cortisol, which makes the protocol easier, even if it has a lower cortisol concentration (2.8–4.4 nmol L–1) than serum (130–830 nmol L–1). Traditional analytical techniques for cortisol detection, such as enzyme-linked immunosorbent assays (ELISA) or liquid chromatography-tandem mass spectrometry (LC-MS/MS), even though highly accurate, often involve complex procedures, extensive sample preparation, and the use of costly reagents. Consequently, there is an increasing demand for innovative sensing technologies that provide rapid, sensitive, and user-friendly alternatives to cortisol detection.

Electrochemical aptasensors have emerged as promising tools for salivary cortisol detection, combining the specificity of aptamers with the high sensitivity and small analytical volume of electrochemical measurements. Aptamers, which are short, single-stranded nucleic acid sequences, can be engineered to have high affinity and selectivity for specific targets such as cortisol. The exploitation of the use of aptamers instead of antibodies includes chemical stability, ease of synthesis, and reduced batch-to-batch variability. Aptamers also enable higher specificity under various environmental conditions, which helps improve sensor robustness. Unlike many aptamer-based optical and colorimetric sensors, ,− the electrochemical aptasensing platform offers superior sensitivity, faster response times, and lower detection limits. , Electrochemical sensors are inherently miniaturizable and amenable to integration with portable devices for point-of-care diagnostics. , Additionally, label-free electrochemical aptasensors have attracted considerable attention due to their exploitation of low cost and simplicity, as they are not covalently modified with a reporter probe. Obviously, electroactive species in the solution are required for the label-free system to operate effectively. The most widely used one, ferri-/ferrocyanide, increased the cost and required additional processes for the biosensing platform. To overcome this challenge, the reagentless electrochemical aptasensor still has the potential to improve the detection performance. The reagentless approach eliminates the need to add redox molecules or a redox label to the solution which is a practical advantage over reagent-based methods. It suppresses one step in the experimental procedure.

Polyaniline (PANI), a well-known conductive polymer, has attracted significant attention as a potential transducer material for a genuinely reagentless electrochemical aptasensor. Its unique properties, which include high electrical conductivity, environmental stability, and ease of functionalization, make it an ideal candidate for providing a robust platform for biomolecule immobilization. − In our work, PANI serves both as a highly conductive polymeric matrix and as a biocompatible interface for efficient aptamer immobilization. − The tailored PANI nanostructure enhances electron transfer kinetics and provides a large surface area for aptamer loading, contributing to improving the sensitivity. Also, the combination of electrochemical detection and PANI-based aptasensing allows for cost-effective fabrication without sacrificing performance, making our sensor suitable for large-scale production and real-world applications.

As a potential tool for stress monitoring, the need for wireless, affordable, and portable electrochemical point-of-care testing (POCT) has emerged. Near-field communication (NFC) technology has recently gained popularity in the field of electrochemical sensors, enhancing their functionality and usability by enabling wireless communication and data transfer at proximity. − By combination of NFC technology with an electrochemical aptasensor, a battery-free power supply can be used, creating a miniaturized POCT platform. Users now have an efficient approach to collecting and evaluating electrochemical data in real time, thanks to the technology, which facilitates the effortless sharing of data between sensors and mobile devices.

In this study, the synergy of a reagentless, label-free electrochemical aptasensor, utilizing a PANI-modified screen-printed electrode, is proposed in combination with a battery-free, NFC-enabled data interface via a smartphone for the one-step detection of cortisol, as illustrated in Figure . This platform creates an accurate, user-friendly, and completely portable system for rapid salivary cortisol detection, opening a new pathway for decentralized, real-time health diagnostics. A cortisol-specific aptamer was immobilized onto a PANI-modified electrode via a glutaraldehyde (GA) cross-linking process (Figure B). The use of PANI not only enables the immobilization step but also facilitates the creation of a redox current. The sensing mechanism is illustrated in Figure D. After one-step loading of the sample and incubating for 30 min, the redox current of PANI is governed by the exchange of cations (such as H+, Na+, and K+) and anions (Cl–) in the electrolyte through the electroactive polymer/electrolyte interface, facilitating redox switching. Among these, chloride ion (Cl–), which is present in phosphate-buffered saline (PBS) and saliva, plays a dominant role in modulating the redox-switching process. In the presence of cortisol in PBS buffer or saliva sample, the binding of the target molecule induces a conformational change in the aptamer, leading to steric hindrance at the polymer/electrolyte interface. The conformational change restricts ion exchange, thereby reducing the charge transfer between PANI and the electrolyte. As a result, a decrease in the PANI current response was observed, which is the basis of the detection mechanism. Using a battery-free NFC potentiostat, the amperometric response was performed and visualized in real time via a smartphone readout. Through this investigation, we aim to make a significant contribution to the advancement of point-of-care diagnostic tools that facilitate noninvasive and efficient monitoring of critical health biomarkers.

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Schematic of the electrochemical aptasensor for cortisol detection. (A) A component of the proposed device. (B) Overall modification steps on the screen-printed graphene electrode (SPGE), (C) procedures for cortisol detection using NFC potentiostat. (D) Chronoamperograms (CA) of blank (blue dashed) and presence of cortisol (blue solid).

2. Experimental Section

2.1. Chemicals, Materials, and Instrumentations

All chemicals used in this work were analytical grade. Cortisol, phosphate buffered saline (PBS) tablet, artificial saliva for pharmaceutical research, and potassium ferricyanide (K3[Fe­(CN)6]) were obtained from Sigma-Aldrich (St. Louis). Cortisone and corticosteroids were provided by Glentham Life Sciences (Wiltshire, U.K.). Aniline and sulfuric acid were purchased from LOBA Chemie (India). Glutaraldehyde was acquired from Thermo Scientific. All aqueous solutions were prepared using ultrapurified water (R ≥ 18.2 MΩ cm), which was produced by a Millipore water purification system.

Carbon graphene ink and silver/silver chloride (Ag/AgCl) ink were obtained from Serve Science (Bangkok, Thailand) and the Gwent group (Torfaen, U.K.), respectively. The screen-printed templates were made by Chaiyaboon Co. Ltd. (Bangkok, Thailand).

Cortisol DNA aptamer (Apt) modified with 5′ amino modifier C6 (5′-amino-C6-AGC AGC ACA GAG GTC AGA TGC AAA CCA CAC CTG AGT GGT TAG CGT ATG TCA TTT ACG GAC C-3′) (61 bases) was purchased from PHUSA Biochem (Vietnam).

Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) for the characterization part were performed with a PalmSens 4 potentiostat/impedance analyzer (PalmSens BV, Netherlands). Chronoamperometry was conducted by the portable potentiostat, which was enabled to control the SIC4341 (Silicon Craft Technology PLC, Bangkok, Thailand) and EmStat4S (PalmSens BV, Netherlands). All electrochemical measurements were performed using a three-electrode system comprising a carbon graphene ink as the working electrode (WE), a counter electrode (CE), and a silver/silver chloride ink as the reference electrode (RE). In all cases, CV and EIS were performed at room temperature (25 ± 2 °C). All of the measurements were carried out in triplicate.

2.2. Fabrication of the Electrochemical Aptasensor

The prototype of the electrochemical aptasensor was demonstrated in Figure A. Three electrodes were fabricated using an in-house screen-printing technique. Carbon graphene ink as working and counter electrodes and Ag/AgCl ink as a reference electrode were screen-printed onto the polyvinyl chloride (PVC) substrate. Then, the SPGE electrode was dried at 60 °C for 30 min.

As illustrated in Figure B, polyaniline (PANI) was modified on the electrode by using an electropolymerization technique. A 30 μL portion of aniline monomer (0.25 M) in 0.5 M sulfuric acid was dropped on the SPGE. The electropolymerization of aniline was performed by cyclic voltammetry (conditions: t equilibrium = 5 s, window potential = −0.8–1.0 V, E step = 0.01 V, scan rate = 0.1 V s–1, scan cycle = 8 cycles). To improve the sensitivity of the detection system, the free amine group on the leucoemeraldine form of PANI was stabilized by dropping 30 μL of sulfuric acid (0.5 M) at the electrode and applying the constant potential of −0.4 V for 50 s.

The procedure for immobilization of Apt onto the PANI was slightly modified from Ranjbar et al. Immobilization of Apt onto the PANI-modified electrode was enabled by using glutaraldehyde (GA) as a cross-linker. The immobilization mechanism proceeds through a two-step Schiff base reaction. A 1 μL portion of glutaraldehyde (0.25%) was loaded onto the electrode and kept for 30 min at room temperature (25 °C) to activate the amine groups of PANI. The GA/PANI/SPGE was washed with Milli-Q water to remove unbound GA and further incubated with Apt (1 μL of 1 μM) for 30 min at room temperature. After the immobilization step was successfully developed, the electrode was washed with Milli-Q water to remove unbound aptamer. The electrochemical aptasensor was stored at 4 °C prior to use.

2.3. NFC-Potentiostat Smartphone App Tag Sensor

The NFC potentiostat was included in a credit-card-sized (5.5 × 8.5 × 0.1 cm3) portable device, as illustrated in Figures A and S1A. The chipset, antenna, and electrode connector comprise this circuit board component. A SIC4341 potentiostat sensor interface chip (NFC type 2) developed by Silicon Craft Technology PLC, Thailand, was used for electrochemical analysis. The SIC4341 sensor interface potentiostat chip is a single-chip solution that enables electrochemical detection via a near-field communication (NFC) system. As seen in Figure S1B, the digital-to-analog converters (DACs) and analog-to-digital converters (ADCs) are integrated with a potentiostat sensor interface circuit. The electrochemical measurement can be operated through the mobile application Chemister, which uses a smartphone processor to convert digital codes from the chip into a current signal related to the concentration value of target analytes. The minimized potentiostat card presents a wireless, battery-free, low-cost, easy-to-use, and portable device because it is a single-chip solution (Figure S1C).

The display on the monitor, which utilizes an NFC potentiostat controlled by a Motorola One with an Android operating system, is depicted in Figure S2. On a smartphone, all operating processes are accessible through the Chemister application. Figure S2A,B displays the Chemister application used to control the SIC4341 NFC-potentiostat circuit. This software offers a range of electrochemical techniques for users to select from. For this experiment, the electrochemical technique of choice was chronoamperometry (CA). The electrode pins IO(0), IO(1), and IO(2) represent the reference electrode (RE), working electrode (WE), and counter electrode (CE), respectively (Figure S2C). The other cortisol detection parameters were set, as shown in Figure S2D.

2.4. Operation of the One-Step Cortisol Detection Using the NFC-Potentiostat Smartphone Detection System

Figure C shows the step stages of the NFC-potentiostat smartphone detection system for cortisol. After the sample (30 μL) was introduced to the electrode, it was allowed to incubate for 30 min. After that, the NFC-potentiostat board was connected to the aptasensor, and chronoamperometry was used to perform the current signal (conditions: t equilibrium = 5 s, applied potential = 0.4 V, T run = 180 s, and T step = 200 ms). By tapping the NFC-potentiostat card, the smartphone was connected. On the smartphone’s screen, the current signal of PANI was observed in real time (Figure D). After being exported as a text file, the raw data were submitted to data processing, which included plotting using Microsoft Excel.

2.5. Application for Detection of Cortisol in Artificial Saliva

To determine cortisol, spiked cortisol in artificial human saliva (provided by Sigma-Aldrich) was incubated on the electrode for 30 min at room temperature. Afterward, the method mentioned above was used for the electrochemical measurement. The concentration of spiked cortisol ranged from 0.3 to 7 nmol L–1. The accuracy of the detection process was evaluated by calculating the recovery efficiency of spiked cortisol in artificial saliva.

3. Results and Discussion

3.1. Electrochemical Characterization of the Battery-Free NFC Potentiostat and Commercial Potentiostat

First, we investigated the electrochemical performance of the NFC potentiostat. Using various concentrations of K3Fe­(CN)6 in 0.1 M KNO3, chronoamperometry was employed to assess the quantitative analysis capabilities of the NFC portable potentiostat under the following conditions. Chronoamperograms of various K3Fe­(CN)6 concentrations, ranging from 0.1 to 5.0 mM, were recorded, as shown in Figure A. As the concentration of K3Fe­(CN)6 increased, the cathodic current also increased. Furthermore, Figure B demonstrates a linear correlation (R 2 = 1) between ΔI and K3Fe­(CN)6 concentrations in the 0.1–5 mM range, confirming the applicability of the NFC portable potentiostat for quantitative studies. In addition, we used the NFC potentiostat to generate chronoamperograms of 1 mM K3Fe­(CN)6 in contrast to the commercial potentiostat (EmStat4S). The distinctive amperograms produced by the two potentiostats are shown in Figure C, which also demonstrate the similarities between them. Furthermore, Figure D displays the ΔI values obtained from both potentiostats using various K3Fe­(CN)6 concentrations, showing good agreement (R 2 = 0.9983) and highlighting the NFC potentiostat’s potential as a quantitative analysis tool.

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(A) Chronoamperograms of K3Fe­(CN)6 with different concentrations using NFC potentiostat (condition: t equilibration = 5 s; E dc = 0.4 V; T run = 120 s; T step = 200 ms), (B) calibration curve of cathodic current (at t = 60 s) of K3Fe­(CN)6 with different concentrations from 0.1 to 5.0 mM, (C) chronoamperograms of 1 mM K3Fe­(CN)6 using NFC potentiostat (blue solid) and conventional potentiostat (EmStat4S, green solid), (D) linear regression comparing the average ΔI via NFC and EmStat4S potentiostats achieved at various concentration of K3Fe­(CN)6 using chronoamperometry (n = 3).

3.2. Characterization of the Apt/PANI/SPGE for Cortisol Detection

The proposed aptasensor was successfully fabricated, as evidenced by subsequent characterization. First, the cyclic voltammogram of PANI polymerization is shown in Figure S3A. Initially, the first cycle of the CV scan for the aniline monomer (blue solid) shows an anodic peak corresponding to the oxidation of aniline to radical cations. Upon successive cycles, the current response (both anodic and cathodic peaks) increases with each scan, indicating continuous growth of an electroactive PANI film on the electrode surface. This increase in the current is a direct signature of the successful polymerization and deposition of a conductive polymer. The appearance of characteristic redox peaks for PANI (e.g., the leucoemeraldine to emeraldine transition and the emeraldine to pernigraniline transition) in subsequent cycles further confirms the formation of the polyaniline structure.

Next, scanning electron microscopy (SEM) was used to characterize the surface morphologies of the SPGE, PANI/SPGE, and Apt/PANI/SPGE. The distribution of graphene sheets on the SPGE is depicted in Figure A. After modification, the fibrous structure of PANI, characterized by a homogeneous distribution of chains over the SPGE surface, is illustrated in Figure B. This morphology can significantly enhance the specific surface area of the electrode. The SEM image of Apt/PANI/SPGE showed an altered electrode morphology, transitioning from a porous to a rough surface after the aptamer was immobilized onto the electrode (Figure C), indicating that the modification was successful. Additionally, Figure S4 also shows the Apt/PANI/SPGE cross-sectional SEM image, illustrating the dense packing by the thickness of approximately 20 μm of the Apt/PANI/SPGE layers over the entire area of the PVC substrate.

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Morphology and electrochemical characterization of electrode modification steps. (A–C) SEM images (5000×) of bare electrode (SPGE), PANI/SPGE, Apt/PANI/SPGE (inset: 500×), (D) cyclic voltammograms (conditions; potential range: −0.6–0.8 V; scan rate: 0.1 V s–1; E step: 0.01 V) and (E) Nyquist plots (conditions; frequency range: 0.01 Hz to 300 kHz; alternating current (AC) potential: 0.1 V) of each fabrication steps performing with 5 mM of ferri/ferro cyanide in 0.1 M KNO3, (F) chronoamperograms of blank (dashed) and 5 nM cortisol (solid) performed with both potentiostats, and (G) linear regression comparing the average ΔI via NFC and EmStat4S potentiostats achieved at three cortisol concentrations using chronoamperometry (error bars, n = 3).

Moreover, the energy-dispersive X-ray spectroscopy (EDX) mapping of the different modified layers of the SPGE is shown in Figure S5. The observed spectra of bare SPGE indicated the elemental distribution of carbon (C) and chlorine (Cl) from the graphene ink (Figure S5A), while the spectrum of nitrogen (N) appeared in PANI/SPGE, confirming the presence of imine sites of PANI on SPGE (Figure S5B). In the case of Apt/PANI/SPGE, the EDX results clearly show a homogeneous distribution of phosphorus signals across the entire sensing area after aptamer immobilization (Figure S5C). This indicates a uniform coverage of the aptamer on the PANI-modified surface, which is critical for consistent and reliable sensor performance.

Subsequently, the electrochemical properties of each step in the fabrication process were investigated by using cyclic voltammetry (5 mM ferro/ferricyanide in 0.1 M KNO3). As shown in Figure D, the anodic (at 0.40 V) and cathodic (at −0.15 V) currents of [Fe­(CN)6]3–/4– increased when the electrode was modified with polyaniline (green solid) compared to the current signal of the bare SPGE (blue solid) due to the conductive property and porosity of the PANI. When the aptamer was immobilized via glutaraldehyde (GA), the oxidation and reduction peak potentials were shifted to 0.6 and −0.2 V, respectively, due to the aptamer’s hindrance on the electrode surface (gray solid). Lastly, the current signal at 0.6 and −0.2 V, respectively, was decreased since the binding between the aptamer and cortisol occurred, which impeded the electron transfer onto the electrode surface. These results indicated that the aptamer was successfully modified on the SPGE.

Next, electrochemical impedance spectra (EIS) were utilized to monitor the modification of the aptamer onto the electrode. The charge transfer resistance (R ct) was achieved by calculating the diameter of the semicircle of the Nyquist plot. As illustrated in Figure E, the R ct of SPGE was found to be 8.95 kΩ (blue dotted line), and the resistance of the Apt/GA/PANI/SPGE decreased to 3.45 kΩ (gray dotted line), which is much higher than that of the PANI/SPGE (2.32 kΩ, green dotted). Additionally, a further increase in R ct (26.76 kΩ) was observed when cortisol was captured by the aptamer onto the electrode surface (yellow dotted). The results implied that the modification was successful.

Control experiments further support the proposed sensing mechanism (Figure S3B). The different cortisol concentration (0.1, 1, 10 nM) was performed on only PANI-modified SPGE without aptamer compared with Apt/PANI/SPGE. For only PANI/SPGE, the current responses were consistently low and remained largely stable, showing negligible change even with increasing concentrations of cortisol (green bar). This indicates that nonspecific physical deposition of cortisol on the PANI layer has a minimal impact on the electrochemical signal. In contrast, our aptasensor (At/PANI/SPGE) exhibited a distinct increase in current response with increasing cortisol concentration. This significant difference in the signal strongly confirms that the observed current changes are primarily due to the specific interaction between cortisol and the aptamer. The underlying mechanism involves the conformational change of the aptamer upon binding with cortisol, which subsequently alters the ion flux at the electrode surface, leading to the measurable electrochemical signal.

CA was also used to measure cortisol with NFC and a conventional potentiostat. Figure F displays the chronoamperometric response to the blank and 5 nM cortisol. The current of the PANI electrode, measured at the 40 s, reveals a high degree of similarity between them. Also, in Figure G, a strong positive linear correlation (R 2 = 0.9974) was observed between the two potentiostats when the ΔI values obtained from the NFC and a conventional potentiostat were plotted against different cortisol concentrations, confirming the sensor’s consistent performance. This suggests that the NFC potentiostat has the potential as a point-of-care (POC) diagnostic tool for cortisol detection.

3.3. Optimization Study of Electrochemical Aptasensor for Cortisol Detection

To assess the performance of the electrochemical aptasensor for cortisol detection using chronoamperometry, several parameters were optimized, including the number of cycles for electropolymerization of PANI, aptamer concentration, incubation time, and applied potential for chronoamperometric detection, all at a cortisol concentration of 10 nM. The effect of a number of cycles for PANI polymerization (2–10 cycles) was studied to control the PANI film thickness. Each condition was immobilized with lysine–Apt (1 μM) and the mixture incubated with cortisol for 30 min. The chronoamperometry technique was performed with a fixed potential of 0.4 V, and data were collected at 40 s. As shown in Figure S6A, the current signal (ΔI; I blank – I analyte) increased with the increase of the number of cycles due to the conductive property of the PANI. When the number of cycles exceeded 8, ΔI decreased. This phenomenon is attributed to the thicker PANI layer, which reduces the apparent diffusion coefficient of counterions and, therefore, decreases the current intensity. As a result, 8 cycles were selected as the optimal condition. Additionally, the concentration of the aptamer was investigated (ranging from 0.5 to 10 μM). As illustrated in Figure S6B, ΔI increased with increasing aptamer concentration, as a higher concentration of aptamer provides more available active sites for cortisol recognition and affects sensitivity. When the concentration of the aptamer is higher than 1 μM, the current decreases to a plateau. It can be explained that the too close packing of aptamer immobilization onto the electrode surface leads to a lower binding affinity, resulting in a drop in sensitivity. Thus, this concentration was selected for further studies. The effect of the incubation time was studied from 5 to 120 min at room temperature. As shown in Figure S6C, the ΔI increased with longer incubation time, as extended incubation time provides higher binding efficiency. The current tended to be stable at 30 min, since the aptamer was saturated by cortisol. Thus, the optimal incubation time was 30 min. Last, the applied potential was optimized using the developed Apt/PANI/SPGE in the presence of 10 nM cortisol. Applied potential was varied at 0.2, 0.4, 0.6, and 0.8 V. Figure S6D demonstrates the increase in ΔI with a more positive applied potential. An applied potential of 0.6 V yields the highest ΔI, remaining stable up to 0.8 V. Therefore, the optimal condition of cortisol detection was a PANI electrodeposition for 8 cycles, an aptamer concentration of 1 μM, an incubation time of 30 min, and an applied potential of 0.6 V.

3.4. Analytical Performance for Cortisol Detection

Using an NFC potentiostat, the analytical performance at various cortisol concentrations was calculated. The developed aptasensor was examined in a 0.1 M PBS buffer solution. Within the concentration range of 0.1 and 10 nM, Figure A shows that the chronoamperometric responses of PANI decreased as the cortisol concentration increased. A calibration plot of the cortisol concentration and current response was achieved (Figure B). The logarithmic cortisol concentration and the ΔI value showed a linear relationship with a correlation coefficient (R 2) of 0.9944 (Figure B, inset) and a limit of detection (LOD) of 27 pM (LOD = 3SDblank/slope), respectively. This range can be used to measure cortisol levels in patients with Addison’s disease and adequately covers the reported biologically expressed range of cortisol in human saliva, with an analysis time of 33 min for the whole assay. We also set the potentiostat of the EmStat4s to the same parameters. The linearity data were comparable to those obtained with the NFC potentiostat, as illustrated in Figure C, and the LOD of 7.9 pM was only slightly lower. It is demonstrable that the NFC potentiostat and aptasensor were effectively combined to create a point-of-care testing platform.

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Analytical performances of electrochemical aptasensor for cortisol detection. (A) Chronoamperograms, (B) calibration curve of cortisol in PBS buffer using an NFC potentiostat, (C) calibration curve of cortisol in PBS buffer using EmStat4S, (D) specificity, and (E) reproducibility (error bars, n = 3).

Next, the performance of our aptasensor was compared with other studies’ cortisol detection designs (Table S1). The proposed platform is reagentless and label-free, since it measures the intrinsic PANI redox signal change based on cortisol target binding. The developed platform allows for a more convenient and straightforward measurement performance compared to other labeled assays and those requiring additional reagents to generate current response signals. Additionally, the performance of battery-free NFC potentiostats is similar to that of other potentiostats. The use of this is typically less expensive (∼$59) than commercial potentiostats (∼$2925). The estimated cost of the device is about $59.16 as detailed in Table S2. Many are designed for low-cost manufacturing and accessibility, making them suitable for resource-limited settings. NFC potentiostats are generally compact and lightweight, making them highly portable and ideal for on-field applications. Their battery-free nature enhances their usability in remote areas, where power sources may be unavailable. They often have straightforward interfaces, allowing users to conduct tests in various environments efficiently, without complex setups. NFC connectivity enables rapid data transfer to smartphones and tablets for efficient analysis.

Additionally, we used a standard solution of several glucocorticosteroids with structurally similar steroids, such as corticosterone and cortisone (10 nM), and six possible interferences in saliva at a concentration comprised in the physiological range, to assess the specificity of the developed cortisol aptasensor. As shown in Figure D, cortisol (10 nM) showed the highest current response, while other interferences (namely, l-glucose (13 mg dL–1), uric acid (3.38 mg dL–1), lactate (1.8 mM), ascorbic acid (0.2 mg dL–1), creatinine (0.12 mg dL–1), and albumin (0.2 mg mL–1)) showed considerably lower current responses. The findings showed that the proposed aptasensor exhibits high specificity for cortisol, which is attributed to the aptamer.

The reproducibility was evaluated by comparing the data obtained from five separately fabricated electrochemical aptasensors. According to the Association of Official Analytical Chemists (AOAC) guidelines, the relative standard deviation (RSD) value of 4.3% (Figure E) is within the acceptable range. This indicates that the developed diagnostic platform is reproducible from the fabrication of the biosensor to the measurement of the signal. Additionally, the aptasensor’s storage stability was initially assessed by placing it in a refrigerator at 4 °C, demonstrating satisfactory performance with over 80% retention during the first 5 days of operation (Figure S7). Longer storage stability is necessary to ensure translational feasibility. To achieve this, it is suggested that future studies investigate more rigorous storage conditions, such as utilizing a dehumidifier or storing the sensor under an inert atmosphere (e.g., N2 gas) to minimize moisture and oxidative degradation. Additionally, vacuum-sealing or employing desiccant materials may further enhance the sensor’s stability over extended periods.

3.5. Artificial Saliva Sample Analysis

Finally, we assessed our aptasensor’s ability to measure cortisol in a clinical sample. Figure shows the calibration curve between the cortisol concentration in artificial saliva and the current response. As the cortisol concentration increased from 0.1 to 10 nM, the ΔI also increased. Additionally, upon comparison of the sensitivity or slope in PBS and artificial saliva, there are no significant differences between the two conditions, as the slope value for saliva remained within an acceptable deviation (RSD = 3.16%) compared to the PBS buffer. It can be concluded that the component in saliva has no effect on cortisol quantification.

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Calibration curve of cortisol in artificial saliva using NFC potentiostat (n = 3).

Moreover, cortisol was spiked in the artificial saliva sample in concentrations ranging from 0.3 to 7 nM. We measured cortisol concentration in the saliva samples using our NFC potentiostat in addition to the EmStat4S. The recovery values, as shown in Table , were in the 95–97% range, which is comparable to the EmStat4S results (91–107%). Furthermore, a 95% confidence level paired t test on the experiment data showed no statistically significant difference. Therefore, our aptasensor accurately detects cortisol, showcasing its potential in artificial saliva, a key step toward biological sample analysis.

1. Cortisol Concentration in the Artificial Saliva Sample was Evaluated by Both Potentiostats.

sample added (nM) found NFC (nM) % recovery NFC found EmStat4s (nM) % recovery EmStat4s
artificial saliva 0.3 0.29 ± 0.04 97 0.32 ± 0.13 107
3.0 2.86 ± 0.33 95 2.73 ± 0.21 91
7.0 6.79 ± 0.53 97 6.89 ± 0.30 98
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4. Conclusions

This work effectively establishes the potential of using PANI as a modified electrode material for the cortisol electrochemical aptasensing platform. An effortless experimental setup can be used to achieve this label-free, reagentless, one-step biosensing, making it possible to easily build a portable device using a smartphone-based battery-free NFC potentiostat for cortisol measurement. Our technology has good sensitivity, a broad linear range, and an LOD of 27 pM. Additionally, it exhibits high specificity, even when other corticosteroids are present. The accurate detection of cortisol in artificial saliva, compared to that of a commercial potentiostat, validated its dependability and offered the possibility of integrating an NFC potentiostat for a point-of-care testing apparatus. Therefore, this development makes it possible to rapidly, affordably, and efficiently perform field analysis of salivary cortisol in real time, especially when sophisticated clinical equipment is unavailable.

Supplementary Material

ao5c06120_si_001.pdf (766.3KB, pdf)

Acknowledgments

The authors gratefully acknowledge the financial support provided by Faculty of Science and Technology, Thammasat University, Contract No. SciGR 20/2567.

Glossary

Abbreviations

NFC

near-field communication

Apt

aptamers

PANI

polyaniline

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c06120.

  • (A) The components of the NFC potentiostat consist of a planar antenna, the NFC microchip (SIC4340/41), and a connector port for the electrode system. (B) SIC4341 NFC potentiostat sensor interface chip block diagram. (C) An image showing the actual setup of the proposed system (Figure S1); images showing the overall operation steps using an NFC potentiostat with an Android smartphone (Figure S2); (A) cyclic voltammograms of PANI using electropolymerization, and (B) the current for cortisol measurement with different concentrations (0.1, 1, 10 nM) using Apt/PANI/SPGE (blue) and only PANI/SPGE (green) (error bars, n = 3) (Figure S3); cross-sectional image of Apt/PANI/SPGE (1000×) (Figure S4); EDX mapping and spectrum of each step of fabrications, including (A) bare SPGE, (B) PANI/SPGE, and (C) Apt/PANI/SPGE (Figure S5); optimization parts for cortisol detection: (A) cycles of electropolymerization of PANI, (B) concentration of aptamer, (c) incubation time, and (D) applied potential (n = 3) (Figure S6); stability of the proposed aptasensor (error bars, n = 3) (Figure S7); comparison with previous works: electrochemical aptasensor to detect cortisol levels in human biofluids (Table S1); estimated cost of the proposed electrochemical aptasensor device (Table S2) (PDF)

S.K.: Conceptualization; project administration; resource; writingreview and editing; formal analysis; investigation. N.T.: Formal analysis; investigation. W.T.: Resource; writingreview and editing. S.B.: Investigation; writingreview and editing. O.C.: Resources. C.S.: Conceptualization; project administration; data curation; formal analysis; investigation; methodology; visualization; writingoriginal draft; funding acquisition; resources; supervision.

The authors declare no competing financial interest.

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ao5c06120_si_001.pdf (766.3KB, pdf)

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