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. 2026 Mar 16;11(12):19692–19702. doi: 10.1021/acsomega.5c13554

Biosensing of Steroid Hormones with 3D Zinc Oxide Tetrapods

Parth Pandit †, Maciej Klein †, Shahnewaz Ali †, Tony Parker ‡,§, Clinton Fookes †,§, Virginie Perlo ‡,§,∥, Marianella Chamorro-Koc ⊥, Graham Kerr §,#, Chamindie Punyadeera ∇, Yogendra Kumar Mishra ○, Ajay K Pandey †,*
PMCID: PMC13044822  PMID: 41939409

Abstract

Accurate monitoring of stress hormones is essential for understanding physiological responses and optimizing human performance. Selective and precise detection of these biomarkers is particularly important for evaluating stress levels in high-demand environments. Herein, we report a multimodal sensor based on zinc oxide tetrapods (ZnO-Ts) for the detection of cortisol and testosterone. The platform integrates fluorescence and electrochemical transduction, enabling a complementary and highly sensitive analysis. ZnO-Ts exhibited fluorescence quenching upon interaction with steroid hormones with detection limits of 0.16 μg/dL for cortisol and 0.25 μg/dL for testosterone. Stern–Volmer and time-resolved photoluminescence analyses revealed analyte-dependent quenching behavior, with cortisol exhibiting a dynamic quenching component and testosterone showing static quenching. A solid-state electrochemical sensor was further fabricated by embedding ZnO-Ts in a poly­(vinyl alcohol) matrix and depositing them onto printed silver electrodes, which demonstrated concentration-dependent electrochemical responses with higher sensitivity toward cortisol. By a combination of optical and electrochemical readouts, this dual-mode sensing strategy provides robust, rapid, and scalable hormone monitoring. The platform’s biocompatibility and duality in identifying two steroidal hormones selectively underscore its potential for wearable diagnostics and point-of-care stress monitoring technologies.

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1. Introduction

Stress triggers a cascade of hormonal changes in the human body, primarily involving cortisol, often termed the “stress hormone,” playing a central regulatory role in metabolism, inflammation, and cognitive function. Testosterone, though traditionally associated with anabolic and reproductive functions, is also modulated by stress and often shows an inverse relationship with chronic stress levels. The dynamic interplay between these hormones significantly influences human performance, decision-making, and overall physiological well-being. As a result, the demand for disease-specific biomarkers and ultrasensitive detection platforms has surged, positioning hormone monitoring as a pivotal and technically demanding field within personalized healthcare. The detection of biomolecules in biological fluids is often hindered by their structural similarity due to their steroidal backbone, chemical complexity, and typically low concentrations. Steroid hormones such as cortisol and testosterone are prime examples, playing crucial roles in stress regulation, metabolic imbalances, and hormonal dysfunction. Accurate monitoring of these hormones is essential for early diagnosis and health assessment, yet their presence in trace levels within complex biological matrices, such as saliva, blood, or urine, poses significant analytical challenges. Athletes monitor cortisol/testosterone ratio to assess recovery and training stress.

Traditional methods for selective detection of biomolecules often rely on complex analytical techniques, including chromatography methods like gas chromatography (GC) and high-performance liquid chromatography (HPLC) and ion mobility spectrometry (IMS), which, despite high precision, are costly, workflow-intensive, and poorly suited for real-time or point-of-care applications. In parallel, electrochemical sensing has emerged as a powerful and complementary approach for hormone detection due to its low power consumption, scalability, and compatibility with portable and wearable devices. Recent electrochemical detection of steroid hormones has primarily employed amperometric, voltammetric, and impedimetric transduction schemes. These sensors have nanostructured electrodes and molecular recognition layers such as antibodies, aptamers, and molecularly imprinted polymers to achieve high sensitivity and selectivity. Despite their strong analytical performance, these platforms often require complex surface functionalization, multistep fabrication, and three-electrode configurations, which can limit the long-term stability and scalability.

To address these limitations, optical sensing strategies, particularly fluorescence quenching mechanisms, have gained increasing attention for biomolecular detection due to their high sensitivity, operational simplicity, and enabling ultralow background signals for detecting low-abundance biomarkers. Conventional fluorescent quenching materials such as graphene, carbon nanotubes, and MoS2 and noble metal nanoparticles such as Au, Ag, and Pt have been widely explored due to their high molar extinction coefficient and optical properties. These materials significantly enhance sensitivity by facilitating fluorescence quenching. However, their quenching efficiency is often constrained by their proximity requirements (1–10 nm) and narrow spectral overlap, limiting broader applicability. These challenges underscore the need for alternative materials such as zinc oxide (ZnO) nanostructures, which offer intrinsic photoluminescence, electrical charge transport, tunable defect states emission, and broadband optical interactions ideally suitable for multimodal sensing strategies.

ZnO nanostructures have emerged as versatile materials due to their high surface area, Zn2+ and O2– terminated polar surfaces, unique room temperature photoluminescence (PL), and tunable morphology. Their wide band gap and n-type conductivity make them well suited for luminescent and sensing technologies. , The 1D ZnO morphology has shown high selectivity toward target chemical molecules, with recent advances in ZnO-based nanomaterials being used for improved sensitivity and selectivity for COVID-19 detection, as well as sensing of urea and lactic acid. Zinc oxide tetrapods (ZnO-Ts), which are composed of four interconnected “nanolegs” forming a unique 3D geometry, offer a high surface-area-to-volume ratio along with distinctive physicochemical properties and have been used in optoelectronic applications, gas sensors, and smart functional coatings. Despite their advantages, integration into biosensing platforms remains largely underexplored in the current literature.

The debate about ZnO defects has been a topic of discussion for a long time, focusing on the dual role of these defects. The defect-induced nonradiative recombination leads to energy loss, reducing the quantum efficiency of devices. ZnO has intrinsic defects such as oxygen vacancies, zinc interstitials, and antisite defects. These defects introduce a continuum of energy states rather than discrete levels, particularly in the near-infrared and visible spectral regions. They create localized energy states within the band gap. These states often act as “traps” for charge carriers, leading to nonradiative recombination where the energy of excited electrons is dissipated as heat rather than light. In biosensing, such defects facilitate emission shifts, fluorescence quenching, and conductivity changes upon analyte interaction. Thus, the same imperfections that hinder luminescent efficiency become advantageous for highly responsive and tunable optical and optoelectronic sensing platforms. These defect properties make ZnO-Ts a versatile material for optoelectronic and optical sensor technologies.

We report a novel approach employing ZnO-Ts as fluorescence quenching probes for the sensitive detection of structurally analogous steroid hormone biomarkers, including cortisol and testosterone. Defect states in ZnO-Ts are characterized using steady-state photoluminescence (PL) measurements, while time-correlated single-photon-counting studies reveal an enhanced defect density in ZnO-Ts and highlight their suitability for selective analyte detection via quenching-based mechanisms. The integration of both optical and electrochemical sensing modalities within a single material platform establishes ZnO-Ts as versatile multimodal sensors for steroid hormone detection.

2. Experimental Section

2.1. Materials and Methods

All reagents used in this study were of analytical grade and used without further purification. All stock solutions were prepared in (absolute) ethanol (ChemSupply Australia). Hydrocortisone (USP, Reference Standard, Sigma-Aldrich) was used as a synthetic cortisol analogue for cortisol, while testosterone (Purum, ≥99.0%, HPLC grade, Sigma-Aldrich) served as the analogue for testosterone. Additional biomarkers like lactate, glucose, estradiol, and progesterone were purchased from Sigma-Aldrich. ZnO-Ts were synthesized at Mads Clausen Institute, University of Southern Denmark. The concentration of the reference ZnO-Ts was fixed at 1 mg/mL in all experiments. All of the experiments have been performed thrice. The concentration ranges of both analytes were fixed at 0.5–3 μg/dL.

2.2. Synthesis of ZnO-Ts

The ZnO tetrapod nanostructures were synthesized using a novel flame transport synthesis (FTS) method. For the ZnO tetrapod synthesis, a ceramic crucible was filled with a 1:2 w/w mixture of precursor Zn microparticle powder and poly­(vinyl butyral) (PVB). The Zn particles are the precursor material, whereas PVB is sacrificed to generate the flame and to help convert the Zn microparticles into atomic vapor. The ceramic crucible filled with precursor mixture is inserted into a simple muffle furnace for the growth process at 900 °C. After a process of 30 min at 900 °C, the heating is turned off and the furnace is left to cool naturally to room temperature. After the process, the crucible is filled with white snowflake-like ZnO micro- and nanoscale tetrapods, which are carefully harvested and used for experimental purposes. These tetrapods are grown in a dedicated furnace (only ZnO growth), so the obtained structures are pure with respect to external impurities. However, due to high-temperature thermodynamic process, these tetrapods exhibit intrinsic defects, which include twin/grain boundaries and also surface structures, such as nanostairs, wrinkles, and ripples, which in turn increase the overall surface area and hence high surface activity. They were stored in an airtight container in a cool environment.

2.3. Optical Studies on Analytes

Absorption spectra were measured using a Cary-60 UV–vis spectrophotometer in the range from 400 to 700 nm. Steady-state photoluminescence (PL) was measured with a Cary Eclipse Fluorescence Spectrometer using a quartz cuvette. For both photoluminescence and absorption measurements, we kept the concentration of optical reporter ZnO-Ts at 1 mg/mL. Each of the analytes was made up to the concentration and was then deliberately added, and the optical characteristics were measured. Time-resolved photoluminescence (TRPL) spectroscopy measurements of ZnO-Ts were carried out in the absence and presence of various concentrations of using pulse excitation at 442 nm and emission at 528 nm in ethanol. The time-resolved photoluminescence dynamics were collected using a DeltaFlex (HORIBA) time-correlated single-photon counting (TCSPC) system. A PDD-920 single-photon detection module collected the signal, which was filtered by a time-domain TDM-1200 monochromator. A picosecond pulsed laser diode, DD-440LB, λ = 440 nm, 100 MHz repetition rate, and 75 ps pulse width, was used as an excitation source. Exponential functions were fitted to extract excited-state lifetimes from the resulting decay curves by using EzTime Software (HORIBA).

2.4. Thin-Film ZnO-Ts Sensor Studies

Poly­(vinyl alcohol) (PVA) solution (10% w/v) was prepared by dissolving 1 g of PVA in 10 mL of deionized water at 80 °C for 1 h. ZnO-Ts (10 mg) were dispersed into the PVA solution and stirred at room temperature for 2 h to obtain a homogeneous nanocomposite suspension. The tetrapod-based nanocomposite was used as a sensing medium. Polyimide substrates were sequentially cleaned with isopropyl alcohol (IPA) and DI water for 10 min, followed by drying in an oven at 80 °C for 30 min. The electrodes were printed onto the substrates using a Voltera V-One PCB printer with Ag ink, and the printed electrodes were soft-baked at 110 °C for 20 min to cure the contact pads. The transparent ZnO-Ts nanocomposite gel was then drop-cast onto the precleaned polyimide substrate between the printed silver electrodes. The interelectrode distance was fixed at 1 mm and the optimized droplet volume was 2.5 μL. Chronoamperometric measurements were carried out using a Keithley Precision Source Meter (2400 Graphical Series, SMU), by applying a constant bias of 1 V across the electrodes. The current response over time was recorded for both analytes. The morphological characterization of ZnO-Ts and ZnO-Ts/PVA was performed by scanning electron microscopy (SEM) using a Phenom Desktop SEM.

3. Results and Discussion

3.1. Sensing Analytes in Bulk Solution

The novel ZnO-Ts are used as optical probes for sensing different analytes that are present in body fluids. This detection method relies on monitoring PL signals upon the addition of analytes at varying concentrations. Since these nanostructures are insoluble in solvents, their morphological and optical properties remain intact. This insolubility allows them to form stable suspensions, which enhance absorbed light intensity and facilitate the formation of aggregates that interact with target analytes. For all experiments, ethanol was selected as the base solvent due to the more uniform dispersion of ZnO-Ts in ethanol than in water, a critical factor for ensuring consistent interactions in sensing applications (Figure A,B).

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Fluorescence quenching study of ZnO-Ts for cortisol and testosterone detection. (A) ZnO-Ts dispersed in ethanol (1 mg/mL) serves as the sensing medium, along with a microscopic image. (B) Fluorescence emission spectra (right) demonstrate quenching upon the addition of cortisol (3 μg/dL; 3 × 10–5 mg/mL) and testosterone (3 μg/dL; 3 × 10–5 mg/mL) under excitation at 442 nm, with a peak emission shift observed around 528 nm. (Analyte concentrations are primarily expressed in μg/dL in accordance with standard ELISA calibration units; equivalent concentrations in mg/mL are provided in parentheses for consistency.)

The absorption curve shows possible interactions between the probe and the analytes. When biomolecules are introduced into the ZnO-Ts solution, changes in the absorbance spectrum occur, indicating that these molecules interact with ZnO-Ts (Figure A). When excited at 442 nm, the emission is observed from 500 to 700 nm with a maximum around 528 nm (Figure B). The fluorescence intensity of ZnO-Ts is reduced drastically with the addition of cortisol and testosterone in a concentration-dependent manner (Figure B,C). The rapid reduction of fluorescence intensity with the increasing concentration of analytes suggests photoinduced energy transfer (PET) from the electron-rich ZnO to electron-deficient analyte molecules.

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(A) UV–vis absorbance spectra of ZnO-Ts (1 mg/mL) in ethanol and upon addition of (3 μg/dL; 3 × 10–5 mg/mL) cortisol and testosterone, illustrating changes in absorbance over the 200–800 nm range. (B) Fluorescence intensity spectra of ZnO-Ts in ethanol under increasing concentrations of cortisol (0.5 to 3.0 μg/dL; 5 × 10–6 to 3 × 10–5 mg/mL). (C) Fluorescence intensity spectra of ZnO-Ts upon addition of testosterone at varying concentrations (0.5 to 3.0 μg/dL; 5 × 10–6 to 3 × 10–5 mg/mL) show similar quenching behavior. (Analyte concentrations are primarily expressed in μg/dL in accordance with standard ELISA calibration units; equivalent concentrations in mg/mL are provided in parentheses for consistency.)

3.2. Stern–Volmer Equation

The sensitivity of fluorescence can be quantified by the Stern–Volmer plot, where I 0 is the initial fluorescence intensity (in the absence of a quencher) and I is the fluorescence intensity in the presence of a quencher. K sv is the quenching constant, which indicates the quenching efficiency. Both analytes show a direct concentration-dependent relationship in the Stern–Volmer plot (Figure ). The linearity of the Stern–Volmer plots indicates efficient quenching; however, Stern–Volmer linearity alone does not distinguish between static and dynamic quenching behavior. The Stern–Volmer plot for cortisol reflects stronger interactions having higher quenching efficiency with better quenching constant, which is 0.91 dL/μg with R 2 = 0.988. It confirms that the quenching process taking place is dynamic in nature (Figure A). In contrast, the Stern–Volmer plot for testosterone shows a much lower slope (0.47 dL/μg) with an R 2 value of 0.995. This suggests that testosterone has the least quenching effect among the analytes studied, indicating a weaker interaction or lower affinity for binding to ZnO-Ts. The lower slope means less efficient quenching, reflecting less efficient energy transfer compared with another analyte (Figure B). TRPL analysis (Figure C) shows an average lifetime decay of 7.51 ns for pristine ZnO-Ts. In the presence of testosterone (3 μg/dL), the lifetime decay is 7.22 and 6.86 ns upon addition of cortisol (3 μg/dL). The implications of these lifetime changes and the underlying interaction mechanisms are discussed in detail in the subsequent sensing mechanism section. Figure D shows the steady-state PL spectra of ZnO-Ts in ethanol with cortisol and testosterone (3 μg/dL), where both analytes cause a reduction in the emission intensity, with a more pronounced quenching observed for cortisol.

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Stern–Volmer plot of analytes. (A) Cortisol showing a linear relationship between fluorescence intensity and quencher concentration of cortisol (μg/dL). (B) Testosterone showing a linear correlation between fluorescence intensity and quencher concentration (μg/dL). (C) TRPL spectra of ZnO-Ts in the presence and absence of analytes (i.e., cortisol and testosterone). IRF represents the instrument response function. (D) Steady-state PL spectra of ZnO-Ts in ethanol (1 mg/mL) and after exposure to testosterone (3 μg/dL; 3 × 10–5 mg/mL) and cortisol (3 μg/dL; 3 × 10–5 mg/mL) confirm the analyte-induced fluorescence quenching. Each plot has an error bar depicting the replicate measurements of n = 3. (Analyte concentrations are primarily expressed in μg/dL in accordance with standard ELISA calibration units; equivalent concentrations in mg/mL are provided in parentheses for consistency).

3.3. Mechanistic Insight into Quenching Behavior

ZnO-Ts is a 3D hexagonal wurtzite crystal structure that functions as a fluorescent probe owing to its wide band gap and intrinsic defect states, which can be harnessed for the fabrication of novel sensors and PL technologies. , ZnO-Ts due to its typical structure and direct band gap of 3.37 eV has various defects, which are characterized via PL studies. , The decrease in PL intensity is associated with an intermolecular photoinduced electron transfer mechanism. The fluorescence quenching behavior of ZnO-Ts in the presence of testosterone and cortisol is analyte dependent, explained by a combination of energy profile, decay time, and molecular proximity between them. The mechanism underlying the selective detection of cortisol using ZnO-Ts can be rationalized through hole injection theory. ZnO with a valence band (VB) energy level of −7.5 eV and a conduction band (CB) energy level of −4.2 eV serves as a photoactive semiconductor capable of generating electron–hole pairs. Upon excitation, ZnO absorbs photons and promotes electrons from VB to CB, leaving behind a positively charged hole in the VB. The photogenerated hole is a highly reactive species that can act as an electron acceptor from nearby donor molecules. Cortisol, a steroid hormone with a HOMO located at −6.17 eV and a LUMO at −1.14 eV, exhibits an energetically favorable alignment for charge transfer. Cortisol can donate an electron from its HOMO, creating hole transfer from ZnO-Ts to cortisol leading to nonradiative recombination, which manifests as measurable fluorescence quenching. This photoinduced electron transfer gives rise to a short-lived charge-transfer intermediate between cortisol and the ZnO-Ts. Importantly, cortisol’s LUMO is significantly higher than ZnO’s CB, making electron transfer from ZnO to cortisol energetically unfavorable. In contrast, testosterone, a structurally similar steroid, has a HOMO at −9.55 eV and a LUMO at −2.84 eV, both of which lie significantly below ZnO’s VB and CB, respectively. This unfavorable alignment rules out both hole and electron transfer pathways. However, ZnO has intrinsic defect states within its band gap. Testosterone might weakly interact with these trap states, altering the decay time or photoluminescence intensity slightly. This overall electron transfer is based on a static quenching mechanism for testosterone (Figure ).

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Schematic illustration of energy profile in ZnO-Ts upon interaction with cortisol. (1) Photoinduced electron–hole pair in the ZnO-Ts. (2) Hole diffusion toward ZnO-Ts/cortisol interface. (3) Hole transfer to cortisol HOMO (which is effectively a hole neutralization in ZnO-Ts valence band via an electron transfer from cortisol HOMO).

To confirm the transfer of energy between the fluorophore and the analytes, TRPL was performed to measure the decay time by monitoring the emission of fluorophore at 528 nm in the absence or presence of the analyte. The decay curves show how the fluorescence intensity decreases over time (in nanoseconds), indicating the lifetime of an excited-state fluorescence before it returns to the ground state. The decay time for testosterone is not changed substantially, while for cortisol, the decay times shorten, indicating faster fluorescence decay and possible charge transfer. The IRF was plotted for both decay time measurements as a reference. The decay profile was fitted by using exponential functions. As shown in Figure C, the decay time for ZnO-Ts in ethanol is 7.51 ns. Upon the addition of cortisol, the lifetime decreased to 6.86 ns, suggesting the presence of an excited-state deactivation pathway consistent with dynamic quenching. In contrast, the decay time in the presence of testosterone (7.22 ns) is comparable to that of bare ZnO-Ts and lies within experimental uncertainty, indicating that testosterone-induced quenching is predominantly static in nature (Figure C). We also studied the simultaneous addition of analytes to observe the quenching pattern. With the addition of analytes, the concentration also increases in the cuvette, which increases the quencher concentration, and hence due to frequent transient collisions, enhanced quenching is observed (Figure S1). Ethanol was added as a negative control, which exhibited the same fluorescence pattern, confirming that the observed quenching arises specifically from the analytes and not from the solvent (Figure S1D). The steady-state PL spectrum shows a decrease in intensity with each analyte added (Figure D). The observed quenching behavior is analyte dependent. For cortisol, the quenching is consistent with a dynamic mechanism, where excited-state interactions between the fluorophore (ZnO-Ts) and the quencher led to increased nonradiative deactivation. The higher the concentration of quenchers, the greater the number of collisions, leading to enhanced quenching. Testosterone shows static-dominated quenching with a minimal lifetime change. The energy level alignment provides a mechanistic explanation for the observed quenching trends and decay dynamics in the ZnO-Ts–analyte interactions. This dual alignment results in significant PL quenching and a notable reduction in the fluorescence decay time, as observed experimentally. Testosterone adsorption perturbs the ZnO-Ts surface dipole environment and defect-related radiative pathways without requiring an efficient excited-state interaction. Accordingly, although direct charge transfer between ZnO-Ts and testosterone is energetically unfavorable, the observed optical response arises from adsorption-induced modulation of ZnO surface states and the local dielectric environment, leading to static quenching without a significant reduction in the PL lifetime. The PL spectra of cortisol and testosterone at different excitation wavelengths also show the same trend in quenching pattern (Figure S1).

3.4. Morphological Characterization

The SEM micrographs show the morphology of the free-standing ZnO-Ts before and after incorporation into the PVA film matrix. The SEM shows the successful synthesis with its characteristic four-branched 3D architecture. The micrographs show that the tetrapods are embedded into the PVA matrix (Figure ). The pod arms show that the morphology of the pods has been intact with the bed. The sharp features are particularly advantageous as they significantly increase the effective surface area and create localized field enhancement sites, thereby facilitating stronger optical and electrochemical interactions with target biomolecules.

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SEM micrographs of the bare ZnO-Ts and ZnO-Ts/PVA nanocomposite films. (A) SEM image of bare ZnO-Ts showing the characteristic 3D morphology. (B, C) SEM images of the ZnO-T/PVA nanocomposite at different magnifications, illustrating the uniform embedding of ZnO tetrapods within the PVA matrix. (D) Higher-magnification SEM image of ZnO-Ts embedded in the PVA matrix. Scale bars: (A) 100 μm, (B) 200 μm, (C) 100 μm, and (D) 50 μm.

3.5. Analytical Performance and Selectivity Studies

A drop of the analyte (2.5 μL) was carefully deposited between the sensor electrodes, and a bias voltage of 1 V was applied across the electrodes and a steady-state current response was measured (Figure A). The baseline response with DI water shows minimal current variation, indicating a low interaction. Upon exposure to testosterone, there is a notable increase in current, while cortisol generates a much higher and more sustained current response, demonstrating the sensor’s sensitivity and selectivity to cortisol relative to testosterone (Figure B). This shows that the zinc oxide tetrapods react stronger with cortisol than with testosterone, which validates our mechanism observed in optical studies. The interferant assay shows the selective response of ZnO-Ts toward cortisol and testosterone. Despite some interferants such as estradiol and progesterone having structural similarity to steroid hormones, their electrochemical responses were negligible compared with cortisol and testosterone. Cortisol produced the highest relative change in current (∼125%), followed by testosterone (∼70%), while other analytes such as lactate, glucose, estradiol, and progesterone induced responses close to the baseline (<5%). All analytes tested were of the same concentration, 1 μg/dL (Figure C). These results confirm that the ZnO-Ts platform exhibits strong selectivity for cortisol, with moderate but distinct sensitivity for testosterone, and excellent discrimination against structurally or functionally unrelated interferants. Long-term stability assessment using 1 μg/dL synthetic steroids revealed a steady current response sustained over 40 days, underscoring the sensor’s robust durability and reproducible performance (Figure D). The current response over time was calculated for both cortisol and testosterone at 0.5–3 μg/dL (Figure S3A,C). The response curve shows the highest current response (∼2.7 μA) is observed for 3 μg/dL cortisol, while the lowest (∼1.2 μA) corresponds to 0.5 μg/dL cortisol. The sensor exhibits a linear concentration-dependent response, with higher cortisol levels generating greater current due to enhanced interaction with the sensing interface. A linear relationship between current and cortisol concentration is observed at a 400 s time window (Figure S3B), with an R 2 value of 0.9880, indicating strong correlation and reliable quantification of cortisol. In contrast, the testosterone response shows a more pronounced initial spike and sharp fall in the current levels (Figure S3C). This shows differing interaction kinetics as compared with cortisol sensing. The calibration curves for the same have also been plotted, which shows a strong correlation between analyte and current response with an R 2 value of 0.9998 (Figure S3D). This current response curve aligns well with optical studies and hence confirms the use of ZnO-Ts as sensing moieties for sensing cortisol and testosterone with high selectivity and sensitivity. The limit of detection (LOD) for each analyte was determined based on the standard criterion signal-to-noise ratio (S/N). The LOD of cortisol was calculated to be 0.16 μg/dL, while for testosterone, it is 0.25 μg/dL (Section S2). These values fall well within the physiological ranges reported for stress-related studies and are comparable to or lower than the detection limits of several biosensors (Table ). The competitive LOD and the key attributes imply that the sensor system is favorable for the detection of both steroidal biomarkers. Moreover, it is sensitive to cortisol, likely due to enhanced binding interactions and energy transfer as described above.

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Electrochemical sensing of cortisol and testosterone using ZnO-Ts. (A) Illustration of ZnO-Ts-PVA suspension prepared for sensing experiments. (B) Chronoamperometric response of the solid-state sensor when exposed to deionized (DI) water, testosterone (1 μg/dL; 1 × 10–5 mg/mL), and cortisol (1 μg/dL; 1 × 10–5 mg/mL). (C) Interferant study showing selectivity with cortisol and testosterone as compared with other analytes, all at the same concentration (1 μg/dL; 1 × 10–5 mg/mL). (D) Stability test of the sensor showing stable current response for over 40 days. Error bars represent the standard deviation obtained from three independently fabricated sensors (n = 3). (Analyte concentrations are primarily expressed in μg/dL in accordance with standard ELISA calibration units; equivalent concentrations in mg/mL are provided in parentheses for consistency).

1. Comparison of Other Cortisol and Testosterone Hormone Detection Platforms with the ZnO-Ts-Based Detection System .

sensor type analytes sample working range (nM) LOD (nM) key attributes refs
ZnO nanorod EIS sensor (electrochemical) cortisol buffer 0.001–10 0.001 antibody based, rigid
paper-based microfluidic devices (electrochemical) cortisol sweat 28–2800 2.8 immunosensor and antibody based
microneedle + AuNP (electrochemical) cortisol buffer ISF 2.8–280 0.28 aptamer-based sensor
MIP-based sensor (electrochemical) testosterone serum 35–3500 35 MIP-based sensor
CNT film electrode (electrochemical) testosterone serum 5.2–5200 52 immunosensor-based sensor
ZnO-Ts multimodal (optical + electrochemical) cortisol and testosterone synthetic solution 13.8–82.8 (C), 17.3–104 (T) 4.42 (C) 8.67 (T) cost-effective, scalable, dual-analyte detection This work
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a

All concentrations were converted from μg/dL and nanomolar (nM) for consistency. Molar concentrations were calculated using the molecular weights of cortisol (362.46 g/mol) and testosterone (288.42 g/mol). C: cortisol; T: testosterone.

3.6. Electrochemical Sensing Mechanism

The increase in current observed upon analyte addition originates from adsorption-induced modulation for the ZnO-Ts/PVA composite rather than the faradaic oxidation of the analyte. In the solid-state sensor, charge transport occurs through a percolated network of n-type ZnO tetrapods bridging the printed silver electrodes under an applied bias of 1 V. The overall resistance of this network is governed by electron depletion layers formed at ZnO surfaces and by potential barriers at the tetrapod–tetrapod interface. Adsorption of neutral, lipophilic analyte molecules such as cortisol and testosterone on the ZnO surface leads to passivation of surface trap states and formation of interfacial dipoles, which effectively screen the surface charge density, resulting in reduced band bending and narrowing of the electron depletion regions. As a result, the effective conduction channel within individual tetrapods and across intertetrapod junctions widens, lowering transport barriers and facilitating enhanced electron percolation through the network. This adsorption-driven conductometric modulation increases the population and mobility of conduction-band electrons, thereby producing an increase in the measured current. Such surface-state-controlled chemiresistive behavior is characteristic of ZnO-Ts nanostructure-based solid-state sensors only.

4. Conclusion

The application of ZnO-Ts nanostructures enabled the successful detection of cortisol and testosterone in both solution and solid-state formats. This nanostructured system showed remarkable selectivity for cortisol and testosterone. These tetrapodal structures interact more with cortisol than testosterone, shown by the high K sv constants of 0.9142 dL/μg and 0.4388 dL/μg. The fluorescence quenching response was found to be analyte dependent, wherein cortisol predominantly induced dynamic quenching, while testosterone exhibited a static quenching mechanism. The LOD for cortisol was determined to be 0.16 μg/dL, compared with 0.25 μg/dL for testosterone, indicating a higher detection sensitivity toward cortisol. Moreover, the favorable binding interactions, together with LOD values that fall within physiologically relevant concentration ranges, underscore the potential of these sensors for practical applications. Furthermore, the fabricated sensor exhibits excellent performance in a compact, low-cost, and rapid-response device configuration, offering a robust pathway toward real-time steroid hormone monitoring.

Supplementary Material

ao5c13554_si_001.pdf (545.2KB, pdf)

Acknowledgments

We acknowledge grant holders Cassandra Pattinson, Simon Smith, and Shannon Edmed, from The University of Queensland, who contributed to the conceptualization, design, and conduct of the Performance PatchWearable Predictive Diagnostic for Warfighter Maintenance Project. YKM acknowledges funding from the ESS lighthouse on hard materials in 3D, SOLID (Danish Agency for Science and Higher Education, grant number 8144-00002B), NANOCHEM, BHJ Fonden, and Fabrikant Mads Clausen Fonden, Denmark.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c13554.

  • Fluorescence and electrochemical characterization of the ZnO-Ts-based multimodal sensor for cortisol and testosterone detection. Fluorescence quenching behavior and emission spectra of cortisol and testosterone at different wavelengths (Figure S1); analytical performance and LOD of each analyte (Section S2); chronoamperometric responses and calibration curves for both hormones (Figure S3) (PDF)

The Performance Patch project was funded by the Australian Government through Defense. The views expressed herein are those of the authors and are not necessarily those of the Australian Government or Defense. Specifically, the following people received funding for the Performance Patch project through a Defense Industry & Innovation Next Generation Technologies Fund by the Australian Government: Prof Tony Parker, Prof Graham Kerr, Prof Chamindie Punyadeera, Prof Ian Stewart, Dr Andrew Hunt, A/Prof Jonathan Peake, Prof Karen Sullivan, Prof Ottmar Lipp, Dr Cassandra Pattinson, Prof Simon Smith, Prof Christian Cook, A/Prof Ajay Pandey, Prof Marianella Chamorro-Koc, Distinguished Prof Kerrie Mengersen, and Prof Clinton Fookes. Chamindie Punyadeera is currently receiving research grant funds from the National Health and Medical Research Council (APP 2002576 and APP 2012560), the Australian Research Council (DP250101156 and IH240100013), the Royal Brisbane Women’s Foundation, Tour De Cure, the Garnett Passe and Rodney Williams Foundation, and the Gallipoli Medical Research Foundation. Parth Pandit was supported by a Mind & Body Performance Patch (sensor development) QUT PhD scholarship.

The authors declare no competing financial interest.

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