Ultra-Low Hydrogen Peroxide Detection via Synergistic Nernst Potential Effect in Organic Electrochemical Transistors

Summary

Here, we present a protocol for ultra-low detection of hydrogen peroxide based on the synergistic Nernst effect. This protocol covers the fabrication of stacked-layer poly(3,4-ethylenedioxythiophene): bromothymol blue (PEDOT: BTB)/polystyrene sulfonate (PEDOT: PSS), and platinum microelectrode (Pt), as well as the determination of the optimal operating point for the as-constructed H₂O₂ sensor.

Abstract

The organic electrochemical transistor (OECT)-based synergistic Nernst potential—generated via the Pt gate electrode catalyzing hydrogen peroxide (H₂O₂) and the interaction between bromothymol blue (BTB) molecules and hydrogen ions (the by-product of H₂O₂ catalysis)—is leveraged for the ultra-low detection of H₂O₂ down to 1.8 × 10^-12 M, with a broad linear detection range from 10^-11 M to 10^-3 M. The formation of this potential is determined by selecting a source-drain voltage (V_DS) and a gate voltage (V_G) of −0.6 V as the optimal operating points, and by adopting the stacked-layer poly(3,4-ethylenedioxythiophene):bromothymol blue/poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:BTB/PEDOT:PSS) as the semiconducting channel material. In addition, relevant verification is provided by characterizing the carrier (de)doping capability of the stacked layers using UV–vis spectroscopy, identifying the optimal operating point via electrochemical measurements, and evaluating the sensing performance of the as-constructed OECT-based H₂O₂ sensor using single-stage constant-voltage scanning. Finally, the OECT-based H₂O₂ sensor is fabricated via a micro-nano manufacturing process, including the preparation of stacked semiconducting layers by spin-coating and the fabrication of microelectrodes via the lift-off process and magnetron sputtering. This methodology can open a broad avenue for the ultra-low detection of analytes through enzyme-catalyzed reactions.

Introduction

Hydrogen peroxide (H₂O₂) is a ubiquitous by-product of numerous enzyme-catalyzed biochemical reactions, including those mediated by lactate oxidase, glutamate oxidase, and glucose oxidase¹. Its pervasive presence in a wide range of physiological and pathological processes^2,3,4 makes it a critical biomolecule for understanding human health and disease states. Therefore, accurate detection of H₂O₂ at ultra-low concentrations is essential for gaining precise insights into bodily conditions, driving significant interest among researchers in developing sensitive detection methodologies with a low limit of detection (LOD).

Traditional H₂O₂ detection methods^5,6, such as spectroscopy, chromatography, and chemiluminescence, have laid the foundation for quantitative analysis but suffer from inherent limitations that hinder their widespread applicability. These techniques typically rely on bulky, expensive instrumentation and require specialized professional expertise, making them impractical for on-site, portable, or point-of-care detection scenarios. Thus, there is an urgent need for alternative detection technologies that combine high sensitivity with miniaturization, low cost, and ease of use.

With the advancement of micro-nano technologies, organic electrochemical transistors (OECTs) have emerged as a promising platform for biosensing applications, owing to their inherent advantages, including miniaturization, low operating voltage, excellent signal amplification capability, and biocompatibility^7,8,9. OECT-based sensors have been increasingly explored for H₂O₂ detection, with a primary focus on modifying H₂O₂-sensitive materials onto microelectrodes to enhance detection sensitivity. For example, Cicoira et al.¹⁰ constructed an OECT-based H₂O₂ sensor with a linear detection range from 5 μM to 10³ μM and a low LOD of 5 μM when Pt was used as the source, drain, and gate materials. Guo et al.¹¹ fabricated an OECT on a flexible poly (ethylene terephthalate)(PET) substrate and a transwell support. The device employed a screen-printed carbon paste electrode modified with carbon nanotubes and Pt nanoparticles as the gate electrode, and PEDOT: PSS as the channel material, exhibiting a linear detection range of 0.5-100 μM and an LOD of 0.2 μM for H₂O₂. Qi et al.¹² reported a donor-acceptor (D-A) type ambipolar single organic mixed ionic-electronic conductor (OMIEC) polymer-based OECT, which exhibited a linear detection range of 0.001-100 μM and an LOD of 1 nM for H₂O₂. The aforementioned H₂O₂ sensor achieved low LOD mainly dependent on the Nernst potential generated by the Pt-based gate electrode catalyzing H₂O₂, which could regulate the electrochemical (de) doping state of the channel layer for utilizing the excellent signal amplification capability. Thus, prior studies in this field have established a solid theoretical and experimental basis for further optimization.

The core goal of this study is to develop a highly controllable and sensitive OECT-based H₂O₂ sensor by further harnessing the Nernst potential, a key mechanism underlying the performance of electrochemical sensors. To achieve this, we propose a synergistic Nernst potential mechanism¹³, which relies on a cascade reaction involving two key components: Pt-based gate electrodes that electrocatalyze H₂O₂, and a stacked semiconductor channel layer (PEDOT: BTB/PEDOT: PSS) that interacts with hydrogen ions generated as a by-product of H₂O₂ catalysis. This design is grounded in established research: bromothymol blue (BTB), a well-known chemical indicator for weak acids and bases¹⁴, has been successfully integrated with PEDOT (as PEDOT: BTB) on the OECT gate or channel layer for pH sensing in prior studies, validating the feasibility of the cascade reaction central to our approach.

Compared to alternative OECT-based H₂O₂ detection methods, our synergistic Nernst Potential strategy offers distinct advantages. Unlike conventional designs that rely solely on the catalytic activity of Pt-based gate electrodes to generate Nernst potential, our approach enhances controllability by coupling this catalytic process with the pH-responsive behavior of the stacked channel layer. This synergy not only improves the sensor's sensitivity but also enhances its stability and tunability, addressing key limitations of existing OECT-based sensors. Furthermore, this technique aligns with the broader trend in OECT-based biosensing, which emphasizes the integration of functional materials and innovative reaction mechanisms to push the boundaries of detection performance.

In the context of broader electrochemical detection literature^{15,16,17,18,19,20,21}, OECTs have become a cornerstone of portable biosensing due to their ability to amplify weak electrochemical signals, making them ideal for low-concentration analyte detection. Our work builds on this foundation by refining the Nernst potential-driven mechanism, offering a new perspective on how to optimize OECT performance for H₂O₂ detection. For readers considering the applicability of this method, our design is particularly well-suited for scenarios requiring high sensitivity, portability, and controllability, such as point-of-care diagnostics, in vitro monitoring, and on-site environmental or biological sample analysis, where traditional methods are impractical. By clarifying the conceptual framework and advantages of our approach, this introduction provides readers with the necessary context to evaluate whether this method aligns with their specific application needs.

Protocol

1. Fabrication and characterization of stacked PEDOT: BTB/PEDOT: PSS semiconductor film

1. Sample preparation
   NOTE: When preparing the single-layer PEDOT: PSS, do Step 1.1.1. When preparing the stacked-layer PEDOT: BTB/PEDOT: PSS, perform Step 1.1.2.
   1. Prepare the single-layer PEDOT: PSS via spin-coating.
      1. Filter the commercial PEDOT: PSS solution using a 0.45 μm filter (polyethersulfone PES).
      2. Take 884 μL of filtered PEDOT: PSS, 1 μL of 0.1 wt% dodecylbenzene sulfonic acid (DBSA), 50 μL of 5 wt% ethylene glycol (EG), and 5 μL of 0.5 wt% 3-glycidoxypropyltrimethoxysilane (GOPS) and mix them thoroughly to form a mixed solution (i.e., PEDOT: PSS precursor).
      3. Stir the PEDOT: PSS precursor overnight using a magnetic stirrer, then sonicate for 5 min
      4. Spin-coat the PEDOT: PSS semiconductor film to bridge the source and drain electrodes using a Spin Coater at a spin speed of 4200 rpm, an accelerated speed of 200 rpm s^-1, and a maintenance time of 60 s.
      5. Bake the film at 130 ℃ for 15 min in an oven, then pipette 10 μL of concentrated sulfuric acid (H₂SO₄) onto the film to remove excess PSS.
      6. Rinse the film subsequently with deionized water, then bake at 130 ℃ for 10 min.
   2. Prepare the stacked-layer PEDOT: BTB/PEDOT: PSS via electrodeposition.
      1. Prepare a solution containing 42 μL of 10 mM 3,4-ethylenedioxythiophene (EDOT), 0.02496 g of BTB, 40 mL of 1 mM phosphate buffer solution (PBS), and 0.4044 g of potassium nitrate (KNO₃). Mix and shake for 10 min until a red solution is obtained.
      2. Electrodeposit PEDOT: BTB onto the spin-coated PEDOT: PSS layer using the cyclic voltammetry (CV) method of a dual potentiostatic electrochemical workstation. Short-circuit the source (S) and drain (D) electrodes to use them as the working electrodes, use a saturated calomel electrode as the reference electrode, a Pt wire as the counter electrode, and the mixed aqueous solution as the electrolyte.
      3. Apply an electric potential ranging from 0 to 1 V on the working electrode at a scan rate of 0.1 V⋅s^-1 for 15 cycles.
      4. Clean the electrodeposited channel with deionized water, then dry at room temperature.
2. Scanning electron microscope (SEM), atomic force microscope (AFM), ultraviolet-visible (UV-vis) spectroscopy, and electrochemical characterization.
   1. Perform morphological characterization of the films using SEM and AFM.
      1. Deposit the single-layer and stacked-layer films onto the Au sheet, respectively, using the Spin Coater at a spin speed of 4200 rpm, an accelerated speed of 200 rpm s^-1, and a maintenance time of 60 s.
      2. Operate the SEM at acceleration voltages of 1 kV and 3 kV in secondary electron mode to observe the surface and cross-sectional morphologies of the as-prepared stacked-layer film, respectively.
      3. Measure the single-layer film (thickness below 115 nm) using a high magnification of 40,000 ×. Measure the stacked-layer film (total thickness 361 nm) using high magnifications of 10,000 × and 40,000 × to elucidate the electrodeposition of the stacked-layer film onto the Au sheet.
      4. Adopt the tapping mode of an AFM to observe the surface roughness variations of the single-layer and stacked-layer films.
      5. Set the AFM scanning range to 5 μm × 5 μm. Record the 3D morphology and calculate the root mean square roughness.
   2. Characterize the photoelectrochemical properties of the stacked layers using UV-vis spectroscopy. Set the wavelength range from 200 nm to 800 nm, optical resolution to 0.73 nm, slit size to 10 μm, integration time to 11 ms, and temperature control to 20–30 °C.
      1. Spin-coat the stacked-layer film onto the indium tin oxide (ITO) at a spin speed of 4200 rpm for 60 s with an accelerated speed of 200 rpm s^-1.
      2. Select pH buffer solutions ranging from pH 3 to pH 9 at intervals of two orders of magnitude as the electrolyte for the spectrophotometer cell, and place the modified ITO electrode in the spectrophotometer cell aligned with the light path.
      3. Use a micro-injection pump to successively add and remove 2 mL of electrolyte solutions with different pH values into the spectrophotometer cell.
      4. Measure and acquire the absorption spectra of the stacked-layer PEDOT: BTB/PEDOT: PSS in each pH solution.
      5. Place a Pt wire in the spectrophotometer cell, and connect the working electrode (WE) of the dual potentiostatic electrochemical workstation to the Pt wire. Apply voltages of +0.6 V and -0.6 V. Connect the control electrode (CE) of the dual potentiostatic electrochemical workstation to the modified ITO electrode.
      6. Prepare H₂O₂ solutions (10^-11–10^-3 M) in 0.1× PBS with a conductivity of 34.39 ± 0.73 mS·cm^-1. Place the modified ITO electrode in the spectrophotometer cell.
      7. Use a micro-injection pump to successively add and remove 2 mL of electrolyte solutions with different H₂O₂ concentrations into the spectrophotometer cell.
      8. Measure and acquire the absorption spectra of the stacked-layer PEDOT: BTB/PEDOT: PSS in different H₂O₂ solutions.
   3. Determine gate operation points using electrochemical measurement.
      1. Conduct CV measurements in 20 mL of H₂O₂ solutions with different concentrations (configured in 0.1× PBS, ranging from 10^-3 M to 10^-11 M at intervals of two orders of magnitude) with the same conductivity of 34.39 ± 0.73 mS cm^-1.
      2. Use a Pt wire as the working electrode, the single-layer film modified on the Au sheet as the counter electrode, an Ag/AgCl electrode as the reference electrode, and H₂O₂ solutions of different concentrations as electrolytes, respectively.
      3. Apply an electric potential ranging from -1 to 1 V on the working electrode at a scan rate of 0.1 V⋅s^-1.
      4. Measure the electrolyte voltage (V_E) in 0.1 × PBS (equivalent to 1 mM PBS) without mixing H₂O₂.
      5. Introduce another Ag/AgCl electrode into the original OECT system (including source, drain, Ag/AgCl gate electrode, and stacked-layer PEDOT: BTB/ PEDOT: PSS semiconductor film), and electrically short-circuit the additional Ag/AgCl electrode and the source electrode in the original OECT system.
      6. Connect an oscillograph to the additional Ag/AgCl electrode, apply a measurement duration of 2 s and a frequency of 10 kHz, and measure the V_E.
      7. Measure the scanning spectra of V_E in 0.1 × PBS with different H₂O₂ concentrations (ranging from 10^-11 M to 10^-3 M at intervals of two orders of magnitude) with the same conductivity of 34.39 ± 0.73 mS cm^-1 under V_G of ±0.6 V and the V_DS of -0.6 V, respectively.
      8. Acquire the middle value of the scanning spectra and use it as the quasi-equivalent value of V_E.

2. Preparation of the H₂O₂ sensor based on the traditional lift-off microfabrication process

1. Grow a 500 nm-thick SiO₂ insulating layer on a three-dimensional (3D) Si substrate, (i.e., 3D silicon oxide layer), then perform ultrasonic treatment with acetone, alcohol, and deionized water for 5 min each in sequence. Dry under nitrogen for 1 min.
2. Spin-coat a layer of negative photoresist ROL-7133 on the silicon oxide layer using a Spin Coater (Step 1: rotation speed 500 rpm, time 25 s, acceleration 100 rpm/s; Step 2: rotation speed 3,000 rpm, time 54 s, acceleration 200 rpm/s; Step 3: rotation speed 4,000 rpm, time 6 s, acceleration 1,000 rpm/s).
3. Fix the photoresist-coated silicon oxide substrate onto the mask using PET tape, then place it on a heating platform at a constant temperature of 110 ℃ for 90 s.
4. Place the photomask flat on the lithography area of the lithography machine, and perform photolithography patterning under the following lithography conditions: light voltage 0.65 V, time 6.5 s.
5. Place the silicon oxide substrate with completed photolithography patterning in AZ-400K developer solution (stock solution: water = 1:4) for 60 s of development. After removal, clean thoroughly with deionized water, then dry using nitrogen gas.
6. Sputter a 20 nm Ti layer and a 100 nm Au layer sequentially on the surface of the silicon oxide substrate using a magnetron sputtering instrument (power 50 W, time 600 s).
7. Soak the substrate in acetone for 10 min, then wash off the excess photoresist.
8. Rinse the prepared microelectrode chips with deionized water for 1 min, then dry with nitrogen for 1 min.
9. Design the channel geometry width-to-length ratio 15:1 and a short channel of 4μm in the photomask
10. Pattern PET tape (1.2 mm in length, 0.6 mm in width) using an ultraviolet laser cut-out machine as a window to align the position of the gate electrode.
11. Sputter a 100 nm Pt layer sequentially on the surface of the gate electrode using a magnetron sputtering instrument (power 50 W, time 500 s).
12. Pattern PET tape (60 μm in length, 15 μm in width) using an ultraviolet laser cut-out machine as a window to align the position of the channel.
13. Spin-coat the channel following Steps 1.1.1 and 1.1.2.

3. Sensing performance of the OECT-based H₂O₂ sensor with the stacked PEDOT: BTB/PEDOT: PSS semiconductor layer

NOTE: When measuring the OECT-based H₂O₂ sensor under a fixed gate voltage of +0.6 V, do Step 3.1. When measuring under a fixed gate voltage of -0.6 V, follow Step 3.2.

1. Measure the output current (I_DS) variations with different H₂O₂ concentrations under a fixed gate voltage of +0.6 V.
   1. Dip 20 μL H₂O₂ solutions with different concentrations (configured in 0.1× PBS, ranging from 10^-3 M to 10^-11 M at intervals of two orders of magnitude) with the same conductivity of 34.39 ± 0.73 mS cm^-1 sequentially on the channel surface.
   2. Connect the source, drain, and gate electrodes of the H₂O₂ sensor to a dual-port Source Meter, respectively.
   3. Apply V_DS of -0.6 V, V_G of +0.6 V, a measure time of 40-150 s to acquire the quasi-relative equilibrium output I_DS, and a measurement duration of 10 s in single-stage constant voltage mode.
   4. Rinse the channel surface with deionized water prior to measuring the next H₂O₂ solution.
   5. Acquire the steady-state I_DS value.
2. Measure the I_DS variations with different H₂O₂ concentrations under a fixed gate voltage of -0.6 V.
   1. Dip 20 μL of H₂O₂ solutions with different concentrations (configured in 0.1× PBS, ranging from 10^-3 M to 10^-11 M at intervals of two orders of magnitude) with the same conductivity of 34.39 ± 0.73 mS cm^-1 sequentially on the channel surface.
   2. Connect the source, drain, and gate electrodes of the H₂O₂ sensor to the dual-port Source Meter, respectively.
   3. Apply V_DS of -0.6 V, V_G of -0.6 V, a measure time of 40-150 s to acquire the quasi-relative equilibrium output I_DS, and a measurement duration of 10 s in single-stage constant voltage mode.
   4. Rinse the channel surface with deionized water prior to measuring the next H₂O₂ solution.
   5. Acquire the steady-state I_DS value.

Results

This paper initially characterizes the stacked-layer PEDOT: BTB/PEDOT: PSS channel material using SEM, AFM, UV-vis spectroscopy, and electrochemical measurement. SEM and AFM images (Figure 1 and Figure 2) reveal that the 241 nm-thick stacked-layer PEDOT: BTB/PEDOT: PSS exhibits abundant granular structures, in contrast to the relatively smooth surface of the 120 nm-thick single-layer PEDOT: PSS film. The root-mean-square roughness (Rq) values ar...

Discussion

The success of the protocol relies on three interdependent critical steps, each validated by experimental characterization. Firstly, the fabrication of the stacked-layer channel material (Protocol 1.1) requires precise control over PEDOT: PSS spin-coating (4200 rpm for 60 s, followed by baking at 130 °C) and PEDOT: BTB electrodeposition (where the short-circuited PEDOT: PSS channel serves as the working electrode, with an electric potential ranging from 0 to 1 V at a scan rate of 0.1 V s⁻^...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10× Phosphate Buffered Saline (PBS)	Lanbolide Trading Co., LTD	P7209	Purity: ≥99%
3,4-ethylenedioxythiophene (EDOT)	Shanghai Aladdin Biochemical Technology Co., Ltd., China	E105649-25g	Purity: ≥99%
3-glycidoxypropyltrimethoxysilane (GOPS)	Shanghai Aladdin Biochemical Technology Co., Ltd., China	G107576	Purity: ≥97%
Developer	Suzhou Yancai Weina Technology Co., Ltd.	AZ 400K
Bromothymol Blue (BTB)	Sinopharm Group Chemical Reagent Co., LTD	B801811-25g	Purity: ≥95%
Deionized water	Shanghai Aladdin Biochemical Technology Co., Ltd., China	W119424	18.2 M cm
Digital microscope	Olympus Corporation of Japan	DSX1000	A cursory observation of the as-prepared OECT's channel
Dodecyl benzene sulfonic acid (DBSA)	Shanghai Aladdin Biochemical Technology Co., Ltd., China	D106550	Purity: ≥90%
Dual potentiostatic electrochemical workstation	Metrohm AG, Switzerland	VIONIC
Dual-port SourceMeter	Tek Technology Co., LTD	Keithley 2636B
Dual-target magnetron sputtering	Shenyang Kejing Automation Equipment Co., LTD	VTC-600-2HD
Ethylene glycol (EG)	Shanghai Aladdin Biochemical Technology Co., Ltd., China	E119700	Purity: ≥99.8%
Hydrogen peroxide (H2O2)	Huize Biochemical Company	EL	Purity: ≥35%
N-methyl1-2-pyrrolidone (NMP)	Sigma-Aldrich (Shanghai) Trading Co., Ltd.	328634	Purity: ≥99.5%
Poly(3,4-ethylenedioxythiophene): polystyrene sulfonate (PEDOT: PSS)	Sigma-Aldrich (Shanghai) Trading Co., Ltd.	739332	Purity: ≥99.7%
Polyethylene terephthalate (PET)	Sigma-Aldrich (Shanghai) Trading Co., Ltd.	GF09063581
Potassium nitrate (KNO3)	Sinopharm Group Chemical Reagent Co., LTD	10017218	Purity: ≥99.0%
Photoresist	Suzhou Yancai Micro-Nano Technology Co., LTD	ROL-7133
Scanning Electron Microscope (SEM)	Carl Zeiss Company of Germany	Gemini Sigma 300
Spin coater	Jiangsu Leibo Scientific Instrument Co., LTD	EZ4
Sulfuric acid (H2SO4)	Sinopharm Group Chemical Reagent Co., LTD	10021608	95.0-98.0%
Atomic Force Microscopy (AFM)	Bruker Corporation of the United States	Bruker Dimension Icon
Ultraviolet laser marking machine	Shanghai Cifang Electrical Technology Co., LTD	RD-JW355
Ultraviolet-visible spectrophotometric spectroscopy (UV-vis)	Ocean Insight Company of the United States	OCEAN-HDX-UV-VIS