Modified Resazurin Ink Testing and the Fluorescence Probe Method for Simple and Rapid Photocatalytic Performance Evaluation

Abstract

Evaluating the air purification performance of photocatalytic materials typically requires complex gas decomposition tests involving expensive analytical equipment and lengthy testing periods. In this study, photocatalytic performance evaluation methods involving resazurin (Rz) ink and fluorescence probe techniques were investigated as alternatives to conventional gas decomposition tests. TiO₂ films with varying performance levels were fabricated by controlling TiO₂ slurry concentration and the amount of photocatalyst deposited through spin coating. Photocatalytic performances of the synthesised films were then evaluated using the acetaldehyde decomposition method, Rz ink test, and fluorescence probe method for measuring OH radical generation. The acetaldehyde decomposition rate constants showed high correlation with both the Rz colour change rate in modified-pH ink (R² = 0.91) and the OH radical concentration (R² = 0.98). Conventional Rz ink testing for high-performance materials showed rapid colour changes, indicating its limited applicability. Our modified-pH Rz ink enabled facile analysis by ensuring controlled reactivity. Both the modified Rz ink method, which enables quantitative evaluation within five minutes even for high-performance materials, and the fluorescence probe method are suitable as reliable screening tools for photocatalytic air purification materials. These simplified evaluation methods will aid in developing more efficient photocatalysts and advancing environmental purification technologies.

1. Introduction

Since the discovery of the water-splitting reaction (known as the Honda–Fujishima effect) in 1972, titanium dioxide (TiO₂) has been widely used as an environmental purification material [1,2,3]. The superhydrophilic and oxidative decomposition properties of TiO₂ enable the fabrication of self-cleaning exterior walls and tiles, while its strong oxidative decomposition capability is utilised in air purifiers [4,5]. With the increasing severity and diversification of indoor environmental issues, modern air purification technologies are expected to meet significantly high standards. Accurate performance evaluation is essential for healthy markets and product development. However, air purification performance assessments require precisely controlled gas concentrations and humidity along with specialised measurement equipment. These factors create significant resource barriers in product development. Therefore, developing rapid and simple evaluation methods is critical and urgent [6].

The resazurin (Rz) ink-based evaluation method for photocatalysts has been standardised by ISO (ISO21066) [7]. Unlike this method [6], the conventional evaluation methods using organic dyes, such as methylene blue, require several hours for measurement and are susceptible to oxygen interference [8,9]. Rz exhibits a distinct colour change from blue to pink upon photocatalytic reduction, showing a half-life of approximately 1 min under UVA (~365 nm) irradiation. The photocatalytic reduction of Rz, which follows zero-order kinetics (with a concentration-independent reaction rate), enables predictable reaction progress and reproducible measurements [10]. Moreover, the reaction rate remains consistent under both aerobic and anaerobic conditions [11], enabling its practical evaluation independent of the measurement environment [6]. The applicability of the conventional Rz ink test, which is typically used for thin films and low-performance photocatalytic materials [12,13], is limited to moderate-performance materials; Rz-based protocols for evaluating high-performance materials, such as those for gas decomposition, require significant refinement and standardisation.

As many of the photocatalytic decomposition processes occur through OH radical attack, estimating the amount of OH radicals formed on the excited photocatalyst surface is a reasonable method for evaluating photocatalytic performance [14,15]. The generated OH radicals have extremely high reactivity and a shorter lifetime [16,17], making direct detection difficult. Therefore, indirect detection methods using trapping agents have become mainstream approaches [18,19,20]. Conventional OH radical measurement by ESR spin trapping uses 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and requires specialised equipment and careful handling [19,21]. Additionally, owing to ESR equipment specifications, only samples of limited shapes and sizes can be analysed, reducing versatility. The fluorescence probe method provides a simpler approach to OH radical measurement. Organic molecules such as terephthalic acid and coumarin effectively react with OH radicals to form products with strong fluorescence [22,23]. The fluorescence probe method used in our study involved sodium terephthalate (NaTA), which specifically reacts with OH radicals to produce 2-hydroxyterephthalic acid (HTA). As HTA emits blue fluorescence under ultraviolet irradiation, the amount of HTA and OH radicals that have reacted can be indirectly quantified from its fluorescence intensity. This simple reaction mechanism enables the evaluation of photocatalytic performance without the need for specialised equipment. An advantage of using terephthalic acid in the fluorescence probe method is that a single isomer is formed in the reaction with OH radicals [15]. Additionally, terephthalic acid does not react with other reactive oxygen species such as superoxide anion, ozone, or HO₂ radicals [24,25,26]. Measurements can be made with simpler equipment and reagents that are easier to handle compared to those used in ESR [27]. As terephthalic acid dissolves only in alkaline solutions [28], there is a challenge that experimental conditions such as pH and solution conductivity change but by using disodium terephthalate, it can be dissolved even in neutral solutions [29,30,31]. Because of its simplicity and versatility, the fluorescence probe method has been widely used in studies related to plasma decomposition [32,33,34] and the photocatalytic decomposition [35,36,37] of persistent organic pollutants in water. In this method, the calculated amount of OH radicals represents the amount that reacted with the reagent and not the total amount [38]. In experiments under specific conditions, the HTA reaction rate in the presence of oxygen has been reported as 35% [39,40]. However, this value may fluctuate depending on environmental conditions and experimental parameters. As the amount of OH radicals calculated by the fluorescence probe method is not an absolute value, it is used for relative evaluations such as comparing photocatalytic performances.

Conventional photocatalyst evaluation methods, particularly, the Rz ink method, face challenges in rapidly and accurately evaluating high-performance materials. In this study, we developed a pH–modified Rz ink method that enables quantitative evaluation of high-performance photocatalytic materials within a short time. Additionally, we systematically verified for the first time the correlation between the fluorescence probe method, which is a simple OH radical detection technique, and acetaldehyde decomposition performance, demonstrating the effectiveness of both methods for evaluating air purification performance. This combined approach significantly streamlines the performance screening process for photocatalytic materials.

The purpose of this study is to verify the effectiveness of the Rz ink test and fluorescence probe method as alternative methods for air purification performance evaluation. Specifically, we conducted the following experiments: (1) fabrication of TiO₂ films with different performance levels by controlling spin-coating conditions and TiO₂ slurry concentration, (2) standard performance evaluation using the conventional acetaldehyde decomposition test, (3) measurement of colour change rate using the modified-pH Rz ink test, (4) measurement of OH radical generation by the fluorescence probe method, and (5) correlation analysis between these methods. Using standard photocatalyst samples with varying performance levels, strong positive correlations between both methods and acetaldehyde decomposition testing were confirmed. Furthermore, the applicability of modified Rz ink for experimentation with high-performance photocatalytic materials was confirmed. This study provides crucial foundational knowledge that is expected to expedite the application of the Rz ink test and fluorescence probe method in preliminary photocatalyst evaluations, such as in screening high-performance materials.

2. Results

2.1. Fabrication and Performance Evaluation of Standard TiO₂ Films

Scanning electron microscopy (SEM) was used for cross-sectional observations of TiO₂ films after exposure to a focused ion beam (FIB) [41]. Film thickness increased with decreasing spin-coating speed and increasing slurry concentration (Figure 1). The average film thickness, measured at six different locations, was 1.82 μm at 1000 rpm (T1), 1.29 μm at 2000 rpm (T1), 1.02 μm at 4000 rpm (T1), 0.36 μm at 4000 rpm (T2), 0.13 μm at 4000 rpm (T5), and 0.07 μm at 4000 rpm (T10). All samples contained internal voids, indicating the formation of porous TiO₂ films. Notably, the observed pores were significantly larger than the crystallite size of TiO₂ in the commercial slurry TKD-701 (6 nm), indicating the formation of secondary particles owing to the aggregation of TiO₂ particles in the slurry during film formation.

X-ray diffraction (XRD) analysis indicated a broad amorphous peak within 15–35° corresponding to the glass substrate (Figure 2a). Analysis of the XRD patterns using the Hanawalt method [42,43] revealed characteristic peaks of anatase TiO₂ at 25.3° (101), 37.8° (004), and 48.0° (200). No rutile peaks were detected, indicating the formation of a pure anatase phase (Figure 2b). These results are consistent with the specifications provided by the slurry manufacturer. Moreover, the intensity of the main diffraction peak at ~25° decreased with decreasing spin-coating speed and solution concentration, consistent with the film thickness variations shown in Figure 1. Notably, because crystalline peaks were below the detection limit, only the amorphous peak corresponding to the glass substrate was detected in the spectrum of the thinnest film (4000 rpm-T10, ~70 nm thick).

Photocatalytic performances of the fabricated films were evaluated through acetaldehyde decomposition tests using the six TiO₂ film samples described in Section 2.1 and Section 2.2. While a minimal reduction in acetaldehyde concentration was observed in the control experiment without TiO₂, significant photocatalytic activity was observed in all systems containing TiO₂ films. Decomposition efficiency increased with decreasing spin-coating speed and increasing slurry concentration (Figure 3a). The most active sample (1000 rpm-T1) decomposed ~90% of the initial acetaldehyde concentration after 60 min, while the thinnest film (4000 rpm-T10) showed ~30% acetaldehyde decomposition. Decomposition kinetics were analysed assuming a pseudo-first-order reaction model for the initial 30 min and a rate constant k₁ (C = C₀ exp(−k₁ t)). High correlation coefficients (R² > 0.99) were observed under all conditions, indicating a consistent reaction mechanism. Furthermore, strong positive correlations were observed between rate constant k₁ and both TiO₂ film thickness (Figure 3b) and anatase TiO₂ (101) peak intensity (Figure 3c).

2.2. Evaluation Method of Photocatalytic Performance Using Rz Ink Tests

The ISO–standardised Rz ink test was used to evaluate the photocatalytic performance of the synthesised samples during acetaldehyde decomposition. Rz ink prepared using standard methods was applied to both samples. While the blue colour remained visible on the bare glass substrate (Figure 4a), it disappeared from the 1000 rpm-T1 sample (Figure 4b). The proportion of the blue component Bt, calculated from recorded images, decreased by ~30% in the 1000 rpm-T1 sample compared with that in the glass substrate. During the ~10 s spin-coating process without UV irradiation, the colour of the sample film changed from blue to pink to colourless, suggesting reduction of the Rz ink on the TiO₂ substrate.

Based on the results shown in Figure 4, the Rz ink used for testing was modified by adding NaOH solution to intentionally slow the photocatalytic reaction rate. Subsequently, the colour of the 1000 rpm-T1 sample, which showed the highest performance in the acetaldehyde decomposition test, was evaluated after ink application. The blue component increased at a pH > 11 (Figure 5). Notably, the sample exhibited the highest Bt value at pH 12.7 and maintained its blue colour for more than 1 h after ink application.

Experiments were conducted with six TiO₂ films of different thicknesses. The Rz ink with added NaOH (pH 12.7) maintained its blue colour regardless of film thickness. In contrast, without NaOH addition, the Bt value of Rz ink decreased with increasing TiO₂ film thickness, leading to progressive decolourisation (Figure 6a,b).

After applying pH–adjusted Rz ink (pH 12.7), the colour changes of the six different TiO₂ samples were monitored under UV irradiation. An analysis of the relationship between the UV irradiation time and red component ratio Rt (Figure 7a–f) showed that the rate of colour change (slope of the fitted line) increased with decreasing spin-coating speed and increasing slurry concentration. This trend is consistent with the results of acetaldehyde decomposition tests. For the 1000 rpm-T1 and 2000 rpm-T1 samples, the Rt increased and then decreased after 15–30 s, indicating a colour transition from blue to pink to colourless. The other samples did not exhibit complete decolourisation. Strong positive correlations were observed between TiO₂ film thickness and the rate of Rz ink colour change (Figure 8a) as well as between the rate constant k₁ of acetaldehyde decomposition and the rate of Rz ink colour change (Figure 8b).

2.3. Evaluation Method of Photocatalytically Generated OH Radicals

Fluorescence measurements using disodium terephthalate (NaTA) were conducted on the six TiO₂ samples. After 1 h of UV irradiation, the fluorescence peaks of the samples showed increasing intensity with decreasing spin-coating speed and increasing slurry concentration (Figure 9a). Moreover, a consistent spectral shape was observed across all conditions, suggesting a uniform reaction mechanism. The OH radical concentrations calculated from the fluorescence intensities exhibited strong positive correlations with both TiO₂ film thickness (Figure 9b) and the rate constant of acetaldehyde decomposition (k₁) (Figure 9c).

3. Discussion

In this study, the photocatalytic performance of TiO₂ films was assessed using acetaldehyde decomposition tests, a modified Rz ink method, and OH radical generation tests.

Cross-sectional observations of the TiO₂ films by FIB-SEM indicated film thicknesses, controlled by the spin-coating speed and slurry concentration, within 70–1820 nm (Figure 1) along with the formation of secondary particles through primary particle aggregation, resulting in porous film formation. This porous structure likely contributes toward photocatalytic activity enhancement through improved reactant diffusion and light scattering/absorption.

XRD analysis confirmed the formation of a pure anatase TiO₂ phase in the synthesised films (Figure 2). The intensity of the main diffraction peak (at ~25°) was correlated with TiO₂ film thickness, indicating that film thickness varied with the deposition conditions, whereas the crystal structure remained constant.

In acetaldehyde decomposition tests, the six TiO₂ films exhibited different reaction rate constants (k₁ values), which showed strong positive correlations with both film thickness and (101) peak intensity (Figure 3). Considering the porous structure of the TiO₂ films, the variation in their acetaldehyde decomposition performance was attributed to their increased effective surface area with increasing film thickness.

Unlike standard Rz ink, which shows limited utility in the evaluation of high-performance photocatalytic materials, Rz ink with a pH of 12.7 enables the quantitative evaluation of such materials by controlling the reaction rate (Figure 4, Figure 5, Figure 6 and Figure 7). An increased pH likely reduces the redox potential of Rz, lowering its reduction rate. This modification enables colour-change tracking under UV irradiation, even for samples with high acetaldehyde decomposition performance. The strong positive correlation between the colour change rate of modified Rz ink and acetaldehyde decomposition activity demonstrates the effectiveness of this method as a simple evaluation technique for air purification (Figure 8). Notably, the correlation coefficient for linear fitting in Figure 8b (R² = 0.91) is slightly lower than that for OH radical measurement (R² = 0.98), which may be attributed to measurement errors inherent to the simplified coating and image capture procedure of the Rz ink method. Notably, the 1000 rpm-T1 and 2000 rpm-T1 samples showed a colour change from blue to pink to colourless. Rz undergoes an irreversible reduction to pink resorufin [44,45], which is further reduced to colourless dihydroresorufin [12,44]. Thus, the second colour change observed in these samples likely results from the formation of dihydroresorufin. Because this is a consecutive reaction, analysing the colouration of the intermediate species (resorufin) may be challenging during high-performance photocatalyst testing. Therefore, while testing such materials, the initial slope shown in Figure 7 may include the secondary transformation from resorufin (pink) to dihydroresorufin (colourless). This secondary transformation could lead to an overestimation of dRt/dt for high-performance samples such as 1000 rpm-T1, contributing to the slightly lower correlation coefficient observed in Figure 8b.

OH radical generation measurements also indicated strong correlations between film thickness and crystallinity (Figure 9). The strong correlation between the acetaldehyde decomposition rate constant and OH radical generation suggests that OH radicals function as the primary active species in the decomposition reaction. Notably, the fluorescence probe method enables simpler measurements than ESR and can be applied to samples of various shapes and sizes. When using the fluorescence probe method, it is important to consider that measurement values may fluctuate due to light-scattering effects. If the NaTA solution used in the test contains TiO₂ particles or fine air bubbles, the excitation light and fluorescence may be reduced by scattering, potentially affecting the accuracy of HTA concentration measurements. However, as measurements with the probe are conducted under static conditions, bubble formation is considered minimal. In this study, TiO₂ was sufficiently immobilised on the glass substrate surface, making light scattering from detached TiO₂ particles unlikely. These considerations suggest that the fluorescence probe method is particularly effective for TiO₂ fixed on substrates such as glass, while special attention must be paid to fluorescence values when analysing powder samples. For powder photocatalyst evaluation, potential solutions include centrifuging the NaTA solution after the photocatalytic reaction to remove particles before fluorescence measurement, or preparing dedicated calibration solutions by dispersing the same amount of TiO₂ powder in the HTA solution used for calibration, followed by centrifugation.

These findings indicate that analyses involving modified Rz ink and fluorescence probe testing can function as highly reliable, effective, and simple alternative evaluation techniques for photocatalytic air purification. Therefore, this study provides meaningful insights regarding photocatalytic performance analysis and proposes evaluation methods with high potential for application in material screening and quality control during photocatalytic product development.

4. Materials and Methods

4.1. Fabrication of TiO₂ Films on Glass Substrates

Microslide glass (S1111, Matsunami Glass Ind., Ltd., Osaka, Japan) was cut into 25 mm × 25 mm pieces and used as the substrate. Four different concentrations of a TiO₂ photocatalytic dispersion (TKD-701, Tayca Corporation, Osaka, Japan), namely, undiluted (T1), 2-fold (T2), 5-fold (T5), and 10-fold (T10), were prepared by dilution with 2-propanol. These TiO₂ solutions were deposited on glass substrates using a two-step spin-coating process; the first step was conducted at 500 rpm for 5 s, and the second step was conducted at 1000, 2000, or 4000 rpm for 10 s. By combining different solution concentrations and spin-coating conditions, six types of TiO₂ films were fabricated, namely, 1000 rpm-T1, 2000 rpm-T1, 4000 rpm-T1, 4000 rpm-T2, 4000 rpm-T5, and 4000 rpm-T10. After deposition, the samples were dried at room temperature (20 ± 5 °C) and pretreated with UV irradiation (1.0 mW/cm²) for 12 h. The UV pretreatment was performed to remove potential organic contaminants from the sample surface and to standardise the initial conditions before testing. This pretreatment was conducted in accordance with JIS and ISO standard testing methods. Under these fabrication conditions, the amount of TiO₂ deposited on glass substrates could be controlled to produce six samples with different photocatalytic performances.

4.2. Structural Analysis of TiO₂ Films

An SEM instrument equipped with an FIB component (FEI Company, Ltd., Hillsboro, OR, USA) was used for the cross-sectional sample preparation and structural analyses of the TiO₂ films fabricated. For cross-sectional analysis, a thin carbon coating was applied onto the sample surface before machining to avoid damage. Subsequently, TiO₂ film cross-sections were prepared by FIB milling of the sample surface at an accelerating voltage of 30 kV. Structural observations were carried out under an accelerating voltage of 2 kV, dwell time of 1 μs, and 32 frame integrations. The θ–2θ scanning XRD patterns of the TiO₂ films were recorded on a Rigaku SmartLab using the Cu Kα line (λ = 1.5406 Å) over a 2θ range of 15–75° with a step size of 0.01° and scan speed of 10°/min. The Hanawalt method was used for phase identification [42,43]. Changes in thickness were estimated from changes in the diffraction intensity of the (101) peak in the anatase phase.

4.3. Photocatalytic Performance Test for Acetaldehyde Decomposition

The photocatalytic performances of the TiO₂ films were evaluated using a modified acetaldehyde decomposition test [46] based on JIS R1757 [47] and ISO 19,652 [48] in a 0.5 L reactor containing four glass samples (25 mm × 25 mm) under UV irradiation (1.0 mW/cm²). The initial concentration of acetaldehyde was set to 100 ppm, and the change in acetaldehyde concentration was monitored for 1 h using a GASERA ONE photoacoustic gas analyser (Gasera, Ltd., Turku, Finland).

4.4. Evaluation of Photocatalytic Performance Using Rz Ink Tests

The photocatalytic performance of the fabricated TiO₂ films was evaluated using a Rz-based photocatalytic indicator ink [7]. To prepare this ink, hydroxyethyl cellulose (750 mg) (HEC; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was dissolved in ultrapure water (49.25 g) with stirring for 12 h at room temperature (20 ± 5 °C). Subsequently, glycerol (5 g) and polysorbate 20 (100 mg) (FUJIFILM Wako Pure Chemical Corporation, Japan) were added with continuous stirring until complete dissolution, followed by the addition of Rz (50 mg) (TCI Co., Ltd., Tokyo, Japan) and stirring for 8 h. To examine the effect of pH on ink colour, NaOH solutions (100 μL) with different concentrations (10 M, 1 M, 0.1 M, 0.01 M, and 0.001 M) were added into this ink. The modified ink was applied to the as-synthesised TiO₂ films by spin coating at 1500 rpm for 10 s. The colour change of the ink under UV irradiation (1.0 mW/cm²) was monitored by recording digital images at intervals of 5–14 s. The RGB values of these images were analysed using ImageJ software ver. 1.54g (Figure 10) [49].

4.5. Evaluation of Photocatalytically Generated OH Radicals

The concentration of the photocatalytically generated OH radicals was measured using the NaTA fluorescence probe method [30,31], whose basic principle is as follows: OH radicals react with TA to produce HTA (Figure 11), and as HTA emits characteristic fluorescence, the amount of HTA produced can be quantified by measuring fluorescence intensity. Moreover, as the reaction proceeds stoichiometrically at a ratio of 1:1, the amount of OH radicals that reacted with TA can be directly estimated from the amount of HTA produced. Here, an NaTA solution (20 mL; 5 mM) (TCI Co., Ltd.) was placed in a Petri dish containing the four photocatalyst samples and irradiated with UV light (1.0 mW/cm²) for 1 h. The fluorescence spectra of these solutions were measured using a SEC2020 UV/vis Spectrometer (ALS Co., Ltd., Tokyo, Japan). A probe combining optical fibres for 310 nm LED excitation light and fluorescence detection was inserted into the test solutions to record data over 400–500 nm. The OH radical concentration was quantified using a calibration curve prepared from standard solutions of 2-hydroxyterephthalic acid (TCI Co., Ltd.).

5. Conclusions

In this study, the effectiveness of using a modified Rz ink test and the fluorescence probe method as simple evaluation techniques for assessing photocatalytic air purification performance was investigated. TiO₂ film samples with varying thicknesses were fabricated by controlling spin-coating speed and slurry concentration to validate the effectiveness of these methods. Notably, adjusting the pH of Rz ink to 12.7 enabled the quantitative evaluation of high-performance photocatalytic materials, which is challenging using standard Rz ink [7]. The proposed method for evaluating OH radical generation uses a fluorescence probe as a simple alternative to ESR. This method maintains a strong correlation with the acetaldehyde decomposition performance of the system. Both modified Rz ink testing and fluorescence probe evaluation methods showed strong correlations with acetaldehyde decomposition performance, confirming their high reliability as alternative evaluation methods for photocatalytic air purification performance. Thus, the evaluation methods proposed in this study have potential for application in material screening and quality control during photocatalytic product development. Future studies will focus on expanding the application of these methods to a wide range of photocatalytic materials and validating them under different evaluation conditions.