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. 2026 Jul 22;11(30):44801–44811. doi: 10.1021/acsomega.6c01270

Application of Eco-Friendly Fluorescent Dyes in Microplastic Detection

Dovilė Motiejauskaitė 1, Karolina Barčauskaitė 1,*
PMCID: PMC13448976  PMID: 42569065

Abstract

The detection of microplastics using fluorescent staining has gained increasing attention due to the need for efficient and cost-effective analytical approaches. This study evaluates the effectiveness of three eco-friendly fluorescent dyescoumarin-6, coumarin-153, and curcuminwhile Nile Red was used as a control, for staining pristine and mechanically and thermally damaged polymer standards under ultraviolet (UV), blue, and green light illumination. Several commonly occurring polymers were analyzed to assess staining efficiency and intensity distribution. The results showed that polypropylene (PP) was the most difficult polymer to stain, with effective fluorescence achieved only using Nile Red and all tested concentrations of coumarin-6. In contrast, polyethylene terephthalate (PET) exhibited the highest staining efficiency, fluorescing under both UV and blue light with all dyes except coumarin-153 at lower concentrations. Optimal staining of high-density polyethylene (HDPE) was achieved using 50 μg mL–1 coumarin-6 under UV illumination, while Nile Red exhibited the highest fluorescence intensity under blue light, regardless of polymer damage. Mechanical and thermal damage generally increased fluorescence intensity and improved uniformity of staining across polymers. However, this trend was not observed for PP, where damage resulted in increased fluorescence asymmetry. Overall, the findings demonstrate that polymer type, dyes, illumination source, and material damage level strongly influence microplastic staining efficiency and should be carefully considered in microplastic analysis.

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1. Introduction

Fluorescent staining of microplastics (MP) is a simple, rapid, inexpensive, and convenient method for studying their distribution in environmental samples. Compared to spectral or chromatographic methods, which can be very expensive, the cost of dyes is lower, although it requires the use of fluorescence microscopy. , This method is also fasterthe staining protocol can take as little as 1 h, while FTIR scanning for a single filter can take up to 9 h, including sample preparation. Fluorescent methods require less expertise than spectral (e.g., FTIR) or gas chromatography–mass spectrometry (GC–MS) methods, which require skilled operators and lengthy preparation processes. Furthermore, fluorescent quantification can be automated using photo analysis software. The main advantages of fluorescent staining are speed, simplicity, and suitability for the analysis of large volumes of environmental samples. However, this is not a perfect analytical method, and it also has a number of disadvantages, such as low selectivity and complex sample preparation. A detailed comparison of the most commonly used analytical techniques is presented in Table .

1. Comparison of Analytical Techniques.

Method Identification Merits Drawbacks References
Optical microscopy Visual (shape, color, size, number) Rapid, low-cost, simple operation, accurate fibers detection No chemical identification, subjective interpretation, work-intensive, strong user bias ,
μ-FTIR Polymer-specific (characterization of chemical bonds and molecular structure), particle number, morphological features (size) Accurate identification without sample destruction, well-established, extensive spectral libraries, AI friendly Size limitations, sample purification required −
μ-Raman Polymer-specific (molecular composition), particle number, morphological features (size, shape) Suitable for submicron particle analysis with high spectral detail, AI friendly Sample purification required, fluorescence interference ,
Pyrolysis–GC–MS Chemical composition (polymers, additives), quantitative data (mass) No size limit, highly accurate Destructive, sample purification required, matrix effect, limitations associated with the analysis of polymers at varying concentrations
Fluorescence microscopy methods Indirect (staining based on hydrophobicity), visual (shape, size, number) Enhanced contrast against background, AI friendly, high throughput False positives, sample purification required, background noise, applicability varies depending on polymer type ,
Nanoparticle-based probes (e.g., quantum dots) Target-specific fluorescence sensing, visual (shape, size, particle count) High photostability, good fluorescence properties, target-specific, selective detection, AI friendly False positives, background noise, still under development ,
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Nile Red (NR) is a lipophilic and metachromatic dye that fluoresces only when bound to hydrophobic materials such as lipids or plastics. It was initially used as a specific fluorescent dye to identify intracellular lipids. NR was later proposed as a promising chemical tool for the visual detection of microplastics in the marine environment. The wide spectrum of fluorescent colors observed in Nile Red-labeled MPs is associated with their polarity. Therefore, Maes et al. (2017) proposed this dye as a potential tool for differentiating MPs based on their polarity. Recent studies using artificial intelligence have demonstrated the ability to classify Nile Red-labeled MPs with 80% accuracy. These results highlight the potential of NR as a valuable tool for accurately identifying and distinguishing between different MP types, thus contributing to the improvement of environmental monitoring and the quantification of MP distribution in the environment. However, NR is not considered environmentally friendly; therefore, the search for natural fluorescent compounds is essential.

Coumarin is an oxygen-containing heterocyclic compound. It was first isolated in 1820 from Tonka beans (Dipteryx odorata). Since coumarins are secondary metabolites of bacteria, plants, and fungi, they are found in many different natural sources: essential oils, fruits, green tea, and other foods. However, coumarins are interesting not only for their biological activity but also for their photosensitivity and ability to fluoresce in the visible light range. , Among the coumarin derivatives, coumarin-6 and coumarin-153 are significant fluorescent dyes. Coumarin-6 has a high quantum yield, which allows efficient conversion of absorbed light into fluorescence. Coumarin-153, due to its sensitivity to the polarity of the environment, is often used as a probe molecule to determine the properties of the microenvironment. , Both compounds can be used to color plastics, especially nonpolar or moderately polar plastics. The use of colorants such as coumarins not only allows plastics to be colored but also enables them to be applied to various technological solutions, such as the detection of microplastics or the labeling of materials using fluorescence microscopy or spectroscopy. , Such applications not only improve the functional properties of plastics but also help in the development of sustainable environmental-monitoring solutions.

Curcumin is the main yellow bioactive compound found in turmeric (C. longa). It is widely used as a spice, food preservative, and colorant. It is a nontoxic, highly promising natural dye with a wide range of biological applications. It absorbs light in the visible spectrum and exhibits fluorescence, and its emission properties are strongly dependent on the polarity of the environment. The photochemical behavior of curcumin, including reactions with oxygen, varies depending on the microenvironment of the molecule, such as polar or nonpolar, protic or aprotic solvents. Curcumin is known to be highly soluble in polar organic compounds such as methanol, dimethyl sulfoxide, and acetonitrile. , Curcumin has been used for centuries as a natural colorant that has no side effects on health or harmful effects on the environment and therefore can be considered a safe alternative in various studies.

Although dye selection is one of the essential steps in applying fluorescence to microplastic analysis, it is also necessary to consider parameters such as the size, shape, color, and polymer composition of the MP particles being analyzed, dye concentration, solvent, and staining temperature. It is known that the detection efficiency of MP decreases with decreasing particle size. Small particles stain less strongly and have a weaker fluorescence intensity, making them more difficult to detect than larger particles. In addition, the shape of MP can affect the staining effect. Studies have shown that fibers are particularly difficult to stain compared to fragments due to their irregular shape. In addition, the color of MP can interfere with staining and quantification. The most used plastics are white, which does not significantly affect the staining efficiency. However, certain polymers, such as black polyvinyl chloride (PVC), are difficult to stain and detect. , The polymer composition of MP determines the interaction between dye and microplastics. Fluorescent staining using lipophilic dyes such as NR is effective in detecting small particles of polyethylene (PE), polypropylene (PP), polystyrene (PS), or polycarbonate (PC) due to their high hydrophobicity, while PVC and polyamide (PA) are more difficult to detect due to their low hydrophobicity. The fluorescence intensity of stained MP is affected by the concentration of the dye. It has been found that the fluorescence intensity first increases up to a certain limit and then decreases. This is due to the aggregation of the dye at high concentrations, which reduces the fluorescence intensity. , Another important aspect when staining MP with fluorescent dyes is the choice of the dye solvent. It is known that the efficiency of the dye depends on the polarity of the solvent. In addition, some solvents can degrade plastic, for example, PS can be dissolved in chloroform and acetone. A solution is often proposed when the dye solvent is acetonemixing it in equal parts with a more plastic-friendly solvent, such as ethanol or methanol. Several studies have been conducted to determine the best solvents for different dyes, so choosing the right one for MP analysis is not difficult. ,,,, Temperature has a significant impact on dyeing efficiency, as the adsorption of dyes on the surface of the MP or the incorporation of dyes into the polymer molecules is temperature-dependent. Fluorescence is only maintained for a short time when microplastics are dyed at room temperature (25 °C). Studies have shown that after 2 months, 73.5% of the MP dyed with NR dyes lost fluorescence due to desorption of dye molecules from the MP surface. Heating helps to enhance dyeing efficiency, ensuring stronger and more stable fluorescence over a long period of time (more than 2 months). The fluorescence intensity increases when the temperature increases from 20 to 50 °C, but it must be lower than the melting point of the polymers. Heating also improves the dyeing of less hydrophobic plastics such as PVC and polyethylene terephthalate (PET). Heating to 50–60 °C is recommended to achieve stronger and more stable dyeing.

The aim of this work was to investigate the applicability of coumarin-6, coumarin-153, and curcumin in microplastic analysis and to assess whether plastic damaged by mechanical and thermal factors can be dyed more effectively using more environmentally friendly dyes. To our knowledge, this is the first study in which coumarin-153 was employed to dye MP particles. Moreover, this study will combine a systematic multidye evaluation with polymer-specific analysis of staining behavior in microplastics. In particular, it integrates fluorescence intensity with image-derived skewness as complementary quantitative metrics, alongside spectroscopic characterization (UV–vis and ATR-FTIR), to provide a more comprehensive assessment of dye performance. This combined approach enables an improved understanding of dye–polymer interactions and their impact on fluorescence-based detection.

2. Materials and Methods

2.1. Reagents

Nile Red (Apollo Scientific Ltd., England), coumarin-153 (Sigma-Aldrich, USA), coumarin-6 (Sigma-Aldrich, India), curcumin (Sigma-Aldrich, China), low-density polyethylene (LDPE) beads, 5 mm (GoodFellow, Hamburg, Germany), high-density polyethylene (HDPE) beads, 2–4 mm (GoodFellow, Hamburg, Germany), polypropylene spheres, 3.18 ± 0.05 mm (PP) (GoodFellow, Cambridge, England), polyamide (nylon 6.6) spheres, 1.59 ± 0.05 mm (PA 6.6) (GoodFellow, Hamburg, Germany), polystyrene microbeads, 250 μm (PS) (Sigma-Aldrich, USA), acetone analytical grade (VWR International, Vienna, Austria), acetonitrile HPLC grade (Sigma-Aldrich, China), methanol HPLC grade (Macron Fine Chemicals, Norway), ethanol 96% (MV GROUP Production, Lithuania), and hexane 97.0% (Sigma-Aldrich, China) were used. The distilled water used in the study was prepared with a Millipore ELIX 100 water purification system.

2.2. Preparation of Standard Microplastic Particles

Automated rotary microtome CUT 6062 (SLEE Medical, Germany) was used to prepare HDPE and LDPE 100 μm thick sheets. Microplastic standard particles were embedded in paraffin using a paraffin embedding system MPS P II (SLEE Medical, Germany). Before embedding, the particles were placed in metal molds and completely infiltrated with molten paraffin to ensure uniform distribution and structural stability. After paraffin infiltration, the blocks were allowed to solidify by placing them on a special surface at −5 °C in the system. The samples were then cut using a microtome. Sections were cut into 100 μm thick sheets. MP particles were separated from the paraffin, and paraffin residues were removed by washing the particles in hexane and then in ethanol. Microplastic sheets were stored in ethanol in a sealed glass container.

PET microplastics were produced by using a water bottle. The bottle was cut into 1–5 mm sheets, and the polymer was confirmed using Raman spectrometry (Ramanstation 400F, PerkinElmer). The Raman instrument was equipped with a 250 mW near-infrared 785 nm laser and a CCD detector. All measurements were performed at 100% laser power, and Raman spectra were collected using an automated Raman focus and resolution of 4 cm–1, 10 accumulations, and 2 s acquisition in the spectral range from 0 to 3300 cm–1. The resulting spectrum was compared with a standard clear PET spectrum from the Plastic Action Center database SLoPP library. The results are presented in Supporting Information S1.

2.3. Microplastics Staining

Nile red dye effectively stains commonly encountered polymers such as PP, low-density polyethylene (LDPE), high-density polyethylene (HDPE), PS, PC, polyurethane (PUR), and PA, although its effectiveness on polyethylene terephthalate (PET) and PVC remains controversial. The NR solution was prepared by mixing 1 mg of NR dye with 50 mL of 99.5% acetone and 50 mL of 97% hexane to achieve a final concentration of 10 μg mL–1. , Nile Red was used as a reference to determine the staining efficiency of the more environmentally friendly dyes: coumarin-6, coumarin-153, and curcumin. Three different dye concentrations were used for staining: 10 μg mL–1, 15 μg mL–1, and 50 μg mL–1, for six different polymers: HDPE, LDPE, PA, PP, PET, and PS. Acetonitrile was used as the solvent for coumarin-153 and curcumin, , while for coumarin-6, a 74.6% acetone-ethyl alcohol mixture in a 1:1 ratio was used. Staining was performed using an ultrasonic bath (VWR, USC-TH) for 45 min at 30 °C, since the tested fluorescent compounds are sensitive to high temperatures. However, according to the work of Lv (2019), MP particles were stained with Nile Red by raising the temperature to 65 °C. Eight units of each polymer were stained in separate chemical beakers by pouring 30 mL of the dye and covering the beakers with aluminum foil. Staining was performed in the dark. To evaluate the staining efficiency, the fluorescence intensity and the skewness of the fluorescence distribution were determined using the ImageJ program.

To evaluate the effectiveness of the dyes in staining environmentally exposed microplastics, PP, PET, LDPE, and HDPE standards were mechanically crushed and thermally treated using a coffee grinder. The dye concentrations used in this study were: NR10 μg mL–1, coumarin-615 μg mL–1, coumarin-15315 μg mL–1, and curcumin15 μg mL–1. Staining was performed under the conditions described above, and fluorescence intensity and fluorescence distribution uniformity (skewness) were assessed.

2.4. Fluorescence Analysis Using Microscopy

Microscopy was performed using a NIKON Eclipse Ni–U fluorescence microscope. Samples were photographed using three types of fluorescence filters: UV (excitation wavelength (EX) 330–380 nm), blue (EX 450–490 nm), and green (EX 510–560 nm) light filters. The ImageJ program was used to measure fluorescence intensity and fluorescence skewness. In order to avoid color distortion in the images obtained, the work was performed in a dark environment. The average fluorescence intensity of the particles was calculated according to the defined mean gray value function in the ImageJ program. A region of interest (ROI) was selected, and the average gray value of all pixels in the selected area was calculated. The average gray value of the background was also calculated. Subsequently, the fluorescence intensity of the particles was recalculated by subtracting the average gray value of the background from the average gray value of the selected particle ROI in each image.

2.5. UV–Vis Spectrophotometric Analysis

UV–vis spectrophotometric analysis was performed to evaluate the optical properties of dye solutions before and after the staining of polymer particles. Only the supernatant dye solutions were analyzed following the staining procedure. Measurements were carried out using a Shimadzu UV-1280 UV–vis spectrophotometer. Spectra were recorded over a wavelength range of 190–800 nm, with a scan speed set to medium and a scan pitch of 1.0 nm. A 1 cm-length glass cuvette was used for all measurements. All spectra were obtained under identical conditions to ensure comparability between samples before and after staining

2.6. Statistical Analysis

All analyses were performed in triplicate, each replicate had 10 units of different polymers separately, and 0.2 mL of PS suspension was added to each replicate. The research data obtained were processed using Microsoft Office Excel. The statistical reliability of the data was assessed using the two-way analysis of variance (ANOVA) method for quantitative characteristics, followed by the Fisher test (LSD), using the RStudio 4.2.2 software package.

3. Results and Discussion

3.1. Changes in UV–Vis Absorbance of Dyes before and after Staining

UV–vis spectra of dye solutions before and after staining did not show a significant decrease in the absorption of most dyes, but the changes were concentration-dependent. In some cases, an increase in absorbance intensity was observed after staining. The spectral data is presented in Figure . It is observed that the absorption of Nile Red at 520 nm increases slightly. An increase in the absorption of coumarin-6 and coumarin-153 is observed at 10 and 15 μg mL–1 concentrations, but the absorption at 50 μg mL–1 concentration does not change. A slightly different effect is recorded with curcumin. Here, a 2-fold effect is observed at 10 μg mL–1 concentration, and the absorption decreases at 420 nm but increases at 280 nm. Dyes at other concentrations showed no change. The observed increase in the absorbance of different dyes may be attributed to changes in dye aggregation state or alterations in the molecular environment following interaction with polymer surfaces. This behavior can be explained by the properties of hydrophobic dyes, as all analyzed dyes possess them, which, depending on their microenvironment, can undergo aggregation and degradation processes. , In addition, the slightly different change in the spectrum of curcumin can also be explained by the fact that although it exhibits hydrophobic properties, it is also more polar (has −OH groups). These results indicate that UV–Vis absorbance alone may not directly reflect dye interaction with polymer surfaces, as spectral changes can also arise from physicochemical transformations of the dye in solution. ATR-FTIR spectra of the dyes before and after the staining procedure, as well as spectra of the polymers before and after staining, are provided in Supplementary Material S5, illustrating whether and how the dyes influence the structure of different polymers. As no major spectral changes were observed, these data are not discussed in detail in the main text.

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Changes in UV–Vis spectra of dyes before and after staining.

3.2. Detection of Fluorescence Using Various Dyes

Very few alternatives to NR have been described in the literature. Several researchers have tested another chemical dye, BODYPI, while others have tried more environmentally friendly alternatives such as Safranin T, coumarin-6, and curcumin to dye polyamide, polystyrene, and polyester fibers. , Coumarin-153 has not been used in microplastic studies to date.

Using four different fluorescent dyes, it was found that plastic fluorescence depends on several factors: polymer type, dye type and concentration, and the excitation wavelength used. Table shows that when using NR, fluorescence is detected almost at all wavelengths for all polymers; however, when UV excitation light is used, fluorescence is not detected with LDPE, PA, and PS polymers. When using more environmentally friendly dyes, fluorescence varies even more, for example, all three alternative dyes do not emit fluorescence under green excitation light. It was also found that curcumin is not effective in dyeing polypropylene.

2. Fluorescence Detection Using Different Fluorescent Dyes .

  Polymer type HDPE LDPE PP PA PS PET
UV (EX 330–380) Undyed polymer – - - + + +
Nile Red 10 μg mL–1 + - + - - +
Coumarin-6 10 μg mL–1 + + + + + +
15 μg mL–1 + - - + + +
50 μg mL–1 + - - + + +
Coumarin-153 10 μg mL–1 – - - + + -
15 μg mL–1 – - - + + -
50 μg mL–1 – - - + + +
Curcumin 10 μg mL–1 – + - + + +
15 μg mL–1 + + - + + +
50 μg mL–1 + - - + + +
Blue (EX 450–490) Undyed polymer – - - + + -
Nile Red 10 μg mL–1 + + + + + +
Coumarin-6 10 μg mL–1 + + + + + +
15 μg mL–1 + + + + + +
50 μg mL–1 + + + + + +
Coumarin-153 10 μg mL–1 – - - - - -
15 μg mL–1 – - - + + -
50 μg mL–1 – - - + + +
Curcumin 10 μg mL–1 + + - + + +
15 μg mL–1 + + - + + +
50 μg mL–1 + + - + + +
Green (EX 510–560) Undyed polymer – - - - - -
Nile Red 10 μg mL–1 + + + + + +
Coumarin-6 10 μg mL–1 – - - - - -
15 μg mL–1 – - - - - -
50 μg mL–1 – - - - - -
Coumarin-153 10 μg mL–1 – - - - - -
15 μg mL–1 – - - - - -
50 μg mL–1 – - - - - -
Curcumin 10 μg mL–1 – - - + - -
15 μg mL–1 – - - + - -
50 μg mL–1 – - - - - -
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a

Note: The symbols + and – indicate positive and negative fluorescence, respectively.

It was found that coumarin-153 stains only polar polymers: PA, PS, and PET, regardless of concentration. We can state this because autofluorescence is lower than fluorescence after staining. This result opens the possibility of using coumarin-153 to determine the polarity of polymers. Until now, NR has been used for this purpose. However, to confirm this, more detailed research is needed. It was found that NR fluoresces differently in blue light depending on the polarity of the polymer: PE and PP fluoresce yellow or green (these are low-polarity polymers); PA and PET fluoresce red (high-polarity polymers), and PS emits orange fluorescence (medium-polarity polymer). Our studies also confirmed this statement (Supporting Information S2). However, under green excitation light, all polymers fluoresce red; therefore, the choice of excitation light is a crucial step in identifying the polarity of polymers.

Our results correlate with a previous study aimed at optimizing the application of coumarin-6 for microplastic staining. In that study, a dye concentration of 10 mg L–1, corresponding to 10 μg mL–1, was used, and the dye was able to stain all polymers analyzed: LDPE, HDPE, PA, PC, PVC, PET, and to a lesser extent, PP and PS. However, in the current study, it was observed that LDPE and PP did not fluoresce under UV light with increasing coumarin-6 concentrations, which may be due to aggregation of coumarin-6 at higher concentrations.

It has been found that some polymers fluoresce under UV excitation light: polyamide, polystyrene, and polyethylene terephthalate, and some under blue light: polyamide and polystyrene. It has also been observed that some dyes negatively affect the fluorescence of these polymers, depending on the concentration used (Supporting Information S3).

NR was not effective in staining LDPE, PA, and PS under UV light, although these polymers fluoresced under blue and green light. Moreover, it is important to emphasize that when analyzing dyed PA and PS polymers, fluorescence was recorded, but it was weaker than the autofluorescence of the polymer itself, so we can consider the dye to be ineffective. Similar results were obtained by other authors. Shim et al. (2016) found that NR is not effective in staining PA, as well as PVC and polyester, which can be explained by the low hydrophobicity of these polymers: NR, being a lipophilic dye, interacts poorly or not at all with more hydrophilic polymers. It was observed that only Nile Red fluoresced under green light, except for PA, where it also fluoresced with curcumin when concentrations of 10 μg mL–1 and 15 μg mL–1 were used. Coumarin-6 at 10 μg mL–1 also stained all polymers and fluoresced under UV and blue light, except for PS under UV light, where fluorescence was weaker than polymer autofluorescence. PP emerged as the most difficult polymer to dye, and only a few dyes were effective: NR and all tested concentrations of coumarin-6. PET was the most effectively dyed polymer, fluorescing under UV and blue light with all analyzed dyes except coumarin-153 at 10 μg mL–1 and 15 μg mL–1 concentrations. The recorded fluorescence under UV and blue excitation light can be found in the Supporting Information S4.

3.3. Fluorescence Distribution Uniformity in Pristine Samples

Skewness is a statistical measure of the skewness of a distribution (the extent to which data deviate from the mean). If the distribution is perfectly symmetrical (Gaussian curve), then the skewness is 0. If the data curves have a longer tail on the right, they are positively skewed (>0). If the tail is longer on the left, the data are negatively skewed (<0). Skewness indicates whether most of the values are concentrated on one side or the other of the mean; − in other words, it determines whether there are more points of intense fluorescence or less intense fluorescence in the area of interest. When analyzing fluorescence data, the skewness index describes the distribution of fluorescence intensity and the heterogeneity of staining. Positive skewness means that most of the polymer surface fluoresces weakly, but there are a few very bright spots, while negative skewness means that most of the polymer surface fluoresces quite brightly, but there are a few nonfluorescent or very weakly fluorescent areas.

It was found that when illuminated with UV light (EX 330–380 nm), regardless of the dye used, PET is dominated by positive asymmetry, i.e., most of the polymer surface fluoresces weakly, but there are a few very bright spots, while PS is negative, i.e., most of the polymer surface fluoresces quite brightly, but there are a few nonfluorescent or very weakly fluorescent areas (Figure ). In PA, almost uniform fluorescence intensity was determined when staining with coumarin-6 dye at a concentration of 15 μg mL–1. When staining PP, it is best to use Nile Red in terms of intensity skewness, and for HDPE, 50 μg mL–1 coumarin-6 is the optimal staining condition.

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Fluorescence intensity skewness when applying different dyes (Nile Red, Coum-6 (coumarin-6), Coum-153 (coumarin-153), Curc (curcumin)) to different polymers (HDPE, LDPE, PP, PET, PA, and PS) using UV (EX 330–380 nm) light.

The findings indicate that when illuminated with blue light (EX 450–490), regardless of the dye and polymer type used, the skewness of the fluorescence intensity is positive or close to 0 (Figure ). When using Nile Red dye, the skewness is almost uniform, and it is close to 0, i.e., the entire polymer surface fluoresces with similar intensity. Also, a fairly uniform intensity of PA and PP polymers was found using coumarin-6 dye regardless of concentration.

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Fluorescence intensity skewness when applying different dyes (Nile Red, Cum-6 (coumarin-6), Cum-153 (coumarin-153), Curc (curcumin)) to different polymers (HDPE, LDPE, PP, PET, PA, and PS) using blue (EX 450–490) light.

3.4. Relationship between Plastic Surface Integrity and Fluorescence

The results showed that when staining HDPE and PET and illuminating them with blue or UV light, the fluorescence intensity is significantly affected by both the level of plastic damage (factor A) and the dye (factor B), as well as their interaction (A × B) (p < 0.1) (Figures and ). The highest fluorescence intensity in blue light was determined using Nile Red, regardless of the level of plastic damage. However, when illuminating with UV radiation, an unexpected trend is observed; when Nile Red is used to dye HDPE and PET plastic, the fluorescence intensity decreases after mechanical and thermal damage. However, a statistically significant decrease is observed only when staining HDPE (see Figure ).

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Influence of plastic damage and dye used on fluorescence intensity under blue spectrum light (EX 450–490 nm). Note: Significance designations: ***very highly significant (p < 0.001), **very significant (p < 0.01), *significant (p < 0.05), .weakly significant (p < 0.1), and an empty spaceinsignificant (p ≥ 0.1). Lowercase letters (a; b) denote the differences between damaged and undamaged plastic when stained with the same fluorescent dye. Uppercase letters (A; B) denote the differences in fluorescence intensity of plastic of the same degree of damage when stained with different fluorescent dyes. Factor Alevel of plastic damage; Factor Bfluorescent dye used.

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Influence of plastic damage and dye used on fluorescence intensity under UV light (EX 330–380 nm). Note: Significance designations: ***very highly significant (p < 0.001), **very significant (p < 0.01), *significant (p < 0.05), .weakly significant (p < 0.1), and an empty spaceinsignificant (p ≥ 0.1). Lowercase letters (a; b) denote the differences between damaged and undamaged plastic when stained with the same fluorescent dye. Uppercase letters (A; B; C) denote the differences in fluorescence intensity of plastic of the same degree of damage when stained with different fluorescent dyes. Factor Alevel of plastic damage; Factor Bfluorescent dye used.

Different trends are seen when dyeing LDPE and PP. When staining these polymers with fluorescent dyes and illuminating them with blue light, only factor B, i.e., the dye used, had a significant effect on the fluorescence intensity. When illuminated with UV light, LDPE staining was significantly affected by both factors and their interaction, whereas PP staining was influenced by both factors, but their interaction was not significant (Figure ).

After analyzing all the results obtained, a clear trend is seen that the fluorescence intensity of mechanically and thermally affected microplastics increases with small deviations, regardless of the dye, polymer, or light used. This conclusion is also confirmed by the work of other scientists, Shim et al. (2016), who studied the applicability of NR for dyeing microplastics. In their work, the scientists claim that since the dye is adsorbed on the plastic surface, polymers with a larger surface area, such as as mesoporous structures or irregular shapes, are dyed more brightly with NR compared to spherical particles. However, the scientists did not measure the fluorescence intensity, the assessment was made only visually. , In this study, a statistically significant increase is seen after staining PET polymers with coumarin-6, coumarin-153, and curcumin dyes and illuminating them with blue light (Figure ). After illuminating the polymers with UV light, an increase is observed in most cases: HDPE when dyed with curcumin dyes; LDPE when dyed with Nile Red, coumarin-6, and coumarin-153; PP when dyed with Nile Red, coumarin-6, and curcumin; PET when dyed with coumarin-6, coumarin-153, and curcumin (Figure ).

The study showed that, although some polymer standards do not fluoresce when stained with fluorescent dyes, weak fluorescence is observed upon mechanical and thermal exposure in some cases. Four out of six such cases were observed with curcumin-153. This was recorded when analyzing LDPE and PET under blue and UV light, and the other two cases were observed when analyzing curcumin-stained PP under blue light and coumarin-6 and curcumin under UV light.

It was found that when illuminated with UV (EX 330–380 nm) light, regardless of the dye used or the polymer analyzed, the asymmetry of the fluorescence intensity of the damaged polymers is closer to 0, i.e., is more uniform than that of pristine microplastic particles (Figure ). These results indicate that when staining microplastics isolated from environmental samples, their fluorescence will be almost uniform, which will allow them to be better detected when observed under a microscope. As a preliminary assessment of environmental sample applicability, selected dyes were tested on particles isolated from river water samples. Representative fluorescence images are provided in Supporting Information S6.

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Fluorescence intensity skewness when applying different dyes (Nile Red, Coum-6 (coumarin-6), Coum-153 (coumarin-153), Curc (curcumin)) to different standard and damaged polymers (HDPE, LDPE, PP, and PET) using UV (EX 330–380 nm) light. Note: * symbol indicates damaged plastic polymer.

Using Nile Red dye, the same trend as discussed earlier is observed (Figure ), and the asymmetry of fluorescence intensity is almost uniform for all of the polymers tested, regardless of whether they are mechanically and thermally damaged or not (Figure ). The asymmetrical results allow us to conclude that mechanical and thermal damage to polymers improves staining efficiency, as the fluorescence is more uniform. However, this conclusion does not apply to PP, since the opposite effect is observed, i.e., the asymmetry of the damaged polymer is positive, with a larger shift from the mean.

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Fluorescence intensity skewness when applying different dyes (Nile Red, Coum-6 (coumarin-6), Coum-153 (coumarin-153), Curc (curcumin)) to different standard and damaged polymers (HDPE, LDPE, PP, and PET) using blue (EX 450–490) light. Note: * symbol indicates damaged plastic polymer.

The selectivity of the staining approach is reflected in the polymer-dependent fluorescence response, where different microplastic types exhibit distinct intensity and skewness values. This indicates that the interaction between the dyes and the polymer surfaces is influenced by the physicochemical properties of the materials. Such behavior is consistent with previous studies demonstrating that fluorescence staining efficiency depends on polymer properties. However, it should be noted that fluorescence-based staining methods may exhibit limited selectivity in complex environmental matrices due to potential interactions with nonplastic materials.

4. Conclusions

The study demonstrated that the efficiency of fluorescent staining for microplastic detection is influenced by several factors: polymer type, dye, excitation light wavelength, and polymer damage. Among the tested polymers, PET was the easiest polymer to stain, as it exhibited the highest fluorescence intensity across most tested conditions, while PP proved to be the most challenging polymer to stain using the selected dyes. Coumarin-6 enabled fluorescence detection across polymers, although the intensity was not uniform among different pristine and damaged polymers. Furthermore, dye concentration was found to play an important role in modulating fluorescence intensity and, in some cases, reducing polymer autofluorescence. Mechanical and thermal damage generally enhanced fluorescence intensity and improved staining uniformity, although this effect was polymer-dependent. Considering the results obtained, we can state that coumarin-153 is not a potential dye for microplastic analysis, as it emits very weak fluorescence and is able to stain only a few polymers (PA, PS, and PET) that already emit autofluorescence. However, coumarin-6 and curcumin may have prospects in MP studies, but more detailed research is necessary. Overall, the findings highlight the need for the careful optimization of staining protocols adapted to specific polymer types and analytical conditions. These findings provide a basis for further improvement of fluorescent staining protocols through selection of dye, its concentration, and illumination conditions for specific polymer types. Future studies should explore the applicability of the tested eco-friendly dyes to environmentally weathered microplastics and complex environmental matrices, as well as their compatibility with automated image analysis and quantitative fluorescence-based detection approaches. Also, the dyes used in this work should be tested together with organic materials to determine their selectivity, and optimization of dyeing conditions is required. Such efforts would contribute to improving the robustness and standardization of microplastic identification methods.

Supplementary Material

ao6c01270_si_001.pdf (2.1MB, pdf)

The data will be made openly available in the Mendeley Data repository after the embargo period under the title Dovile PhD Manuscript_1.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c01270.

  • S1: identification of nonstandard polymers; S2: fluorescence color emitted by Nile Red when illuminating different polymers with blue light (EX 450–490); S3: autofluorescence of polymers and the effect of dyes under different excitation light; S4: fluorescence color emitted by four different fluorescent dyes when illuminating different polymers with blue and UV light; S5: ATR-FTIR spectrum of dyes and polymers before and after staining; S6: staining of microplastic particles isolated from river water (PDF)

D.M.: Writingoriginal draft, Visualization, Methodology, Investigation, Formal analysis. K.B.: Writingreview and editing, Data curation, Conceptualization.

This work was funded by the Research Council of Lithuania under grant P-ST-24–53.

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao6c01270_si_001.pdf (2.1MB, pdf)

Data Availability Statement

The data will be made openly available in the Mendeley Data repository after the embargo period under the title Dovile PhD Manuscript_1.

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