Optimizing the ratio of Coomassie and Methylene blue dyes for a cost-effective and rapid staining of PET, PVC, PP, PS, LLDPE, LDPE, and HDPE

The ubiquitous presence of plastic brought on by the extensive use of plastic products calls for e�cient and rapid plastic detection methods for the detection and evaluation of pollution. The commonly used Nile red dye takes many hours and is expensive while also not equally e�cient across all the common plastic waste. To address this, we investigated the staining e�ciency and optimized the ratio of a combined dye of Coomassie brilliant blue and Methylene blue. In the optimisation process, Methanol-based Coomassie and Methylene blue dyes effectively stained the PET, PP, PS, LLDPE, LDPE, and HDPE plastics without compromising the plastic's integrity. Image analysis showed a generally better staining e�cacy compared to Nile red. Given the cost-effectiveness, e�ciency, and accessibility of the blue dyes in labs, the optimized ratio of the blue dyes makes it suitable for large-scale plastic staining across the six tested types of plastic, replacing Nile red.


Introduction
Plastic products have become ubiquitous due to their durability and low cost [1].The "Plastic Europe" data showed that the world produced 390.7 million tons of plastic in 2021, when the COVID-19 pandemic peaked [2].China is the largest plastic producer in the world, reaching up to 32% of the global plastic production [2].However, the exponential growth of plastic production has brought about major environmental concerns regarding plastic wastes [3,4], especially when they are not generally biodegradable, taking many years to decompose.With plastics leeching into soil, water, and livestock, detecting small plastic particles and even nano-scale micro-plastics has become a priority for scienti c researchers and environmentalists [5].
The detection methods of tiny plastic particles are classi ed into three categories: Microscopy, Spectroscopy, and Thermal analysis.Microscopy includes microscopic visual inspection, Scanning Electron Microscopy (SEM), and Scanning Electron Microscope coupled with energy-dispersive X-ray Spectroscopy (SEM-EDS).Spectroscopy includes Fourier Transform Infrared Spectrometer (FTIR) and Raman Spectroscopy.Thermal cracking includes Thermal Extraction Desorption-Gas Chromatography-Mass Spectrometry (TED-GC-MS) and Pyrolysis-Gas Chromatography-Mass Spectrometry (Py-GC-MS).A summary of the common methods is shown in Table 1.• Simple sample preparation • Di cult to characterise particles below 100 µm.
• Large misidenti cation: 20% error for normal plastics and 70% error for transparent plastics.
• Often need to couple the microscope with other techniques, such as Spectroscopy • Time-consuming and labourintensive [6,7] Fluorescence Microscopy Distribution Biological samples like tissue, cells, bacteria, etc.
• Easy • Useful strategy for white and transparent plastics • Can track the digestion, uptake, etc., in a biodome.
• Can detect smaller particles than optical microscopy • Sample needs to be uorescent or stained.• Elemental analysis of particles if coupled with EDS.
• Easy to distinguish plastics from organic matters • It is time-consuming to analyse all the particles on a lter.
• Heavy metals and microorganisms adsorbed on the surface of plastics can be interfering factors [7,11] Raman Spectroscopy

Size, Characterization
Samples have less impurities and uorescence • Can detect small plastics < 20 µm • Non-destructive analysis of materials • Lack of particle size information [13] Methods such as Py-GC-MS, FTIR, Raman spectroscopy, and others are often limited by high costs, complex sample pretreatment, and time-consuming procedures.Thus, visual inspection is still the primary method for detecting small plastic particles and microplastics.A recent review of the methods to detect microplastics in water and sediment (N = 40) showed that a signi cant proportion (32.5%) were based on visualisation methods [14].Given the wide adoption of the visualization method, Nile red, a common uorescent dye used to dye lipids and proteins in cell biology, has become increasingly popular.For staining plastics, Nile red is typically used within the concentration range of 1 µg/mL to 1000 µg/mL (1 mg/mL) for staining times ranging between 5 minutes to 66 hours [15].
Despite its useful application, the high cost of Nile red limits its widespread use.To overcome this, the lower costing Coomassie brilliant blue and Methylene blue could be used as alternatives, especially as a combination.While Nile red powder can be a couple of hundred US dollars per gram, Coomassie brilliant blue R250 powder is around US$1 and Methylene blue powder is around ~ US$2.Both Coomassie brilliant blue and Methylene blue are commonly used dyes in chemistry and cell biology, making them available in most laboratories, including in school labs.Coomassie brilliant blue is a triphenylmethane dye that is widely used for protein staining in polyacrylamide gels [16,17,18], while Methylene blue is a thiazine dye that has been used for staining a variety of biological specimens, such as bacteria, fungi, and blood cells [19].
Thus, even though the Nile red uorescence staining method addresses the issues of slow spectral methods and the challenge of detecting small plastic particles (< 20 µm), the problem of high detection costs in terms of required consumables remain.Furthermore, the requirement of a uorescence microscope for visualization limits its use.Therefore, this study aims to optimise the combination of both Coomassie brilliant blue and Methylene blue for various types of plastic staining to provide a simple, e cient, and inexpensive solution for plastic detection.

Staining of microplastics.
Microplastic PET, PVC, PP, PS, LLDPE, LDPE, and HDPE were purchased from Guangyuansuhua (Guangdong, China).All seven types of microplastics had an identical diameter of 150 µm.Filter papers were purchased from Changde BKMAM Biotechnology Co., Ltd.(Hunan, China).The mesh size of the lter papers was less than 120 µm.

Optimization of Solvent
To ensure optimal staining effectiveness, various organic solvents were tested according to the approach previously described by Maes et al. [15].Acetone and Methanol were chosen to assess the impact of different solvents on the staining e cacy of Nile red dye, Coomassie brilliant blue, and Methylene blue (referred to as the CM blue dye hereafter).Nile red and the CM blue dye (comprising of Coomassie brilliant blue to Methylene blue mass ratio of 1:1) were dissolved in Acetone and Methanol, respectively, to reach a concentration of 1 mg/mL.Plastic fragments were incubated in a 200 µL solution of Nile red and CM blue dyes in both Acetone and Methanol solvents separately for 30 minutes at room temperature before the plastics were removed and rinsed with distilled water.

Validation of CM Blue Dye Staining Method in Plastics Fragments
In the preliminary test, Coomassie brilliant blue and Methylene blue were mixed 1:1 by mass in Methanol to examine whether the blue dye could stain plastic fragments.For the CM blue dye groups, ve types of plastic fragments (PET, PP, PS, PE, and HDPE) were put into different centrifuge tubes, and 200 µL blue dye of 1 mg/mL concentration was added into each centrifuge tube to immerse the plastic fragments completely.Similarly, 200 µL of Nile red in Methanol with a concentration of 1 mg/mL was used for the ve types of plastic fragments.200 µL of Methanol with dye was used as a control group to compare the staining effect of dyes on plastic fragments.All plastic fragments were washed with distilled water after a 30-minute incubation at room temperature.Subsequently, the stained microplastics were ltered and washed with distilled water.The microplastics were collected and oven-dried at 80 ℃ for one hour.

Optimization of Blue Dye Proportion
Nile red solution in Methanol staining was compared with CM blue dye in Methanol as a control group to stain the seven types of microplastics (PET, PVC, PP, PS, LLDPE, LDPE, and HDPE).50 mg of microplastics was incubated with 200 µL Methanol and Nile red solution in Methanol at 1 mg/mL concentration for 30 minutes, respectively.Following the incubation, the stained microplastics were placed on lter paper and washed with distilled water.
After ltration, all microplastics were collected and oven-dried at 80 ℃ for one hour.All treatments were performed as three independent replicates.

Data Analysis and Statistics
ImageJ software was used to quantify the staining effect on the plastic.The grey value of the picture was measured by removing the background from the staining results of the plastic particles.To examine the variations in grey values between different groups following the plastic staining, Independent-Samples T-Test on independent samples was performed using SPSS version 26 (IBM Statistical Package).

Effect of Dye Solvents on Plastics
Figure 1 shows the effect of the dye solvents on plastic samples.When Acetone was used as a solvent, there was a clear alteration of the PS sample regardless of whether Nile red or CM blue dye was used.In contrast, plastic samples dyed with Methanol did not show shape alterations.Thus, Methanol was chosen as the solvent for the subsequent experiments.

CM Blue Dye Staining E cacy on Plastics
Figure 2 illustrates the staining results obtained by incubating the plastics with the CM blue dye and Nile red at a concentration of 1 mg/mL for the same duration.Visual examination showed a comparable staining effect between the CM blue dye and that of Nile red.

Optimization of the CM Blue Dye C: M Ratio
Figure 3 illustrates the staining effects of blue dye with different ratios of Coomassie brilliant blue to Methylene blue on the various plastic particles.Low proportions of Coomassie brilliant blue, e.g., C: M = 0:10, did not show observable staining on the plastic particles.As the amount of portion of Methylene blue increased, the staining effect was more obious across the various plastics.The Nile red dye demonstrated superior staining on PVC than all the CM blue dye combinations.
The average grey values of each plastic particle after staining with different proportions of Coomassie brilliant blue: Methylene blue in blue dye (C: M ratio) are summarised in Table 2. Notably, the minimum grey value of PVC plastic occured at the C: M ratio of 5:5; the minimum grey value of PET plastics occurred at the C: M ratio of 7:3; the minimum grey values of PP, LDPE, and HDPE plastics took place at the C: M ratio of 8:2; and the minimum grey values of PS and LLDPE plastics were observed at C: M ratio of 10:0.
The staining effect of Nile red solution (Methanol) on PVC particles was the most prominent, displaying signi cant differences from the Methanol group (p < 0.05).However, the grey values of the other Nile red groups (PET, PP, PS, LLDPE, LDPE, and HDPE) did not exhibit signi cant differences compared to the Methanol groups (p > 0.05).This indicated that the staining effect of Nile red on PET, PP, PS, LLDPE, LDPE, and HDPE plastic particles was negligible.
In addition, for plastic particles other than PS, low portions of Coomassie brilliant blue in the blue dye yielded higher grey values than the Methanol control group.The results based on grey values were consistent with the visual observations.Table 2 The average grey value of each plastic particle after staining (C = Coomassie brilliant blue, M = Methylene blue, Concentration = 1 mg/mL).

CM Blue Dye on Microplastics
Visual observation of the staining of the Nile red and CM blue dye on the microplastics showed more consistent dark staining by the CM blue dye than Nile red in Fig. 4.
Grey value measurements were utilised for a more quantitative analysis of the staining effects and presented in Table 3. Except for PP, all microplastics in the Nile red groups (PET, PVC, PS, LLDPE, LDPE, and HDPE) showed signi cant differences in grey values compared to the unstained Methanol groups (p < 0.05).Similarly, the grey values of microplastics in all CM blue dye groups demonstrated signi cant differences from those in the unstained groups (p < 0.05).However, by comparing the grey values, it was observed that blue dye at the optimal ratios (PET, C: M = 7:3; PVC, C: M = 5:5; PP, LDPE, and HDPE, C: M = 8:2; PS and LLDPE, C: M = 10:0) exhibited a better staining effect on microplastics compared to Nile red.This nding is consistent with the results obtained from the experiments with plastic particles.It is worth noting that the CM blue dye showed a better staining effect on PVC microplastics compared to Nile red at a C: M ratio of 5:5, which differed from the staining results obtained from the experiments with plastic particles, probably due to post-production treatment of the regular shaped plastics.The use of the optimised CM blue dye for plastic staining offers several advantages over existing methods.Firstly, it is more cost-effective than Nile red staining.At the optimal C: M ratios, the cost of using blue dye to detect PVC, PET, PP, LDPE, HDPE, PS, and LLDPE is about 1% that of Nile red.The availability and affordability of Coomassie brilliant blue and Methylene blue make the CM blue dye a more practical option for large-scale plastic detection projects, with the dyes responding likely to the different polymer natures.Secondly, the CM blue staining method required only 30 minutes of incubation time, signi cantly reducing the time requirements of other detection methods, such as spectroscopy or thermal analysis.This e ciency is particularly valuable when dealing with many samples.Furthermore, the CM blue dye staining did not require uorescence microscopy or other high-end equipment for visualisation.In fact, for microplastics characterised by their small size (< 5 mm) [5], a simple light microscope may already be su cient.
Given the usage of both Coomassie and Methylene blue dyes in biology, it is necessary to note the limitation of looking for microplastics in tissues using the CM blue staining method.Organic compounds present can also lead to staining and can be found in many plastic wastes.While this can be mitigated by some cleaning and sample pre-treatment, such processes could be tedious and add to process costs.Nonetheless, the problem is also present with the more expensive Nile red alternative, thus the continued need for acid, alkali, or other digestions to degrade biotic substances without signi cantly altering the chemical or structural integrity of plastic particles [20].
It should be noted that the plastics used in everyday products may have additional additives or treatments, such as colouring in the product that may affect the staining results.A glimpse of such differences could be found in the slight differences in results here between the microplastics and the plastic particles.Thus, there may be room for further optimisations based on the additives and colouring to yield more visually observable staining.
Nonetheless, the demonstration of our analysis here using grey values and image processing from photos also demonstrates the possibility of automated detection of microplastics to be used for environmental monitoring of a range of applications, including water samples, sediments, soils, and biota.Such a piece of automated equipment could be built based on devices built on Microcontroller kits and 3D printing for the detection of blue particles or light absorption for an on-the-go quanti cation and detection [21,22] or even smartphone applications leveraging on image processing of smartphone photos [23,24] for higher throughput and quantitative processing.

Conclusions
With the cost of 1% of Nile red, we optimized the ratio of Coomassie brilliant blue and Methylene blue dyes in Methanol for a more cost-effective, faster, and e cient staining to detect plastic particles and microplastics that could be used for a wide range of environmental monitoring applications.The

2. 1 . 1
Evaluating the staining of irregular-shaped plastic fragments.Polyethylene Terephthalate (PET) was collected from plastic mineral water bottles produced by Hangzhou Dingjin Food Co., Ltd.(Zhejiang, China).Polypropylene (PP) and Polystyrene (PS) were collected from the food packaging and fast-food boxes, respectively.Both were produced by Ruikang Houseware Co., Ltd.(Zhejiang, China).Polyethylene (PE) was collected from the food packaging bags made by Green Password Houseware Co., Ltd.(Zhejiang, China).High-Density Polyethylene (HDPE) was collected from the bottled milk bottle produced by Yiming Food Co., Ltd.(Zhejiang, China).All the plastics were cut into small pieces using scissors.The length and width of PET fragments were approximately 5.5 × 4.0 mm; PP fragments were 5.0 × 3.5 mm; PS fragments were 4.5 × 3.5 mm; PE fragments were 6.0 × 5.0 mm; HDPE fragments were 4.5 × 3.5 mm.2.1.2Staining of regular-shaped plastic particles.PET, PP, PS, LLDPE, LDPE, and HDPE plastic particles were all purchased from Usolf (Shandong, China).PVC plastic particles were purchased from Xinxicheng (Guangdong, China).The length, width, and height of PET particles were 3.3 × 2.7 × 2.0 mm, respectively; PP particles were 4.7 × 3.7 × 2.5 mm; PS particles were 4.0 × 3.0 × 3.3 mm; LLDPE particles were 5.0 × 4.3 × 2.5 mm; LDPE particles were 4.7 × 3.3 × 3.5 mm; and HDPE particles were 4.3 × 4.0 × 3.5 mm; The bottom surface of PVC particles had a diameter of 3.0 mm and a height of 3.0 mm.

For
better homogenous staining, regular-shaped plastic was used in the cut plastics.The CM Blue dye was sub-divided into 11 groups according to the proportion of Coomassie brilliant blue to Methylene blue from C: M = 0:10 to C: M = 10:0 (C = Coomassie brilliant blue, M = Methylene blue).Nile red and CM blue dye storage solutions were prepared at 10 mg/mL in Methanol and diluted ten times to the working concentration of 1 mg/mL.For the CM blue dye, the total mass of Coomassie brilliant blue and Methylene blue was 1 mg per 1 mL of powder in Methanol.All plastic particles were added to the 200 µL solution of Methanol, Nile red in Methanol, and CM blue dye in Methanol separately for 30 minutes at room temperature before rinsing with distilled water.All experiments were performed in three independent technical replicates.2.2.4The Staining Effect of CM Blue Dye on MicroplasticsTo test the staining effect of blue dye on microplastics, seven types of microplastics were used in this experiment.Individual microplastics were stained with the optimized blue dye ratio: PET (C: M = 7:3), PVC (C: M = 5:5), PP (C: M = 8:2), PS (C: M = 10:0), LLDPE (C: M = 10:0), LDPE (C: M = 8:2), and HDPE (C: M = 8:2).200 µL of 1 mg/mL CM blue dye submerged ~ 50 mg of the mentioned types of microplastics at room temperature for 30 minutes.

4 .
Discussions and Future PerspectivesThis study sought evaluate a more cost-effective and convenient way of staining plastics by optimising and evaluating the ratio of Coomassie brilliant blue and Methylene blue dyes compared to the Nile red dye.By testing the common types of plastics commonly found in products, we found different plastics to exhibit different optimal (Coomassie brilliant blue: Methylene blue) ratios in the combined blue dye.PVC exhibited the best staining results at C: M = 5:5 (mass) in the combined dye; PET at C: M = 7:3 (group); PP, LDPE, and HDPE at a C: M ratio of 8:2 (mass); and PS and LLDPE at C: M = 10:0 (mass).These optimised C: M ratios provide guidelines for enhancing the staining e cacy for different plastic types, emphasising the importance of tailoring the dye composition based on the speci c plastic.While initial tests suggested the lower Coomassie dyes to be better for staining, this was the case only for PS and LLDPE and for the other cases, some portion of Coomassie brilliant blue dye complimented the staining by Methylene blue, e.g., PVC. Figures

Figure 1 Effect
Figure 1

Figure 2 CM
Figure 2

Table 1
Summary of the plastics determination technologies.

Table 3
Average grey value of each microplastic after staining (Concentration = 1 mg/mL).