Environmental Chemistry Letters

https://doi.org/10.1007/s10311-022-01545-3

ORIGINALPAPER

Rapid detection of nanoplastics and small microplastics by Nile‑Red

staining and flow cytometry
Angelica Bianco1,4 · Luca Carena2 · Nina Peitsaro3 · Fabrizio Sordello2 · Davide Vione2 · Monica Passananti1,2

Received: 20 July 2022 / Accepted: 10 November 2022

© The Author(s) 2022

Abstract
Microplastics are of rising health concerns because they have been detected even in remote and pristine environments, from
the Artic snow to the Marianne Trench. The occurrence and impact of nanoplastics in ecosystems is almost unknown, in
particular due to analytical limitations such as very small sizes that fall below detection limits of current techniques. Here
we take advantage of a common interference in analytical flow cytometry to develop a method for the quantification of the
number of plastic particles in the 0.6–15 µm size range. Plastic particles are stained with the lipophilic dye Nile-Red then
detected by flow cytometry, a method regularly used in biology for rapid quantification of fluorescent cells. We found that
sample analysis lasts 90 s, which is hundreds of times faster than the analysis of filter portions by micro-Raman and other
spectroscopic techniques. Our method is highly efficient in detecting polyethylene, with staining efficiency higher than 70%
and signal linearity with concentration. Staining efficiency up to 96% was observed for polyvinylchloride and for polystyrene.

Keywords Flow cytometry · Nanoplastics · Microplastics · Nile-Red

Introduction the environment is almost unknown, because their size is

below the instrumental detection limit of the techniques cur-
Production of synthetic plastics derived from fossil fuels rently used for microplastics, such as micro Fourier trans-
continues rising, while the mismanagement of plastic waste form infrared (µFT-IR) and micro-Raman spectroscopy
has recently caused serious pollution problems (Padervand (µRaman), yet nanoplastics are most probably more danger-
et al. 2020). Plastics undergo a slow process of erosion in ous than microplastics due to the properties of nanomaterials
the environment, leading to the breakdown of large plastic (Sharma et al. 2022).
debris into microplastics, less that 1 mm-sized (Thompson Nanoplastics have been detected in water and in soil by
et al. 2004) and even nanoplastics, less that 1000 nm-sized pyrolysis–gas chromatography-mass spectrometry, which
(Gigault et al. 2018; Dawson et al. 2018; González-Pleiter gives the concentration in mass of plastic polymers (Hurley
et al. 2019). The occurrence and impact of nanoplastics in and Nizzetto 2018; Ter Halle et al. 2017; Wang et al. 2019;
Xu et al. 2022; Materić et al. 2022). However, to the best
of our knowledge, there is not yet any study that reports
* Monica Passananti on nanoplastics number concentration in a specific environ-
[email protected] ment. To analyze micro- and nanoplastics in the environ-
1
Institute for Atmospheric and Earth System Research/ ment, there are several steps to follow: sampling, sample
Physics, Faculty of Science, University of Helsinki, treatment, concentration and/or separation, identification,
00014 Helsinki, Finland and quantification (Razeghi et al. 2021; Huang et al. 2022).
2
Dipartimento di Chimica, Università di Torino, Via Pietro After sampling, a purification step is needed for the analysis
Giuria 5, 10125 Turin, Italy of plastic particles, which usually consists in the digestion
3
Department of Biochemistry and Developmental Biology, of the organic matrix. However, nanoplastics are not inert to
Faculty of Medicine, University of Helsinki, 00014 Helsinki, the digestion treatments (Bianco et al. 2020); therefore, more
Finland research is needed in this field (Cai et al. 2021).
4
Present Address: Laboratoire de Météorologie Physique, Quantification is the final step of the analysis. µFT-IR
UMR 6016, CNRS, Université Clermont Auvergne, and µRaman, scanning electron microscope (SEM) and
63178 Aubière, France

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transmission electron microscope (TEM) are widely used paper (Whatman). The use of plastic labware was avoided,
to measure the sizes or concentrations in studies into micro and glassware was carefully rinsed with Milli-Q water.
and nanoplastics (Sarau et al. 2020; Lê et al. 2021; Zhou Experiments were performed under a bench hood.
et al. 2021; Li et al. 2022). Dynamic light scattering and
nanoparticle tracking analysis can also characterize the size Instruments
of nanoparticles, by measuring fluctuations in scattered light
intensity due to the particle Brownian movement (Frisken Dimensional and shape characterization was performed with
2001; Hernandez et al. 2019). However, dynamic light scat- dynamic light scattering. These measurements were carried
tering and nanoparticle tracking analysis have considerable out on Nile-Red suspensions in methanol, using an ALV
limitations when analyzing polydisperse and non-spherical NIBS (not invasive back scattering) apparatus (ALV, Lan-
nanoplastics in field samples, and have been mostly used for gen, Germany), equipped with a correlator mod ALV5000,
high-concentration and monodisperse nanoplastics. and measuring scattered light for at least 20 s at 298 K.
The detection and quantification of nanoplastics and Fluorescence excitation–emission matrix spectra were
small microplastics is still a challenging task, and research is taken with a Varian Cary Eclipse spectrofluorimeter, using
needed to improve the actual methods and develop new ones. a fluorescence quartz cuvette with 1.000 cm optical path
Interestingly, nanoplastics and small microplastics are well length. Excitation–emission matrix spectra were obtained
known to give interferences in bacterial cell counting carried with 600 nm ­min–1 scan rate, and the slit width on both exci-
out by dye staining and flow cytometry, because they interact tation and emission (5 or 10 nm) was a compromise between
with lipophilic dyes. In this work we take advantage of this spectral resolution and signal intensity.
characteristic, by testing the detection and quantification of Flow cytometry analysis was performed with a BD Accuri
plastic particles of different polymers, in the size range of C6 flow cytometer (BD Biosciences), using 35 µL ­min–1
200–20,000 nm, by Nile-Red staining and flow cytometry flow rate and volume of analysis of 100 µL. Analysis of each
detection. This technique is routinely used in biology for sample requires 90 s and is rapid compared to the analysis
single cell analysis, and it enables a quick and cheap quan- of filters by µRaman and µFT-IR, which take hours for the
tification of fluorescent particles. This work has the goal of analysis of a small portion of a filter. The 488 nm laser of the
showing that flow cytometry is suitable for the final steps of cytometer was used for excitation, and the emitted fluores-
nanoplastics and small microplastics analysis, i.e., detection cence signal was detected at 530/540 nm. These wavelengths
and quantification. It also provides insight into the limits of were chosen on the basis of the experiments performed by
the method, and the associated need to still develop suitable Dutta et al. (1996).
sampling and purification protocols.

Results and discussion

Experimental
Determination of the concentration of Nile‑Red
Chemicals
Dyes, and in particular Nile-Red, are used for staining
Nile-Red (CAS 7385–67-3, technical grade), ­H2O2 (30% in microplastics (Dutta et al. 1996; Erni-Cassola et al. 2017;
water), and methanol were purchased from Sigma Aldrich; Costa et al. 2021), and for helping in discrimination between
polystyrene particles in water were purchased from Thermo plastic debris and non-plastic particles and fragments. The
Fischer Scientific, with diameter of 600 nm, 700 nm, and standard procedure in the use of Nile-Red foresees the dis-
1, 2, 3, 4, 5, 6, 10, and 15 µm, with uncertainty on the size solution of the dye in acetone or methanol, followed by
below 3%; polyethylene particles (740–4990 nm) were pur- addition of few drops of the dye solution, to cover each fil-
chased from Cospheric; polytetrafluoroethylene, with aver- ter containing microplastics. The analysis of the filters is
age particle dimensions of 3 µm, was purchased from Poly- performed after a contact time, ranging from 30 to 60 min
sciences Inc; polyvinylchloride H850D high expansion rate (Rumin et al. 2015; Erni-Cassola et al. 2017). In this work
spheres, with particles smaller than 36 µm, was purchased we aimed to analyze aqueous nanoplastics suspensions, thus
from Kureha. All reagents were used as received, without the staining protocol had to be adapted. Previous works
further purification. Water used was of Milli-Q quality. Fur- report that the solubility of Nile-Red in water is around
ther polystyrene particles were produced, by grinding poly- 100 ~ 200 mg ­L–1. However, preliminary flow cytometry
styrene macroplastics in an agate ball mill for successive measurements on Nile-Red solutions in Milli-Q water,
cycles (6 × 30 min), with interruptions to avoid temperature with concentration below the solubility limit, showed that
increase. Particles with diameter smaller than 25 µm were Nile-Red forms aggregates (Figure S1). Fortunately, these
separated by suspension in methanol, and filtration on filter aggregates are not fluorescent, as shown in Figure S2a for an

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Environmental Chemistry Letters

excitation–emission matrix fluorescence spectrum of Nile- polystyrene beads calibration. The size range thus obtained
Red alone. Furthermore, aggregates dissolve upon addition is in excellent agreement with the commercial product infor-
of plastic particles, presumably because the dye molecules mation (0.74–4.99 µm), which suggests that the polyethylene
are preferably adsorbed on plastics. Figure S2b depicts the particles were well stained and fluorescent. In the volume
excitation–emission matrix spectrum of Nile-Red with poly- analyzed (100 µL) there are 1.12 × ­105 particles, of which
vinylchloride, showing much lower Rayleigh scattering sig- (8.23 ± 0.16) × ­104 are fluorescent. It is worth noting that the
nal compared to Nile-Red alone. Rayleigh scattering is the extrapolation of these values gives a concentration of fluo-
main linear feature in these spectra, due to light scattering rescent particles of the order of 8 × ­105 fluorescent particles
by suspended particles. The same result was confirmed by ­mL−1, which is not far from the known initial concentration
flow cytometry (Figure S3). A Nile-Red concentration of in the sample (1 × ­106 part ­mL−1).
10 mg ­L–1 was chosen for the following experiments, as a To better test the repeatability of the analysis, and the
good compromise between staining efficiency and reduction relationship between the theoretical and measured amount
in aggregate formation. of particles, suspensions with different concentrations of
polyethylene nanoparticles were analyzed in triplicate. The
Experiments on synthetic plastics results are presented in Figure S5, clearly showing a linear
relationship with R ­ 2 = 0.96, and coefficient of variation of
Figure S4 shows good linearity of the flow cytometry signal triplicates lower than 10%. This calibration shows that the
(scattered light), when calibrated with unstained polystyrene method can be applied for a quantitative analysis of small
beads with size ranging from 0.6 to 15 µm. Then, an aque- microplastics and nanoplastics, in the number concentration
ous suspension of polyethylene nanoplastics, with average range of ­104–106 particles ­mL−1.
dimension of ~ 3 µm, was spiked with Nile-Red to a final Commercially available and monodisperse polystyrene
dye concentration of 10 mg ­L–1. After 30 min, the suspen- micro- and nanoparticles of 1, 3, and 5 µm in diameter were
sion was analyzed by flow cytometry. Figure 1a shows the stained with Nile-Red, using the same procedure explained
scatter plot, reporting the side scatter, which is the light scat- before but with two different concentrations of Nile-Red,
tered by suspended particles at right angles with respect to i.e., 10 and 90 mg ­L–1. As a result, polystyrene particles of
the laser beam, as a function of the forward scatter, which 1 and 3 µm were not stained by the dye, while 5 µm beads
is light scattered in a forward direction. The scatter plot were stained only at the highest Nile-Red concentration.
identifies two distinct regions, and the red dots highlight This finding could be a consequence of the functionaliza-
the fluorescent particles that make up 73.4% of the total, as tion of polystyrene particles, which is required to maintain
shown in Fig. 1b. These particles range in size between 0.5 them in stable suspension in water. For this reason, we also
and 5 µm, as reported by the blue dots in Fig. 1a, obtained by obtained polystyrene particles from commercial objects. A

Fig. 1  a Side scatter (SSC-A), which is the light scattered by sus- obtained, between 0.5 and 5 µm, is in excellent agreement with the
pended particles at right angles with respect to the laser beam, as a commercial product information (0.74–4.99 µm). b Histogram of
function of the forward scatter (FSC-A), which is light scattered counts versus total cell fluorescence FL2-A, depicting the percentage
in a forward direction, for all the particles detected in the sample of particles that show fluorescence, which confirms that the polyeth-
(black), or for fluorescent polyethylene particles (red). The blue dots ylene particles were well stained and fluorescent
are obtained by calibration with polystyrene beads. The size range

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compact-disk cover was firstly manually fragmented into with Milli-Q water, added with Nile-Red, and then ana-
small pieces, and then grinded into a ball mill, following lyzed (see Scheme S2). The organic matter present in river
the procedure described in Sect. 2.1. The resulting methanol water is likely to interact with plastic particles (Boldrini
suspension of polystyrene particles, with size lower than et al. 2021), and thus interfere with the analysis of nano
25 µm, was evaporated and resuspended in Milli-Q water, and microplastics. Therefore, we applied a simple purifica-
then stained with Nile-Red, and finally analyzed with flow tion protocol: organic matter was digested with H ­ 2O2 (as
cytometry. Results show that particles had sizes ranging described in SI-S1), and ­H2O2 residues were eliminated with
from nanometric to micrometric, with maximum diameter ­FeSO4 or peroxidase. To obtain a reference blank, Milli-Q
between 2 and 4 µm, and that most of the particles were water was treated in the same way. The full list of samples
stained (96.6%) (Fig. 2). that were treated and analyzed by flow cytometry is reported
The experiments on polystyrene highlight the limita- in Table S1. The samples in which peroxidase was used to
tion of the methodology combining dye staining with flow quench residual H ­ 2O2 are not shown, because the fluores-
cytometry: it cannot be applied to plastic particles function- cence signal of peroxidase hid the signal of Nile-Red on
alized with hydrophilic functions, especially in the case of plastics. A comparison of the results, obtained by analyzing
monodisperse polystyrene, where surface functionalization Vantaa River water with and without polyethylene, is shown
is applied to obtain stable water suspensions. On the other in Fig. 3.
hand, flow cytometry was able to detect polystyrene particles The number of particles detected in the polyethylene-
obtained from a daily life object, containing additives and spiked samples was lower (~ 46%) compared to the theo-
antioxidants, in addition to allowing for the detection of non- retical number of particles added to the samples. Still, these
functionalized nano and microplastics made of polyethylene results demonstrate as a proof-of-concept that it is possible
(Fig. 1) and polyvinylchloride [data not shown]. to detect small microplastics and nanoplastics also in com-
The same approach was tested on polytetrafluoroethylene plex media, by using a simple and fast methodology. On the
as well, but these particles were not efficiently stained by other hand, there could be several reasons for the observed
Nile-Red. quantification problems, which are most likely associated
with the digestion process and the interference with organic
Experiments on natural samples and inorganic matter (Matijaković Mlinarić et al. 2022).
Indeed, digestion could degrade some of the smallest plas-
The main challenge of this work was to test the flow cytom- tic particles, which are not inert to the hydroxyl radicals
etry performance in a complex natural matrix. To this pur- that could be generated by H ­ 2O2, in the presence of some
pose, river water was sampled and spiked with polyethylene river water solutes such as Fe species, occurring naturally
particles, to obtain a final concentration of 6 × ­106 part ­mL–1. or added as ­FeSO4 to eliminate ­H2O2 (Bianco et al. 2020;
Vantaa river (Helsinki, Finland) water was filtered on glass Atugoda et al. 2022). Another effect that could influence
fiber filters, and nano and microplastics of polyethylene the number of detected particles might be their interaction
were deposited on these filters, as explained in SI-S1. As a with dissolved organic matter, as shown in previous stud-
first approach, the material deposited on the filters was just ies (Chen et al. 2018). Small plastic particles may in fact
extracted by ultrasounds in methanol. The suspension thus form aggregates with dissolved organic matter, and facilitate
obtained was evaporated under fume hood, reconstituted dissolved organic matter assembly (Chen et al. 2018). This

Fig. 2  a Histogram of counts

versus total cell fluorescence
FL2-A, showing that 96.6%
of polystyrene particles were
stained by Nile-Red and fluo-
rescent. b Side scatter (SSC-A)
versus forward scatter (FSC-A)
of all the particles detected
in the sample (black), with poly-
styrene fluorescent particles in
red. The blue dots are obtained
by calibration with polysty-
rene beads. Results show that
particles had sizes ranging from
nanometric to micrometric, with
maximum diameter between 2
and 4 µm

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Environmental Chemistry Letters

Fig. 3  Side scatter (SSC-A) versus forward scatter (FSC-A) plot of all both samples, as well as the theoretical concentration of polyethylene
the particles detected in Vantaa River water, with (red) and without particles, calculated with the linear regression presented in Figure S5.
(black) addition of polyethylene (PE) particles, after digestion with The number of particles detected in the polyethylene-spiked samples
­H2O2. Polystyrene standards are reported in blue. This plot demon- was lower than the theoretical number of particles added to the sam-
strates that the detection method can be applied also to environmen- ples, because of aggregation of particles or loss during the digestion
tal samples. The table presents the values of the detected particles for process

for river water in the absence and in the presence of poly-

ethylene particles, indicating that polyethylene potentially
induces the aggregation of organic matter and the formation
of larger particles.
The results presented in this article clearly show that the
new methodology consisting in staining with Nile-Red and
detection by flow cytometry is rapid and efficient for the
analysis of pure plastic particles (polyethylene), plastic parti-
cles produced from a daily life object (polystyrene compact-
disk cover), and plastic particles in environmental samples,
such as river water.

Conclusion

We have designed a new methodology for the detection of

small microplastics and nanoplastics. Plastic particles were
Fig. 4  Box plots of the forward scatter and side scatter parameters,
stained with the lipophilic dye Nile-Red and analyzed by
calculated for river water samples with and without polyethylene par-
ticles. The bottom and top lines of the boxes correspond to the 25th flow cytometry. The detection efficiency was tested on poly-
and 75th percentiles, respectively. The middle line represents the ethylene, which is the most common polymer and accounts
median. The ends of the whiskers represent the 10th and 90th percen- for more than 30% of all produced plastics (Plastics—The
tiles. Results indicate that polyethylene potentially induces the aggre-
Facts 2021). Good results were also obtained with polyvi-
gation of organic matter and the formation of larger particles
nylchloride, and with plastic fragments derived by a com-
mercial polystyrene object. Unfortunately, Nile-Red could
phenomenon is confirmed by the trends observed in Fig. 4, not efficiently stain either polytetrafluoroethylene, or poly-
which reports the forward scatter and the side scatter values styrene nanobeads bearing a functionalized hydrophilic

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 Environmental Chemistry Letters

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tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 10311-0​ 22-0​ 1545-3. s41467-​018-​03465-9
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