# Low-cost highlighter nanoparticles for highly efficient multistep excitation energy transfer

Jonathan Piard, Ilaria Degano, Clément Doré, Jean-Frédéric Audibert, Chloé Grazon, Clémence Allain, Loïc Bertrand

► To cite this version:

Jonathan Piard, Ilaria Degano, Clément Doré, Jean-Frédéric Audibert, Chloé Grazon, et al. Low-cost highlighter nanoparticles for highly efficient multistep excitation energy transfer. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy [1994-..], 2026, 352, pp.127477. 10.1016/j.saa.2026.127477. hal-05537216

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Submitted on 4 Mar 2026

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# Low-cost highlighter nanoparticles for highly efficient multistep excitation energy transfer

Jonathan Piard<sup>a,*</sup>, Ilaria Degano<sup>b,D</sup>, Clément Doré<sup>a</sup>, Jean-Frédéric Audibert<sup>a,D</sup>, Chloé Grazon<sup>c,D</sup>, Clémence Allain<sup>a,D</sup>, Loïc Bertrand<sup>a,D,*</sup>

## HIGHLIGHTS

* Multistep energy transfer is first reported in highlighter ink.

* Near-unity efficiency multistep energy transfer is measured among highest recorded.

* First report of 130–150 nm stable monodisperse dye-loaded nanoparticles in commercial highlighter ink.

* Detailed dye composition is identified.

* Efficient use of orange highlighter as water–ethanol sensor.

## GRAPHICAL ABSTRACT

<figure><img src="image_4.png" /></figure>

## ARTICLE INFO

Dataset link: https://doi.org/10.5281/zenodo.18189694

**Keywords:**

Nanomaterials

Multistep excitation energy transfer

Low-cost commercial products

Water–ethanol sensing

## ABSTRACT

Efficient excitation energy transfer (EET) is essential for energy harvesting, optoelectronics, luminescence bioimaging and photothermal medicine. Recent advances in optimised multistep EET systems have expanded the potential of these applications, offering large Stokes shifts, long-range energy transfer, and a broader tuning range for excitation and emission. However, the energy transfer efficiency of these systems rarely exceeds 90%, suggesting room for further improvement. Seeking low-cost solutions to this global problem, we show that a worldwide commercial orange highlighter ink produces both direct and multistep EET with near-unity efficiency (94.4%) among the highest recorded in the literature. We reveal that this efficiency results from a stable monodisperse dispersion of dye-loaded polymer nanoparticles in a water–glycerol medium. We identified the main dyes involved, elucidated the two-step energy transfer sequence, and demonstrated that orange highlighter particle dispersions can serve as an efficient water–ethanol sensor through monitoring variations in EET efficiency. This work presents a low-cost and readily available material exhibiting exceptionally high multistep EET efficiency, offering broad application potential.

## 1. Introduction

Energy transfer in multichromophoric systems occurs via two main mechanisms [1]: Förster resonance energy transfer (FRET), which relies on dipole–dipole interactions [2], and Dexter electron exchange [3]. At distances < 1 nm, both mechanisms can coexist, leading to complex

energy dynamics [4]. In such cases, excitation energy transfer (EET) has been shown to adequately describe the overall energy migration within the system [4]. The development of EET systems has become one of the most promising topics in the fields of analytical chemistry [5–8], chemical biology [4,9–12] and material science [13,14]. Recent

<footnote><sup>a</sup> Université Paris-Saclay, ENS Paris-Saclay, CNRS, PPSM, 4 avenue des Sciences, 91190 Gif-sur-Yvette, France</footnote>

<footnote><sup>b</sup> University of Pisa, Department of Chemistry and Industrial Chemistry, via Moruzzi, 13, 56124, Pisa, Italy</footnote>

<footnote><sup>c</sup> Univ. Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255, F-33400 Talence, France</footnote>

<footnote>* Corresponding authors.
E-mail addresses: jonathan.piard@ens-paris-saclay.fr (J. Piard), loic.bertrand@ens-paris-saclay.fr (L. Bertrand).</footnote>
advances have focused on multistep EET systems, which offer several advantages over conventional single-step EET [15,16]: (i) they allow large Stokes shifts without spectral overlap between donor and acceptor, (ii) they enable long-range energy transfer (> 10 nm) [17–19], (iii) they provide both a broader excitation and emission tuning range, and (iv) they better mimic natural light-harvesting processes [20,21]. Multistep EET has been explored in various fields, including artificial photosynthesis [20,22–24], biosensing [25–27], optoelectronics [28] and fluorescence imaging [29]. Researchers have developed strategies based on supramolecular assemblies [15,20,30,31], nanoencapsulation [32–35] or covalently linked fluorophores within dendritic [36], polymeric [37] or biomolecular matrices [38,39]. However, current approaches often suffer from complex synthesis, high reagent consumption, and suboptimal yields, limiting their practical application. While the highest reported efficiencies demonstrate sequential energy transfer reaching up to 95% for biomolecular scaffold-based systems built from supramolecular peptide nanotubes [40], the transfer energy efficiency of the most efficient multistep EET systems rarely exceeds 90%. To address this global issue, we explore inexpensive and widely available products, exemplified by a commercial orange highlighter ink that exhibits combined direct and multistep excitation energy transfer with near-unity EET efficiency.

This study provides new insights into the chemical composition of fluorescent inks, paving the way for a more complete chemical understanding of their optical properties. We emphasise the potential of the formulation identified as a versatile, cost-effective, and commercially available material exhibiting exceptionally efficient multistep excitation energy transfer (EET), suitable for a broad range of applications. As a proof of concept, we demonstrate the use of orange highlighter as a low-cost water–ethanol sensor.

## 2. Experimental section

### 2.1. Materials

Over the two years of this study, dozens of highlighters from each of three commercial highlighter references (refs 70/54, 70/24 and 70/56; Stabilo Boss Original Arty Desk Set, Schwanhäusser Industrie, Heroldsberg, Germany) were tested, with no noticeable variation in the results. Spectroscopic properties were evaluated in solution. For solutions, deionised water (resistivity > 18.2 MΩ cm, Millipore Milli-Q system, Merck) and absolute ethanol (> 99.9%, v/v, Carlo Erba) were used.

For comparison, commercially available dyes were employed: Basic Yellow 40 (BY40, CAS 29556-33-0) obtained from Gruppo Colle (Italy) and BVDA (Netherlands); Basic Red 1:1 (BR 1:1, CAS 3068-39-1) from TCI (Japan); Basic Violet 11 (BV11, CAS 2390-63-8) from Alfa Chemistry (USA); Fluorescein and Rhodamine B (> 95%) from Sigma-Aldrich (USA). All reagents were used as received, without further purification.

### 2.2. Sample preparation

The tip of each commercial highlighter was dipped in a solution comprising 25 ml of purified water (resp. ethanol) for 2–10 s at room temperature. The samples prepared from refs 70/24, 70/56 and 70/54 are referred to as SBY, SBP and SBO, respectively. Samples prepared with SBY, SBP, and SBO in water are designated SBY_W, SBP_W, and SBO_W, while those prepared in ethanol are designated SBY_E, SBP_E, and SBO_E, respectively. The same assignment was used for samples prepared from commercial BY40, BR 1:1, and BV11 dyes, referred to as BY40_W, BR 1:1_W, and BV11_W in water, and as BY40_E, BR 1:1_E, and BV11_E in ethanol. These solutions were either analysed directly or diluted to achieve an absorbance of less than 0.1 ($\lambda_{exc}$ = 420, 500, 510 or 550 nm according to the sample).

### 2.3. Methods

**Absorption and emission spectroscopy (steady-state measurements).** All absorption spectra were recorded at room temperature using a UV–visible spectrophotometer (UVmc², Safas, Monaco). Emission and excitation fluorescence spectra in solution were collected at room temperature using a spectrofluorometer (Cary Eclipse, Agilent Technologies, USA). Identical parameters were used for all spectra (excitation / emission slits: 5 nm / 5 nm, averaging time: 0.1 s, step size: 1 nm, scan step: 1 nm, PMT voltage: 292 V). In all cases, emission spectra were recorded from ($\lambda_{exc}$ + 10) nm to 700 nm with $\lambda_{exc}$ at 420, 500, 510 or 550 nm, according to the sample. Excitation spectra were recorded from 350 to ($\lambda_{em}$–10) nm with $\lambda_{em}$ at 500, 570 or 610 nm, according to the sample. No corrections (standard source profile and detector efficiency) were applied to the excitation and emission spectra.

**Time-resolved fluorescence.** Fluorescence lifetime decay curves were measured using time-correlated single-photon counting (TCSPC) with femtosecond laser excitation using a Ti:Sapphire laser (Tsunami, Spectra-Physics) pumped by a doubled Nd laser (Millennia Xs, Spectra-Physics). Light pulses at 780 nm from the oscillator were modulated by an acousto-optic crystal at a repetition rate of 4 MHz, then frequency-doubled to 390 nm using a nonlinear crystal. Fluorescence photons were detected at a 90° angle through a monochromator and a polariser set to the magic angle, using a photomultiplier (MCP R3809U, Hamamatsu) connected to the TCSPC module (SPC-130-EMN, Becker & Hickl). The fluorescence data were analysed with the Globals software package (Laboratory for Fluorescence Dynamics, University of California, Irvine, USA), which includes reconvolution analysis and global nonlinear least-squares minimisation. The excitation and emission wavelengths were selected based on previous experiments at 420 nm or 500 nm for excitation, and 490 nm, 550 nm or 590 nm for emission. A long-pass filter in emission was added.

**Transmission electron microscopy.** Dry diameters of the particles composing the inks were determined by transmission electron microscopy (TEM). In short, undiluted samples were deposited on positively charged carbon-membrane coated copper grids. The particles were imaged on a Hitachi H7650 electron microscope (80 kV). The images were analysed using a Python segmentation analysis program (code available upon request) to extract the mean nanoparticle diameters.

**Photography.** Samples were illuminated with a Liasimi UV pocket lamp for violet illumination (irradiation between 375 and 425 nm) and Daphomeu RGB LED light for green illumination (irradiation between 500 and 550 nm).

**Förster radius calculation.** The fluorescence quantum yields (QY) of BY40 and BR 1:1 in water were determined using fluorescein in 0.1 M NaOH (QY of 0.95 [1,41]) and Rhodamine B in ethanol (QY of 0.70 [42]) as reference standards, respectively. Calibration curves at 525 nm for BR 1:1 and 558 nm for BV11 in water were established based on three replicates for each concentration. Förster radii were obtained from PhotochemCAD 2.1 Software. Refractive index was 1.33 (for water) and orientation factor fixed to 0.66 (for solution).

**Determination of ethanol level.** A concentrated SBO_W stock solution was prepared, and 2.5 mL ethanol/water mixtures with varying ethanol volume fractions (v/v) were made. The refractive index of each mixture was first measured using a Novex Abbe refractometer. Subsequently, 10 µL of the stock solution was added to each mixture. Absorption spectra, emission spectra ($\lambda_{exc}$ = 420 nm), and excitation spectra ($\lambda_{em}$ = 610 nm) were then recorded. All measurements were performed in triplicate. To evaluate the validity of the calibration curve, ethanol (96.0°, Aldrich), a commercial gin (Gin Mare, 42.7°), and a commercial vodka (Absolut, Pernod Ricard, 40.0°) were analysed.
## 3. Results and discussion

### 3.1. Unexpected emission of the orange highlighter ink

Despite their widespread use, the relationship between highlighter inks' chemical composition and their optical behaviour remains only partly elucidated [43–53]. The core of this study concerns the optical properties of an orange commercial highlighter (ref. 70/54, see *Meth-ods*), compared to yellow (ref. 70/24) and pink (ref. 70/56) references. Initially, the tips of the highlighter pens were immersed in water to extract the ink. Within seconds, the water became coloured and slightly opalescent (Fig. 1A). Dispersions of the orange, yellow, and pink inks were referenced as SBO, SBY, and SBP, respectively, followed by W (water) or E (ethanol) according to the solvent (see *Materials*).

The absorption spectrum of SBY_W and SBP_W exhibited one band at 440 nm and two bands at 533 and 565 nm, respectively, resulting in a yellow and pink colouration under visible light (Fig. 1A, Fig. 1D, Supplementary Tab. S1.1).

The SBO_W absorption spectrum exhibited three distinct bands at 440, 533, and 565 nm, yielding an orange colouration (Fig. 1A, Fig. 1D Supplementary Tab. S1.1). This spectrum combines the features of SBY_W and SBP_W. Similarly, the absorption spectrum of the orange ink in ethanol (SBO_E) exhibited three bands at 435, 530, and 555 nm, resulting in an orange hue with a slight hypochromic shift (Fig. 1A, Fig. 1D, Supplementary Fig. S1.1B, Supplementary Tab. S1.2). This spectrum also combines the absorption profiles of SBY_E and SBP_E (Supplementary Fig. S1.1B, Supplementary Tab. S1.2).

The emission spectrum of the yellow ink SBY_W ($\lambda_{exc}$ = 420 nm) exhibited a band at 495 nm, consistent with its green emission under these conditions (Fig. 1B, Fig. 1D, Supplementary Tab. S1.1). SBP_W remained non-fluorescent under violet light (Fig. 1B) while emitting orange fluorescence under green light (Fig. 1C). The absence of emission from SBP_W is consistent with its absorption spectrum, which lacks any significant absorption bands in the violet (Fig. 1D). The orange fluorescence observed under green light is consistent with the emission band at 585 nm when excited at 510 nm or 550 nm (Fig. 1D, Supplementary Tab. S1.1).

Assuming that SBO_W combines the fluorescence properties of SBY_W and SBP_W, as it does for absorption, only green fluorescence from SBY_W was expected under violet light, since SBP_W neither absorbs nor emits light (Fig. 1B,D). However, surprisingly, SBO_W exhibited an orange fluorescence (Fig. 1B). This emission was strikingly similar to that of SBP_W under green light (Fig. 1C). This is all the more unexpected given that, in ethanol, SBO_E, as anticipated, emitted the expected green fluorescence (Fig. 1B), highlighting the presence of a distinctive photophysical behaviour in water. Indeed, the emission spectrum of SBO_E featured a single band at 489 nm ($\lambda_{exc}$ = 420 nm), resulting in the observed green fluorescence (Fig. 1D, Supplementary Tab. S1.2). In turn, upon excitation at 420 nm, SBO_W exhibited an emission spectrum with a maximum at 585 nm, and no emission around 490 nm (Fig. 1D, Supplementary Tab. S1.1). The emission observed in SBY_W was completely absent, and was instead replaced by the emission of SBP_W.

Surprised by such behaviour, we sought to better understand the origin of their optical properties and to investigate the ink composition (dyes and formulation), suspecting energy transfer processes from SBY to SBP dyes.

### 3.2. Dye identification

Dye identification was first performed using HPLC-ESI-Q-ToF and HPLC-DAD, which revealed the dye combinations present in the three inks, SBY, SBP, and SBO (Supplementary Fig. S5.1–S5.5, Supplementary Tab. S5.1).

Results showed that SBY contained only one yellow dye, Basic Yellow 40 (BY40) (Fig. 2), consistent with its excitation spectrum in water

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** (A, B) Photograph of SBY_W, SBP_W, SBO_W and SBO_E under visible (A) and violet illumination (B). (C) Photograph of SBP_W under green illumination. (D) Absorption, emission ($\lambda_{exc}$ = 420 or 550 nm) spectra of SBY_W, SBP_W, SBO_W and SBO_E.</figcaption></figure>

and ethanol ($\lambda_{eml}$ = 510 nm) closely matching the absorption spectrum, aside from minor scattering-related deviations (Supplementary Fig. S1.1A,B).

In contrast, SBP was primarily composed of Basic Red 1:1 (BR 1:1) and Basic Violet 11 (BV 11) (Fig. 2), alongside other pink dyes such as Rhodamine B (RhB), Basic Violet 11-N-Et (BV 11-Et), and Basic Violet 11:1 (BV 11:1). SBO was found to contain all the aforementioned dyes (Supplementary Fig. S5.5), with BY40 accounting for 93.8% of the dye content, BR 1:1 for 3.6%, and BV11 for 2.6% (details on quantification are provided in the Supplementary Text S5.4). These results are consistent with previous studies on similar pink and orange highlighter inks, which identified the same main dye components [53].
<figure><img src="image_6.png" /><figcaption>Fig. 2. Chemical structures of Basic Yellow 40, Basic Red 1:1, and Basic Violet 11.</figcaption></figure>

Having identified the dyes involved, it is important to examine their formulation and environment, as these factors can affect their interactions and spatial arrangement, ultimately influencing their optical properties and EET.

### 3.3. Chemical formulation

The chemical formulation was then investigated using analytical pyrolysis. Pyrograms of all samples (Supplementary Fig. S6.1) were qualitatively identical, except for few peaks associated with the specific dye(s). The main components (Supplementary Tab. S6.1) were subsequently grouped according to their material type and interpreted following the available literature on the analysis of felt tip pen inks [47, 54,55]. Based on the detection of the monomers and oligomers typical of the pyrolysis profile of ethylhexylacrylate (peaks #6, #7, #8, #11, #14 and #16) and of glycerol (#13), we can hypothesise that the inks are composed of polymeric particles of polystyrene and polyacrylates dispersed in water/glycerol media, as reported for other pen inks (felt tip: Van Der Werf et al. [55]). The MidIR spectra are consistent with the presence of glycerol and water (Supplementary Fig. S7.1). Several peaks due to the pyrolysis of an acrylonitrile-styrene copolymer were also detected (e.g. the monomers acrylonitrile, #2, and styrene, #9, the dimers and trimers). This copolymer forms a rigid material, suggesting that it was likely used for the highlighter's felt-tip or container, which may have formed particles dispersed within the paint layer, or may have dissolved within it. The presence of polyacrylonitrile (PAN), which is sometimes added in felt tip pen ink formulations to enhance resistance and transparency [47,55] may be suggested by the detection of methylacrylonitrile (#4) and benzenepropanenitrile (#15). Due to the co-presence of the acrylonitrile-styrene copolymer, the final identification of PAN is unfortunately not possible. At retention times higher than 21 min, the pyrograms feature pyrolysis products related to BY40 for SBO and SBY samples, and to xanthenes for the SBP and SBO samples.

Finally, TEM analysis revealed that the above mentioned particles of polystyrene and polyacrylates are spherical with an average diameter of approximately 130–140 nm (Supplementary Fig. S3.1, Supplementary Text S3). These particle sizes were further confirmed by NTA and DLS measurements, which indicated hydrodynamic diameters of around 125 nm and 155 nm, respectively (Supplementary Tab. S4.1, Supplementary Text S4). In particular, SBO_W contained nanoparticles with a monodisperse size distribution, showing average diameters of 134 nm (FWHM = 34 nm) by TEM (Fig. 3, Supplementary Text S3), 123 nm by NTA (span: 0.36), and 152 nm by DLS (polydispersity index (PDI): 0.020). The observation of scattering effects in the absorption spectra of SBY_W, SBP_W, SBO_W is consistent with the presence of a dispersion (Fig. 1D and Supplementary Fig. S1.1A).

These findings indicate that commercial highlighter inks contain polystyrene and polyacrylate nanoparticles in a water/glycerol medium, with the dyes most likely encapsulated within the nanoparticles and in close proximity to one another.

### 3.4. Comparison with pure dyes: first multistep EET evidences

The steady-state absorption and emission spectra of SBY_W (Fig. 1D) closely matched those of BY40_W (Fig. 4A, B), while BR1:1_W exhibited absorption and emission bands around 525 nm and 550 nm, respectively, and BV11_W showed red-shifted absorption and emission bands centred near 560 nm and 580 nm (Fig. 4A, B, Table 1).

This allows for unambiguous assignment of the absorption bands in both SBO_W and SBP_W, with those at 533, and 565 nm attributed to BR 1:1, and BV11, respectively, and the additional band at 440 nm in SBO_W assigned to BY40 (Fig. 4A).

All these absorption bands can be attributed to allowed $\pi \rightarrow \pi^*$ electronic transitions, in agreement with TD-DFT calculations performed on structurally similar molecules such as coumarins (for BY40) [56,57] and rhodamines (for BR 1:1 and BV11) [58,59].

The BR 1:1/BV11 ratio can then be estimated by HPLC-DAD analysis as 4/3 for SBO and 8/3 for SBP by integrating the corresponding peak areas in the chromatograms (Supplementary Text S5.2, Supplementary Fig. S5.1). These data correlate with the A(533 nm)/A(565 nm) ratio in the SBO_W and SBP_W absorption spectra and the A(530 nm)/A(555 nm) ratio in SBO_E (Fig. 1D), with values of 4/3 for SBO sample and 6/3 for SBP_W.

However, surprisingly, for SBO_W only the emission behaviour of BV11 was observed upon excitation at 420 nm (Fig. 4B) and 500 nm (Supplementary Fig. S1.1D), with an emission band centred around 585 nm. Excitation of BR1:1_W and BV11_W at 420 nm also resulted in an almost complete lack of fluorescence (Fig. 4B). This suggests the occurrence of direct EET from BY40 (fluorescence quantum yield of 0.055 ± 0.006) to both BR 1:1 (molar absorption coefficient of 8.488 ± 0.027 × 10⁴ L mol⁻¹ cm⁻¹ at 525 nm, Supplementary Fig. S2.3A) and BV11 (molar absorption coefficient of 8.913 ± 0.015 × 10⁴ L mol⁻¹ cm⁻¹ at 559 nm, Supplementary Fig. S2.3B), with corresponding Förster radii of 3.6 nm and 3.4 nm, respectively (Supplementary Fig. S2.4B, Table 1). Beyond the BY40-to-BR 1:1 transfer, energy is also transferred from BR 1:1 to BV11, leading to a multistep EET.

Further evidence of such process in SBO_W was observed in the excitation spectrum ($\lambda_{em}$ = 610 nm), which showed signals at 440 nm and 535 nm, corresponding to the absorption regions of the BY40 and BR 1:1 donors, respectively (Fig. 4A).

In addition, for SBP_W, only the emission behaviour of BV11 was observed, with an emission band centred around 585 nm upon excitation at both 500 and 550 nm (Fig. 1D, Supplementary Fig. S1.1A). A Förster radius of 5.5 nm in water was calculated based on the emission properties of BR 1:1 (fluorescence quantum yield of 0.38 ± 0.02) and the absorption properties of BV11, allowing a very high EET efficiency (Supplementary Fig. S2.4B, Table 1) between these two dyes. The strong similarity between the excitation spectrum at 610 nm and the absorption spectrum of both SBO_W and SBP_W (Supplementary Fig. S1.1A) in the absorption region of BR 1:1 and BV11 (500–600 nm) further supports the assumption of a direct EET between BR 1:1 and BV11.

Such direct (BY40-BR1:1, BY40-BV11) and multistep EET (BY40-BR 1:1-BV11) processes supports the exclusive emission behaviour of SBO_W upon excitation at 420 nm (Fig. 4B) and can be further evidenced by the behaviour of SBY_E, SBP_E and SBO_E samples and time-resolved measurements in both water and ethanol.
<figure><img src="image_7.png" /><figcaption>Fig. 3. TEM image (A) and size distribution (B) of SBO_W. Scale bar: 1 µm.</figcaption></figure>

**Table 1**

Results from steady-state measurements ($\lambda_{max}$ in absorption and emission), absorption coefficient, fluorescence quantum yield, and Förster radius for BY40_W, BR 1:1_W, and BV11_W. *Value not determined.

<table><thead><tr><th>Sample</th><th>λ<sub>max</sub> (abs)/nm</th><th>λ<sub>max</sub> (fluo)/nm</th><th>c(λ<sub>max</sub>)/(M<sup>-1</sup> cm<sup>-1</sup>)</th><th>φ<sub>f</sub></th><th>Förster radius R<sub>0</sub>(i) for compound i/nm</th></tr></thead><tbody><tr><td>BY40_W</td><td>440</td><td>493</td><td>-*</td><td>0.055 ± 0.006</td><td>3.6 (BR 1:1); 3.4 (BV11)</td></tr><tr><td>BR 1:1_W</td><td>525</td><td>552</td><td>8.488 ± 0.027 × 10<sup>4</sup></td><td>0.38 ± 0.02</td><td>3.6 (BY40); 5.5 (BV11)</td></tr><tr><td>BV11_W</td><td>559</td><td>581</td><td>8.913 ± 0.015 × 10<sup>4</sup></td><td>-*</td><td>3.4 (BY40); 5.5 (BR 1:1)</td></tr></tbody></table>

<figure><img src="image_8.png" /><figcaption>Fig. 4. (A) Absorption spectra of SBO_W, BY40_W BR1:1_W and BV11_W samples and excitation spectrum ($\lambda_{em}$ = 610 nm) of SBO_W. (B) Emission ($\lambda_{exc}$ = 420, 500 or 550 nm) spectra of samples SBO_W, BY40_W, BR1:1_W and BV11_W in water.</figcaption></figure>

### 3.5. Water vs ethanol behaviour: additional multistep EET evidences

NTA measurements in ethanol, showed that nanoparticles were preserved, yet with a higher mean particle size of 215 nm (SBY_E), 182 nm (SBP_E) and 184 nm (SBO_E) (Tab. S4.1). The steady-state absorption and emission spectra of SBY_W (Fig. 1B, Supplementary Tab. S1.1) and SBY_E (Supplementary Fig. S1.1B, Supplementary Tab. S1.2) closely matched those of BY40_W (Fig. 4A, Supplementary Fig. S2.2A, Supplementary Tab. S2.1) and BY40_E (Supplementary Fig. S2.2B, Supplementary Tab. S2.1), respectively. Time-resolved measurements are similar between SBY_E and BY40_E (Fig. 5, Supplementary Tab. S1.2, Supplementary Fig. S2.4A, Supplementary Tab. S2.1). Yet, the amplitude-averaged lifetime of BY40_W was markedly shorter than that of SBY_W, 0.11 ns vs 1.41 ns (Fig. 5, Supplementary Tab. S1.1, Supplementary Fig. S2.4A, Supplementary Tab. S2.1).

These observations strongly indicate dye solubilisation in ethanol in the SBY_E sample and encapsulation of the dyes within the nanoparticles, where the local solvent environment differs from water. In addition, SBP_E showed excitation-dependent emission, with a band at 552 nm when excited at 500 nm and another at 571 nm when excited at 550 nm (Supplementary Fig. S1.1B, Supplementary Tab. S1.2). A similar behaviour was observed for SBO_E with bands measured at 552 nm ($\lambda_{exc}$ = 500 nm), and 575 nm ($\lambda_{exc}$ = 550 nm) (Supplementary Fig. S1.1B, Supplementary Tab. S1.2). The emission bands around 550 and 570 nm can be assigned to BR 1:1 and BV11 respectively, based on the results obtained for BR 1:1 and BV11 in water and ethanol with $\lambda_{max}$ values around 551–552 nm and 578–581 nm respectively (Fig. 4B, Supplementary Tab. S2.1, Supplementary Fig. S2.2B). The excitation spectra of SBP_E and SBO_E also varied with the emission wavelength, showing bands at 438 nm for $\lambda_{em}$ = 500 nm, 530 nm for
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** (Top) Fluorescence lifetime decay ($\lambda_{exc} = 420$ nm and $\lambda_{em} = 490$ nm) of samples SBY_W and SBO_W. (Bottom) Fluorescence lifetime decay ($\lambda_{exc} = 420$ nm and $\lambda_{em} = 490$ nm) of SBY_E and SBO_E.</figcaption></figure>

$\lambda_{em} = 570$ nm, and 553 nm for $\lambda_{em} = 610$ nm (Supplementary Fig. S1.1B, E, Supplementary Tab. S1.2). Excitation of one dye leads to its own emission, thereby highlighting the absence of EET in ethanol. These findings corroborate a full solubilisation of the dyes, which disrupts dye-dye interactions and consequently leads to the loss of EET.

Time-resolved fluorescence measurements of SBO and SBP in water and ethanol supported this hypothesis, while also confirming both direct and multistep EET processes in water (Fig. 5, Supplementary Fig. S1.2, Supplementary Text S1). The fluorescence lifetime decay curve for SBO_W at 490 nm ($\lambda_{exc} = 420$ nm) showed a tri-exponential behaviour with a markedly shorter amplitude-averaged lifetime of 80 ps, compared to 1.41 ns for SBY_W, which also exhibited tri-exponential behaviour (Fig. 5, Supplementary Tab. S1.1). Furthermore, at 590 nm (excited at 420 nm), SBO_W displayed a single negative pre-exponential factor associated with a 70 ps time constant, indicating a rising lifetime (Supplementary Fig. S1.2, Supplementary Tab. S1.1). This substantial reduction in donor lifetime (BY40), combined with the observed increase in acceptor emission lifetime (BR 1:1 and BV11), provides compelling evidence of efficient EET (direct and/or multistep). The observation of a rising component in the fluorescence lifetime decay curve at 590 nm (excited at 500 nm) for both SBO_W and SBP_W (Supplementary Fig. S1.2A, Supplementary Tab. S1.1) is a confirmation of the presence of an additional EET interaction between BR 1:1 and BV11.

The fluorescence lifetime decay curves for SBO_E at 490 nm ($\lambda_{exc} = 420$ nm) displaying a tri-exponential behaviour (0.18, 0.73, and 2.00 ns) with amplitude-averaged lifetimes of 0.44 ns (Fig. 5, Supplementary Tab. S1.2) which was in close proximity to SBY_E (0.36 ns) but markedly distinct from SBY_W (1.41 ns). The fluorescence lifetime decay curve at 590 nm ($\lambda_{exc} = 420$ and 500 nm) also displayed tri-exponential behaviour with amplitude-averaged lifetimes of 1.44 ns (0.22, 0.84, and 3.83 ns) and 2.83 ns (0.19, 2.04, and 3.73 ns) respectively, with no rise time observed in the last case (Supplementary Fig. S1.2B, Supplementary Tab. S1.2).

Dyes can therefore be considered independent and do not interact with each other. The high EET efficiency observed for SBO_W likely

arises from short intermolecular distances between dyes within the nanoparticles, enabling both efficient direct and multistep EET.

Based on NTA, TEM, HPLC, and absorption data, the average intermolecular distance between dye molecules is estimated to be in the range of 1.3–1.5 nm (Supplementary Text S8.6). Specifically, the distance between BY40 and BR 1:1 or BV11 is approximately 1.3 nm, while the distance between BR 1:1 and BV11 is around 3.2 nm when estimated using the Onsager radii. These distances are roughly half of the corresponding Förster radii (Table 1), indicating efficient energy transfer between dyes.

The tri- or tetra-exponential behaviour observed for SBY_W, SBP_W, and SBO_W (Supplementary Fig. S1.2A, Supplementary Tab. S1.1) further supports dye encapsulation within polymeric nanoparticles, which generate heterogeneous local environments and diverse molecular arrangements, leading to several relaxation pathways. Moreover, the environment is markedly different from bulk water or ethanol, making direct comparison challenging between SBY_E, SBP_E, and SBO_E samples and the corresponding pure dyes in water (BY40_W, BR 1:1_W, and BV11_W). In ethanol, BY40 exhibits bi-exponential decay with a major short-lived component ($\tau \approx 0.20$ ns), in contrast to the mono-exponential lifetimes of ~2.4–2.7 ns observed for Coumarin 6 [60] and the pure BR 1:1 and BV11 dyes (Supplementary Fig. S2.4A, Supplementary Tab. S2.1). SBY_E showed a similar two-exponential behaviour, slightly modified by the presence of glycerol, while SBP_E and SBO_E, being mixtures of several dyes, display more complex multi-exponential decays that make direct comparison with the pure dyes difficult. These differences likely reflect interactions between dyes, minor contributions from BR 1:1 or BV11, and heterogeneity in local environments, but they do not challenge the effective release of the dyes in ethanol.

### 3.6. A near unity energy transfer efficiency for multistep EET in SBO_W

The integration of the results obtained leads to a clear EET mechanism (see schematic representation in Fig. 6).

The close proximity and spectral overlap between BY40, BR 1:1 and BV11 provides favourable conditions for EET, like in SBP_W (Fig. 6A). In SBO_W, both direct (BY40-BR1:1, BY40-BV11) and multistep EET (BY40-BR 1:1-BV11) are involved (Fig. 6B). However, other mechanisms cannot be entirely excluded, in particular Dexter energy transfer and homo-FRET (primarily involving the pink dyes, BR 1.1 and BV11). Amplitude-averaged lifetimes, obtained from fluorescence lifetime decay curves at 490 nm ($\lambda_{exc} = 420$ nm), allow for a comparison of the emission behaviour of BY40 (donor) in SBY and SBO samples and a quantification of the EET efficiency (Supplementary Text S8). The latter was determined to be 94.4 ± 0.1% (Supplementary Tab. S8.1) for SBO_W. An efficiency of 97.2 ± 0.1% was determined from steady-state (Supplementary Text S8). This extremely high quantum efficiency, close to unity, is among the highest EET efficiencies ever reported in the literature for a multistep energy transfer system [15,21,40].

Given that the polymeric nature of nanoparticles ensures a generally random orientation of molecules, inadequate molecular orientation could explain the inability to achieve unity for the orientation factor ($\kappa$). This factor was estimated at 0.496 for fixed molecules with random orientation [61].

In ethanol (SBO_E), the EET efficiency was estimated at 0.3 ± 0.4% from time-resolved fluorescence measurements and at 22.1 ± 6.8% from steady-state measurements (Supplementary Tab. S8.1). The emission behaviour of SBO_E and SBP_E suggests that the dyes act independently, without EET, even though the nanoparticles remain present. This suggests that the dyes may have left the nanoparticles and entered the solvent phase for both samples (Fig. 6C, D). This leads to the loss of EET due to the increase in distance between the two dyes.
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Schematic representation of the emission behaviour correlating with the observed EET efficiency in water (A: SBP_W, B: SBO_W) and ethanol (C: SBP_E, D: SBO_E).</figcaption></figure>

### 3.7. Stability and photostability of the SBO_W emulsion

The stability and photostability of the dispersion and its multistep energy transfer were examined to start evaluating potential applications (Supplementary Text S9). Zetametry measurements revealed a markedly negative ζ-potential value for all inks between –34 and –41 mV (–40 mV for SBO_W) in water and –38 to –45 mV in ethanol (Supplementary Tab. S4.1), indicating an exceptional stability of the dispersion in both solvent.

Such stability of SBO_W was further demonstrated by absorption, emission, DLS, and ζ-potential measurements over time, showing minimal variation over 600 min (Supplementary Fig. S9.1 and S9.2). The ζ-potential decreased slightly in absolute value but remained highly negative (< –20 mV). The photostability of the SBO_W nanoparticles under violet light irradiation was investigated. Absorption decreases slightly, with complete discolouration expected after 16 h based on the evolution at 440 nm (Supplementary Fig. S9.3A). Emission is modestly affected, showing an increase at 495 nm and a decrease at 583 nm, likely due to different degradation kinetics of the dyes within the nanoparticles (Supplementary Fig. S9.3B). Despite this progressive colour loss, the mean size and PDI of the nanoparticles, as measured by DLS, remained unchanged, indicating that their structural integrity is preserved under light exposure (Supplementary Fig. S9.4).

### 3.8. Orange highlighter as a water-ethanol sensor

All these results pave the way for potential applications of the SBO_W dispersion, such as its use as a fluorescent probe. Only a simple proof of concept is explored in the present paper.

Fluorescent probes are widely used for alcohol sensing [62–64]. The notable difference between the emission properties of SBO_W and

SBO_E highlights the potential of SBO as an EET-based water-ethanol ratio sensor (Fig. 7A). EET efficiency could be readily assessed from steady-state spectra using two fluorescence intensity ($I_f$) ratios: (i) the emission ratio $I_f(490 \text{ nm}) / I_f(585 \text{ nm})$, and (ii) the excitation ratio $I_f(440 \text{ nm}) / I_f(530 \text{ nm})$.

The results showed a pronounced increase in the emission ratio at ethanol concentrations above 50°, while the excitation ratio changed most significantly below 50° (Fig. 7B). These calibration curves demonstrate that SBO_W acts as a water-ethanol sensor based on EET, employing either the excitation ratio- or emission-based ratiometric fluorescence method. To validate the sensor's reliability, a 96.0° ethanol sample was analysed: the excitation ratio-based method yielded an accurate value of 97.2 ± 1.2°, while the emission ratio-based method gave a close value of 98.2 ± 1.8° (Fig. 7B). Commercial gin and vodka were then tested to evaluate the sensor's performance in more complex matrices. The excitation ratio-based method accurately determined the ethanol content for the gin sample (41.1 ± 2.7° vs 42.7°). In contrast, the vodka sample showed a larger deviation (21.0 ± 6.6° vs 40°). Similarly, the refractive index-based calibration curve gives accurate results for pure 96° ethanol, while slight deviations are observed for gin (38.0 vs 42.7°) and vodka (41.0 vs 40.0°). These results show that sample matrix can affect measurement reliability by altering fluorescence properties. Nevertheless, SBO_W can be used as a water-ethanol sensor, provided that preliminary tests are performed to verify the calibration curve in the intended substrate. These findings are promising and suggest that SBO_W could be employed as a low-cost, stepwise EET-based fluorescence sensor for quantifying alcohol content, with potential applications extending to life science systems, as already demonstrated for FRET sensors in the literature [65]. For long-term performance of this sensor, the stock solution should be kept in the dark (e.g., wrapped in aluminum foil) to preserve photostability (Supplementary Fig. S9.3).

## 4. Conclusion

We show that the formulation of a commercial orange highlighter ink, consists of a stable monodisperse dispersion of dye-loaded nanoparticles (134 nm) in aqueous media. This combination in the orange ink enabled the occurrence of both direct and multistep EET, where BY40 acts as the initial donor, transferring energy to both BR 1:1 and BV11, and, in the multistep case, passing through BR 1:1 before reaching BV11. This system achieves a near-unity multistep EET efficiency in water (94.4%), ranking among the highest ever reported.

This provides a low-cost and readily available material exhibiting high multistep excitation energy transfer (EET) efficiency, suitable for a wide range of applications. Such properties could benefit fields including energy harvesting, optoelectronics, solar cells, photothermal medicine, chemical biology, materials science, and education.

As a proof of concept, the orange highlighter acts as an efficient and low-cost water-ethanol sensor, with the EET efficiency decreasing to 0.3% in ethanol, indicating a complete loss of energy transfer in this medium.

This article highlights the interest of researching advancements in everyday products. Bio-inspiration and the recently proposed paleo-inspiration approach [66] demonstrate how convergent evolution can offer valuable concepts and material solutions for serendipitous discoveries of high-value formulations. Here, the manufacturer's search for a universal platform capable of integrating numerous dyes must have been the key factor that made it possible to combine three different dyes in nanodroplets, thereby enabling highly efficient EET, while providing a stable system in solution (ink storage). Existing market products tend to present fewer regulatory challenges for new applications while also benefiting industrial scalability and cost efficiency. This work therefore advocates for a renewed focus of chemists on reanalysing the physical properties of available commercial products.
<figure><img src="image_11.png" /><figcaption>**Fig. 7.** (A) Emission spectra ($\lambda_{exc}$ = 420 nm) and excitation spectra ($\lambda_{em}$ = 610 nm) of SBO_W (0°) and SBO_E (100°). (B) Evolution of the refractive index $nD^{25}$, emission ratio (490/585) and excitation ratio (440/530) for different ethanol fraction (v/v), with results shown for 96° ethanol, as well as commercial gin and vodka.</figcaption></figure>

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

The experiments were conducted during a “Pedagogical Innovation Period” awarded to J.P., who extends his gratitude to Université Paris-Saclay and ENS Paris-Saclay for their trust and support in providing this opportunity. The Centre for Instrument Sharing of the University of Pisa (CISUP) is acknowledged for providing the Py-GC-MS instrumentation. The authors would like to express their gratitude to G. Clavier, R. Méallet, and M. Auvray for their invaluable contributions during the initial discussions on this project. The authors thank M. Vincent and M. Lecoq for their contributions to the initial experiments with undergraduate students, which provided preliminary evidence of EET in orange highlighters. They acknowledge B. Cheklal, N. Delaveau, and P. Varin for conducting supplementary investigations during an undergraduate project-based teaching activity. L.B. and J.P. thank Professor Keitaro Nakatani (PPSM) for years of frequent discussions on the development of their research. ID thanks Dr Jacopo La Nasa (University of Pisa) for his insights on polymeric formulations.

## Appendix A. Supplementary data

Supplementary material related to this article can be found online at https://doi.org/10.1016/j.saa.2026.127477.

## Data availability

All relevant data for evaluating the conclusions are available in the paper, Supplementary Material, and the Zenodo repository https://doi.org/10.5281/zenodo.18189694.

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