Conventional analytical techniques, including chromatographic and spectroscopic methods, have delivered important insights into acrolein chemistry, but they come with practical burdens. Many require harsh reaction conditions and labor-intensive sample preparation, and they often rely on indirect detection of acrolein-derived protein adducts or acrolein-amine conjugates. In complex biological matrices, these approaches suffer from limited chemical selectivity and reduced sensitivity, making convenient and accurate quantification difficult in both research and clinical settings. Fluorescence-based methods have therefore emerged as attractive alternatives, offering high sensitivity, operational simplicity and the possibility of direct readout.

Existing fluorescent probes, however, still leave room for improvement. Naphthalimide-based probes optimized for well-defined matrices such as food extracts show strong fluorescence and good biocompatibility but depend strongly on pH, solvent composition and incubation time, which restricts their quantitative reliability in complex samples like tobacco smoke. Nanomaterial-based platforms that combine sensing with acrolein scavenging are useful for threshold indication and removal assessment but are poorly suited to precise, reproducible quantification. Activity-based probes that collectively target lipid-derived electrophiles, including acrolein and 4-hydroxynonenal, can map the overall electrophilic environment but cannot selectively measure acrolein alone. These gaps motivated a Japanese research team to refine a distinctly chemical solution.

Writing in the open-access journal Results in Chemistry, Masayuki Takamatsu, Hiroya Tsuchida, Hinako Nabetani, Kazuya Kabayama, Ambara R. Pradipta, Katsunori Tanaka, Koichi Fukase and Atsushi Shimoyama report a second generation of pyrene-based fluorescent probes with enhanced chemoselectivity for acrolein. The work builds on a striking piece of chemistry the group had uncovered earlier: acrolein reacts with biologically relevant amines to form characteristic eight-membered heterocycles through a formal imino [4+4] cycloaddition. That unusual dimerization is the entire engine of the new sensing strategy.

The first-generation probe, Probe A, featured an ethanolamine unit as the reactive handle attached to a pyrene fluorophore. When acrolein triggered dimerization of Probe A, two pyrene units were brought into close proximity, and the monomer emission near 375 nanometers gave way to a red-shifted band near 475 nanometers, the signature of pyrene excimer emission. Pyrene is uniquely suited to this role because two pyrene units held close together in an appropriate excited-state geometry produce exactly this red-shifted excimer glow. Rather than acting as a passive label, pyrene functions as a mechanism-linked reporter: the fluorescence change is tied directly to covalent dimer formation, not to reversible binding of the analyte.

To improve on Probe A, the team designed four new analogues, Probes B through E, by systematically modifying the 1,2-amino alcohol motif that carries the reactive amine. Probe B repositioned the primary amine from a terminal carbon to an internal carbon of the amino alcohol, introducing steric bulk around the reactive site. Probe C replaced the hydroxy group with an additional amino group, creating a 1,2-diamine scaffold. Probes D and E were serine-derived 1,2-amino alcohol analogues chosen for synthetic accessibility and to test how linker length between the amino alcohol and the pyrene unit influences excimer formation. All four probes were synthesized from readily available protected amino acid derivatives using coupling agents such as DMT-MM and standard protection-deprotection chemistry.

Time-course experiments revealed clear structure-activity relationships. When each probe was reacted with one equivalent of acrolein in chloroform and the excimer emission at 475 nanometers monitored over 48 hours, all three of Probe A, Probe B and Probe C plateaued after roughly 40 hours, and mass spectrometry confirmed dimer formation in each case. But the trajectories differed. Probe B, with its internal amine, responded more slowly at first, likely because forming the conjugated imine intermediate with acrolein is less favorable in its altered amine environment. Probe C reached only about 60 percent of the plateau intensity of the other two, apparently because the eight-membered aminal dimers it forms are less stable and partially decompose over time.

The chemoselectivity comparison proved decisive. The team challenged each probe with a panel of structurally related alpha,beta-unsaturated aldehydes: methacrolein, crotonaldehyde and sorbaldehyde, which introduce alpha,beta-substitution and extended conjugation respectively. After 24 hours, the relative preference for acrolein followed the order Probe B greater than Probe D approximately equal to Probe E, far exceeding Probe A. The first-generation probe showed only moderate selectivity because its terminal primary amine readily forms conjugated imines with bulkier unsaturated aldehydes as well, and those dimers also emit excimer fluorescence, masking the acrolein signal. The more sterically hindered amine environments of the second-generation amino alcohol probes discriminated against these larger aldehydes, exactly as the design concept predicted.

Acetic acid emerged as a surprising and powerful additive. When the probes were treated with acrolein in the presence of 23 millimolar acetic acid, the emission responses of the serine-derived Probes D and E increased markedly, while those of Probes A and B decreased. The authors propose that acetic acid facilitates activation of the aldehyde or of imine and iminium-type intermediates, promoting dimer formation, while protonation of the more basic amines in Probes A and B suppresses their nucleophilicity. Under the optimized chloroform-acetic acid conditions, Probe D and Probe E both reached a plateau within 24 hours, faster than the first-generation system, and Probe D showed the strongest overall response.

Probe D was accordingly selected as the lead candidate for quantitative evaluation. With the probe fixed at 1.0 millimolar and acrolein varied from 0.033 to 1.1 millimolar, the excimer intensity at 475 nanometers showed an excellent linear relationship after 24 hours, with a coefficient of determination of 0.9985. The authors are careful to note that a formal limit of detection was not calculated, so the lowest concentration examined, 33 micromolar, should not be read as the detection limit; previously reported tethering and ratiometric methods have achieved submicromolar limits, though direct comparison is complicated by very different conditions and media. The contribution here is not record sensitivity but chemoselectivity, synthetic practicality and compatibility with an extraction-based workflow.

That workflow was put to the test with biological samples. The researchers lysed RAW264.7 macrophage-like cells with Triton X-100 and sonication, extracted acrolein into chloroform, and reacted the extract with Probe D under the optimized conditions. When identical amounts of acrolein were extracted from buffer alone or from buffer mixed with cell lysate, comparable excimer intensities resulted, showing that major co-extracted components did not suppress the reaction or the fluorescence readout. The team then treated cells with hydrogen peroxide at 0, 62 and 125 millimolar for three hours to induce oxidative stress, extracted the lysates, and observed a concentration-dependent increase in the red-shifted fluorescence response, consistent with more extractable acrolein-derived reaction products.

The authors are appropriately measured about what this means. The extraction-based experiment does not constitute direct intracellular imaging or real-time cellular sensing, and it does not establish acrolein as the exclusive contributor among all extractable electrophilic components. Still, the demonstration that a simple covalent dimerization reaction, read out through pyrene excimer emission, can survive the jump from pure chloroform solutions to cell-lysate extracts is a meaningful step toward practical acrolein analysis. Further optimization aimed at stabilizing the dimeric products and improving fluorescence efficiency may push sensitivity further. For now, the study shows that rearranging a single amino group within a 1,2-amino alcohol motif, and letting a touch of acetic acid assist the chemistry, can turn an unusual [4+4] cycloaddition into a sharper, more selective fluorescent eye on one of biology’s most damaging aldehydes.

Subject of Research: Development of second-generation pyrene excimer fluorescent probes for chemoselective detection of acrolein via imino [4+4] cycloaddition dimerization

Article Title: An improved pyrene excimer fluorescent probe with enhanced chemoselectivity for acrolein detection

Article References: Takamatsu, M., Tsuchida, H., Nabetani, H., Kabayama, K., Pradipta, A. R., Tanaka, K., Fukase, K., & Shimoyama, A. (2026). An improved pyrene excimer fluorescent probe with enhanced chemoselectivity for acrolein detection. Results in Chemistry, 31, Article 103962. https://doi.org/10.1016/j.rechem.2026.103962

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103962

Keywords: acrolein, pyrene excimer, fluorescent probe, chemoselectivity, imino [4+4] cycloaddition, 1,2-amino alcohol, serine-derived probe, oxidative stress, lipid peroxidation, cell lysate extraction, fluorescence spectroscopy, Results in Chemistry