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. 2026 Sep 28;11(40):61744–61750. doi: 10.1021/acsomega.6c07587

Monodisperse Polymer Optical Microresonators as a Versatile Platform for Protein-Conjugated Biocompatible Optical Probes

Honoka Seki †, Kariana K Dewi †, Tomoyuki Tsujimoto †, Sooyeon Kim ‡,§,∥,*, Asuma Kubono ‡, Yuichi Taniguchi ‡,§, Yohei Yamamoto †,∥, Hiroshi Yamagishi †,⊥,#,*
PMCID: PMC13670325  PMID: 42889879

Abstract

Optical microresonators have attracted increasing attention as next-generation miniature optical probes for biological sensing, but their practical applications remain limited by insufficient biocompatibility and limited chemical functionalizability at the surface. Here, we report polymer-based optical microresonators (MR) as a surface-functionalizable and biocompatible platform for covalently anchoring biofunctional molecules. Highly spherical and monodisperse polystyrene MR were prepared by inkjet printing and conjugated with guest molecules using Sulfo-SANPAH as a heterobifunctional cross-linker. This strategy enabled the stable surface conjugation of multiple classes of biomolecules, including ovalbumin proteins, IgG antibodies, and polylysine, even after repeated washing. MR retained sharp peaks of the whispering-gallery-mode resonance even after conjugation and immersion in water and a buffer solution, indicating their applicability to biologically relevant environments. Furthermore, IgG-conjugated MR were internalized by macrophage cells without causing visible cellular damage. These results establish polymer microresonators as a promising platform for biofunctional and biocompatible optical probes capable of operating in aqueous and cellular environments.

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Introduction

Whispering-gallery-mode (WGM) optical microresonators have attracted increasing attention in biosensing as emerging devices particularly suited for quantitative measurements in complex media. WGM microresonators are micrometer-scale objects with highly symmetric geometries that confine light within their bodies through total internal reflection at their periphery. , The confined light gives rise to sharp and periodic resonances in transmission or emission spectra, and the spectral positions of these resonances are highly sensitive to changes in the physical and chemical properties of the surrounding medium. Because the signals can be read out through remote optical excitation and collection of emission, WGM microresonators can operate even in complex media under in situ conditions. They can therefore serve as precise sensors for temperature, pressure, humidity, and chemical concentration. −

The detection of chemical concentrations in biological environments using WGM resonators is particularly valuable because their precision and reproducibility often surpass those of conventional molecular fluorescent probes. Whereas conventional fluorescent molecular probes typically enable quantitative concentration measurements down to the μM or sub-μM range, , advanced optical microresonator sensors, particularly high-Q or plasmon-enhanced WGM systems, have achieved fM- or even single-molecule-level detection in selected cases. ,

In parallel to these demonstrations, techniques for introducing optical microresonators into biological environments have also progressed. Early achievements included the intracellular incorporation of inorganic resonators and dye-doped polystyrene resonators, enabling laser emission inside living cells , barcode-type cell tagging, − and the detection of intracellular mechanical changes. , More recent technologies have further improved the biointegration of microresonators by tailoring their surfaces. Silica encapsulation is an established strategy for improving the stability and biocompatibility of inorganic or semiconductor resonator particles. − The outermost SiO2 layer can be further functionalized using silane coupling reagents to anchor biomolecules. Electrostatic adsorption is another authentic methodology, where polyionic molecules are immobilized on particle surfaces through electrostatic interactions. −

Nonetheless, the biocompatibility and versatility of existing methods remain limited. Silane coupling is mainly applicable to hydroxylated or oxide surfaces and often requires prior formation of a silica or oxide interface. The additional SiO2 layer can reduce the sensitivity of microresonators to external changes. The immobilization of bioactive molecules on a rigid inorganic oxide surface can also compromise their functions in some cases, because molecular orientation, surface density, linker structure, and nonspecific adsorption can affect molecular functions. Electrostatic adsorption is simpler, but it is mainly applicable to charged molecules or polyionic substances and is intrinsically dynamic. Therefore, desorption can occur upon changes in pH, ionic strength, or competing biomolecules.

As a solution to these problems, here we report the combination of polymer-based optical microresonators (MR) and Sulfo-SANPAH as a versatile platform for covalently anchoring guest biocompatible molecules on the surface of microresonators (Figure a). Sulfo-SANPAH is a heterobifunctional cross-linker bearing an amine-reactive N-hydroxysuccinimide (NHS) ester at one end and a photoactivatable nitrophenyl N3 group at the other. The NHS ester enables conjugation with primary amines in biomolecules, whereas the nitrophenyl N3 group can be photoactivated to form covalent bonds with polymer surfaces (Figure b). Given its inherent versatility, we believe that this reaction could be applied not only to conventional biorelated devices but also to microspherical resonators. Using this approach, we successfully conjugated ovalbumin proteins, polylysine peptide, and IgG antibodies onto the surface of polymer microresonators. The cross-linking was stable even after intensive washing for 5 cycles. We also found that the conjugation process did not deteriorate the optical resonance performance in water or buffer solution. Moreover, the protein-conjugated polymer resonators could be internalized into living cells without causing visible cellular damage.

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(a) Schematic representations of the synthetic procedure for MRGuest and the procedure of the cell experiments. (b) Guest conjugation workflow onto the surface of MR mediated by Sulfo-SANPAH.

Results and Discussion

Synthetic Procedures for Peptide-Conjugated MRs

Polymer microresonators were prepared according to a previous report from our group (Figure a). A solution of polystyrene (PS, 1.0 mg mL–1) in 1,2-dichloroethane (C2H4Cl2) was loaded into an inkjet printhead and ejected as microdroplets with an average diameter of 57.2 μm onto an aqueous poly­(vinyl alcohol) solution (PVA, 0.5 wt %) in a PTFE dish. After incubation at 25 °C for 1 day, the droplets solidified through evaporation of C2H4Cl2 to yield spherical PS particles (MR). The resulting MR was washed with water five times to remove residual impurities. The number of MR particles produced in a single batch was estimated to be approximately 4.2 × 105 provided that the droplet ejection frequency was 120 Hz and the ejection time was 1 h.

The aqueous suspension of MR was then conjugated with proteins using Sulfo-SANPAH as a heterobifunctional cross-linker (Figure b). Prior to conjugation with MR, an aqueous solution of Sulfo-SANPAH (60 μL, 50.8 μM) was mixed with an aqueous solution of ovalbumin as a model compound (240 μL, 11.1 μM), in a microtube and stirred overnight at 4 °C in the dark, allowing the Sulfo-NHS ester moiety to react with amino groups on the protein. Unreacted Sulfo-SANPAH was removed by dialysis three times, where the mixture was transferred into a cellophane dialysis tube and dialyzed against stirred phosphate-buffered saline (PBS) for 1 h. The dialyzed solution was then mixed with the aqueous suspension of MR and irradiated with UV light at 365 nm for 30 min at 4 °C. Upon UV irradiation, the nitrophenyl azide moiety of Sulfo-SANPAH generates reactive nitrene species, which can form covalent bonds with the polymer surface through reactions with aromatic rings. The resulting suspension was washed with water five times to remove unreacted proteins and linkers, affording ovalbumin-conjugated MR (MR ovalbumin ). The same procedure was also applied to the conjugation of normal mouse IgG and polylysine, yielding the corresponding guest-conjugated MR Guest (MR IgG , MR polylysine ).

Morphological and Chemical Characterizations of MRs

The morphology and surface chemistry of MR and MR ovalbumin were investigated by optical microscopy (OM), fluorescence microscopy (FM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). OM images showed that the pristine MR possessed a highly spherical morphology (Figure a). The diameter (D) histogram, calculated from SEM images, showed a monodisperse distribution with an average diameter of 6.7 μm with a standard deviation (σ) of 0.4 μm (Figure a). The surface of MR was smooth as identified by the root-mean-square (RMS) roughness as small as 3.39 (Figure a).

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(a) Optical microscopy and scanning electron microscopy (SEM) images of MR together with a plot of ΔGray Value along the yellow dashed line in the SEM image and a histogram of D of MR. (b) Optical microscopy and SEM images of MRovalbumin together with a plot of ΔGray Value along the yellow dashed line in the SEM image. (c) Fluorescent microscopy images (λex = 500 nm) of MR and MRovalbumin as well as MRs conjugated with ovalbumin but without Sulfo-SANPAH (SS) and without UV irradiation during the reaction. Insets show the corresponding bright field images. Black scale bars in the insets: 10 μm. (d) Relative elemental contents of carbon and nitrogen together with their ratio detected for MR, MRovalbumin , MRIgG , and MRpolylysine by energy-dispersive X-ray spectroscopy.

The successful conjugation of ovalbumin was directly visualized by FM using Texas Red-labeled ovalbumin. MR ovalbumin exhibited red fluorescence under excitation at 500 nm, whereas pristine MR appeared dark under the same conditions (Figure c). Control experiments, in which MR was treated by the same procedure but without either Sulfo-SANPAH or UV irradiation, also gave dark images (Figure c). These results support the covalent conjugation of ovalbumin onto MR through the Sulfo-SANPAH linker. After ovalbumin conjugation, MR ovalbumin became slightly rougher than pristine MR (Figure b). The root-mean-square roughness of MR albumin increased to 5.72, presumably due to the conjugated protein layer. Consistently, the EDX spectra of MR ovalbumin showed increased N content compared with that of pristine MR (Figure d), further supporting successful protein conjugation. Similar increases in N contents were also observed for MR IgG , MR polylysine by EDX analysis, indicating that the target biomolecules were successfully conjugated to MR.

We also found that the present method is applicable to microspheres ( PVK MR) made from polyvinylcarbazole (PVK). PVK MRs were prepared and conjugated with ovalbumin using the same procedure described above. Successful conjugation was confirmed by the red fluorescence of ovalbumin observed on the PVK MR surface and by the increase in the N/C ratio in the EDX profiles after conjugation (Figure S1).

WGM Properties of MRs under Water

The optical resonance properties of MR and MR ovalbumin were investigated by microscopic photoluminescence spectroscopy (μ-PL). μ-PL spectra were measured using a home-built inverted microscope equipped with an objective lens (100×, NA 1.45), excitation laser (λ = 405 nm, Δ = 250 ns, f = 20 kHz), and a spectrometer (Figure a). For the WGM measurements, MR and MR ovalbumin were doped with a fluorescent dye (9,10-bis­(phenylethynyl)­anthracene, BPEA). The BPEA-doped MR ( BPEA MR) was prepared by inkjet printing using a C2H4Cl2 solution of PS containing BPEA (13.2 μM) as the precursor solution. The remaining preparation procedure was identical to that described above. The morphology of the resulting BPEA MR was mostly identical to that of MR (Figure S2).

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(a) A schematic representation of the μ-PL setup. (b–e) μ-PL spectra of BPEAMR in air (b), BPEAMRovalbumin in air (c), BPEAMRovalbumin in H2O (d), and BPEAMRovalbumin under PBS. (f) A bar chart of Q-factor of BPEAMR in air (i), BPEAMR in H2O (ii), BPEAMR in PBS (iii), BPEAMRovalbumin in air (iv), BPEAMRovalbumin in H2O (v), BPEAMRovalbumin in PBS (vi). Error bars represent standard deviation (n = 3).

Upon photoexcitation, BPEA MR exhibited green emission from the doped BPEA, and the μ-PL spectra showed sharp periodic peaks superimposed on the broad spontaneous emission band of BPEA (Figure b). These sharp peaks were assigned to transverse-electric and transverse-magnetic WGM resonances, as supported by the linear relationship between the free spectral range and the inverse particle diameter (Figures S3 and S4).

Importantly, the sharp resonance peaks were largely retained after protein conjugation and even after immersion in water or buffer solutions (Figure c–e). The Q-factor, which represents the sharpness of the resonance peaks and is defined as λ/Δλ (Δλ: full-width at half-maxima of the peak), decreased from 1.5 × 103 to 1.4 × 103 after protein conjugation (Figure f), while the former value is ambiguous since the value is close to the instrumental limitation (Figure S5). We also observed WGM spectra under water and found that the Q-factor decreased from 1.5 × 103 to 1.4 × 103 after immersion in water (Figure f), indicating that efficient light confinement was partially deteriorated due to the relatively high refractive index of proteins and water in comparison to that of air. The BPEA-doped MR ovalbumin ( BPEA MR ovalbumin ) also showed WGM peaks with Q-factor of 1.3 × 103 under PBS (Figure f). While the Q-factors were deteriorated than those observed under atmosphere, they are still sharp and visible and are promising for the sensing in biological environments. Analogously, BPEA MR IgG and BPEA MR polylysine also showed sharp WGM peaks under pure water and PBS (Figure S6). We also confirmed whether BPEA doping affected Q-factor and found no obvious adverse effect (Figure S7), while this conclusion remains tentative because of the instrumental resolution limit. We found that the Q-factor decreased with increasing surface roughness. BPEA MR with higher RMS roughness resulting from surface conjugation exhibited lower Q-factors (Figure S8).

Biocompatibility of MRs

The biocompatibility and cellular uptake of MRs were evaluated using phagocytic cells. For this experiment, MRs were synthesized by a previously reported miniemulsion method instead of the inkjet process in order to increase the quantity of particles. Briefly, a CHCl3 solution of PS (100 μL, 5 mg mL–1) and BPEA (66.1 μM) was added to an aqueous solution of sodium dodecyl sulfate (SDS, 2 mL, 46.8 mM) and vigorously stirred with a homogenizer for 90 s. The resulting suspension was left to stand at 25 °C for 2 days to allow evaporation of CHCl3, followed by washing with water to yield BPEA MR with an average diameter of 2.3 ± 0.76 μm (Figure S9). The morphology of the resulting BPEA MR was mostly identical to that of MR although BPEA MR showed a much broader size distribution due to the emulsification process. BPEA MR was then conjugated with IgG to afford BPEA MR IgG following the procedure described above and was utilized for the biological experiments. This design was inspired by previous reports showing that IgG-coated foreign targets are well recognized by immune cells. , Both BPEA MR and BPEA MR IgG prepared by the miniemulsion method exhibited WGM resonance peaks in PBS, although the peaks were broader than those observed for MRs prepared by the inkjet method (Figure S9).

Prior to the addition of BPEA MR IgG , J774A.1 macrophages were seeded in wells and activated with lipopolysaccharide (LPS). , An aqueous suspension of BPEA MR IgG was then added to the cells and incubated for 3 h. Unbound BPEA MR IgG was removed with extensive washing, and the BPEA MR IgG -containing cells were subjected to further characterization. For confocal fluorescence imaging, the plasma membranes were stained with PlasMem Bright Red, followed by fixation with 4% paraformaldehyde.

Confocal fluorescence imaging clearly visualized the internalization of BPEA MR IgG into macrophages, with the particles appearing as green spheres inside the cells (Figures a, b and S10). Each macrophage engulfed multiple BPEA MR IgG particles in the cytoplasm, with an average number of beads per cell of 16.8 ± 10.3 (n = 19). Some macrophages engulfed more than 30 particles plausibly due to the large excess of BPEA MR IgG added to the culture medium (approximately 150 μg mL–1). It is worth highlighting that internalization of multiple beads did not cause any apparent morphological damage such as bleb formation or cell shrinkage. A cell viability assay further confirmed the high biocompatibility of BPEA MR ovalbumin , BPEA MR IgG , and BPEA MR polylysine (>98%, Figure c).

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A confocal microscopic image (a) and its magnified view (b) of macrophages (J774A.1, red) with BPEAMRIgG (green). White square indicates the area magnified in (b). (b) From xz and yz sectional images along the yellow lines, most of the BPEAMRIgG were localized inside of the cell, while some were strongly adsorbed on the membrane. (c) A bar chart of cell viability of macrophages with and without BPEAMRGuest treatment (i–iii and iv, respectively). Error bars represent standard deviation (n = 3).

Engulfment efficiency was evaluated by flow cytometry under the same incubation conditions as those used for confocal fluorescence imaging. LPS-activated macrophages were incubated with BPEA MR IgG for 3 h, collected by trypsinization, and subjected to flow-cytometry measurements. The flow-cytometry plots showed that approximately 93% of macrophages internalized or strongly bound BPEA MR IgG on their surface (Figure S11). We also found that BPEA MR IgG internalized into the cells are smaller than the as-prepared BPEA MR (Figure S12). Considering that BPEA MR smaller than 6 μm hardly showed WGM peaks (Figure S13), the optimum size of BPEA MR under the conditions tested should be 6–10 μm.

Altogether, these results demonstrate that BPEA MR Guest exhibits sufficiently low cytotoxicity and high engulfment efficiency. The low cytotoxicity was attributed not only to the biocompatibility of BPEA MR Guest but also to the resistance of macrophages to oxidative stress. , The efficient engulfment of BPEA MR was in line with a previous report showing that 71% and 36.1% of macrophages engulfed 1.7–2.2 μm and 2.5–4.5 μm polystyrene beads, as determined by flow cytometry. The markedly higher cellular association and engulfment efficiency observed in this study is plausibly attributable to the IgG/protein-functionalized surface of BPEA MR. These results demonstrate the effectiveness of our probe design strategy, whereby biomolecular surface conjugation enhances macrophage recognition and uptake.

Conclusions

In this work, we developed polymer microresonators as a versatile platform for covalently anchoring biomolecules onto optical resonator surfaces. Using Sulfo-SANPAH as a heterobifunctional cross-linker, ovalbumin, IgG, and polylysine were successfully conjugated to polystyrene microresonators, as confirmed by fluorescence microscopy, scanning electron microscopy, and elemental analysis. The biomolecule-conjugated resonators retained clear WGM resonances after surface functionalization and even in aqueous media, including water and PBS. Although the Q-factors decreased slightly after conjugation and immersion in aqueous media, sharp periodic resonance peaks remained observable, supporting the applicability of these resonators to optical sensing under biologically relevant conditions. Furthermore, IgG-conjugated resonators were efficiently internalized by macrophages without causing significant cytotoxicity. Further optimization of the protein conjugation conditions will be essential for reducing surface roughness and thereby improving the Q-factor of the resonators.

These results establish polymer microresonators as a flexible molecular-engineering platform whose surfaces can be readily functionalized with diverse organic and naturally derived molecules to construct biofunctional optical probes capable of operating in aqueous and cellular environments. Several studies have demonstrated the potential of biocompatible microresonators and microlasers for cell tracking, optical barcoding, and the sensing of biological, chemical, and physical parameters within living cells. − In the present study, we developed an efficient method for conjugating proteins of interest to polymer-based microresonators without compromising their optical properties. Furthermore, the resulting biofunctionalized microresonators could be internalized by living cells without inducing acute cytotoxicity. Unlike lasing, optical resonance does not require intense optical pumping and can be monitored with mild excitation conditions, thereby enabling long-term live-cell imaging with negligible phototoxicity. These properties could expand the applications of microresonators beyond intracellular optical emitters to intracellular biosensors and traceable reporters of specific biological processes involving the conjugated proteins. Taken together, this study demonstrates the potential of polymer-based microresonators as biofunctional and biocompatible platforms for intracellular sensing and functional studies in living cells.

Supplementary Material

ao6c07587_si_001.pdf (775.7KB, pdf)

Acknowledgments

This work was financially supported by FOREST (JPMJFR232J), PRESTO (JPMJPR25J4), and CREST (JPMJCR20T4) from JST; KAKENHI (JP26H01324, JP26H00697, JP24H00470, JP24K01306, and JP23KK0099) from JSPS; the 2025 research grant from the Amano Institute of Technology; and the Kurita Water and Environment Foundation (25A003). We also thank the iCeMS Analysis Center at Kyoto University for their assistance with the LSM880 with Airyscan and FACS Aria II systems, and the One-stop Sharing Facility Center for Future Drug Discoveries in the Graduate School of Pharmaceutical Sciences at the University of Tokyo.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c07587.

  • Materials and methods, synthesis and characterization, spectroscopy, and microscopy data (PDF)

S.K., Y.Y., and H.Y. conceived the idea and arranged the experiments; H.S., K.K.D., and T.T. conducted the synthesis and spectroscopy; H.S., S.K., A.K. and Y.T. conducted cell experiments; S.K., Y.Y., and H.Y. prepared the manuscript with feedback from the other authors.

The authors declare no competing financial interest.

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