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ma=86400 Frequency locked whispering evanescent resonator (FLOWER) for biochemical sensing applications | Nature Protocols
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Frequency locked whispering evanescent resonator (FLOWER) for biochemical sensing applications

Nature Protocols (2025)Cite this article

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Abstract

Sensitive, rapid and label-free biochemical sensors are needed for many applications. In this protocol, we describe biochemical detection using FLOWER (frequency locked optical whispering evanescent resonator)—a technique that we have used to detect single protein molecules in aqueous solution as well as exosomes, ribosomes and low part-per-trillion concentrations of volatile organic compounds. Whispering gallery mode microtoroid resonators confine light for extended time periods (hundreds of nanoseconds). When light circulates within the resonator, a portion of the electromagnetic field extends beyond the cavity, forming an evanescent field. This field interacts with bound analytes resulting in a change in the cavity’s effective refractive index, which can be tracked by monitoring shifts in the resonance wavelength. The surface of the microtoroid can be functionalized to respond specifically to an analyte or biochemical interaction of interest. The frequency-locking feature of frequency locked optical whispering evanescent resonator means that the instruments respond to perturbations in the surface by very rapidly finding the new resonant frequency. Here we describe microtoroid fabrication (4–6 h), how to couple light into these devices using tapered optical fibers (20–40 min) and procedures for coupling antibodies as well as G-protein coupled receptors to the microtoroid’s surface (from 1 h to 1 d depending on the target analyte). In addition, we describe our liquid handling perfusion system as well as the use of a rotary selector valve and custom fluidic chamber to optimize sample delivery. Step-by-step details on how to perform biosensing experiments and analyze the data are described; this takes 1–2 d.

Key points

  • FLOWER tracks the resonance wavelength shift of a resonator caused by the perturbation of adsorbed analytes. Combining FLOWER with microtoroid resonators can enable single molecule detection and zeptomolar sensitivity.

  • The silica surface of the microtoroid resonator can be functionalized using well established protocols to achieve binding specificity. Two procedures describe their fabrication and use in example analytical applications.

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Fig. 1: WGM microtoroid resonator and its properties.
Fig. 2: Schematic of the protocol workflow.
Fig. 3: Schematic of PDH locking technique used for high-speed frequency modulation.
Fig. 4: Schematic for optical fiber taper station.
Fig. 5: Overview of the biochemical sensing station.
Fig. 6: Schematic of CO2 laser reflow system.
Fig. 7: How to align the center of the laser and the microscope’s FOV.
Fig. 8: Overview of the microtoroid fabrication process.
Fig. 9
Fig. 10
Fig. 11: Optical fiber tapering process.
Fig. 12: Fluidic chamber assembly from Steps 153–159.
Fig. 13: Spike-protein RBD for B.1.1.529 omicron binding to hACE2 in tricine buffer.
Fig. 14: hACE2 and spike-RBD-His wild-type (WT) binding sensorgram.
Fig. 15: Relative wavelength shift and initial slope extracted from Fig. 14.

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Data availability

Source data displayed in Figs. 13–15 and the setting file to use with the Digilock Frontend can be found in Supplementary Information of this paper. Source data are provided with this paper.

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Acknowledgements

We acknowledge support in part from NIH R35GM137988.

Author information

Authors and Affiliations

  1. Wyant College of Optical Sciences, The University of Arizona, Tucson, AZ, USA

    Sartanee Suebka, Adley Gin & Judith Su

  2. Department of Biomedical Engineering, The University of Arizona, Tucson, AZ, USA

    Judith Su

Authors
  1. Sartanee Suebka
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  2. Adley Gin
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  3. Judith Su
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Contributions

FLOWER was invented by J.S. S.S. wrote the manuscript with input from all authors. A.G. wrote the microtoroid fabrication and DOPC lipid vesicle fabrication and functionalization section. All authors read and approved the manuscript.

Corresponding author

Correspondence to Judith Su.

Ethics declarations

Competing interests

J.S. owns a financial stake in Femtorays Technologies, which develops label-free molecular sensors. All other authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Arunas Ramanavicious and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Key references

Gin, A. et al. npj Biosensing 1, 9 (2024): https://doi.org/10.1038/s44328-024-00009-8

Gin, A. et al. Nat. Commun. 15, 7445 (2024): https://doi.org/10.1038/s41467-024-51320-x

Hao, S. et al. Light. Sci. Appl. 13, 195 (2024): https://doi.org/10.1038/s41377-024-01536-9

Kim, S.-K. et al. ACS Pharmacol. Transl. Sci. 7, 348–362 (2024): https://doi.org/10.1021/acsptsci.3c00197

Xu, Y. et al. ACS Appl. Mater. Interfaces 16, 5120–5128 (2024): https://doi.org/10.1021/acsami.3c16012

Extended data

Extended Data Fig. 1

First fluidic chamber design.

Extended Data Fig. 2

Optical fiber a before and b after tapering.

Extended Data Fig. 3

Fluidic chamber holder assembly.

Extended Data Fig. 4

Protocol to find CO2 laser reflow system focal plane.

Extended Data Fig. 5

CO2 laser thermal reflow setup.

Extended Data Fig. 6

SEM images of microtoroids.

Extended Data Fig. 7

DOPC lipid vesicle functionalization equipment and vesicle extrusion.

Extended Data Fig. 8

Transmission efficiency during tapering a single mode optical fiber.

Extended Data Fig. 9

Sensorgram for various versetamide concentrations and 10 nM spike-RBD-WT mixture in PBS.

Supplementary information

Reporting Summary

Supplementary Software 1

Setting file to be used with DigiLock Frontend software.

Source data

Source Data Fig. 13

a, Sensorgram of hACE2-spikeRBD binding experiment. b, Binding curve data. The wavelength shift at equilibrium was obtained by fitting data in a with a one-site specific binding equation.

Source Data Fig. 14

hACE2 and spike-RBD-His wild type binding sensorgram with the use of regeneration buffer.

Source Data Fig. 15

Relative wavelength shift and initial slope extracted from Source Data Fig. 14.

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Suebka, S., Gin, A. & Su, J. Frequency locked whispering evanescent resonator (FLOWER) for biochemical sensing applications. Nat Protoc (2025). https://doi.org/10.1038/s41596-024-01096-7

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