Surface Functionalization of Photonic Life Marker Chips for Asymmetric Mach–Zehnder Interferometry (aMZI)-Based Biosensing

Abstract

One of the primary goals of extraterrestrial life detection is the qualitative identification and quantitative analysis of biosignatures that can provide direct or indirect evidence of extraterrestrial life and help reveal biological activity or prebiotic processes.^{1, 2} Current methods for extraterrestrial life detection mainly include mass spectrometry,^{3, 4} Raman spectroscopy,^{5, 6} and emerging microfluidic chip-based biosensing techniques.^{7, 8} However, extraterrestrial samples often contain high concentrations of salts and diverse minerals,^{9, 10} which may make sensitive and selective detection of biosignatures challenging owing to strong background signals. Therefore, there is a need for in situ analytical techniques that can secure biomolecule detection performance with high selectivity, strong resistance to interference, and low detection limits. The Life Marker Chip / Origin of Life Marker Chip (LMC_OOL) project aims to develop an efficient biosignature detection platform for planetary exploration missions. Driven by this aim, this study focuses on the development of efficient chemical surface modification strategies for immobilizing biorecognition elements onto photonic chips for asymmetric Mach–Zehnder interferometry (aMZI), which is essential for the selective detection of target biosignature molecules.

The aMZI photonic chip is a highly sensitive refractive index-based sensing platform that has attracted widespread attention due to its label-free nature and its ability to provide sensitive, in-situ and real-time measurements.¹¹ In this study, the tested aMZI chip consists of a high-refractive-index silicon nitride (Si₃N₄) waveguide layer and a silicon dioxide (SiO₂) cladding. During fabrication, part of the SiO₂ cladding is removed to form a “sensing window”, which exposes the waveguide directly to the liquid environment in sensing applications. During operation, the input light is split into a reference arm and a sensing arm. The reference arm functions as an internal reference and is generally isolated by the SiO₂ cladding layer. In comparison, the sensing arm, which is functionalized with bioreceptors that are able to specifically recognize the target analyte, is typically exposed to the matrix environment. When target molecules bind to the receptors on the sensing surface, they cause a local change in refractive index, which changes the light propagation phase and optical path length in the waveguide. This change is measured as a wavelength shift in the interference spectrum, with a resolution down to the picometer (pm) level.^11-13

To analyze target biosignatures (such as D/L-phenylalanine, D/L-histidine, and guanine) accurately, a sensitive and selective receptor layer is required on the waveguide surface. In our work, poly-L-lysine (PLL) is used as a surface modification layer for Si₃N₄ substrates. The positively charged amine groups in PLL can strongly attach to the Si₃N₄ surfaces, which become negatively charged upon activation using UV/ozone, oxygen plasma, or piranha solution. Residual unbound amine groups of the PLL can be further functionalized through various chemical approaches, such as coupling reactions with N-hydroxysuccinimide (NHS) ester crosslinkers, enabling the covalent immobilization of recognition elements (e.g., aptamers) onto the chip surface. This PLL-based method is simple, stable, and widely used, and provides a reliable surface for detecting low-concentration biomarkers using aMZI in complex environments.^{11, 13}

Considering the potential damage of the extreme radiation environment in space to organic coatings, we are also developing molecularly imprinted polymer (MIP) materials based on a siloxane backbone as an alternative recognition strategy. By using target molecules as templates during polymerization, the resulting MIP materials form binding sites that match the target molecules, allowing selective recognition.¹⁴ Combined with the high sensitivity of aMZI optical detection, this hybrid approach provides a promising platform for detecting trace biomarkers and will be useful for future extraterrestrial exploration.

Here, we will present the initial results of the project, with e.g., sensor sensitivity tests, the fabrication of PLL-based sensing layers, the evaluation of D/L-phenylalanine sensing, and the first trial with MIPs usage.

 

References

[1] V. Abrahamsson and I. Kanik, Frontiers in Astronomy and Space Sciences, vol. 9, 2022.

[2] M. A. Sephton, A. Steele, F. Westall, and F. Schubotz, Proceedings of the National Academy of Sciences, vol. 122, no. 2, p. e2404256121, 2025.

[3] P. V. Johnson, L. W. Beegle, and I. Kanik, in Mass Spectrometry Handbook, pp. 389–405, 2012.

[4] A. Riedo, C. de Koning, A. H. Stevens, C. S. Cockell, A. McDonald, A. C. López, V. Grimaudo, M. Tulej, P. Wurz, and P. Ehrenfreund, Astrobiology, vol. 20, no. 10, pp. 1224–1235, 2020.

[5] K. Uckert, A. Parness, N. Chanover, E. J. Eshelman, N. Abcouwer, J. Nash, R. Detry, C. Fuller, D. Voelz, R. Hull, D. Flannery, R. Bhartia, K. S. Manatt, W. J. Abbey, and P. Boston, Astrobiology, vol. 20, no. 12, pp. 1427–1449, 2020.

[6] M. W. Sandford, A. K. Misra, T. E. Acosta-Maeda, S. K. Sharma, J. N. Porter, M. J. Egan, and M. N. Abedin, Applied Spectroscopy, vol. 75, no. 3, pp. 299–306, 2021.

[7] J. Kim, E. C. Jensen, A. M. Stockton, and R. A. Mathies, Analytical Chemistry, vol. 85, no. 16, pp. 7682–7688, 2013.

[8] M. F. Mora, F. Greer, A. M. Stockton, S. Bryant, and P. A. Willis, Analytical Chemistry, vol. 83, no. 22, pp. 8636–8641, 2011.

[9] F. Postberg, S. Kempf, J. Schmidt, N. Brilliantov, A. Beinsen, B. Abel, U. Buck, and R. Srama, Nature, vol. 459, no. 7250, pp. 1098–1101, 2009.

[10] F. Tosi et al., Nature Astronomy, vol. 8, no. 1, pp. 82–93, 2024.

[11] M. J. Goodwin, G. A. J. Besselink, F. Falke, A. S. Everhardt, J. J. L. M. Cornelissen, and J. Huskens, ACS Applied Bio Materials, vol. 3, no. 7, pp. 4566–4572, 2020.

[12] T. Chalyan, R. Guider, L. Pasquardini, M. Zanetti, F. Falke, E. Schreuder, R. G. Heideman, C. Pederzolli, and L. Pavesi, Biosensors, vol. 6, no. 1, 2016.

[13] S. Aphrham, M. Verheijden, and J. Huskens, Langmuir, vol. 41, no. 17, pp. 11205–11214, 2025.

[14] J. J. BelBruno, Chemical Reviews, vol. 119, no. 1, pp. 94–119, 2019.