(276a) Award Submission: Employing Novel Metasurface Nanopillars for the Detection of Antimicrobial Resistances Via Beta-Lactamase Activity

Metasurfaces and metamaterial nanotechnology have undergone exponential growth in advancements which has opened the topic to a variety of mediums. This field has seen great interest in use as biosensors specifically, because of their high customizability, sensitivity, and often nondestructive sensing strategies.[1] Through precise control of the dimensions of subwavelength features, a variety of novel effects can be observed based on particle interactions. Due to alterations in the surrounding refractive index, or from plasmon resonance in the presence of photons, the unique surface properties of the nanoscale structures can undergo detectable changes.[2],[3] Fabrication of the novel silicon nanopillars described herein allowed for production of the intricate geometries capable of producing a desirable reflectance spectrum for the sensitive and specific detection of bound analytes, namely antibiotics and enzymes related to antimicrobial resistances (AMR). With the ever-expanding use of antibiotics in healthcare, combating bacterial resistance has emerged as a forefront issue.[4] Agar dilutions have become the established gold standard for the identification of antibiotic resistance among bacteria colonies.[5] This technique is extremely accurate and capable of identifying AMR in many bacteria samples simultaneously but is labor-intensive and expensive to perform with the equipment and trained personnel required. To overcome these issues, detecting the presence of β-lactamase enzymes allows for a simpler and cheaper strategy for observing antibiotic resistance to β-lactam class antibiotics in bacteria. Incorporating metasurface technologies for AMR detection will offer higher sensitivity than popular lateral flow assays, while maintaining the rapid result capabilities.

The 1 cm² samples used in this work were covered with the periodic nanopillar array, which consisted of 150 nm, 180 nm, or 200 nm diameter nanopillars at a customizable edge-to-edge distance varying from 155 nm to 240 nm. The height of the nanopillars were controlled at 110 nm or 210 nm by adjusting etching time and plasma composition, which allows for greater control over the pillar shape and the final spectra of the metasurface. This metasurface resulted from a facile fabrication process that only required DUV lithography with a negative photoresist/BARC layer, and a metal etch process with an accompanying strip step. The nanofeatures produced here pushed the capabilities of common DUV equipment, lending to specialized pillars that have been sparsely investigated. Through the combination of metallic nanoparticles with the nanopillars, simulations have suggested that a novel double-peak reflectance spectrum with a narrow FWHM below 10 nm can be observed, which is comparable to experimental findings for the silicon metasurface. These sharp peaks and valleys would increase the sensitivity of the overall system, resulting in simple and rapid detection of the antibiotic signatures.

The initial assay was developed for the sensing of target antibiotics via binding with the surface of the silicon nanopillars and surrounding planar surface. The silicon had been functionalized by a self-assembling monolayer consisting of an 11-aminoundecyltriethoxysilane (AUTES) monomer, which was confirmed by contact angle measurements. This molecule coated the silicon surface with a terminal amino group that preferentially bound to the carboxylic compound existing within the β-lactam antibiotic.[6] The antibiotic acted as a linker in this assay, allowing for specialized BSA-coated gold nanoparticles (BSANS) with a glutaraldehyde linker to be adhered to the functionalized silicon features. This glutaraldehyde molecule was essential as a spacer between the antibiotic and BSA, preventing the cephalexin antibiotic from being enveloped in the protein’s binding domains. The coupling of the gold to silicon by the AUTES-antibiotic-glutaraldehyde chain allowed for plasmon resonant effects to be detected as 7 nm peak wavelength shifts in the double-peak reflectance spectra at picomolar concentrations of BSANS with nanomolar concentrations of cephalexin present. Alterations to the assay can be done for the detection of β-lactam-resistant bacteria through degradation of the antibiotic by the β-lactamase enzyme, preventing capture of nanoparticles on the nanopillar surface. By functionalizing the nanopillars with PBPs, which would only bind to intact β-lactam antibiotics, the presence of AMR can thereby be observed. The AUTES monomer in this assay would be replaced by a (3-Mercaptopropyl) triethoxysilane (MPTES) monomer which has a terminal thiol group, as opposed to an amino group, which is preferable for the capture of the PBP. Extensions of this work would lead to detection of these enzymes in a complex media such as milk and urine, where the BSA coating on the nanoparticles will aid in preventing non-specific protein absorption while identifying antibiotics and enzyme presence in a high noise environment. The metasurface technology developed here, in combination with the biological assay, would assist in further advancing AMR detection by simplifying the sensing strategy and speeding up result time compared to popular methods, while maintaining the high sensitivity and specificity required for clinical applications.

References:

[1] J. Hong, M. Su, and K. Zhao et al., Biosensors 13, 327 (2023).

[2] T. Zhou, W. Ji, H. Fan et al., Biosensors 13, 681 (2023).

[3] J. Hu, F. Safir, K. Chang et al., Nature Communications 14, (2023).

[4] R. Uchil, G. Kohil et al., Journal of Clinical and Diagnostic Research 8, 7 (2014)

[5] M. Weinstein, J. Patel, B. Limbago et al., Clinical and Laboratory Standards Institute M07 11, 38 (2018)

[6] Q. Li, T. Zhang, and L. Bian, Journal of Chromatography B 1014, 90 (2016).