Nanoneedle-based biosensing in cells

Background: Nanoneedles are high aspect ratio nanostructures with a distinctive bio-interface[1]. Their unique, though not fully understood, interactions with cells allow them to sense intracellular conditions with remarkable efficiency, typically causing less toxicity and disturbance than traditional probes[2, 3]. By establishing long-term, reversible interfaces with cells, nanoneedles can monitor biological functions over several days. Their nanoscale size, combined with their assembly into large, ordered, dense arrays, enables them to track the functions of extensive cell populations and generate functional maps with submicron spatial resolution.

Intracellularly, nanoneedles detect electrical activity in complex excitable networks, as well as measure concentrations, functions, and interactions of biomolecules in situ. Extracellularly, they can detect cellular forces with piconewton sensitivity or efficiently sort rare cells based on their membrane receptors. Nanoneedles are versatile tools for studying a wide range of biological systems, from individual cells and biological fluids to tissues and living organisms [4-6].;;

This PhD research aims to expand the understanding of fundamental cellular processes by engineering the geometry and surface chemistry of nanoneedle arrays. Utilising nanofabricated nanostructures, it seeks to elucidate how mechanical and physical cues can direct cell behaviour and coordinate cellular functions, including extra and intracellular biosensing. Sensing biomolecules, their interactions, and broadly intracellular conditions have the unprecedented ability to elucidate intracellular state of large number of cells with single-cell or even sub-cell resolution and without significant perturbation.

;Research Gaps

;In my nanoneedle-based sensing project, a significant gap lies in the fact that each cell type and sensing modality constitutes an almost unique system due to the vast differences in cell morphology, size, mechanical properties, and the signalling networks at play. This variability, combined with the limited understanding of the fundamental principles governing cell–nanoneedle interactions, necessitates nearly de novo optimisation of nanoneedle systems for each specific cell target, as well as for each sensing application. This presents a critical challenge for developing broadly applicable and efficient nanoneedle technologies. Moreover, designing multifunctional nanoneedles capable of detecting multiple cellular parameters simultaneously could reduce the need for separate optimization steps.;Methodology.

;Methodology:

;Aim 1: Nanofabrication (at the Melbourne Centre for Nanofabrication (MCN));The first aim of this project is dedicated to the nanofabrication of nanostructures with specific topographies. Optimisation of these nanostructures will focus on their shape, material, and dimensional properties. Of particular interest are nanoneedles, which are conical in shape, and nanotubes, which are hollow and tubular, constructed from silicon or polymer materials. Critical dimensions such as height, diameter, tip size, and spacing (pitch) will be finely tuned. These nanostructures will be crafted using advanced nanofabrication tools at MCN and subsequently analysed through electron microscopy for detailed characterization.;

Aim 2: Surface chemistry ;Chemical functionalisation is a parameter available to modulate critical cellular processes such as the sensing modalities by regulating the interaction between desirable cargo and targeted cell. Modifying the surface of nanoneedle devices via—physical adsorption and covalent/non-covalent bonding—can play a vital role in enhancing their efficiency in terms of the targeted cellular application. The interaction between these nanodevices, their carried substances and the target biological systems is influenced by the surface functionalisation techniques employed. ;

Aim 3: Nanoneedle-based sensing in cells;Demonstrate a step-change in intracellular biosensing. We will identify optimal combinations of the three variables (topography, surface chemistry, and interfacing) for efficient NT-mediated biosensing, by immobilising fluorogenic antibodies to NTs to bind and detect cytokines from the cell interior in just one step, allowing quantitative analysis of CAR-T cell potency;The essence of the live-cell assay will be the use of nanoneedles that are functionalised with targeted fluorogenic antibodies, enabling direct biosensing of intracellular cytoplasmic cytokines. We will use cysteine–maleimide chemistry to covalently bind fluorogenic antibodies against each of the targeted cytokines (IFN-γ, TNF-α) to an array of nanoneedles. ;

;Impact and Outcome;The specific outcome of this project will be the development of optimized nanoneedle-based platforms for intracellular biosensing, capable of directly detecting cytokines like IFN-γ and TNF-α within live cells. The impact will be a significant advancement in biosensing technology, enabling real-time, quantitative monitoring of cellular processes such as CAR-T cell potency, with potential applications in immunotherapy, disease diagnostics, and precision medicine. This platform will enhance our ability to study and manipulate cellular behaviour with unprecedented accuracy and efficiency.;"