Protein Binding Assessment of Immobilised Nanobody using Interferometric Nanoporous Platform

Protein Binding Assessment of Immobilised Nanobody using Interferometric Nanoporous Platform | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 14 July 2025 V1 Latest version Share on Protein Binding Assessment of Immobilised Nanobody using Interferometric Nanoporous Platform Authors : Cheryl Suwen Law 0000-0002-3276-8052 , Jayden Revink , Joel Lee , Juan Wang , Andrew Abell , Fiona Whelan 0000-0002-0791-6850 , and Abel Santos 0000-0002-5081-5684 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175248374.49334158/v1 220 views 170 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Protein sensors are key tools for infectious disease diagnosis and monitoring. The medical diagnostics field is now rapidly identifying key protein markers for the detection of a range of conditions, from early cancer development through to assessment of dementia risk a decade prior to symptoms onset. Many point-of-use diagnostic tools form part of standard protocols for small molecule sensing in healthcare, including glucose and ketone body sensors. Label-free optical biosensors have emerged as reliable detection tools that provide high versatility and adaptability to detect a broad range of target analytes. Herein, we combined nanoporous anodic alumina (NAA) photonic chip technology with tailor-engineered ‘nanobodies’—recombinant variable domains of heavy-chain-only antibodies—to achieve high affinity binding to human serum albumin (HSA). We monitored dynamic shifts in the optical fingerprint of nanobody-conjugated NAA platforms in real time when these were exposed to target proteins, through reflectometric interference spectroscopy (RIfS). We performed a comprehensive characterisation of the sensing performance, where binding mechanisms were elucidated through kinetic profiles. The nanobody-conjugated NAA protein sensor demonstrated good affinity, selectivity, and sensitivity towards HSA. Our analysis revealed a sensitivity of 27.3 ± 3.2 nm µM–1 and low limit of detection of 16.3 ± 1.6 µM, which are well-below the ranges for diagnosis of medical conditions such as nephrotic syndrome. The obtained results revealed that the RIfS-based protein sensor integrated with nanobody-modified NAA has promising potential to develop point-of-care platforms for clinical diagnosis of diseases. Protein Binding Assessment of Immobilised Nanobody using Interferometric Nanoporous Platform Cheryl Suwen Law 1,2 * , Jayden Revink 1,2 , Joel Lee 3 , Juan Wang 1,2 , Andrew D. Abell 2,4 , Fiona Whelan 3* , and Abel Santos 1,2* 1 School of Chemical Engineering, The University of Adelaide, 5005 Adelaide, Australia. 2 Institute for Photonics and Advanced Sensing, The University of Adelaide, 5005 Adelaide, Australia. 3 Department of Molecular and Biomedical Sciences, The University of Adelaide, 5005 Adelaide, Australia. 4 Department of Chemistry, The University of Adelaide, 5005 Adelaide, Australia. *E-mails: [email protected] ; [email protected] ; [email protected] Abstract : Protein sensors are key tools for infectious disease diagnosis and monitoring. The medical diagnostics field is now rapidly identifying key protein markers for the detection of a range of conditions, from early cancer development through to assessment of dementia risk a decade prior to symptoms onset. Many point-of-use diagnostic tools form part of standard protocols for small molecule sensing in healthcare, including glucose and ketone body sensors. Label-free optical biosensors have emerged as reliable detection tools that provide high versatility and adaptability to detect a broad range of target analytes. Herein, we combined nanoporous anodic alumina (NAA) photonic chip technology with tailor-engineered ‘nanobodies’—recombinant variable domains of heavy-chain-only antibodies—to achieve high affinity binding to human serum albumin (HSA). We monitored dynamic shifts in the optical fingerprint of nanobody-conjugated NAA platforms in real time when these were exposed to target proteins, through reflectometric interference spectroscopy (RIfS). We performed a comprehensive characterisation of the sensing performance, where binding mechanisms were elucidated through kinetic profiles. The nanobody-conjugated NAA protein sensor demonstrated good affinity, selectivity, and sensitivity towards HSA. Our analysis revealed a sensitivity of 27.3 ± 3.2 nm µM –1 and low limit of detection of 16.3 ± 1.6 µM, which are well-below the ranges for diagnosis of medical conditions such as nephrotic syndrome. The obtained results revealed that the RIfS-based protein sensor integrated with nanobody-modified NAA has promising potential to develop point-of-care platforms for clinical diagnosis of diseases. Keywords: Nanoporous anodic alumina, nanobody, human serum albumin, reflectrometric interference spectroscopy. INTRODUCTION Sensitive, specific and quantitative protein detection is a key target for the development of point-of-use diagnostics in industries such as healthcare, biosecurity, environmental monitoring, and food safety and traceability. Precise protein sensing and quantification is dependent on a detection entity that contributes specificity; a measurable binding event; and a transduction mechanism for monitoring and quantifying the target analyte. The fast uptake of lateral flow immunochromatographic assay (or lateral flow tests, LFTs) for the detection of SARS–CoV2 infections worldwide illustrates the power of simple point-of-use diagnostics. [1] At the other end of the protein detection systems are laboratory-based analytics such as ELISA (enzyme-linked immunosorbent assay), with labour-intensive and long experimental timelines, taking between 3–8 hours to complete. [2] Despite these differences, the majority of protein detection methods leverage the binding specificity of animal-produced, complex monoclonal antibodies. However, these essential binding entities are both expensive to produce and prone to inconsistent performance. [3] Antibody-based detection methods such as LFTs and ELISAs are dependent on the correct selection and precise production of specific antibodies. Purification of monoclonal antibodies, derivatisation of hybridoma cell lines, and scale-up of antibody production require extensive expertise and substantial capital investment. Additionally, the variability in tissue culture conditions, cell line maintenance, complex biomolecule purification processes and delicate storage conditions contribute to the limited consistency over the market lifetime of these binding moieties. [4,5] So, recent efforts in bioprocess engineering have focused on addressing the shortcomings of existing antibody production. Modularisation of antibodies has heralded a new era of specific antigen binding capability in the form of single antigen-binding domains of heavy chain-only antibodies that circulate in the Camelidae family—V H H antibodies, or ‘nanobodies’ (Nb). [6] In this process, following immunisation with a target protein, B-lymphocytes are isolated from blood; mRNA is extracted and antibody–antigen binding domain cDNAs are amplified and ligated into phage display vectors, generating a library of nanobody coding sequences. The highest affinity antigen binding can then be selected by phage display for isolation of genes coding for high-affinity nanobodies, which can be expressed in bacteria and purified using standard chromatographic techniques, with increased stability across a range of temperatures and pH (Fig. S1). [7] Building on this established method, structural proteomics approaches are being deployed to identify, model and test Nb-antigen complex specificity and affinity. [8] Randomisation of surface binding loops to create random nanobody libraries is now obviating the need for animal-based production. [9] In silico design of binding surfaces, teamed with high stringency elimination of off-target binding, is now producing high-affinity antigen binding stable nanobodies. [10] Given their small size, high stability, tailorable specificity and affinity, and readily scalable production in bacterial culture; nanobodies are an ideal binding moiety for the development of point-of-use protein sensors. The combination of nanobody binders with solid-state platforms provides a versatile approach to engineer a range of novel protein sensors. In particular, optical-based protein sensors have been long envisaged for label-free detection and quantification of biomolecules in the field of biology, medicine, and biotechnology. These platforms provide simplicity, rapid response, and a non-invasive means of interrogating binding events occurring on solid surfaces. [11,12] Of all solid-state platforms, optical transducers based on nanoporous anodic alumina (NAA) thin films are a model optical material to develop a broad range of sensing systems for multiple target analytes. [13] The nanopores of NAA, which can be precisely engineered through anodisation, function as nanocontainers to accommodate biological molecules and enable tuneable chemical selectivity and bio-specificity through surface chemistry engineering. [14] Thin films of NAA can be integrated with various optical techniques such as reflectometric interference spectroscopy (RIfS). The interaction of a NAA thin film with a white light beam results in the generation of a characteristic interferometric spectrum featuring well-resolved fringes associated with the Fabry–Pérot effect. When binding events occur inside the nanopores of the NAA film, fringes in the RIfS spectrum undergo shifts, which can then be resolved to monitor molecular interactions. The simplicity, reliability and stability of RIfS sensing systems, together with the versatility of NAA and its tailorable surface chemistry make NAA-based RIfS systems a highly attractive platform to engineer highly sensitive qualitative and quantitative optical platforms for a broad range of analyte molecules such as circulating tumour cells, DNAs, and small molecules. [13–15] Fig.1. Overview of the development of our label-free nanobody-conjugated interferometric protein sensor. (a) Engineering of nanoporous anodic alumina (NAA) sensing platforms by the two-step anodisation process in oxalic acid electrolyte, followed by surface chemistry modification involving gold coating on the top surface and functionalisation with an amine functional layer on the inner surface of nanopores. (b) An AlphaFold model of a high affinity HSA binding camelid-derived single variable heavy chain domain (Nb80) 20 ; variable complementarity-determining regions (CDR1-3) are highlighted (pink, blue, and green). (c) Illustration of the flow cell system integrating reflectometric interference spectroscopy (RIfS) and chemically modified NAA platforms used for the binding assessment of Nb80 to proteins. (d) Representative RIfS spectrum of a NAA platform upon interaction with white light (left) used to monitor shifts in effective optical thickness (ΔOT eff ) of the thin film in response to different analytical solutions (right) (NB: (i) surface chemistry engineering of NAA and (ii) real-time detection of protein molecules). However, to the best of our knowledge, the combination of nanobodies with nanoporous interferometric thin films remains unexplored to date. Motivated by this, herein we present a RIfS-based nanobodies-conjugated NAA interferometric sensor for real-time, in-situ detection of proteins (Fig. 1). Nbs were engineered to target serum albumins (SA), predominantly human serum albumin (HSA), because of its importance in clinical diagnosis as an indicator for kidney damage, diabetes, cardiovascular and liver diseases. [16–19] Following production and purification, Nbs were site-specific immobilised on the inner surface of the nanopores of NAA interferometers to provide target recognition capability for HSA. Nb-conjugated NAA interferometers were exposed to model solutions of HSA of varying concentrations. Dynamic changes in the effective optical thickness of the thin films were used as a label-free optical transduction mechanism for identification and quantification of the target protein. The Nb-conjugated NAA platforms were also exposed to different types of SA and other proteins to characterise the affinity and selectivity of the proposed sensing system. Our findings demonstrate the capability of the NAA-based RIfS system combined with nanobodies as biorecognition element for protein sensing, which have potential implications for real-life applications in food industry and medical diagnosis. MATERIALS AND METHODS Materials High purity (99.9997%) aluminium (Al) foils with a nominal thickness of 320 µm were supplied by Goodfellow Cambridge Ltd. (UK). Oxalic acid (H 2 C 2 O 4 ), perchloric acid (HClO 4 ), chromic acid (H 2 CrO 4 ), imidazole, Trizma base, 1,4-dithiothreitol (DTT), human serum albumin (HSA), bovine serum albumin (BSA), mouse serum albumin (MSA), phosphate buffered saline (PBS), γ-globulin from human blood (γG), transferrin from human blood plasma (TFN), and (3-aminopropyl)triethoxysilane (APTES) were purchased from Sigma-Aldrich (Australia). Luria Broth (LB) Bacto tryptone and Bacto yeast extract, Ampicillin, (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide) (EDC) and N- hydroxysuccinimide (NHS) were purchased from Thermo Fisher Scientific Inc. (Australia). 85% wt/wt phosphoric acid, ethanol (C 2 H 5 OH, EtOH) and 30 wt% hydrogen peroxide (H 2 O 2 ) were purchased from ChemSupply (Australia). Carboxylic acid-functionalized methoxy polyethylene glycol of 5000 Da (mPEG–COOH) was obtained from Nanocs Inc. (USA). Nb80 gene fragment and Escherichi coli NEB 5α were purchased from Integrated DNA Technologies Inc. (USA) and New England Biolabs (Australia), respectively. Ultrapure water (18.2 MΩ) Milli-Q (Australia) was used in the preparation of aqueous solutions used in this study. Cloning A bacterial expression codon optimised gene fragment of Nb80 was synthesised (gBlock; IDT DNA) with flanking regions of homology to a modified pET3a vector encoding a His tag and tobacco etch virus protease site ( pET3a-HisTEV ) (see Supporting Information —Gene Synthesis). [ 20] pET3a-HisTEV vector was linearised in two fragments with primer pairs 1/2 and 3/4 (Supporting Information —Table S1); template plasmid DNA was removed by DpnI digest; and the synthesised gene fragment was cloned in-frame with the HisTEV tag sequence by three fragment Gibson Assembly and electroporated into electrocompetent E. coli NEB 5 α (New England Biolabs) selected on LB agar supplemented with 100 µg mL –1 ampicillin. [ 21] The pET3a-HisTEV-Nb80 plasmid was amplified, and sequence verified prior to expression and purification of Nb80. Protein Purification pET3a-HisTEV-Nb80 was transformed into chemically competent E. coli BL21 (DE3) and plated on LB agar ampicillin (100 µg mL –1 ) incubated at 37 °C for 18 h. 50 mL of LB Ampicillin 100 µg mL –1 (LB Amp ) was inoculated with a single colony and grown at 37 °C for 18 h under shaking at 180 rpm, and sub-cultured at 1:50 into 2 L of LB Amp and incubated at 37 °C and 180 rpm to an optical density at 600 nm (OD 600 ) of 0.6. Protein expression was induced with 0.1 mM IPTG at 18 °C with shaking 90 rpm for 18 h. Cells were harvested via centrifugation 4,000 × g for 20 min at 4 °C, supernatant discarded, and the cell pellet resuspended in lysis buffer (50 mM Tris, 500 mM NaCl, 20 mM imidazole, 2.5 mM DTT, pH 7.5). Cells were lysed on ice by sonication with an Autotune CV334 Ultrasonic Processor equipped with a standard probe (136 mm × 13 mm; Sonics and Materials, US) using 20 × 10 s pulses with 50 s intervals. Cell debris was removed by centrifugation (40,000 × g , 20 min, 4 °C) and the supernatant loaded onto an Ni charged 5 mL His-trap FF column (Cytiva) pre-equilibrated in lysis buffer at a flow rate of 3 mL min –1 . The column was washed with 100 mL of lysis buffer followed by gradient elution from 20 mM imidazole to 250 mM imidazole over 20 column volumes. Elution was monitored at a wavelength of λ = 280 nm and peak fractions analysed by SDS-PAGE with Coomassie staining. Fractions containing Nb80 were pooled and concentrated over a 5K MWCO centrifugal filter. Concentrated Nb80 was dialysed into size-exclusion chromatography (SEC) buffer (phosphate buffered saline, 2.5 mM DTT, pH 7.4) with 3K MWCO SnakeSkin™ Dialysis Tubing (Thermo Fisher Scientific). The protein was concentrated to a volume of 5 mL and injected onto a HiLoad 16/600 Superdex 200 pg size exclusion column pre-equilibrated in SEC buffer and eluted at a flow rate of 1 mL min –1 . Peak fractions were analysed by SDS-PAGE with Coomassie staining and pure fractions pooled and concentrated for subsequent experiments; 200 µL aliquots of purified Nb80 were flash-cooled in liquid N 2 and stored at –80 °C. A model of Nb80 was predicted using the AlphaFold2 (Jumper et al., 2021) CoLab notebook. [ 22] Fabrication of Nanoporous Anodic Alumina Interferometers NAA-based Fabry–Pérot interferometers were produced by the two-step electrochemical oxidation process. [ 23] 1.5 × 1.5 cm 2 square Al substrates were cut and cleaned via sonification in EtOH and ultrapure water for 5 min each, and then dried under air stream. Prior to anodisation, Al substrates were electropolished in a mixture of HClO 4 : EtOH (1:4 v:v ) at 20 V and 5 °C for 3 min under stirring condition. The first anodisation step was performed in an aqueous electrolyte of 0.3 M oxalic acid at 40 V and 6 °C for 20 h, using an electrochemical reactor with a circular window of 1 cm diameter and controlled stirring rate of 300 rpm. The resulting NAA layer that acted as a sacrificial anodic oxide film was chemically removed by wet chemical etching in an aqueous mixture of 0.2 M H 2 CrO 4 and 0.4 M H 3 PO 4 at 45°C for 24 h. The second anodisation step was then carried out using the same anodising condition for 2 h instead. Lastly, NAA were pore-widened in an aqueous solution of 5 wt % H 3 PO 4 at 35 °C for 15 min. Surface Chemistry Modification of NAA Interferometers The inner surface of NAA interferometers was chemically modified with APTES following a well-established protocol. [24,25] Prior to silanisation, NAA were immersed in 30 wt % H 2 O 2 at 90 °C for 10 min for hydroxylation, followed by drying under air stream. They were subsequently functionalized with APTES by chemical vapour deposition under vacuum at 110 °C for 3 h and washed with ultrapure water to remove any unbound APTES molecule. To enhance light interference, a layer of ~5 nm thick gold (Au) was deposited on the top surface of NAA using a sputter coater equipped with a film thickness monitor (Sputter Coater 108auto, Cressington, USA). [26] Real-time Binding Assessment of Nb80–proteins in NAA using RIfS The RIfS-based optical sensor setup comprised a tungsten light source (LS-1LL, Ocean Optics, USA) coupled to a bifurcated optical probe, a miniature spectrometer (Ocean HDX, Ocean Insight, USA), and a transparent plexiglass microfluidic flow cell. White light from the tungsten source with an illumination spot of 2 mm was focused on the surface of NAA packed in the flow cell by the optical probe. The reflected light from the illuminated spot was collected and transferred to the spectrometer via the collection fiber of the probe. The RIfS spectra were recorded in the wavelength range of 400–1000 nm at an interval of 0.5 min, integration time of 50 ms, and 10 average measurements. The optical spectra were then processed by applying fast Fourier transform (FFT) in Igor Pro library (Wavemetrics, USA) to estimate effective optical thickness (OT eff ) of NAA according to Equation 1: OT eff = 2 n eff L P cos θ (1) where n eff is the effective refractive index of NAA, L P is the physical thickness of NAA (nanopore length), and θ is the angle of incidence/refraction of light, which is θ = 0° in this case. [ 27] Prior to real-time sensing acquisition, 50 µg mL –1 Nb80 in 10 mM of PBS was mixed with 10-fold molar excess of EDC to Nb80 and 2.5-fold molar excess of NHS to EDC. The mixture was incubated for 1 h at room temperature to activate the carboxylates of Nb80. The carboxylate moiety of mPEG–COOH (1 wt % in PBS) were activated using the same protocol. The real-time, in-situ binding between Nb80 and HSA was monitored through changes in OT eff (ΔOT eff ) of APTES-modified NAA interferometers under dynamic flow conditions. Chemically modified NAA interferometers were sandwiched in a custom-made flow cell and exposed to different analytical solutions of target biomolecules injected into the system at a constant flow rate of 100 µL min –1 controlled by a syringe pump (Fusion Touch, Chemyx, USA). A stable baseline was first established by introducing 10 mM PBS (pH 7.4) into the flow system for 15 min, followed by activated Nb80 (50 µg mL –1 in PBS) for 60 min, and then PBS for 15 min to remove any physisorbed Nb80 from the inner surface of the NAA interferometers. Nb80-functionalised NAA interferometers were then exposed to activated mPEG-COOH (1 wt% ) for 30 min to block the remaining active amine functional groups on the surface. This step was followed by a subsequent 15 min of PBS washing. After functionalisation of the inner surface of the NAA interferometers, solutions of various concentrations of HSA were flowed through the system (i.e., 2.26, 1.88, 1.51, 1.13, 0.75, and 0.38 µM). These solutions were prepared from a stock solution of 15.1 µM HSA in 10 mM pH 7.4 PBS. After 60 min of continuous flow of HSA, fresh PBS was flowed over the NAA platforms for 15 min to wash off loosely bound HSA molecules for the establishment of the final ΔOT eff associated with Nb80–HSA interactions. Using the same protocol, the binding affinity of Nb80 to other serum albumins (i.e., 0.75 µM MSA and BSA) and protein families (i.e., 50 µg mL –1 of γG and TFN) was also assessed based on real-time ΔOT eff monitoring. Structural Characterisation of NAA Interferometers The structural features of NAA interferometers were characterised by images acquired using a field-emission gun scanning electron microscope (FEG-SEM FEI Quanta 450). FEG-SEM images were analysed using ImageJ software (public domain program developed at the RSB of the NIH). [28] Solution Characterisation of Nb80 Complex Formation with Serum Albumins, γ-globulin, and transferrin Solutions of HSA (0.4 mg mL –1 ), MSA (1.6 mg mL –1 ) and BSA (0.4 mg mL –1 ) in SEC buffer were incubated with an equal concentration ( w / v ) of Nb80 mixed at 1:1 v / v and equilibrated at room temperature for 2 h; 500 µL samples were applied to a Superdex 200 Increase 10/300 GL SEC column (Cytiva) pre-equilibrated in SEC buffer and eluted at a flow rate of 0.5 mL min –1 with an ÄKTA pure chromatography system (Cytiva). Control mixtures comprising 1:1 v / v ratios of 0.4 mg mL –1 Nb80 with 0.4 mg mL –1 γG; and 0.4 mg mL –1 TFN, respectively, were analysed as above. Control samples of purified Nb80; serum albumins; γ-globulin and transferrin were also separated by analytical SEC. Chromatograms of isolated proteins and mixed samples were overlaid to determine the relative peak positions. Fractions corresponding to peaks of Nb80; serum albumins; γ-globulin; transferrin and complexes thereof were analysed by SDS-PAGE (NuPAGE Bis-Tris Mini Protein Gels, 4−12%, 1 mm; NuPAGE MES SDS Running Buffer, Invitrogen) and Coomassie blue staining. 3. RESULTS AND DISCUSSION 3.1 Structure of NAA Interferometers Anodisation of Al substrates in 0.3 M H 2 C 2 O 4 at its self-ordering potential of 40 V yielded the growth of arrays of anodic alumina nanopores driven by the electric field-induced migration of ionic species (i.e., H + , Al 3+ , O 2– , and HO – ). As illustrated in Fig. 2a, NAA interferometers have distinct geometrical characteristics such as the nanopore length (L P ), nanopore diameter (D P ), interpore distance (D Int ), and thickness of barrier oxide layer (τ BOL ). The cross-sectional FEG–SEM view of a representative NAA interferometer shown in Fig. 2b revealed the propagation of alumina nanopores perpendicular to the underlying aluminium substrate with straight coherency, from top to bottom, forming a nanoporous anodic film of L P = 4.5 ± 0.03 µm. The bottom side of each straight cylindrical nanopore was closed by a hemispherical barrier oxide layer with a thickness of τ BOL = 32.6 ± 3.4 nm, as depicted in Fig. 2c. The top FEG–SEM view of the NAA interferometer in Fig. 2d shows a homogenous distribution of self-organised nanopores within their grain domain boundaries across the surface of the anodic film. These nanopores arranged in hexagonal cells (Fig. 2e) and had an average D P of 62.8 ± 5.9 nm and D Int of 100.4 ± 6.8 nm. Fig. 2. Structural characterisation of nanoporous anodic alumina (NAA) interferometers. (a) Schematic of the idealised structure of a NAA interferometer characterised by the pore length (L P ), interpore distance (D Int ), pore diameter (D P ), and thickness of barrier oxide layer (τ BOL ). (b) Cross-sectional FEG-SEM view image of a NAA platform showing an array of straight cylindrical nanopores that grow perpendicularly to the underlying aluminium substrate (scale bar = 1.5 µm). (c) A magnified view of the bottom tips of the nanopores of a NAA interferometer closed by a hemispherical anodic oxide layer (scale bar = 500 nm). (c) Top FEG-SEM view image of a NAA platform depicting a homogenous distribution of self-organised nanopores arranged into domains across the surface (scale bar = 1 µm). (d) A magnified view of nanopores following a honeycomb-like hexagonal cell arrangement (scale bar = 500 nm). Note that these NAA platforms were treated by pore widening in 5 wt% phosphoric acid for 15 min at 35°C. 3.2 Binding Assessment of HSA to Nb80-modified NAA Interferometers The surface chemistry modification and sensing stages for the development of Nb80-based NAA interferometric sensors are detailed in Fig. 3a. To engineer the surface chemistry of the NAA platforms for the immobilisation of Nb80, as-produced NAA interferometers were first hydroxylated in H 2 O 2 solution for the generation of hydroxyl (–OH) groups. The –OH groups on the inner surface of NAA facilitated the attachment of APTES molecules via siloxane bond (Si–O–Si) formed from the condensation reaction between silanol groups on APTES molecules and –OH groups on the surface of anodic alumina (Al 2 O 3 ). [ 29] The APTES-functionalised NAA platforms were then sandwiched in a custom-made flow system and different analytical solutions were introduced sequentially to build a HSA sensing system based on Nb80-conjugated NAA interferometers. Note that Nb80 was selected from literature with characterised high-affinity binding to HSA and MSA. [ 20] The coding sequence for Nb80 was synthesised and cloned into a bacterial expression vector. Affinity tagged Nb80 was expressed and isolated from E. coli lysate by standard affinity and size exclusion chromatography to high purity (Fig. S2). For Nb80 to couple with the amine (–NH) moieties of the APTES functional layer covalently bound inside the alumina nanopores, the carboxylate (–COOH) groups of Nb80 were activated via carbodiimide crosslinker chemistry. This involved the exposure of Nb80 to EDC catalysed by the addition of NHS, where the C-terminus of Nb80 reacted with EDC to form Nb80 with an amine-reactive (dry-stable) NHS ester of –COOH groups. [ 30] The presence of NHS ester governed the efficient conjugation of Nb80 to the primary –NH groups of APTES inside the NAA platforms via the formation of amide crosslinks. Following the immobilisation of Nb80, mPEG–COOH activated by the same carbodiimide chemistry was introduced to block any free functional group on the NAA interferometers and increase the selectivity to HSA molecules. The final sensing stage was the binding assessment of HSA molecules by Nb80-modified NAA interferometers. The build-up of molecules inside the nanopores of the NAA platforms after each sensing stage caused a shift in the interference pattern and effective refractive index of the anodic thin films. Hence, the RIfS spectra of NAA interferometers red shifted, as shown in Fig. 3b. According to Equation 1, the increase in effective refractive index increases OT eff and red-shifts the position of the band in the FFT spectra derived from the RIfS spectra in Fig. 3c. [ 31] An intense FFT band was observed in the range of OT eff = 17500–18000 nm, with a concurrent red shift in OT eff across the sensing stages. Fig. 4a shows the real-time change of OT eff (ΔOT eff ) recorded through the surface chemistry engineering and sensing stages in APTES-modified NAA interferometric platforms. At time (t) = 0 min, PBS was introduced into the flow sensing system to establish a stable baseline. The binding of activated Nb80 to APTES-modified NAA platforms in the flow system via carbodiimide crosslinking induced an exponential change in ΔOT eff , where the plateau indicated a saturation of Nb80 bound on the APTES functional molecules covering the inner surface of NAA interferometers. ΔOT eff then decreased slightly due to the removal of loosely bound Nb80 during the washing of the NAA platforms with PBS. The subsequent increase in ΔOT eff was associated with the exposure of the NAA platforms to mPEG–COOH solution to reduce non-specific adsorption of HSA onto APTES molecules. [ 32,33] When the injecting solution was switched to PBS, ΔOT eff returned to the previous value of ~25 nm. Based on the value of ΔOT eff before and after the injection of mPEG (labelled as III in Fig. 4a), all APTES molecules available on the NAA platforms were occupied by Nb80 or blocked by mPEG. When the analytical HSA solution was flowed into the sensing system, HSA molecules might orientate to expose their binding sites to interact with Nb80 molecules immobilised onto the NAA surface. Since Nb80 is a small molecule with a long complementarity-determining region 3 (CDR-3), it forms unique hypervariable finger-like structures to recognise cavities or hidden epitopes on target molecules. [ 34] Previous high-throughput docking and clustering analysis of Nb80 unique to HSA reveals the existence of five epitopes on HSA, with three dominant, non-linear/conformational epitopes and two minor epitopes. [ 35] HSA is known to be made up of a repeating pattern of three α-helical homologous domains (I, II, and III), with each domain divided into two sub-domains (A and B) connected by an extended loop. [ 36] The domains and sub-domains of HSA are capable of binding to metabolites and chemical compounds through their hydrophobic and positively-charged residues. Fig. 3. Surface chemistry engineering of nanobody-conjugated NAA interferometers for real-time detection of HSA molecules. (a) Schematics describing the stages occurring inside the nanopores of NAA interferometers through the surface chemistry engineering and sensing stages, starting from the silanisation of the oxide surface (Stage I), followed by the immobilisation of C-terminus activated Nb80 (Stage II), then blocking of Nb80–unbound surfaces with methoxy polyethylene glycol (Stage III), and lastly the exposure to HSA molecules (Stage IV). (b) Shift in the RIfS spectra of a NAA sensing platform corresponding to each binding stage, with the inset showing a magnified view of the red shift in the fringe maxima and minima in the highlighted region. (c) The bands derived from RIfS spectra by fast Fourier transform represents the ΔOT eff of the NAA platform after each stage, which is red-shifted when subjected to each binding interaction, as observed in the inset. As identified by previous epitope mapping analysis, HSA epitopes are located on domains I and III, as well as in between domains I and II, where Nb80 displayed a higher binding preferential over concave epitopes. [ 20] Therefore, HSA molecules might self-arrange so that their epitopes align with Nb80 for effective interaction with a substantial overlap on Sudlow’s binding sites (sub-domains IIA and IIIA). [ 20] Another possible site of interaction is on HSA residues, where they would form salt bridges with the CDR residues of Nb80 (e.g. D60, R107 Fig. 3b) that are surface-exposed to the PBS solution, while the other parts of Nb80 would be anchored on the surface of the alumina nanopores. [ 20] Furthermore, the interaction of HSA–Nb80 could occur via tyrosine (Y106, Fig. 3b) at the centre of the CDR-3 loop of Nb80, with the insertion of its aromatic side chain into the specific pockets on HSA epitopes. This is facilitated by extra flexibilities provided by serine and glycine tucking away from the CDR-3 centre to facilitate the orientation of tyrosine side chain in the HSA epitope pockets. [ 35] It is also likely that multiple Nb80 molecules docked into a single HSA molecule since HSA has multiple sites to accommodate a small molecule like Nb80. As a result, the effective interaction of HSA with Nb80 immobilised on the inner surface of NAA interferometers via these interaction mechanisms would be responsible for the increase in effective refractive index of the NAA film and the concomitant ΔOT eff induced by that change. A final PBS washing step was performed to determine the total ΔOT eff . This step was characterised by a slight decrease in ΔOT eff , which was attributed to the detaching of loosely bound HSA complexes formed on the surface of Nb80-functionalised NAA intereferometers. [ 37] Fig. 4b reveals that the trends in ΔOT eff followed a similar pattern when Nb80-modified NAA platforms were subjected to different concentrations of HSA ([HSA]) and the subsequent PBS washing step. However, the total ΔOT eff was demonstrated to be dependent on [HSA], where ΔOT eff increased with [HSA] until reaching the saturation point. Fig. 4c summarises the values of ΔOT eff versus [HSA]. It is apparent that, from [HSA] = 0.0 to 1.51 µM, ΔOT eff reached a maximum of 38.0 ± 0.3 nm associated with an increased frequency of Nb80–HSA interactions enhanced by the higher number of available HSA molecules per unit volume. This also accounted for the rapid increase in ΔOT eff observed at high [HSA] attributed to the high frequency of binding interactions between HSA and Nb80. At low [HSA], the increase in ΔOT eff was limited by the availability of HSA molecules to interact with Nb80. Therefore, the ΔOT eff curve featured a smaller slope with time, denoting a weak kinetic binding interaction. Fig. 4. Binding assessment of Nb80-functionalised NAA interferometric sensors to HSA molecules in solutions of varying concentration. (a) Real-time ΔOT eff of silanised NAA platforms (I) in response to exposure to a 50 µg mL –1 solution of activated Nb80 molecules (II), 1 wt % mPEG–COOH (III), and HSA solution (IV). Note that PBS was introduced at the start of the process to establish a baseline, and in between stages for washing. (b) Compilation of ΔOT eff of Nb80-modified NAA platforms over time recorded during the adsorption/desorption of HSA (highlighted region in (a)) in solutions with varying concentration. (c) Langmuir fitting of ΔOT eff –[HSA] plot for the determination of the equilibrium binding constant. (d) Association and dissociation curves correlated to HSA–Nb80 interactions derived from the ΔOT eff –time plot. (e) Fitted association curves of HSA to Nb80-modified NAA interferometers at different [HSA] for the determination of exponential time constant, k s . (f) The fitted dissociation curves of HSA obtained from Nb80-modified NAA interferometers exposed to different [HSA] for the determination of dissociation rate constant, k d . A linear fitting within the region of [HSA] from 0 to 1.51 µM established the performance of the proposed protein sensor, the sensitivity, low limit of detection, and linearity of which were determined to be 27.3 ± 3.2 nm µM –1 , 16.3 ± 1.6 µM, and 0.95, respectively (Fig. S3a). A further increase of [HSA] to the range of 1.88 to 2.26 µM, however, did not red-shift ΔOT eff any further as the Nb80–HSA reaction was limited by the saturation of Nb80 molecules available on the inner surface of NAA interferometers. Eventually, the system reached its saturation point at this stage, despite the high in-flux of HSA molecules. This was reinforced by the increasing pattern of ΔOT eff at [HSA] = 1.51, 1.88, and 2.26 µM, where the initial red shift of ΔOT eff occurred at different rate but eventually reached a plateau in ΔOT eff . The numerical fitting of experimental data in Fig. 4c revealed that this binding interaction could be precisely described by a Langmuir isotherm model (See S4 in Supporting Information). Data analysis revealed a strong correlation between the experimental data and the theoretical model, with a linearity (R 2 ) of 0.87. [ 38] The equilibrium dissociation constant (K d ), equilibrium association constant (K a ), and maximum ΔOT eff (ΔOT max ) based on the adapted Langmuir model were determined to be (10.6 ± 5.4) × 10 –7 M, (0.10 ± 0.03) × 10 7 M –1 , and 56.2 ± 16.8 nm, respectively. The affinity of Nb80 to HSA in this study is lower than that of previously reported values (i.e., K d = 166 × 10 –12 M). 20 This discrepancy could be due to the restricted molecular orientation of Nb80 molecules as they were immobilised on a solid support, limiting the accessibility of HSA epitopes to Nb80 paratopes. In contrast, when Nb80 molecules are not bound to a solid support, they can rotate freely and dock themselves into HSA epitopes without any hindrance, hence exhibiting higher affinity to HSA molecules in the liquid matrix. To determine the kinetic rate constants (S5– Supporting Information), the time-dependent ΔOT eff curves were characterised according to association and dissociation curves, with the former related to the adsorption of HSA to Nb80, and the latter to the desorption of HSA from Nb80 during PBS washing (Fig. 4d). It was assumed that the kinetics were not limited by diffusion in the time scale over which the data were fitted. [ 38.39] Fig. 4e compiles the association curves of Nb80–HSA binding fitted numerically to Equation S13. By evaluating the association curves using Equation S13, the exponential time constant (k s ) at each [HSA] was determined, as summarised in Table S2. As k s was linearly correlated to [HSA], [ 39,40] the slope of k s –[HSA] plot in Fig. S3b revealed the association rate constant (k a ) of Nb80–HSA binding to be 540.8 ± 130.0 M –1 s –1 . The evaluation of desorption data in Fig. 4f using Equation S16 yielded an average dissociation rate constant (k d ) of (2 ± 0.5) × 10 –4 s –1 (values of k d at each [HSA] are provided in Table S2). The equilibrium association constant, K a —ratio of k a to k d —was calculated to be (2.7 ± 0.4) × 10 6 M –1 , which is smaller than that quantified through the Langmuir model. The discrepancies in the values of K a could be associated with: (i) restricted diffusion of molecules in the nanopores, [ 41–45] (ii) steric hindrance of binding sites induced by the adsorbed Nb80, [ 40,46] (iii) multiple-stage binding, [ 47] and (iv) surface heterogeneity-induced multiple binding interactions. [ 48] The equilibrium dissociation constant (K ­d ) of (0.37 \(\pm\) 0.09) × 10 –6 M for immobilised Nb80–HSA is comparable to that of HSA–monoclonal antibody (1.2 × 10 –6 M, 1.1 × 10 –7 M, 10.4 × 10 –6 M and 5.4 × 10 –6 M determined by capillary zone electrophoresis, alternating current electrophoresis, isothermal titration calorimetry and piezoelectric immunosensor, respectively). [ 49,50] Note that the evaluation of rate constants are influenced by the detection technique, therefore the difference in the values of K ­d . The details of the Langmuir plot and kinetic analysis are provided in S4 and S5 (Supporting Information). 3.3 Affinity Assessment of Nb80-modified NAA Interferometers to Serum Albumins BSA and MSA are structurally similar to HSA, sharing a 72–76 % sequence identity. [ 51,52] Therefore, Nb80–modified NAA platforms were assessed against BSA and MSA molecules to benchmark the selectivity of this system to HSA. Fig. 5a depicts the real-time ΔOT eff of a NAA platform with immobilised Nb80 when exposed to 1.13 µM of serum albumins (SAs: HSA, BSA and MSA). After establishing a stable baseline with PBS, ΔOT eff red-shifted over time upon injecting SAs in the flow cell system. After a given time, the system eventually stabilised, reaching a saturation point. PBS was then introduced into the flow sensing system to acquire the final ΔOT eff associated with the binding event and remove loosely bound SA molecules from the Nb80-functionalised NAA platform. Based on the quantified values of ΔOT eff induced upon exposure to solutions of different SAs in Fig. 5b (i.e., ΔOT eff = 24.8 ± 1.2 nm for HSA, 23.2 ± 1.2 nm for MSA, and 20.0 ± 1.0 nm for BSA), it is inferred that Nb80-conjugated NAA platforms had stronger affinity to SA in the order of BSA < MSA < HSA. The difference in the affinity of Nb80 to SA stemmed from the slight deviation in the sequences and structures of the binding epitopes on SA molecules. [ 51] Fig. 5. Affinity assessment of Nb80–conjugated NAA platforms to different types of serum albumins (SA). (a) Real-time ΔOT eff in response to the binding of bovine (BSA), mouse (MSA), and human serum albumin (HSA) to Nb80 molecules immobilised on NAA interferometers. (b) ΔOT eff as a function of type of SA. (c) Fitted association curves of Nb80 molecules to different SA molecules. (d) The fitted dissociation curves of SA molecules from Nb80-modified NAA. Nonetheless, the relatively conserved epitopes shared between HSA and MSA could still be targeted by Nb80 molecules. Epitope mapping by structural proteomics has generated a model of Nb80 in complex with HSA, with two critical salt bridges forming between HSA K383/Nb80 D60; and HSA E400/Nb80 R107. [ 20] In light of this, similar to HSA, MSA preserves a Lys at amino acid residue 383; and a Glu at residue 400, which could support salt bridge formation with Nb80. This is consistent with the observed small difference in ΔOT eff induced by Nb80 interacting with MSA and HSA. Conversely, BSA has charge reversals at positions, E382 (equivalent to HSA K383) and K399 (equivalent to HSA E400) (Fig. S6), demonstrated to eliminate binding by Nb80 when such charge reversals were reconstituted in HSA K383D; E400R . [ 20] Furthermore, there are two regions localised on the subdomain IB of BSA that are significantly different from that of HSA in terms of exposed residues on the protein surface. [ 52] This could affect the interaction of BSA with Nb80, and together with the key charge reversals, explain the lower binding affinity recorded for this system. The results obtained align well with previous reports. [ 45] The binding affinity of Nb80 evaluated through ΔOT eff in Nb80-functionalised NAA interferometers can also be expressed as a function of association and dissociation rate constants through the numerical fittings of the association and dissociation curves. Fig. 5c shows the association curves related to the binding of SA to Nb80-modified NAA platforms. The exposure to HSA triggered a rapid increase in ΔOT eff compared to that of MSA and BSA. Interestingly, the ΔOT eff of Nb80-modified NAA interferometers increased at a similar slope when exposed to BSA and MSA for the first 30 min. This can be attributed to the common epitopes in both SA molecules, which could be targeted first by Nb80 molecules. By numerically fitting the association curves to Equation S12, k a for Nb80 to 1.13 µM of BSA, MSA and HSA were calculated to be 360.1 ± 72.0, 308.2 ± 77.0, and 540.8 ± 130.0 M –1 s –1 , respectively. The dissociation curves in Fig. 5d show a decrease in ΔOT eff when unbound SA molecules were washed off from Nb80-modified NAA interferometers with PBS. The dissociation of SA–Nb80 complexes determined using Equation S15 occurred at k d = (3 ± 1) × 10 –4 s –1 for BSA, (2 ± 1) × 10 –4 s –1 for MSA, and (2 ± 0.5) × 10 –4 s –1 for HSA. Derived from the values of k a and k d , the equilibrium constant K a increased in the order of BSA (1.5 ± 0.2) × 10 6 M –1 < MSA (1.7 ± 0.3) × 10 6 M –1 < HSA (2.7 ± 0.4) × 10 6 M –1 , which aligned with the affinity of Nb80 exhibited towards different SAs established by ΔOT eff . Fig. 6. Analytical size exclusion chromatography (SEC) analysis of Nb80 and SA co-elution. SEC chromatograms (a–c; y -axis absorbance at λ = 280 nm (mAu) of 100 µg (a, c) or 400 µg (b) purified protein. Control samples of Nb80 (grey, (a–c)); and (a) HSA, (b) MSA and (c) BSA, are shown in blue. Mixtures of Nb80 and SAs in ratios 1:1 wt / wt (a) Nb80/HSA; (b) Nb80/MSA and (c) Nb80/BSA are shown in red. Nb80-dependent shifts in the elution volume of HSA and MSA were observed (a–b), while the BSA peak (b) showed an elution volume consistent with the BSA-alone control. The ability of SA molecules to form complexes with Nb80 immobilised on the NAA platforms was further interrogated by the characterisation of these interactions with target antigens for HSA, MSA and control serum antigen BSA in solution by analytical SEC. Following incubation of 1:1 wt / wt mixtures of Nb80 with HSA (Fig. 6a), MSA (Fig. 6b) and BSA (Fig. 6c), analytical SEC elution profiles were compared with control elution profiles for each protein in isolation. Clear shifts in the elution volume of HSA (Fig. 6a) and MSA (Fig. 6b) were evident following incubation with purified Nb80. The peak shift to an earlier elution volume indicated the formation of a complex of higher molecular weight than the control serum albumin samples alone. SDS-PAGE analysis of peak fractions of HSA/Nb80 and MSA/Nb80 showed a band of a size consistent with Nb80 alone (17.5 kDa) co-eluting with both HSA and MSA (Fig. S7a and d). In contrast, the BSA control showed no significant change in the elution volume, indicating that no complex was formed with Nb80, in agreement with published data. [ 18] This was also reflected by the absence of a co-eluted band of ~17.5 kDa in SDS-PAGE analysis of peak fractions from mixed samples BSA:Nb80 (Fig. S7c). 3.4 Selectivity Assessment of Nb80-modified NAA Interferometers to Various Families of Proteins Nanobodies are known for their small size and unique binding domains, which enable specific recognition of cryptic epitopes on target molecules. [ 53,54] Fig. 7. Selectivity assessment of Nb80-conjugated NAA interferometers to different families of proteins. Real-time ΔOT eff revealed a negligible interaction between immobilized Nb80 molecules and: (a) γ-globulin and (b) human serum transferrin consistent with analytical SEC analysis of Nb80 co-elution experiments. Isolated control samples (100 µg) of (c, d) Nb80, (c) γ-globulin, and (d) transferrin, were eluted from a Superdex 200 analytical column. Mixtures of 1:1 wt / wt (c) Nb80/γ-globulin and (d) Nb80/transferrin, showed no significant shifts in the elution volume of γ-globulin and transferrin. To validate the selectivity of Nb80 towards SAs, Nb80-functionalised NAA interferometers were subjected to two different types of human proteins: γ-globulin (γG) and human serum transferrin (TFN). Fig. 7a shows that no ΔOT eff was observed when 0.75 µM γG was flown over the Nb80-modified NAA platforms. A similar trend was observed in Fig. 7b, which indicated no effective interaction of TFN (0.75 µM) with Nb80 molecules functionalised on the inner surface of NAA interferometers. This was in strong contrast with the values of ΔOT eff associated with the Nb80–HSA interaction shown in Fig. 4a, which revealed a substantial and immediate red shift in ΔOT eff upon exposure to HSA molecules. This suggests that the shape and conformation of Nb80 molecules are tailor-engineered to HSA molecules for highly specific recognition of HSA epitopes. γG and TFN molecules do not possess epitopes of similar structure or surface residues to those of HSA, and are molecules with significantly different size, shape and configuration to those of HSA. On one hand, γG is a multi-chained molecule (molecular weight of ~155–160 kDa) made up of heavy and light polypeptide chains with disulfide bridges crosslinking between them. 55,56 On the other hand, TFN is a glycoprotein composed of a single polypeptide chain (molecular weight of ~80 kDa) with two globular lobes of alternating α-helical and β-sheet segments connected by a short randomly coiled-peptide chain to form a bilobal structure. [ 57,58] Therefore, the convex paratope of Nb80 was unable to access the cavities or clefts on γG and TFN molecules due to the conformational difference. Additional SEC analyses of control mixtures of Nb80 with γG and TFN in Fig. 7c and d also showed no change in the SEC elution volume of either control protein, indicating the absence of complex formation in solution for these controls. In agreement with the analytical SEC, SDS-PAGE analysis of peak fractions from mixed samples of γG:Nb80 and TFN:Nb80 did not show a co-eluted band of ~17.5 kDa, supporting the observation that these control proteins did not interact with Nb80 (Fig. S7b). CONCLUSIONS This study presented the first demonstration of integrating nanobodies into NAA-based RIfS system for the development of a protein sensor. The highly HSA-selective nanobody Nb80 was expressed in bacteria and purified to homogeneity. Purified Nb80 was then strategically crosslinked onto NAA interferometers via its C-terminus and its interaction with HSA was monitored in situ via the real-time shift in RIfS fingerprints of NAA. The sensing performance of the proposed Nb80-based RIfS sensor was characterised by a proportional change in effective optical thickness in response to increasing concentrations of HSA, which revealed a sensitivity of 27.3 ± 3.2 nm µM –1 and low limit of detection of 16.3 ± 1.6 µM. The sensitivity of this system exceeded the circulating concentrations of serum albumin in normal adults (i.e., ~600 µM) and in disease states such as nephrotic syndrome, where the circulating concentration drops to the range 100–360 µM 59 highlighting the potential of this system for real-life medical diagnostics. The proposed protein sensor also demonstrated a high selectivity and affinity to HSA when tested against a range of proteins such as mouse and bovine serum albumins, γ-globulin and transferrin. The high selectivity of the sensor was attributed to its paratopes with unique conformation tailored to the epitopes of HSA molecules. This was in good agreement with analytic SEC and SDS-PAGE results, which denoted negligible complex formation of Nb80 with proteins other than HSA. In summary, this study provides new opportunities to develop portable, reliable and selective RIfS-based protein sensors by harnessing nanobodies as a sensing element. The proposed system could be deployed as a complementary tool to benchmark analytical techniques for point-of-care clinical and medical diagnostics. Author Contributions Cheryl Suwen Law: conceptualisation, methodology, data curation, formal analysis, investigation, visualisation, writing–original draft, writing–review and editing. Jayden Revink: data curation, formal analysis, writing–review and editing. Joel Lee: data curation, formal analysis, writing–review and editing. Juan Wang: data curation, formal analysis, writing–review and editing. Andrew D. Abell: writing–review and editing, funding acquisition. Fiona Whelan: conceptualisation, methodology, data curation, formal analysis, investigation, writing–original draft, writing–review and editing, supervision, project administration, and funding acquisition. Abel Santos: methodology, funding acquisition, supervision, project administration, and writing–review and editing. All authors have given approval to the final version of the manuscript. ACKNOWLEDGEMENT The authors would like to acknowledge and pay their respects to the Kaurna-Adelaide people, the Traditional Custodians of the land on which the work was performed. The authors thank the support provided by the Australian Research Council through grants DP220102857 and DP230103062, School of Chemical Engineering, School of Biological Sciences, Institute for Photonics and Advanced Sensing (IPAS), The University of Adelaide. The authors acknowledge the instruments and expertise of Microscopy Australia at Adelaide Microscopy, The University of Adelaide, enabled by NCRIS, university, and state government support. F.W. was supported by the Ramsay Fellowship in Applied Science. C.S.L. thanks the support provided by The University of Adelaide and IPAS through her Future Making Fellowship. CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. ORCID Cheryl Suwen Law https://orcid.org/0000-0002-3276-8052 Andrew D. Abell https://orcid.org/0000-0002-0604-2629 Fiona Whelan https://orcid.org/0000-0002-0791-6850 Abel Santos https://orcid.org/0000-0002-5081-5684 REFERENCES 1. J. Budd, B.S. Miller, N.E. Weckman, D. Cherkaoui, D. Huang, A.T. Decrux, N. Fongwen, G-R. Han, M. Broto, C.S. Estcourt, J. Gibbs, D. Pillay, P. Sonnenberg, R. Meurant, M.R. 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Keywords human serum albumin nanobody nanoporous anodic alumina reflectrometric interference spectroscopy Authors Affiliations Cheryl Suwen Law 0000-0002-3276-8052 Adelaide University View all articles by this author Jayden Revink The University of Adelaide View all articles by this author Joel Lee The University of Adelaide View all articles by this author Juan Wang The University of Adelaide View all articles by this author Andrew Abell The University of Adelaide View all articles by this author Fiona Whelan 0000-0002-0791-6850 The University of Adelaide View all articles by this author Abel Santos 0000-0002-5081-5684 [email protected] The University of Adelaide View all articles by this author Metrics & Citations Metrics Article Usage 220 views 170 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Cheryl Suwen Law, Jayden Revink, Joel Lee, et al. Protein Binding Assessment of Immobilised Nanobody using Interferometric Nanoporous Platform. 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