Affimer reagents as tool molecules to modulate platelet GPVI-ligand interactions and specifically bind GPVI dimer.

Glycoprotein (GP)VI plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22 and D18 bound GPVI with the highest affinities (KD in the nM range). These Affimers inhibited GPVI-CRP-XL/collagen interactions, CRP-XL/collagen induced platelet aggregation and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and ADP. D22 but not M17/D18 displaced nanobody2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1-domain, while M17 targets a site on the D2-domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody Fab-fragment. D18 targets a new region on the D2-domain. We found that D18 is a stable non-covalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2-domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2-domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.

Photoactive imaging and therapy for colorectal cancer using a CEA-Affimer conjugated Foslip nanoparticle.

Theranostic nanoparticles hold promise for simultaneous imaging and therapy in colorectal cancer. Carcinoembryonic antigen can be used as a target for these nanoparticles because it is overexpressed in most colorectal cancers. Affimer reagents are synthetic proteins capable of binding specific targets, with additional advantages over antibodies for targeting. We fabricated silica nanoparticles using a water-in-oil microemulsion technique, loaded them with the photosensitiser Foslip, and functionalised the surface with anti-CEA Affimers to facilitate fluorescence imaging and photodynamic therapy of colorectal cancer. CEA-specific fluorescence imaging and phototoxicity were quantified in colorectal cancer cell lines and a LS174T murine xenograft colorectal cancer model. Anti-CEA targeted nanoparticles exhibited CEA-specific fluorescence in the LoVo, LS174T and HCT116 cell lines when compared to control particles (p < 0.0001). No toxicity was observed in LS174T cancer mouse xenografts or other organs. Following photo-irradiation, the anti-CEA targeted particles caused significant cell death in LoVo (60%), LS174T (90%) and HCT116 (70%) compared to controls (p < 0.0001). Photodynamic therapy (PDT) at 24 h in vivo showed a 4-fold reduction in tumour volume compared to control mouse xenografts (p < 0.0001). This study demonstrates the efficacy of targeted fluorescence imaging and PDT using Foslip nanoparticles conjugated to anti-CEA Affimer nanoparticles in in vitro and in vivo colorectal cancer models.

Therapeutic drug monitoring of immunotherapies with novel Affimer-NanoBiT sensor construct.

Concentration-therapeutic efficacy relationships have been observed for several therapeutic monoclonal antibodies (TmAb), where low circulating levels can result in ineffective treatment and high concentrations can cause adverse reactions. Rapid therapeutic drug monitoring (TDM) of TmAb drugs would provide the opportunity to adjust an individual patient's dosing regimen to improve treatment results. However, TDM for immunotherapies is currently limited to centralised testing methods with long sample-collection to result timeframes. Here, we show four point-of-care (PoC) TmAb biosensors by combining anti-idiotypic Affimer proteins and NanoBiT split luciferase technology at a molecular level to provide a platform for rapid quantification (<10 minutes) for four clinically relevant TmAb (rituximab, adalimumab, ipilimumab and trastuzumab). The rituximab sensor performed best with 4 pM limit of detection (LoD) and a quantifiable range between 8 pM-2 nM with neglectable matrix effects in serum up to 1%. After dilution of serum samples, the resulting quantifiable range for all four sensors falls within the clinically relevant range and compares favourably with the sensitivity and/or time-to-result of current ELISA standards. Further development of these sensors into a PoC test may improve treatment outcome and quality of life for patients receiving immunotherapy.

"Affimer" synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation.

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease.

Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition.

Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.

Enzyme - Switch sensors for therapeutic drug monitoring of immunotherapies.

Therapeutic monoclonal antibodies (TmAb) have emerged as effective treatments for a number of cancers and autoimmune diseases. However, large interpatient disparities in the pharmacokinetics of TmAb treatment requires close therapeutic drug monitoring (TDM) to optimise dosage for individual patients. Here we demonstrate an approach for achieving rapid, sensitive quantification of two monoclonal antibody therapies using a previously described enzyme switch sensor platform. The enzyme switch sensor consists of a ß-lactamase - ß-lactamase inhibitor protein (BLA-BLIP) complex with two anti-idiotype binding proteins (Affimer proteins) as recognition elements. The BLA-BLIP sensor was engineered to detect two TmAbs (trastuzumab and ipilimumab) by developing constructs incorporating novel synthetic binding reagents to each of these mAbs. Trastuzumab and ipilimumab were successfully monitored with sub nM sensitivity in up to 1% serum, thus covering the relevant therapeutic range. Despite the modular design, the BLA-BLIP sensor was unsuccessful in detecting two further TmAbs (rituximab and adalimumab), an explanation for which was explored. In conclusion, the BLA-BLIP sensors provide a rapid biosensor for TDM of trastuzumab and ipilimumab with the potential to improve therapy. The sensitivity of this platform alongside its rapid action would be suitable for bedside monitoring in a point-of-care (PoC) setting.

Affimers targeting proteins in the cardiomyocyte Z-disc: Novel tools that improve imaging of heart tissue.

Dilated Cardiomyopathy is a common form of heart failure. Determining how this disease affects the structure and organization of cardiomyocytes in the human heart is important in understanding how the heart becomes less effective at contraction. Here we isolated and characterised Affimers (small non-antibody binding proteins) to Z-disc proteins ACTN2 (α-actinin-2), ZASP (also known as LIM domain binding protein 3 or LDB3) and the N-terminal region of the giant protein titin (TTN Z1-Z2). These proteins are known to localise in both the sarcomere Z-discs and the transitional junctions, found close to the intercalated discs that connect adjacent cardiomyocytes. We use cryosections of left ventricles from two patients diagnosed with end-stage Dilated Cardiomyopathy who underwent Orthotopic Heart Transplantation and were whole genome sequenced. We describe how Affimers substantially improve the resolution achieved by confocal and STED microscopy compared to conventional antibodies. We quantified the expression of ACTN2, ZASP and TTN proteins in two patients with dilated cardiomyopathy and compared them with a sex- and age-matched healthy donor. The small size of the Affimer reagents, combined with a small linkage error (the distance from the epitope to the dye label covalently bound to the Affimer) revealed new structural details in Z-discs and intercalated discs in the failing samples. Affimers are thus useful for analysis of changes to cardiomyocyte structure and organisation in diseased hearts.

Validation of a 3D perfused cell culture platform as a tool for humanised preclinical drug testing in breast cancer using established cell lines and patient-derived tissues.

3D cell culture models of cancer are currently being developed to recapitulate in vivo physiological conditions and to assess therapeutic responses. However, most models failed to incorporate the biochemical and biophysical stimuli from fluid flow. In this study, a three-dimensional scaffold, SeedEZ was applied within the PerfusionPal perfused culture system to investigate how perfusion, and blood-like oxygen delivery influenced breast cancer cell growth and their responses to a commonly used breast cancer drug tamoxifen. Our results showed that breast cancer cells could be maintained over 3 weeks in PerfusionPal with increased cell viability compared to static 3D culture in fully humanised conditions. This platform also supported examining the effect of tamoxifen on breast cancer cell lines and in primary patient-derived breast cancer samples. Future work is warranted to further the adaption for fully humanised assessment of drug effectiveness in a patient personalized approach with the aim to reduce the burden of animal use in cancer research and increase the degree of human pre-clinical data translation to clinic.

VelcroVax: a "Bolt-On" Vaccine Platform for Glycoprotein Display.

Having varied approaches to the design and manufacture of vaccines is critical in being able to respond to worldwide needs and newly emerging pathogens. Virus-like particles (VLPs) form the basis of two of the most successful licensed vaccines (against hepatitis B virus [HBV] and human papillomavirus). They are produced by recombinant expression of viral structural proteins, which assemble into immunogenic nanoparticles. VLPs can be modified to present unrelated antigens, and here we describe a universal "bolt-on" platform (termed VelcroVax) where the capturing VLP and the target antigen are produced separately. We utilize a modified HBV core (HBcAg) VLP with surface expression of a high-affinity binding sequence (Affimer) directed against a SUMO tag and use this to capture SUMO-tagged gp1 glycoprotein from the arenavirus Junín virus (JUNV). Using this model system, we have solved the first high-resolution structures of VelcroVax VLPs and shown that the VelcroVax-JUNV gp1 complex induces superior humoral immune responses compared to the noncomplexed viral protein. We propose that this system could be modified to present a range of antigens and therefore form the foundation of future rapid-response vaccination strategies. IMPORTANCE The hepatitis B core protein (HBc) forms noninfectious virus-like particles, which can be modified to present a capturing molecule, allowing suitably tagged antigens to be bound on their surface. This system can be adapted and provides the foundation for a universal "bolt-on" vaccine platform (termed VelcroVax) that can be easily and rapidly modified to generate nanoparticle vaccine candidates.

Fibrin-glycoprotein VI interaction increases platelet procoagulant activity and impacts clot structure.

BACKGROUND: The glycoprotein VI (GPVI) signaling pathway was previously reported to direct procoagulant platelet activity through collagen binding. However, the impact of GPVI-fibrin interaction on procoagulant platelet development and how it modulates the clot structure are unknown. OBJECTIVES: To determine the effect of GPVI-fibrin interaction on the platelet phenotype and its impact on the clot structure. METHODS: Procoagulant platelets in platelet-rich plasma clots were determined by scanning electron microscopy (wild-type and GPVI-deficient murine samples) and confocal microscopy. Procoagulant platelet number, clot density, clot porosity, and clot retraction were determined in platelet-rich plasma or whole blood clots of healthy volunteers in the presence of tyrosine kinase inhibitors (PRT-060318, ibrutinib, and dasatinib) and eptifibatide. RESULTS: GPVI-deficient clots showed a higher nonprocoagulant vs procoagulant platelet ratio than wild-type clots. The fiber density and the procoagulant platelet number decreased in the presence of Affimer proteins, inhibiting GPVI-fibrin(ogen) interaction and the tyrosine kinase inhibitors. The effect of GPVI signaling inhibitors on the procoagulant platelet number was exacerbated by eptifibatide. The tyrosine kinase inhibitors led to an increase in clot porosity; however, no differences were observed in the final clot weight, following clot retraction with the tyrosine kinase inhibitors, except for ibrutinib. In the presence of eptifibatide, clot retraction was impaired. CONCLUSION: Our findings showed that GPVI-fibrin interaction significantly contributes to the development of procoagulant platelets and that inhibition of GPVI signaling increases clot porosity. Clot contractibility was impaired by the integrin αIIbß3 and Btk pathway inhibition. Thus, inhibition of GPVI-fibrin interactions can alleviate structural characteristics that contribute to a prothrombotic clot phenotype, having potential important implications for novel antithrombotic interventions.

Affimers and nanobodies as molecular probes and their applications in imaging.

Antibodies are the most widely used, traditional tool for labelling molecules in cells. In the past five to ten years, many new labelling tools have been developed with significant advantages over the traditional antibody. Here, we focus on nanobodies and the non-antibody binding scaffold proteins called Affimers. We explain how they are generated, selected and produced, and we describe how their small size, high binding affinity and specificity provides them with many advantages compared to antibodies. Of particular importance, their small size enables them to better penetrate dense cytoskeletal regions within cells, as well as tissues, providing them with specific advantage for super-resolution imaging, as they place the fluorophore with a few nanometres of the target protein being imaged. We expect these novel tools to be of broad interest to many cell biologists and anticipate them becoming the tools of choice for super-resolution imaging.

Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line.

Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent-an Affimer protein-that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.

Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay.

C. difficile infection (CDI) is a leading healthcare-associated infection with a high morbidity and mortality and is a financial burden. No current standalone point-of-care test (POCT) is sufficient for the identification of true CDI over a disease-free carriage of C. difficile, so one is urgently required to ensure timely, appropriate treatment. Here, two types of binding proteins, Affimers and nanobodies, targeting two C. difficile biomarkers, glutamate dehydrogenase (GDH) and toxin B (TcdB), are combined in NanoBiT (NanoLuc Binary Technology) split-luciferase assays. The assays were optimized and their performance controlling parameters were examined. The 44 fM limit of detection (LoD), 4-5 log range and 1300-fold signal gain of the TcdB assay in buffer is the best observed for a NanoBiT assay to date. In the stool sample matrix, the GDH and TcdB assay sensitivity (LoD = 4.5 and 2 pM, respectively) and time to result (32 min) are similar to a current, commercial lateral flow POCT, but the NanoBit assay has no wash steps, detects clinically relevant TcdB over TcdA, and is quantitative. Development of the assay into a POCT may drive sensitivity further and offer an urgently needed ultrasensitive TcdB test for the rapid diagnosis of true CDI. The NanoBiTBiP (NanoBiT with Binding Proteins) system offers advantages over NanoBiT assays with antibodies as binding elements in terms of ease of production and assay performance. We expect this methodology and approach to be generally applicable to other biomarkers.

Purification and Analysis of Circulating Lipid Particles.

Lipid particles found in circulating extracellular fluids such as blood or lymph are essential for cellular homeostasis, metabolism and survival. Such particles provide essential lipids and fats which enable cells to synthesize new membranes and regulate different biochemical pathways. Imbalance in lipid particle metabolism can cause pathological states such as atherosclerosis. Here, elevated low-density lipoprotein (LDL) accumulation leads to fat-filled lesions or plaques in arterial walls. In this chapter, we provide a detailed set of protocols for the rapid and safe purification of lipid particles from human blood using high-speed ultracentrifugation. We provide a detailed set of assays for further analysis of the biochemical and cellular properties of these lipid particles. By combining these assays, we can better understand the complex roles of different lipid particles in normal physiology and disease pathology.

Affinity purification of fibrinogen using an Affimer column.

BACKGROUND: Fibrinogen is an abundant plasma protein with an essential role in blood coagulation and haemostasis thus receiving significant research interest. However, protein purification is time consuming and commercial preparations often have protein contaminants. The aim of this study was to develop a new method to purify high quality and functional fibrinogen. METHODS: Fibrinogen-specific Affimer protein, isolated using phage display systems, was immobilised to SulfoLink resin column and employed for fibrinogen purification from plasma samples. Fibrinogen was eluted using a high pH solution. Commercial human fibrinogen was also further purified using the Affimer column. Fibrinogen purity was determined by SDS-PAGE and mass spectrometry, while functionality was assessed using turbidimetric analysis. RESULTS: Affimer-purified fibrinogen from human plasma showed purity at least comparable to commercially available preparations and was able to form physiological fibrin networks. Further purification of commercially available fibrinogen using the Affimercolumn eliminated multiple contaminant proteins, a significant number of which are key elements of the coagulation cascade, including plasminogen and factor XIII. CONCLUSIONS: The Affimercolumn represents a proof of concept novel, rapid method for isolating functional fibrinogen from plasma and for further purification of commercially available fibrinogen preparations. GENERAL SIGNIFICANCE: Our methodology provides an efficient way of purifying functional fibrinogen with superior purity without the need of expensive pieces of equipment or the use of harsh conditions.

Protein-conjugated microbubbles for the selective targeting of S. aureus biofilms.

Staphylococcus aureus (S. aureus) is an important human pathogen and a common cause of bloodstream infection. The ability of S. aureus to form biofilms, particularly on medical devices, makes treatment difficult, as does its tendency to spread within the body and cause secondary foci of infection. Prolonged courses of intravenous antimicrobial treatment are usually required for serious S. aureus infections. This work investigates the in vitro attachment of microbubbles to S. aureus biofilms via a novel Affimer protein, AClfA1, which targets the clumping factor A (ClfA) virulence factor - a cell-wall anchored protein associated with surface attachment. Microbubbles (MBs) are micron-sized gas-filled bubbles encapsulated by a lipid, polymer, or protein monolayer or other surfactant-based material. Affimers are small (∼12 kDa) heat-stable binding proteins developed as replacements for antibodies. The binding kinetics of AClfA1 against S. aureus ClfA showed strong binding affinity (KD = 62 ± 3 nM). AClfA1 was then shown to bind S. aureus biofilms under flow conditions both as a free ligand and when bound to microparticles (polymer beads or microbubbles). Microbubbles functionalized with AClfA1 demonstrated an 8-fold increase in binding compared to microbubbles functionalized with an identical Affimer scaffold but lacking the recognition groups. Bound MBs were able to withstand flow rates of 250 µL/min. Finally, ultrasound was applied to burst the biofilm bound MBs to determine whether this would lead to biofilm biomass loss or cell death. Application of a 2.25 MHz ultrasound profile (with a peak negative pressure of 0.8 MPa and consisting of a 22-cycle sine wave, at a pulse repetition rate of 10 kHz) for 2 s to a biofilm decorated with targeted MBs, led to a 25% increase in biomass loss and a concomitant 8% increase in dead cell count. The results of this work show that Affimers can be developed to target S. aureus biofilms and that such Affimers can be attached to contrast agents such as microbubbles or polymer beads and offer potential, with some optimization, for drug-free biofilm treatment.

Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models.

Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5-7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.

Piggybacking on the Cholera Toxin: Identification of a CTB-Binding Protein as an Approach for Targeted Delivery of Proteins to Motor Neurons.

A significant unmet need exists for the delivery of biologic drugs such as polypeptides or nucleic acids to the central nervous system for the treatment and understanding of neurodegenerative diseases. Naturally occurring bacterial toxins have been considered as tools to meet this need. However, due to the complexity of tethering macromolecular drugs to toxins and the inherent dangers of working with large quantities of recombinant toxins, no such route has been successfully exploited. Developing a method where a bacterial toxin's nontoxic targeting subunit can be assembled with a drug immediately prior to in vivo administration has the potential to circumvent some of these issues. Using a phage-display screen, we identified two antibody mimetics, anticholera toxin Affimer (ACTA)-A2 and ACTA-C6 that noncovalently associate with the nonbinding face of the cholera toxin B-subunit. In a first step toward the development of a nonviral motor neuron drug-delivery vehicle, we show that Affimers can be selectively delivered to motor neurons in vivo.

RAS-inhibiting biologics identify and probe druggable pockets including an SII-α3 allosteric site.

RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. Here, we identify two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signaling pathways with distinct isoform and mutant profiles. Affimer K6 binds in the SI/SII pocket, whilst Affimer K3 is a non-covalent inhibitor of the SII region that reveals a conformer of wild-type RAS with a large, druggable SII/α3 pocket. Competitive NanoBRET between the RAS-binding Affimers and known RAS binding small-molecules demonstrates the potential to use Affimers as tools to identify pharmacophores. This work highlights the potential of using biologics with small interface surfaces to select unseen, druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.

Reagentless Affimer- and antibody-based impedimetric biosensors for CEA-detection using a novel non-conducting polymer.

Polyoctopamine (POct), an amine-functionalised non-conducting polymer, as the transducer layer in an electrochemical biosensor, is presented. This polymer offers versatile covalent coupling either through thiol linker conjugation, carboxyl or aldehyde functional groups without the requirement of pre- or post-surface activation. The colorectal cancer biomarker carcinoembryonic antigen (CEA) was selected as the target analyte, whilst an antibody and a synthetic binding protein, an Affimer, were used as distinct bioreceptors to demonstrate the versatility of polyoctopamine as a transducer polymer layer for oriented immobilisation of the bioreceptors. The electrodeposited polymer layer was characterised using cyclic voltammetry, electrochemical impedance spectroscopy, and on-sensor chemiluminescent blotting. The performance of optimised POct-based biosensors were tested in spiked human serum. Results showed that the electropolymerisation of octopamine on screen printed gold electrode generates a thin polymer film with low resistance. Close proximity of the immobilised bioreceptors to the transducer layer greatly enhanced the sensitivity detection. The sensitivity of the smaller monomeric bioreceptor (Affimer, 12.6 kDa) to detect CEA was comparable to the dimeric antibody (150 kDa) with limit of detection at 11.76 fM which is significantly lower than the basal clinical levels of 25 pM. However, the Affimer-based sensor had a narrower dynamic range compared to the immunosensor (1-100 fM vs. 1 fM - 100 nM, respectively). All electrochemical measurements were done in less than 5 min with small sample volumes (10 µl). Hence, polyoctopamine features a simple fabrication of impedimetric biosensors using amine-functionalisation technique, provides rapid response time with enhanced sensitivity and label-free detection.