Enhanced Biosensor Platforms for Detecting the Atherosclerotic Biomarker VCAM1 Based on Bioconjugation with Uniformly Oriented VCAM1-Targeting Nanobodies

Abstract

Surface bioconjugation of biomolecules has gained enormous attention for developing advanced biomaterials including biosensors. While conventional immobilization (by physisorption or covalent couplings using the functional groups of the endogenous amino acids) usually results in surfaces with low activity, reproducibility and reusability, the application of methods that allow for a covalent and uniformly oriented coupling can circumvent these limitations. In this study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1), an atherosclerotic biomarker, is engineered with a C-terminal alkyne function via Expressed Protein Ligation (EPL). Conjugation of this nanobody to azidified silicon wafers and Biacore™ C1 sensor chips is achieved via Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) “click” chemistry to detect VCAM1 binding via ellipsometry and surface plasmon resonance (SPR), respectively. The resulting surfaces, covered with uniformly oriented nanobodies, clearly show an increased antigen binding affinity, sensitivity, detection limit, quantitation limit and reusability as compared to surfaces prepared by random conjugation. These findings demonstrate the added value of a combined EPL and CuAAC approach as it results in strong control over the surface orientation of the nanobodies and an improved detecting power of their targets—a must for the development of advanced miniaturized, multi-biomarker biosensor platforms.

Highly sensitive electrochemical biosensor based on redox - active monolayer for detection of anti-hemagglutinin antibodies against swine-origin influenza virus H1N1 in sera of vaccinated mice

“…The application of His-tag chemistry for immobilization bioanalytical active molecules on the surface of redox active monolayers deposited on the gold electrodes has solicited vivid interest among scientists. This method allows to keep the stable and oriented immobilization of sensing element onto solid support with maintaining their biological activity [ 36 , 37 ]. In this type electrochemical biosensors, the procedure for immobilization of the specific recognition elements involved the interaction between transition metal centers forming complexes on the electrode surface and nitrogen atoms from the histidine–tag present.…”

Early changes in the urine proteome in a rat liver tumor model

“…The details of the differential proteins are shown in Table S2. Four proteins (B2MG, VCAM1, HA11, LG3BP) were altered at all four time-points, and the trend was consistent at each time-point ( [16] atherosclerosis [17] P01891 [22] nasopharyngeal carcinoma [23] Q9H008 [24] cervical cancer [25] Q13228 Selenium-binding protein 1(SBP1) ↓ -0.15 0.15 0.05 0.009 tissue(biomaker) [26] prostate cancer [27] P40925 [28] non-small cell lung cancer [29] O75874 [33] bladder [34] ，lung [35] P20472 [28] P26038 Moesin(MOES) ↓ -0.13 0.45 -0.00027 breast cancer [36] Q5T2W1 Na(+)/H(+) exchange regulatory cofactor NHE-…”

Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

“…As we have seen, the great stability of Nbs adds to a better performance and extend the shelf-life of biosensors ( 54 ), but their small size (4 nm × 2.5 nm × 3 nm) also contributes to obtaining higher coating density on the sensor surface, which, together with their oriented immobilization has been shown to improve the biosensors performance ( 80 ). Controlled immobilization of Nbs onto biosensor surfaces have been accomplished by C-terminal alkyne function via Expressed Protein Ligation ( 81 ) (see below) and in vivo biotinylated Nbs bound to streptavidin ( 50 ). Also, the compact size of Nbs enabled their direct use as templates to synthesis highly derivatized gold nanoparticles that were incorporated into microelectrodes and used to detect the analyte by changes on the tunneling current of the gold-antibody networks.…”

“…The fusion protein was then immobilized onto chitin-agarose and self-cleavage was induced by incubation with cysteine-alkyne, releasing the Nb modified with a clickable alkyne function ( 106 ). Using this procedure, biosensors for detecting this atherosclerosis biomarker were generated using a click chemistry reaction that enable the oriented immobilization of the modified Nb onto azide-derivatized silicon baffles or surface plasmon resonance chips ( 81 ).…”