Abstract
The early detection of the human immunodeficiency virus (HIV) is of paramount importance to achieve efficient therapeutic treatment and limit the disease spreading. In this perspective, the assessment of biosensing assay for the HIV-1 p24 capsid protein plays a pivotal role in the timely and selective detection of HIV infections. In this study, multi-parameter-SPR has been used to develop a reliable and label-free detection method for HIV-1 p24 protein. Remarkably, both physical and chemical immobilization of mouse monoclonal antibodies against HIV-1 p24 on the SPR gold detecting surface have been characterized for the first time. The two immobilization techniques returned a capturing antibody surface coverage as high as (7.5 ± 0.3) × 1011 molecule/cm2 and (2.4 ± 0.6) × 1011 molecule/cm2, respectively. However, the covalent binding of the capturing antibodies through a mixed self-assembled monolayer (SAM) of alkanethiols led to a doubling of the p24 binding signal. Moreover, from the modeling of the dose-response curve, an equilibrium dissociation constant KD of 5.30 × 10−9 M was computed for the assay performed on the SAM modified surface compared to a much larger KD of 7.46 × 10−5 M extracted for the physisorbed antibodies. The chemically modified system was also characterized in terms of sensitivity and selectivity, reaching a limit of detection of (4.1 ± 0.5) nM and an unprecedented selectivity ratio of 0.02.
Highlights
One of the main features of a biosensing platform is combining a high sensitivity with selectivity in the binding interactions between immobilized biorecognition species and the target analyte [1,2]
The characterization of both physiosorbed and covalently immobilized anti-p24 capturing antibodies on a gold surface was assessed via surface plasmon resonance (SPR) characterization
The presence of the noble metal thin film (50 nm gold layer) causes partial loss of the reflected light by exciting the metal surface electrons. This produces an evanescent wave that propagates along the interface between the dielectric and the metal layer [24,25]
Summary
One of the main features of a biosensing platform is combining a high sensitivity with selectivity in the binding interactions between immobilized biorecognition species and the target analyte [1,2]. The immobilization of bioreceptors to a surface always results in the reduction or loss of mobility. By using antibody fragments or protein G mediated immobilization, a more efficient capture of the bio-recognition element has been observed, improving the sensitivity of immunosensing platforms [9,10]. Physical immobilization is suited to deposit biorecognition elements on various surfaces. It does not require any additional coupling reagents or chemical modification of the biomolecules, being cost-effective and faster than other immobilization techniques. HIV p24 antibodies (anti-p24) by physisorption and chemical deposition through self-assembled monolayers on a 0.42 cm wide gold detecting interface were characterized with surface plasmon resonance (SPR) for the first time. The detection efficacies toward human immunodeficiency virus (HIV-1) p24 capsid proteins were compared utilizing the SPR real-time monitoring of the bio-affinity reactions
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