Tackling reproducibility in microcantilever biosensors: a statistical approach for sensitive and specific end-point detection of immunoreactions

Abstract

aIn the biomedical field, end-point detection bioassays such as enzyme-linked immunosorbent assays (ELISAs) are essential tools because of their simplicity, high-throughput, and suitability for their use at the point-of-care. End-point bioassays are significantly constrained by the need of sample labeling with fluorescent or colorimetric tags for subsequent detection. A promising strategy to overcome these limitations is to harness recent advances in label-free biological nanosensors. Here we analyse the potential of nanomechanical biosensors based on surface stress for the label-free end-point detection of horseradish peroxidase. We address the variability of the sensor responsethrough the analysis of 1012 cantilevers with different antibody surface densities, two blocking strategies based on polyethylene-glycol (PEG) and bovine serum albumin (BSA) and stringent controls. The study reveals that the performance of the assay critically depends on both antibody surface density and blocking strategies. We find that the optimal conditions involve antibody surface densities near but below saturation and blocking with PEG. We find that the surface stress induced by the antibody-antigen binding is significantly correlated to the surface stress generated during the antibody attachment and blocking steps. The statistical correlation is harnessed to identify immobilization failure or success, and thus enhancing the specificity and sensitivity of the assay. This procedure enables achieving a rate of true positives and true negatives of 90% and 91% respectively. The detection limit is of 10 ng/mL (250 pM) that is similar to detection limit obtained in our ELISA

Detection of cancer biomarkers in serum using a hybrid mechanical and optoplasmonic nanosensor

“…antibodies and detection antibodies, respectively, using a procedure that ensures optimal recognition efficiency and ultralow fouling capability 10 . The materials and methods used in the functionalization and sandwich assays are detailed in Supplementary Section 1.…”

“…Nanotechnology provides biosensors with unprecedented levels of sensitivity that can be used to monitor biomarkers predicted by proteomics. However, such nanosensors have significant issues regarding specificity and reproducibility, and are thus not yet ready for biomarker screening in blood [8][9][10] . These problems occur, in part, because of the extreme difficulty in detecting low-abundance protein biomarkers among plasma proteins, which can have concentrations that are around seven orders of magnitude higher (albumins, for example, have concentrations of ∼40 mg ml −1 ).…”

Hydration Induced Stress on DNA Monolayers Grafted on Microcantilevers

“…However, their application to clinical diagnosis will also demand for a method capable of providing hundreds of measurements in a short time, as well as manageable preparation steps for the microcantilever sensors that can still provide reproducible mechanical patterns. 9,10 All the above reasons highlight the need to pursue in depth understanding of the hybridization process and of the conformations of the DNA layers.…”

“…15,16 Recent theoretical and experimental work has highlighted the fact that even slightly different experimental conditions, such as ionic strength, temperature or pH variations during immobilization, may lead to largely divergent mechanical effects. 17,18 The final strand density in self-assembled monolayers plays a critical role in nanomechanical sensing 9 and it is indeed far from being fully controllable. 19,[20][21][22][23] One singularly limiting difficulty has been to accomplish the anchoring of the ssDNA probes with a well-defined uniform density and a standing up conformation.…”

Hybrid Integrated Label-Free Chemical and Biological Sensors

“…PEG is a protein-resistant and anti-fouling polymer with low toxicity and high biocompatibility [58]. Studies on cantilevers with both BSA and PEG layers revealed that PEG is more successful in depression of nonspecific binding [83,237]. Yen et al [238] recently demonstrated the application of ethanolamine as the blocking agent in the detection of C-reactive protein (CRP).…”