Fig 1.
Schematic representation of the p24 sandwich immunoassay.
(a) The top surface of the cantilever is functionalized with capture antibodies against HIV-1 p24 antigen (top schematic). Antifouling molecules are immobilized on the bottom surface of the cantilever and voids between the antibodies to minimize nonspecific interactions. The cantilever is then immersed in the human serum sample to allow specific binding of p24 to the cantilever surface (middle schematic). Finally, the p24 antigen captured on the cantilever is specifically linked to 100-nm-diameter gold nanoparticles that carry detection antibodies. (b) Scanning electron microscopy image of the silicon microcantilever arrays used in this work. (c). Schematics of the 96-well microtiter plate format, in which the immunoassays were carried out.
Fig 2.
Nanomechanical and optoplasmonic transduction of the immunoreaction product.
(a) Schematics of the optical beam deflection method for measuring the microcantilever vibration. A laser beam is focused onto the cantilever free end region. The deflection of the reflected beam due to the cantilever vibration is measured by a position-sensitive photodetector. The microcantilever is driven by a piezoelectric actuator beneath the microcantilever array chip. (b) Resonance frequency peaks (bottom graphs) of the first three vibration modes of a silicon cantilever before (dotted lines) and after (solid lines) the immunoreaction assay for a blank human serum sample (control) and for 5 10−4 pg/mL of p24 in human serum. Top panels represent the vibration mode shapes. The relative variations of the resonance frequencies are shown beside the resonance frequency peaks. (c) Schematics of the multiple pathways for light scattering by the gold nanoparticles bound to the microcantilever. (d) Darkfield optical images of the microcantilevers analyzed in (b) after the immunoassays. The images show the microcantilever and chip preclamping regions. At the right zoomed images of regions at the preclamping and the microcantilever are also shown.
Fig 3.
Nanomechanical and optoplasmonic detection of p24 in human serum.
Top graph: added mass inferred from the changes in the frequency response of the first three vibration modes of the microcantilevers. Bottom graph: scattering signal obtained from processing the darkfield images of the microcantilever after the immunoassay. The small symbols represent results of single microcantilevers. Big symbols and error bars represent the mean and error of all results at each concentration. Dashed line represents the limit of blank as defined in the text.
Fig 4.
(a) Probability of the nanomechanical and optoplasmonic signals being above the Limit of Blank as a function of p24 antigen concentration in human serum. The grey dashed line represents the 95% probability that determines the Limit of Detection. (b) Estimation of viral load in blood during the first five weeks after HIV infection [1,3]. The limits of detection and the periods of detection of the fourth-generation immunoassays (4th G EIA), nucleic acid amplification testing methods (NAAT) and the presented optomechanoplasmonic method (OMP) are indicated.