Quantitative epitope analysis reveals drastic 63% reduced immuno-affinity and 60% enhanced transmissibility for SARS-CoV-2 variants.

SARS-CoV-2 is the cause of a global pandemic that has led to more than 4 million deaths, continues to spread and holds the world in a tight grip. The virus has developed substantial mutations that undermine the efficacy of current vaccines and monoclonal antibody therapies. Semi-quantitative immuno - and neutralization assays are unable to provide direct quantitative insights about the minute variations of emerging mutants. Here, we develop a quantitative assay that enables synchronous screening of emerging variant epitopes with single amino acid resolution. We report on specific label-free quantitative nanomechanical analysis of pseudovirus spike interaction with ACE2 receptors. Within minutes, we can characterize the B.1.1.7 variant transmissibility due to its 63% increased binding, and measure a 60% reduced efficacy of antibodies towards B.1.351 and P.1 variants. Our technology can assist vaccine development studies, with focus on comparing protection patterns and novel vaccine candidates and tracking of immunity over time.

Multimodal real-time frequency tracking of cantilever arrays in liquid environment for biodetection: Comprehensive setup and performance analysis.

We present a nanomechanical platform for real-time quantitative label-free detection of target biomolecules in a liquid environment with mass sensitivity down to few pg. Newly fabricated arrays of up to 18 cantilevers are integrated in a micromachined fluidic chamber, connected to software-controlled fluidic pumps for automated sample injections. We discuss two functionalization approaches to independently sensitize the interface of different cantilevers. A custom piezo-stack actuator and optical readout system enable the measurement of resonance frequencies up to 2 MHz. We implement a new measurement strategy based on a phase-locked loop (PLL), built via in-house developed software. The PLL allows us to track, within the same experiment, the evolution of resonance frequency over time of up to four modes for all the cantilevers in the array. With respect to the previous measurement technique, based on standard frequency sweep, the PLL enhances the estimated detection limit of the device by a factor of 7 (down to 2 pg in 5 min integration time) and the time resolution by more than threefold (below 15 s), being on par with commercial gold-standard techniques. The detection limit and noise of the new setup are investigated via Allan deviation and standard deviation analysis, considering different resonance modes and interface chemistries. As a proof-of-concept, we show the immobilization and label-free in situ detection of live bacterial cells (E. coli), demonstrating qualitative and quantitative agreement in the mechanical response of three different resonance modes.

Nanotechnological immunoassay for rapid label-free analysis of candidate malaria vaccines.

Malaria is a life-threatening epidemic disease with half of the world's population at risk. Although its incidence rate has fallen since 2010, this ratio dramatically stalled between 2014 and 2018. New fast and optimized tools in vaccine analysis and seroconversion testing are critically needed. We developed a clinical diagnostic device based on piezo-actuated nanoresonators that perform as quantitative in situ calibrated nano-bio sensors for specific detection of multiple target molecules in serum samples. The immunoassay successfully diagnoses humoral immune responses induced by malaria vaccine candidates and reveals the timeline and stage of the infection. We applied the newly developed strategy to a variety of different samples, from pure antibody/vaccine solutions, to blood samples from clinical trials on both naïve and pre-exposed malaria volunteers from sub-Saharan countries. Our nanomechanical assay provides a direct one-step label-free quantitative immunoassay that is on par with the gold-standard, multi-step enzyme-linked immunosorbent assay (ELISA). We achieve a limit of detection of few pg ml-1, or sub-pM concentrations. The 6 µl sample volume allows more than 50 experiments from one finger prick. Furthermore, we simultaneously detected multiple analytes by differential functionalization of multiple sensors in parallel. The inherent differential read-out with in situ controls reduces false positive results. Due to the faster turnaround time, the minimal volume required and the automatized handling system, this technique has great potential for miniaturization and routine diagnostics in pandemic emergencies.

Towards personalised rapid label free miRNA detection for cancer and liver injury diagnostics in cell lysates and blood based samples.

Advances in prevention, diagnosis and therapy are coupled to innovation and development of new medical tools, leading to improved patient prognosis. We developed an automatic biosensor platform that could provide a non-invasive, rapid and personalised diagnosis using nanomechanical cantilever sensors. miRNA are involved in gene expression and are extractable biomarkers for multiple diseases. We detected specific expression patterns of miRNA relevant to cancer and adverse drug effects directly in cell lysates or blood based samples using only a few microliters of sample within one hour. Specific miRNA hybridisation to the upper cantilever surface induces physical bending of the sensor which is detected by monitoring the position of a laser that reflects from the sensors surface. Internal reference sensors negate environmental and nonspecific effects. We showed that the sensitivity of label free cantilever nanomechanical sensing of miRNA surpasses that of surface plasmon resonance by more than three orders of magnitude. A cancer associated miRNA expression profile from cell lysates and one associated with hepatocytes derived from necrotic liver tissue in blood-based samples has been successfully detected. Our label free mechanical approach displays the capability to perform in relevant clinical samples while also obtaining comparable results to PCR based techniques. Without the need to individually extend, amplify or label each target allowing multitarget analysis from one sample.