Inexpensive High-Throughput Multiplexed Biomarker Detection Using Enzymatic Metallization with Cellphone-Based Computer Vision.

Multiplexed biomarker detection can play a critical role in reliable and comprehensive disease diagnosis and prediction of outcome. Enzyme-linked immunosorbent assay (ELISA) is the gold standard method for immunobinding-based biomarker detection. However, this is currently expensive, limited to centralized laboratories, and usually limited to the detection of a single biomarker at a time. We present a low-cost, smartphone-based portable biosensing platform for high-throughput, multiplexed, sensitive, and quantitative detection of biomarkers from single, low-volume drops (<1 µL) of clinical samples. Biomarker binding to spotted capture antigens is converted, via enzymatic metallization, to the localized surface deposition of amplified, dry-stable, silver metal spots whose darkness is proportional to biomarker concentration. A custom smartphone application is developed, which uses real-time computer vision to enable easy optical detection of the deposited metal spots and sensitive and reproducible quantification of the biomarkers. We demonstrate the use of this platform for high-throughput, multiplexed detection of multiple viral antigen-specific antibodies from convalescent COVID-19 patient serum as well as vaccine-elicited antibody responses from uninfected vaccine-recipient serum and show that distinct multiplexed antibody fingerprints are observed among them.

Evidence that small proteins translocate through silicon nitride pores in a folded conformation.

The interaction of three proteins (histidine-containing phosphocarrier protein, HPr, calmodulin, CaM, and maltose binding protein, MBP) with synthetic silicon nitride (SiN(x)) membranes has been studied. The proteins which have a net negative charge were electrophoretically driven into pores of 7 and 5 nm diameter with a nominal length of 15 nm. The % blockade current and event duration were measured at three different voltages. For a translocation event it was expected that the % block would be constant with voltage whilst the event duration would decrease with increasing voltage. On the basis of these criteria, we deduce that MBP whose largest dimension is 6.5 nm does not translocate whereas up to 40% of CaM molecules can translocate the 7 nm pore as can a majority of HPr molecules, with some translocations being observed for the 5 nm pore. For translocation events the magnitude of the % blockade current is consistent with a folded conformation of the proteins surrounded by a hydration shell of 0.5-1.0 nm.

Single-molecule bonds characterized by solid-state nanopore force spectroscopy.

Weak molecular interactions drive processes at the core of living systems, such as enzyme-substrate interactions, receptor-ligand binding, and nucleic acid replication. Single-molecule force spectroscopy is a remarkable tool for revealing molecular scale energy landscapes of noncovalent bonds, by exerting a mechanical force directly on an individual molecular complex and tracking its survival as a function of time and applied force. In principle, force spectroscopy methods can also be used for highly specific molecular recognition assays, by directly characterizing the strength of bonds between probe and target molecules. However, complexity and low throughput of conventional force spectroscopy techniques render such biosensing applications impractical. Here we demonstrate a straightforward single-molecule approach, suitable for both biophysical studies and molecular recognition assays, in which a approximately 3 nm silicon nitride nanopore is used to determine the bond lifetime spectrum of the biotin-neutravidin complex. Thousands of individual molecular complexes are captured and dissociated in the solid-state nanopore under constant applied forces, ranging from 400 to 900 mV, allowing us to extract the location of the energy barrier that governs the interaction, mapped at Deltax approximately 0.5 nm. These results highlight the capacity of a solid-state nanopore to detect and characterize intermolecular interactions and demonstrate how this could be applied to rapid, highly specific molecular detection assays.