Measurement of Protein Kinetics Using a Liquid Phase-Based Biosensing Platform.

Macromolecular association is crucial to many fields in biomedical sciences, including drug development, gene editing, and diagnostics. In particular, protein-protein association and dissociation rate constants are typically determined using surface plasmon resonance systems, which require costly instrumentation and cumbersome procedures (e.g., blocking, washing, and separation). Herein, we demonstrate that protein-binding constants can be readily determined using a real-time biosensing platform facilitated by graphene oxide-modified microwell plates and fluorophore-labeled proteins, where the fluorescent probes remain highly fluorescent during protein association, whereas fluorescent bioprobes that are not associated with their counterparts are quenched by graphene oxide. Binding data of three pairs of proteins were systematically determined employing this single-step platform and compared with those data reported by the suppliers or the literature, suggesting that this approach is comparable and consistent with the existing ones. Such pairs include (i) human immunoglobulin G (H-IgG)-fluorophore-labeled anti-H-IgG, (ii) prostate-specific antigen (PSA)-quantum dot-labeled anti-PSA, and (iii) anti-RBD-fluorophore-labeled SARS-CoV-2 spike receptor-binding domain recombinant protein. We also offer an open-source software that automatically determines the binding kinetics constants of proteins. This Technical Note introduces a simple, yet effective, platform to determine relevant information on protein kinetics, which can be performed using a microwell plate reader and economical materials like graphene oxide. We foresee a new generation of diagnostics based on our affordable protein kinetics analysis.

Real-Time Photoluminescent Biosensing Based on Graphene Oxide-Coated Microplates: A Rapid Pathogen Detection Platform.

Pathogenic bacterial contamination is a major threat to safety, human health, and ecosystems. Herein, we report an advantageous single-step, wash-free, and real-time bacterial detection platform operating with a single antibody. Escherichia coli was detected as a model analyte. This technology is based on graphene oxide-coated microplates (GOMs) and photoluminescent bioprobes (PLBs). On the one hand, using nonradiative energy transfer, GOMs are conceived to deactivate the photoluminescence of those PLBs that are not experimenting immunoreactions via antibody-bacterial membrane affinity. On the other hand, those PLBs experimenting immunoreactions preserve their photoluminescence because of both (i) the distance between the complex (PLBs-bacteria) and GOMs and (ii) the low affinity between the same complex and GOMs. With an optimal analytical performance of ∼30 min, the resulting bacterial detection platform was demonstrated to be fast and highly sensitive, exhibiting a limit of detection of ∼2 CFU mL-1. Industrial real samples were also successfully analyzed in a widely used format that is amenable to high-throughput applications. Moreover, the proposed technology is highly transformative, as graphene oxide is able to quench different fluorophores, and other analytes can be detected by simply changing the specific antibody.

Facile Determination of COVID-19 Seroconversion via Nonradiative Energy Transfer.

Serological tests are crucial in a pandemic scenario, since they are a valuable tool to spot those citizens with potential immunity, specific regions with herd immunity or particular at-risk populations, as well as acquired immunity after vaccination. Hence, high-throughput, fast, cost-effective, and straightforward technologies facilitating interrogation of COVID-19 seroconversion are an existing need. Herein, we developed an innovative assay for the determination of COVID-19 seroconversion. Fluorophore-labeled SARS-CoV-2 spike receptor-binding domain recombinant protein (F-RBD) was discovered to operate as a bioprobe that emits a strong fluorescence upon COVID-19 antibody detection; however, F-RBD fluorescence was deactivated by graphene oxide-decorated surfaces when COVID-19 antibodies are absent in the sample. With a cost of less than 0.5 USD per test (at laboratory scale), the biosensing system offers optimum results within 42 min. To demonstrate that this technology is technically sound in a relevant environment, 34 human serum samples were analyzed and clearly differentiated, requiring a tiny amount of serum (1 µL to be later diluted in saline buffer).

Microwell plates coated with graphene oxide enable advantageous real-time immunosensing platform.

Immunoassays are fundamental analytical tools in molecular diagnostics, therapy monitoring and drug discovery. Nevertheless, they often take around 6 h and require cumbersome procedures. We introduce a breakthrough in immunosensing based on the photoluminescence quenching capabilities of graphene oxide (GO) and the versatile format offered by the famous microwell plates. Taking advantage of the highly efficient non-radiative energy transfer occurring between photoexcited fluorophores (donors) and GO (acceptor), we discovered that flurophore-labelled antibodies (Fl-Abs) are quickly and strongly quenched by the studied GO-coated microwell, whereas Fl-Abs complexed with the respective analyte are weakly quenched by the same surface due to the low affinity between the GO-coated surface and the relatively long distance between these photoluminescent complexes and the GO-coated surface. In doing so, we developed a conceptually innovative single-step immunosensing platform, avoiding blocking, separation and washing steps and exploiting a single antibody. Importantly, the biosensing response can be interrogated in real time. This leads to an advantageous immunodetection phenomenon which is observable in few minutes (e.g. 5 min). The resulting highly transformative biosensing platform operates with different photoluminescent agents and different analytes. Besides, this biosensing platform was demonstrated to operate with real samples of human urine doped with different concentrations of prostate specific antigen.