Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into easy-to-use point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a "mix-and-measure" homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout that can be detected using a basic digital camera. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. We also introduce the use of a calibrator luciferase that provides a robust ratiometric signal, allowing direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. RAPPID combines ratiometric bioluminescent detection with antibody-based target recognition into an easy-to-implement standardized workflow, and therefore represents an attractive, fast, and low-cost alternative to traditional immunoassays, both in an academic setting and in clinical laboratories for point-of-care applications.

while caveolae-mediated endocytosis is mainly responsible for the transport of vesicular cargos. [2] In many viruses, the genetic material is recognized and packaged during the protein capsid formation, [3] and others may uptake noncognate cargo after the formation of capsids. [4] In the latter case, openings in the protein shell are required for the cargo trafficking, and selected substrates can be targeted through regulating the pore properties. [5] Alternative to the pore engineering, the interior surface of capsids can also be engineered to control the substrate influx via electrostatic or hydrophobic interactions. [6] Continuous efforts have been devoted to elucidating the naturally occurring internalization in an attempt to understand this fundamental biological process and eventually achieve biomedical goals. [7] Artificial protocells prepared from polymers (polymersomes), [8] inorganic nanoparticles (colloidosomes), [9] and proteinpolymer conjugates (proteinosomes) [10] have recently emerged as a robust study model owing to their semipermeable membrane structures. Incidentally, these artificial protocells exhibit cell-mimicking behavior, i.e., spatial positioning, [11] extracellular signal responsiveness [12] and predatory behavior. [9a] In spite of their potential, the biomedical applications of such protocells have been scarcely exploited. [13] Here, we investigated the potential of artificial proteinosomes for heparin scavenging. [10] Heparin is a widely used anticoagulant agent in many clinical applications. [14] However, Heparin is a commonly applied blood anticoagulant agent in clinical use. After treatment, excess heparin needs to be removed to circumvent side effects and recover the blood-clotting cascade. Most existing heparin antidotes rely on direct heparin binding and complexation, yet selective compartmentalization and sequestration of heparin would be beneficial for safety and efficiency. However, such systems have remained elusive. Herein, a semipermeable protein-based microcompartment (proteinosome) is loaded with a highly positively charged chitosan derivative, which can induce electrostaticsdriven internalization of anionic guest molecules inside the compartment. Chitosan-loaded proteinosomes are subsequently employed to capture heparin, and an excellent heparin-scavenging performance is demonstrated under physiologically relevant conditions. Both the highly positive scavenger and the polyelectrolyte complex are confined and shielded by the protein compartment in a time-dependent manner. Moreover, selective heparinscavenging behavior over serum albumin is realized through adjusting the localized scavenger or surrounding salt concentrations at application-relevant circumstances. In vitro studies reveal that the cytotoxicity of the cationic scavenger and the produced polyelectrolyte complex is reduced by protocell shielding. Therefore, the proteinosome-based systems may present a novel polyelectrolyte-scavenging method for biomedical applications.

DNA has emerged as an attractive medium for archival data storage due to its durability and high information density. Scalable parallel random access to information is a desirable property of any storage system. For DNA-based storage systems, however, this still needs to be robustly established. Here we report on a thermoconfined polymerase chain reaction, which enables multiplexed, repeated random access to compartmentalized DNA files. The strategy is based on localizing biotin-functionalized oligonucleotides inside thermoresponsive, semipermeable microcapsules. At low temperatures, microcapsules are permeable to enzymes, primers and amplified products, whereas at high temperatures, membrane collapse prevents molecular crosstalk during amplification. Our data show that the platform outperforms non-compartmentalized DNA storage compared with repeated random access and reduces amplification bias tenfold during multiplex polymerase chain reaction. Using fluorescent sorting, we also demonstrate sample pooling and data retrieval by microcapsule barcoding. Therefore, the thermoresponsive microcapsule technology offers a scalable, sequence-agnostic approach for repeated random access to archival DNA files.

The rational design and implementation of synthetic, orthogonal mammalian communication systems has the potential to unravel fundamental design principles of mammalian cell communication circuits and offer a framework for engineering of designer cell consortia with potential applications in cell therapeutics and artificial tissue engineering. We lay here the foundations for the engineering of an orthogonal, and scalable mammalian synthetic intercellular communication platform that exploits the programmability of synthetic receptors and selective affinity and tunability of diffusing coiled-coil (CC) peptide heterodimers. Leveraging the ability of CCs to exclusively bind to a selected cognate receptor, we demonstrate orthogonal receptor activation, as well as Boolean logic computations. Next, we reveal synthetic intercellular communication based on synthetic receptors and secreted multidomain CC ligands and demonstrate a minimal, three-cell population system that can perform distributed AND gate logic. Our work provides a modular and scalable framework for the engineering of complex cell consortia, with the potential to expand the aptitude of cell therapeutics and diagnostics.