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Of various molecular diagnostic assays, the real-time reverse transcription polymerase chain reaction is considered the gold standard for infection diagnosis, despite critical drawbacks that limit rapid detection and accessibility. To confront these issues, several nanoparticle-based molecular detection methods have been developed to a great extent, but still possess several challenges. In this study, a novel nucleic acid amplification method termed nanoparticle-based surface localized amplification (nSLAM) is paired with electrochemical detection (ECD) to develop a nucleic acid biosensor platform that overcomes these limitations. The system uses primer-functionalized Fe 3 O 4 -Au core-shell nanoparticles for nucleic acid amplification, which promotes the production of amplicons that accumulate on the nanoparticle surfaces, inducing significantly amplified currents during ECD that identify the presence of target genetic material. The platform, applying to the COVID-19 model, demonstrates an exceptional sensitivity of ∼1 copy/μL for 35 cycles of amplification, enabling the reduction of amplification cycles to 4 cycles (∼7 min runtime) using 1 fM complementary DNA. The nSLAM acts as an accelerator that actively promotes and participates in the nucleic acid amplification process through direct polymerization and binding of amplicons on the nanoparticle surfaces. This ultrasensitive fast-response system is a promising method for detecting emerging pathogens like the coronavirus and can be extended to detect a wider variety of biomolecules.

Programmable coassembly of multicomponent nanoparticles (NPs) into heterostructures has the capability to build upon nanostructured metamaterials with enhanced complexity and diversity. However, a general understanding of how to manipulate the sequence-defined heterostructures using straightforward concepts and quantitatively predict the coassembly process remains unreached. Drawing inspiration from the synthetic concepts of molecular block copolymers is extremely beneficial to achieve controllable coassembly of NPs and access mesoscale structuring mechanisms. We herein report a general paradigm of kinetic pathway guidance for the controllable coassembly of bivalent DNA-functionalized NPs into regular block-copolymer-like heterostructures via the stepwise polymerization strategy. By quantifying the coassembly kinetics and structural statistics, it is demonstrated that the coassembly of multicomponent NPs, through directing the specific pathways of prepolymer intermediates, follows the step-growth copolymerization mechanism. Meanwhile, a quantitative model is developed to predict the growth kinetics and outcomes of heterostructures, all controlled by the designed elements of the coassembly system. Furthermore, the stepwise polymerization strategy can be generalized to build upon a great variety of regular nanopolymers with complex architectures, such as multiblock terpolymers and ladder copolymers. Our theoretical and simulation results provide fundamental insights on quantitative predictions of the coassembly kinetics and coassembled outcomes, which can aid in realizing a diverse set of supramolecular DNA materials by the rational design of kinetic pathways.

Background: Determination of the fate of nanoparticles (NPs) in a biological system, or NP biodistribution, is critical in evaluating an NP formulation for nanomedicine. Current methods to determine NP biodistribution are greatly inadequate, due to their limited detection thresholds. Herein, proof of concept of a novel method for improved NP detection based on in situ polymerase chain reaction (ISPCR), coined "nanobarcoding," is demonstrated., Methods: Nanobarcoded superparamagnetic iron oxide nanoparticles (NB-SPIONs) were characterized by dynamic light scattering, zeta potential, and hyperspectral imaging measurements. Cellular uptake of Cy5-labeled NB-SPIONs (Cy5-NB-SPIONs) was imaged by confocal microscopy. The feasibility of the nanobarcoding method was first validated by solution-phase PCR and "pseudo"-ISPCR before implementation in the model in vitro system of HeLa human cervical adenocarcinoma cells, a cell line commonly used for ISPCR-mediated detection of human papilloma virus (HPV)., Results: Dynamic light-scattering measurements showed that NB conjugation stabilized SPION size in different dispersion media compared to that of its precursor, carboxylated SPIONs (COOH-SPIONs), while the zeta potential became more positive after NB conjugation. Hyperspectral imaging confirmed NB conjugation and showed that the NB completely covered the SPION surface. Solution-phase PCR and pseudo-ISPCR showed that the expected amplicons were exclusively generated from the NB-SPIONs in a dose-dependent manner. Although confocal microscopy revealed minimal cellular uptake of Cy5-NB-SPIONs at 50 nM over 24 hours in individual cells, ISPCR detected definitive NB-SPION signals inside HeLa cells over large sample areas., Conclusion: Proof of concept of the nanobarcoding method has been demonstrated in in vitro systems, but the technique needs further development before its widespread use as a standardized assay.