Capillarity-Driven Enrichment and Hydrodynamic Trapping of Trace Nucleic Acids by Plasmonic Cavity Membrane for Rapid and Sensitive Detections.

Small-reactor-based polymerase chain reaction (PCR) has attracted considerable attention. A significant number of tiny reactors must be prepared in parallel to capture, amplify, and accurately quantify few target genes in clinically relevant large volume, which, however, requires sophisticated microfabrication and longer sample-to-answer time. Here, single plasmonic cavity membrane is reported that not only enriches and captures few nucleic acids by taking advantage of both capillarity and hydrodynamic trapping but also quickly amplifies them for sensitive plasmonic detection. The plasmonic cavity membrane with few nanoliters in a void volume is fabricated by self-assembling gold nanorods with SiO2 tips. Simulations reveal that hydrodynamic stagnation between the SiO2 tips is mainly responsible for the trapping of the nucleic acid in the membrane. Finally, it is shown that the plasmonic cavity membrane is capable of enriching severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genes up to 20 000-fold within 1 min, amplifying within 3 min, and detecting the trace genes as low as a single copy µL-1. It is anticipated that this work not only expands the utility of PCR but also provides an innovative way of the enrichment and detection of trace biomolecules in a variety of point-of-care testing applications.

Bubble-free diatoms polymerase chain reaction.

Polymerase chain reaction (PCR) in small fluidic systems not only improves speed and sensitivity of deoxyribonucleic acid (DNA) amplification but also achieves high-throughput quantitative analyses. However, air bubble trapping and growth during PCR has been considered as a critical problem since it causes the failure of DNA amplification. Here we report bubble-free diatom PCR by exploiting a hierarchically porous silica structure of single-celled algae. We show that femtoliters of PCR solution can be spontaneously loaded into the diatom interior without air bubble trapping due to the surface hydrophilicity and pore structure of the diatom. We discover that a large pressure gradient between air bubbles and nanopores rapidly removes residual air bubbles through the periodically arrayed nanopores during thermal cycling. We demonstrate the DNA amplification by diatom PCR without air bubble trapping and growth. Finally, we successfully detect DNA fragments of SARS-CoV-2 with as low as 10 copies/µl by devising a microfluidic device integrated with diatoms assembly. We believe that our work can be applied to many PCR applications for innovative molecular diagnostics and provides new opportunities for naturally abundant diatoms to create innovative biomaterials in real-world applications.

Quantum Electrodynamic Behavior of Chlorophyll in a Plasmonic Nanocavity.

Plasmonic nanocavities have been used as a novel platform for studying strong light-matter coupling, opening access to quantum chemistry, material science, and enhanced sensing. However, the biomolecular study of cavity quantum electrodynamics (QED) is lacking. Here, we report the quantum electrodynamic behavior of chlorophyll-a in a plasmonic nanocavity. We construct an extreme plasmonic nanocavity using Au nanocages with various linker molecules and Au mirrors to obtain a strong coupling regime. Plasmon resonance energy transfer (PRET)-based hyperspectral imaging is applied to study the electrodynamic behaviors of chlorophyll-a in the nanocavity. Furthermore, we observe the energy level splitting of chlorophyll-a, similar to the cavity QED effects due to the light-matter interactions in the cavity. Our study will provide insight for further studies in quantum biological electron or energy transfer, electrodynamics, the electron transport chain of mitochondria, and energy harvesting, sensing, and conversion in both biological and biophysical systems.

Water-Wettable Open Plasmonic Nanocavities for Ultrasensitive Molecular Detections in Multiple Phases.

Plasmonic nanocavities between metal nanoparticles on metal films are either hydrophobic or fully occupied by nonmetallic spacers, preventing molecular diffusion into electromagnetic hotspots. Here we realize water-wettable open plasmonic cavities by devising gold nanoparticle with site-selectively grown ultrathin dielectric layer-on-gold film structures. We directly confirm that hydrophilic dielectric layers of SiO2 or TiO2, which are formed only at the tips of gold nanorod via precise temperature control, render sub-10 nm cavities open to the surroundings and completely water-wettable. Simulations reveal that spontaneous wetting in our cavities is driven by the presence of tip-selective hydrophilic layer and tendency of minimizing high energy air/water interface inside the cavities. Our plasmonic cavities show significant Raman enhancement of up to 4 orders of magnitude higher than those of conventional ones for molecules in various media. Our findings will offer new opportunities for sensing applications of plasmonic nanocavities and have huge impacts on cavity plasmonics.

Sensitive and Direct Optical Monitoring of Release and Cellular Uptake of Aqueous CO from CO-Releasing Molecules.

Dynamics of release and cellular uptake of aqueous CO from CO-releasing molecules (CORMs) significantly affect signaling and cell viability. So far, it has been mainly observed by IR, UV-visible, and fluorescence techniques, which suffer from poor sensitivity and slow response time. Here, we show how to directly probe the mass transfer of aqueous CO from CORMs to cells using a fluidic chamber integrated with live cells and Raman reporters of large-area Au@Pd core-shell nanoparticle assembly to emulate a physiologically relevant microenvironment. We sensitively and directly detect CO release from trace CORMs of as low as 100 nM by measuring the Raman transitions of CO via rapid chemisorption onto the surface of the Au@Pd nanoparticles. By using our method, we successfully observe the dynamics of CO release from CORM-2 despite its very short half-life. We also reveal that the initial rate of CO release from CORM-3 is dramatically decreased by tens to hundreds of times when exposed to physiologically relevant pH variations from 7.4 to 2.5, which can be attributed to the acid hydrolysis of the CO ligand. CORM-2 tends to quickly release CO regardless of pH, probably because of its rapid cleavage into two monomeric Ru complexes by the co-solvent. The decrease in the initial rate at lower temperatures is more significant for CORM-3 than for CORM-2. Finally, we observe that the cellular uptake of aqueous CO from CORM-3 by lung cancer cells is approximately 2 times higher than that of normal lung cells.

Optical Detection of Small Metabolites for Biological Gas Conversion by using Metal Nanoparticle Monolayers Produced by Capillary-Assisted Transfer.

Detection of small metabolites is essential for monitoring and optimizing biological gas conversion. Currently, such detection is typically done by liquid chromatography with offline sampling. However, this method often requires large equipment with multiple separation columns and is at risk of serious microbial contamination during sampling. Here we propose real-time optical detection of small metabolites using uniform plasmonic nanoparticles monolayers produced by capillary-assisted transfer. We reproducibly fabricate metal nanoparticles monolayers with a diameter of ∼1 mm for the detection of acetate, butyrate, and glucose by a glass capillary tube. Metal nanoparticles monolayers are not only uniform in terms of average interparticle distance but also structurally stable under dynamic fluidic conditions. The monolayers resistant to fluid shear stress with surface-enhanced Raman scattering are able to reversibly monitor the concentration of acetate and sensitively detect acetate and glucose at levels as low as 10 µM, which is more than 2 orders of magnitude lower than the concentration range of typical biological gas conversion. In addition, structurally similar metabolites such as acetate and butyrate, when mixed, become distinguishable by our method.

Plasmonic bacteria on a nanoporous mirror via hydrodynamic trapping for rapid identification of waterborne pathogens.

A rapid, precise method for identifying waterborne pathogens is critically needed for effective disinfection and better treatment. However, conventional methods, such as culture-based counting, generally suffer from slow detection times and low sensitivities. Here, we developed a rapid detection method for tracing waterborne pathogens by an innovative optofluidic platform, a plasmonic bacteria on a nanoporous mirror, that allows effective hydrodynamic cell trapping, enrichment of pathogens, and optical signal amplifications. We designed and simulated the integrated optofluidic platform to maximize the enrichment of the bacteria and to align bacteria on the nanopores and plasmonic mirror via hydrodynamic cell trapping. Gold nanoparticles are self-assembled to form antenna arrays on the surface of bacteria, such as Escherichia coli and Pseudomonas aeruginosa, by replacing citrate with hydroxylamine hydrochloride in order to amplify the signal of the plasmonic optical array. Owing to the synergistic contributions of focused light via the nanopore geometry, self-assembled nanoplasmonic optical antennas on the surface of bacteria, and plasmonic mirror, we obtain a sensitivity of detecting E. coli as low as 102 cells/ml via surface-enhanced Raman spectroscopy. We believe that our label-free strategy via an integrated optofluidic platform will pave the way for the rapid, precise identification of various pathogens.

General and programmable synthesis of hybrid liposome/metal nanoparticles.

Hybrid liposome/metal nanoparticles are promising candidate materials for biomedical applications. However, the poor selectivity and low yield of the desired hybrid during synthesis pose a challenge. We designed a programmable liposome by selective encoding of a reducing agent, which allows self-crystallization of metal nanoparticles within the liposome to produce stable liposome/metal nanoparticles alone. We synthesized seven types of liposome/monometallic and more complex liposome/bimetallic hybrids. The resulting nanoparticles are tunable in size and metal composition, and their surface plasmon resonance bands are controllable in visible and near infrared. Owing to outer lipid bilayer, our liposome/Au nanoparticle shows better colloidal stability in biologically relevant solutions as well as higher endocytosis efficiency than gold nanoparticles without the liposome. We used this hybrid in intracellular imaging of living cells via surface-enhanced Raman spectroscopy, taking advantage of its improved physicochemical properties. We believe that our method greatly increases the utility of metal nanoparticles in in vivo applications.