Single-Molecule Electrical and Spectroscopic Profiling Protein Allostery Using a Gold Plasmonic Nanopore.

Direct structural and dynamic characterization of protein conformers in solution is highly desirable but currently impractical. Herein, we developed a single molecule gold plasmonic nanopore system for observation of protein allostery, enabling us to monitor translocation dynamics and conformation transition of proteins by ion current detection and SERS spectrum measurement, respectively. Allosteric transition of calmodulin (CaM) was elaborately probed by the nanopore system. Two conformers of CaM were well-resolved at a single-molecule level using both the ion current blockage signal and the SERS spectra. The collected SERS spectra provided structural evidence to confirm the interaction between CaM and the gold plasmonic nanopore, which was responsible for the different translocation behaviors of the two conformers. SERS spectra revealed the amino acid residues involved in the conformational change of CaM upon calcium binding. The results demonstrated that the excellent spectral characterization furnishes a single-molecule nanopore technique with an advanced capability of direct structure analysis.

Probing Multidimensional Structural Information of Single Molecules Transporting through a Sub-10 nm Conical Plasmonic Nanopore by SERS.

Probing the orientation and oxygenation state of single molecules (SMs) is of great importance for understanding the advanced structure of individual molecules. Here, we manipulate molecules transporting through the hot spot of a sub-10 nm conical gold nanopore and acquire the multidimensional structural information of the SMs by surface enhanced Raman scattering (SERS) detection. The sub-10 nm size and conical shape of the plasmonic nanopore guarantee its high detection sensitivity. SERS spectra show a high correlation with the orientations of small-sized single rhodamine 6G (R6G) during transport. Meanwhile, SERS spectra of a single hemoglobin (Hb) reveal both the vertical/parallel orientations of the porphyrin ring and oxygenated/deoxygenated states of Hb. The present study provides a new strategy for bridging the primary sequence and the advanced structure of SMs.

Single-cell thermometry with a nanothermocouple probe.

Herein, a nanopipette-based thermocouple probe that possesses high temperature resolution, rapid response, good reversibility and stability was constructed and successfully applied for single-cell temperature sensing. Different intracellular temperatures were observed in diverse types of cells, which reveals differences in their metabolism levels. Temperature responses of cancer and normal cells against various exogenous drugs were also demonstrated. The spatially resolved temperature sensing of three-dimensional cell culture models unveils the existence of their inner temperature gradients. This work would facilitate drug screening and disease diagnosis.

Ammonia-Induced Anomalous Ion Transport in Covalent Organic Framework Nanochannels.

More anomalous transport behaviors have been observed with the rapid progress in nanofabrication technology and characterization tools. The ions/molecules inside nanochannels can act dramatically different from those in the bulk systems and exhibit novel mechanisms. Here, we have reported the fabrication of a nanodevice, covalent organic frameworks covered theta pipette (CTP), that combine the advantages of theta pipette (TP), nanochannels framework, and field-effect transistors (FETs) for controlling and modulating the anomalous transport. Our results show that ammonia, a weak base, causes a continuous supply of ions inside covalent organic framework (COF) nanochannels, leading to an abnormally high current depending on the ionic/molecular size and the pore size of the nanochannel. Furthermore, CTP can distinguish different concentrations of ammonia and have all of the qualities of a nanosensor.

Surface-Roughened SERS-Active Single Silver Nanowire for Simultaneous Detection of Intracellular and Extracellular pHs.

The simultaneous and accurate detection of intracellular pH (pHi) and extracellular pH (pHe) is essential for studying the complex physiological activities of cancer cells and exploring pH-related therapeutic mechanisms. Here, we developed a super-long silver nanowire-based surface-enhanced Raman scattering (SERS) detection strategy for simultaneous sensing of pHi and pHe. A surface-roughened silver nanowire (AgNW) with a high aspect ratio is prepared at a nanoelectrode tip using a Cu-mediated oxidation process, which is then modified by pH-sensitive 4-mercaptobenzoic acid (4-MBA) to form 4-MBA@AgNW as a pH sensing probe. With the assistance of a 4D microcontroller, 4-MBA@AgNW is efficient in simultaneously detecting pHi and pHe in both 2D and 3D culture cancer cells by SERS, with minimal invasiveness, high sensitivity, and spatial resolution. Further investigation proves that the surface-roughened single AgNW can also be used in monitoring the dynamic variation of pHi and pHe of cancer cells upon stimulation with anticancer drugs or under a hypoxic environment.

Super-Long SERS Active Single Silver Nanowires for Molecular Imaging in 2D and 3D Cell Culture Models.

Establishing a systematic molecular information analysis strategy for cell culture models is of great significance for drug development and tissue engineering technologies. Here, we fabricated single silver nanowires with high surface-enhanced Raman scattering activity to extract SERS spectra in situ from two-dimensional (2D) and three-dimensional (3D) cell culture models. The silver nanowires were super long, flexible and thin enough to penetrate through multiple cells. A single silver nanowire was used in combination with a four-dimensional microcontroller as a cell endoscope for spectrally analyzing the components in cell culture models. Then, we adopted a machine learning algorithm to analyze the obtained spectra. Our results show that the abundance of proteins differs significantly between the 2D and 3D models, and that nucleic acid-rich and protein-rich regions can be distinguished with satisfactory accuracy.

Free-Standing Single Ag Nanowires for Multifunctional Optical Probes.

Miniaturized and manipulable optical probes are the foundation for developing in situ characterization devices in confined space. We developed two methods for fabricating free-standing single Ag nanowires (AgNWs) directly at the tip of a glass capillary either by chemical or electrochemical reduction. The electrochemical nature of both methods resulted in a rapid growth rate of AgNWs up to 1.38 µm/s and a controllable length from 5 to 450 µm. The AgNWs with a unique anisotropic structure allow localized surface plasmon resonance and surface plasmon waveguides in the radial direction and axial direction, respectively. We verified the possibility of using single AgNWs as an optical dispersion device and waveguide probe. By controlling the experimental conditions, rough-surface AgNWs with high surface-enhanced Raman scattering (SERS) activity were also fabricated. These SERS-active probes also exhibited advantages in acquiring molecular information from a single living cell.