Nanoconfinement and Crowding Enhanced Single-Molecule Detection of Small Molecules with Nanopipettes.

Glass nanopipettes have gained widespread use as a versatile single-entity detector in chemical and biological sensing, analysis, and imaging. Its advantages include low cost, easy accessibility, simplicity of use, and high versatility. However, conventional nanopipettes based on the volume exclusion mechanism have limitations in detecting small biomolecules due to their small volume and high mobility in aqueous solution. To overcome this challenge, we have employed a novel approach by capitalizing on the strong nanoconfinement effect of nanopipettes. This is achieved by utilizing both the hard confinement provided by the long taper nanopipette tip at the cis side and the soft confinement offered by the hydrogel at the trans side. Through this approach, we have effectively slowed down the exit motion of small molecules, allowing us to enrich and jam them at the nanopipette tip. Consequently, we have achieved high throughput detection of small biomolecules with sizes as small as 1 nm, including nucleoside triphosphates, short peptides, and small proteins with excellent signal-to-noise ratios. Furthermore, molecular complex formation through specific intermolecular interactions, such as hydrogen bonding between closely spaced nucleotides in the jam-packed nanopipette tip, has been detected based on the unique ionic current changes.

Anticancer Drug Doxorubicin Spontaneously Reacts with GTP and dGTP.

Here, we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 °C or above yields a covalent product: the doxorubicin-GTP or -dGTP conjugate where a covalent bond is formed between the C14 position of doxorubicin and the 2-amino group of guanine. Density functional theory calculations show the feasibility of this spontaneous reaction. Fluorescence imaging studies demonstrate that the doxorubicin-GTP and -dGTP conjugates cannot enter nuclei although they rapidly accumulate in human SK-OV-3 and NCI/ADR-RES cells. Consequently, the doxorubicin-GTP and -dGTP conjugates are less cytotoxic than doxorubicin. We also demonstrate that doxorubicin binds to ATP, GTP, and other nucleotides with a dissociation constant (Kd) in the sub-millimolar range. Since human cells contain millimolar levels of ATP and GTP, these results suggest that doxorubicin may target ATP and GTP, energy molecules that support essential processes in living organisms.

Development of multifunctional nanopipettes for controlled intracellular delivery and single-entity detection.

The intracellular delivery of biomolecules and nanoscale materials to individual cells has gained remarkable attention in recent years owing to its wide applications in drug delivery, clinical diagnostics, bio-imaging and single-cell analysis. It remains a challenge to control and measure the delivered amount in one cell. In this work, we developed a multifunctional nanopipette - containing both a nanopore and nanoelectrode (pyrolytic carbon) at the apex - as a facile, minimally invasive and effective platform for both controllable single-cell intracellular delivery and single-entity counting. While controlled by a micromanipulator, the baseline changes of the nanopore ionic current (I) and nanoelectrode open circuit potential (V) help to guide the nanopipette tip insertion and positioning processes. The delivery from the nanopore barrel can be facilely controlled by the applied nanopore bias. To optimize the intracellular single-entity detection during delivery, we studied the effects of the nanopipette tip geometry and solution salt concentration in controlled experiments. We have successfully delivered gold nanoparticles and biomolecules into the cell, as confirmed by the increased scattering and fluorescence signals, respectively. The delivered entities have also been detected at the single-entity level using either one or both transient I and V signals. We found that the sensitivity of the single-entity electrochemical measurement was greatly affected by the local environment of the cell and varied between cell lines.

Differentiation of metallic and dielectric nanoparticles in solution by single-nanoparticle collision events at the nanoelectrode.

In this work, we demonstrate a highly effective method to generate and detect single-nanoparticle (NP) collision events on a nanoelectrode in aqueous solutions. The nanoelectrode of a nanopore-nanoelectrode nanopipette is first employed to accumulate NPs in solution by dielectrophoresis (DEP). Instead of using amperometric methods, the continuous individual NP collision events on the nanoelectrode are sensitively detected by monitoring the open-circuit potential changes of the nanoelectrode. Metallic gold NPs (GNPs) and insulating polystyrene (PS) NPs with various sizes are used as the model NPs. Due to the higher conductivity and polarizability of GNPs, the collision motion of a GNP is different from that of a PS NP. The difference is distinct in the shape of the transient potential change and its first time derivative detected by the nanoelectrode. Therefore, the collision events by metallic and insulating NPs on a nanoelectrode can be differentiated based on their polarizability. DEP induced NP separation and cluster formation can also be probed in detail in the concentrated mixture of PS NPs and GNPs.

Conjugating Daunorubicin and Doxorubicin to GTP by Formaldehyde to Overcome Drug Resistance.

Daunorubicin and doxorubicin are among the most potent anti-cancer drugs and bind to DNA through intercalation. In this paper, we demonstrate that formaldehyde can efficiently and specifically conjugate daunorubicin and doxorubicin to GTP, resulting in the formation of daunorubicin-GTP-1 and doxorubicin-GTP-1 conjugates. The linkage occurs between the 2-NH2 of guanine and the 3'-NH2 of daunosamine. We characterized these daunorubicin/doxorubicin-GTP conjugates using various methods, including UV-Vis, fluorescence, CD, FT-IR, and mass spectrometry. Our results also indicate that these daunorubicin/doxorubicin-GTP conjugates bind to DNA via intercalation. Furthermore, we observed rapid accumulation of these conjugates in human cancer cells and observed cytotoxic effects in both doxorubicin-sensitive SK-OV-3 and doxorubicin-resistant NCI/ADR-RES cells, suggesting that these daunorubicin and doxorubicin derivatives can overcome doxorubicin resistance.

Membrane Remodeling of Human-Engineered Cardiac Tissue by Chronic Electric Stimulation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show immature features, but these are improved by integration into 3D cardiac constructs. In addition, it has been demonstrated that physical manipulations such as electrical stimulation (ES) are highly effective in improving the maturation of human-engineered cardiac tissue (hECT) derived from hiPSC-CMs. Here, we continuously applied an ES in capacitive coupling configuration, which is below the pacing threshold, to millimeter-sized hECTs for 1-2 weeks. Meanwhile, the structural and functional developments of the hECTs were monitored and measured using an array of assays. Of particular note, a nanoscale imaging technique, scanning ion conductance microscopy (SICM), has been used to directly image membrane remodeling of CMs at different locations on the tissue surface. Periodic crest/valley patterns with a distance close to the sarcomere length appeared on the membrane of CMs near the edge of the tissue after ES, suggesting the enhanced transverse tubulation network. The SICM observation is also supported by the fluorescence images of the transverse tubulation network and α-actinin. Correspondingly, essential cardiac functions such as calcium handling and contraction force generation were improved. Our study provides evidence that chronic subthreshold ES can still improve the structural and functional developments of hECTs.

Detecting Individual Proteins and Their Surface Charge Variations in Solution by the Potentiometric Nanoimpact Method.

Label-free detection and analysis of proteins in their natural form and their dynamic interactions with substrates at the single-molecule level are important for both fundamental studies and various applications. Herein, we demonstrate a simple potentiometric method to achieve this goal by detecting the native charge of protein in solution by utilizing the principle of single-entity electrochemistry techniques. When a charged protein moves near the vicinity of a floating carbon nanoelectrode connected to a high-impedance voltage meter, the distinct local electrostatic potential changes induced by the transient collision event of protein, also called the "nanoimpact" event, can be captured by the nanoelectrode as a potential probe. This potentiometric method is highly sensitive for charged proteins, and low-molecular-weight proteins less than 10 kDa can be detected in low-salt-concentration electrolytes. By analyzing the shape and magnitude of the recorded time-resolved potential change and its time derivative, we can reveal the charge and motion of the protein in the nonspecific protein-surface interaction event. The charge polarity variations of the proteins at different pH values were also successfully probed. Compared with synthetic spherical nanoparticles, the statistical analysis of many single-molecule nanoimpact events revealed a large variation in the recorded transient potential signals, which may be attributed to the intrinsic protein dynamics and surface charge heterogeneity, as suggested by the finite element method and molecular dynamic simulations.

Pigmentation Affects Elastic Fiber Patterning and Biomechanical Behavior of the Murine Aortic Valve.

The aortic valve (AoV) maintains unidirectional blood distribution from the left ventricle of the heart to the aorta for systemic circulation. The AoV leaflets rely on a precise extracellular matrix microarchitecture of collagen, elastin, and proteoglycans for appropriate biomechanical performance. We have previously demonstrated a relationship between the presence of pigment in the mouse AoV with elastic fiber patterning using multiphoton imaging. Here, we extended those findings using wholemount confocal microscopy revealing that elastic fibers were diminished in the AoV of hypopigmented mice (KitWv and albino) and were disorganized in the AoV of K5-Edn3 transgenic hyperpigmented mice when compared to wild type C57BL/6J mice. We further used atomic force microscopy to measure stiffness differences in the wholemount AoV leaflets of mice with different levels of pigmentation. We show that AoV leaflets of K5-Edn3 had overall higher stiffness (4.42 ± 0.35 kPa) when compared to those from KitWv (2.22 ± 0.21 kPa), albino (2.45 ± 0.16 kPa), and C57BL/6J (3.0 ± 0.16 kPa) mice. Despite the striking elastic fiber phenotype and noted stiffness differences, adult mutant mice were found to have no overt cardiac differences as measured by echocardiography. Our results indicate that pigmentation, but not melanocytes, is required for proper elastic fiber organization in the mouse AoV and dictates its biomechanical properties.

Single-Entity Approach to Investigate Surface Charge Enhancement in Magnetoelectric Nanoparticles Induced by AC Magnetic Field Stimulation.

Magneto-electric nanoparticles (MENPs), composed of a piezoelectric shell and a ferromagnetic core, exhibited enhanced cell uptake and controlled drug release due to the enhanced localized electric field (surface charge/potential) and the generation of acoustics, respectively, upon applying alternating current (AC) magnetic (B)-field stimulation. This research, for the first time, implements an electrochemical single-entity approach to probe AC B-field induced strain mediated surface potential enhancement on MENP surface. The surface potential changes at the single-NP level can be probed by the open circuit potential changes of the floating carbon nanoelectrode (CNE) during the MENP-CNE collision events. The results confirmed that the AC B-field (60 Oe) stimulation caused localized surface potential enhancement of MENP. This observation is associated with the presence of a piezoelectric shell, whereas magnetic nanoparticles were found unaffected under identical stimulation.

Observing dynamic molecular changes at single-molecule level in a cucurbituril based plasmonic molecular junction.

In recent years, surface enhanced Raman spectroscopy (SERS) has emerged as a prominent tool for probing molecular interaction and reaction with single-molecule sensitivity. Here we use SERS to investigate the dynamic changes of the cucurbit[7]uril (CB[7]) based plasmonic molecular junctions in solution, which are spontaneously formed by the adsorption of gold nanoparticles (GNPs) at the CB[7] modified gold nanoelectrode (GNE) surface. The typical fingerprint Raman peaks of CB[7] are very weak in the SERS spectra. However, chemically enhanced peaks are prominent in the spectra due to the charge transfer across the metal-molecule interface through specific noncovalent interactions between the gold atoms and CB[7] or its guest molecule. We first investigated the selectively enhanced and greatly shifted C[double bond, length as m-dash]O peak of CB[7] in the SERS spectra. Based on the bias-dependent changes of the C[double bond, length as m-dash]O peak, we found the gold-carbonyl interaction was strengthened by the positive bias applied to the GNE, resulting in stable CB[7] junctions. Next, we found the CB[7] junction could also be stabilized by the inclusion of a guest molecule amino-ferrocene, attributed to the interactions between gold adatoms and the cyclopentadienyl ring of the guest molecule. Because this interaction is sensitive to the orientation of the guest molecule in the cavity, we revealed the rotational motion of a guest molecule inside the CB[7] cavity based on the dynamic spectral changes of the cyclopentadienyl ring peak.

Simultaneous Ionic Current and Potential Detection of Nanoparticles by a Multifunctional Nanopipette.

Nanopore sensing-based technologies have made significant progress for single molecule and single nanoparticle detection and analysis. In recent years, multimode sensing by multifunctional nanopores shows the potential to greatly improve the sensitivity and selectivity of traditional resistive-pulse sensing methods. In this paper, we showed that two label-free electric sensing modes could work cooperatively to detect the motion of 40 nm diameter spherical gold nanoparticles (GNPs) in solution by a multifunctional nanopipette. The multifunctional nanopipettes containing both nanopore and nanoelectrode (pyrolytic carbon) at the tip were fabricated quickly and cheaply. We demonstrated that the ionic current and local electrical potential changes could be detected simultaneously during the translocation of individual GNPs. We also showed that the nanopore/CNE tip geometry enabled the CNE not only to detect the translocation of single GNP but also to collectively detect several GNPs outside the nanopore entrance. The dynamic accumulation of GNPs near the nanopore entrance resulted in no detectable current changes, but was detected by the potential changes at the CNE. We revealed the motions of GNPs both outside and inside the nanopore, individually and collectively, with the combination of ionic current and potential measurements.