Glass Nanopipette Research Articles

Solid-State Glass Nanopipettes: Functionalization and Applications.

Solid-state glass nanopipettes provide a promising confined space that offers several advantages such as controllable size, simple preparation, low cost, good mechanical stability, and good thermal stability. These advantages make them an ideal choice for various applications such as biosensors, DNA sequencing, and drug delivery. In this review, we first delve into the functionalized nanopipettes for sensing various analytes and the methods used to develop detection means with them. Next, we provide an in-depth overview of the advanced functionalization methodologies of nanopipettes based on diversified chemical kinetics. After that, we present the latest state-of-the-art achievements and potential applications in detecting a wide range of targets, including ions, molecules, biological macromolecules, and single cells. We examine the various challenges that arise when working with these targets, as well as the innovative solutions developed to overcome them. The final section offers an in-depth overview of the current development status, newest trends, and application prospects of sensors. Overall, this review provides a comprehensive and detailed analysis of the current state-of-the-art functionalized nanopipette perception sensing and development of detection means and offers valuable insights into the prospects for this exciting field.

Rectifying artificial nanochannels with multiple interconvertible permeability states

Transmembrane channels play a vital role in regulating the permeation process, and have inspired recent development of biomimetic channels. Herein, we report a class of artificial biomimetic nanochannels based on DNAzyme-functionalized glass nanopipettes to realize delicate control of channel permeability, whereby the surface wettability and charge can be tuned by metal ions and DNAzyme-substrates, allowing reversible conversion between different permeability states. We demonstrate that the nanochannels can be reversibly switched between four different permeability states showing distinct permeability to various functional molecules. By embedding the artificial nanochannels into the plasma membrane of single living cells, we achieve selective transport of dye molecules across the cell membrane. Finally, we report on the advanced functions including gene silencing of miR-21 in single cancer cells and selective transport of Ca2+ into single PC-12 cells. In this work, we provide a versatile tool for the design of rectifying artificial nanochannels with on-demand functions.

Rare Earth-Carbon Dots Nanocomposite-Modified Glass Nanopipettes: Electro-Optical Detection of Bacterial ppGpp.

As an important alarmone nucleotide, guanosine 3'-diphosphate-5'-diphosphate (ppGpp) can regulate the survival of bacteria under strict environmental conditions. Direct detection of ppGpp in bacteria with high sensitivity and selectivity is crucial for elucidating the role of ppGpp in bacterial stringent response. Herein, the terbium-carbon dots nanocomposite (CDs-Tb) modified glass nanopipet was developed for the recognition of ppGpp. The CDs-Tb in glass nanopipette preserved their fluorescence properties as well as the coordination capacity of Tb3+ toward ppGpp. The addition of ppGpp not only led to the fluorescence response of CDs-Tb but also triggered variations of surface charge inside the glass nanopipet, resulting in the ionic current response. Compared with nucleotides with similar structures, this method displayed good selectivity toward ppGpp. Moreover, the dual signals (fluorescence and ionic current) offered a built-in correction for potential interference. Apart from the high selectivity, the proposed method can determine the concentration of ppGpp from 10-13 to 10-7 M. Taking advantage of the significant analytical performance, we monitored ppGpp in Escherichia coli under different nutritional conditions and studied the relationship between ppGpp and DNA repair, which is helpful for overcoming antibiotic resistance and promoting the development of potential drugs for antibacterial treatment.

Single-Cell Caspase-3 Measurement Using a Biomimetic Nanochannel.

Direct single-cell caspase-3 (Casp-3) analysis has remained challenging. A study of single-cell Casp-3 could contribute to revealing the fundamental pathogenic mechanisms in Casp-3-associated diseases. Here, a biomimetic nanochannel capable of single-cell sampling and ionic detection of intracellular Casp-3 is devised, which is established upon the installment of target-specific organic molecules (luc-DEVD) within the orifice of a glass nanopipette. The specific cleavage of luc-DEVD by Casp-3 could induce changes of inner-surface chemical groups and charge properties, thus altering the ionic response of the biomimetic nanochannel for direct Casp-3 detection. The practical applicability of this biomimetic nanochannel is confirmed by probing intracellular Casp-3 fluctuation upon drug stimulation and quantifying the Casp-3 evolution during induced apoptosis. This work realizes ionic single-cell Casp-3 analysis and provides a different perspective for single-cell protein analysis.

Dual-signal readout sensing of ATP content in single dental pulp stem cells during differentiation via functionalized glass nanopipettes

Adenosine triphosphate (ATP) is regarded as the “energy currency” in living cells, so real-time quantification of content variation of intracellular ATP is highly desired for understanding some important physiological processes. Due to its single-molecule readout ability, nanopipette sensing has emerged as a powerful technique for molecular sensing. In this study, based on the effect of targeting-aptamer binding on ionic current, and fluorescence resonance energy transfer (FRET), we reported a dual-signal readout nanopipette sensing system for monitoring ATP content variation at the subcellular level. In the presence of ATP, the complementary DNA-modified gold nanoparticles (cDNAs-AuNPs) were released from the inner wall of the nanopipette, which leads to sensitive response variations in ionic current rectification and fluorescence intensity. The developed nanopipette sensor was capable of detecting ATP in single cells, and the fluctuation of ATP content in the differentiation of dental pulp stem cells (DPSCs) was further quantified with this method. The study provides a more reliable nanopipette sensing platform due to the introduction of fluorescence readout signals. Significantly, the study of energy fluctuation during cell differentiation from the perspective of energy metabolism is helpful for differentiation regulation and cell therapy.

Nanoscale visualization of the anti-tumor effect of a plasma-activated Ringer's lactate solution.

Plasma-activated Ringer's lactate solutions (PALs), which are Ringer's lactate solutions treated with non-thermal atmospheric-pressure plasma, have an anti-tumor effect and can be used for chemotherapy. As the anti-tumor effect of the PAL is influenced by the cell-treatment time, it is necessary to monitor the structural changes of the cell surface with non-invasive, nanoscale, and time-lapse imaging to understand the anti-tumor effect. In this study, to characterize the anti-tumor effect of the PAL, we used scanning ion conductance microscopy (SICM), using glass nanopipettes as probes, to visualize the structural changes of the cell surface. SICM time-lapse topographic imaging visualized a decrease in the movement of lamellipodia in normal cells and cancer cells after the PAL treatment. Furthermore, in normal cells, protrusive structures were observed on the cell surface. Time-lapse imaging using SICM allowed us to characterize the differences in the morphological changes between the normal and cancer cells upon exposure to the PAL.

Dual-current signal high-sensitivity detection of telomerase using signal amplified DNA functionalized metal-organic frameworks in glass nanopipettes

As a biomarker for early cancer diagnosis, the quantitative and sensitive detection of telomerase is very hot topic in the field of biological detection. In this work, we reported a novel telomerase assay strategy-based glass nanopipettes (GNs) which were constructed with DNA functionalized metal-organic frameworks (MOFs, PCN-224, C144H112N12O64Zr15) as composite probes (DNA-PCN-224, DNA-PCN-224-MB (methylene blue)). The composite probes provided more probe binding sites, so the aggregation behavior of the composite probes would occur obviously in nanopores restricted domain space under the recognition and amplification of telomerase. The relative size of the GNs reduced due to the size effect of MOFs. This size effect exhibits a good signal-to-noise ratio in large pores, resulting in signal amplification. With different modifications of the GNs, on the one hand, the reduction of the relative pore size amplified the ionic current signal; on the other hand, the electrolytic current signal further verified the reliability of this signal amplification, which originated from the transfer of electrons occurring on the gold layer by the electroactive substance MB. The former quantified the assay, allowing a telomerase detection range of 0–10,000 Hela cells/mL and a detection limit of 100 Hela cells/mL. This work provided a detection platform for detecting related telomerase diseases and opened a new detection method of double signals.

Glass Nanopipette-Based Plasmonic SERS Platform for Single-Cell MicroRNA-21 Sensing during Apoptosis.

As one of the most widely distributed microRNAs, microRNA-21 (miRNA-21) significantly regulates target genes' expression levels and participates in many cellular and intercellular activities, and its abnormal expression is always related to some diseases, especially cancer. Hence, detecting miRNA-21, as a biomarker, at the single-cell level helps us to reveal cell heterogeneity and expression level variation during the state change of cells. In this study, we constructed a gold nanoparticles nanomembrane (AuNPs-NM)-modified plasmonic glass nanopipette (P-nanopipette) surface-enhanced Raman scattering (SERS) sensing platform to sensitively detect content variation of the intracellular miRNA-21 during the electrostimulus (ES)-induced apoptosis process. The cytoplasm-located miRNA-21 was first extracted by using the extraction DNA (HP1)-modified P-nanopipette through a hybridization chain reaction (HCR). The nanopipette was then incubated with a labeling DNA (HP2) and reporter 4-MBA-modified Raman tag. The Raman signal (collected from the tip area near the orifice within 1 μm) showed a good response to the content variation of intracellular miRNA-21 under ES, and the proposed single-cell SERS detection platform provides a simple way to study intracellular substance change and evaluate cancer treatment outcomes.

A Fast and Reversible Responsive Bionic Transmembrane Nanochannel for Dynamic Single-Cell Quantification of Glutathione.

Biological channels can rapidly and continuously modulate ion transport behaviors in response to external stimuli, which play essential roles in manipulating physiological and pathological processes in cells. Here, to mimic the biological channels, a bionic nanochannel is developed by synergizing a cationic silicon-substituted rhodamine (SiRh) with a glass nanopipette for transmembrane single-cell quantification. Taking the fast and reversible nucleophilic addition reaction between glutathione (GSH) and SiRh, the bionic nanochannel shows a fast and reversible response to GSH, with its inner-surface charges changing between positive and negative charges, leading to a distinct and reversible switch in ionic current rectification (ICR). With the bionic nanochannel, spatiotemporal-resolved operation is performed to quantify endogenous GSH in a single cell, allowing for monitoring of intracellular GSH fluctuation in tumor cells upon photodynamic therapy and ferroptosis. Our results demonstrate that it is a feasible tool for in situ quantification of the endogenous GSH in single cells, which may be adapted to addressing other endogenous biomolecules in single cells by usage of other stimuli-responsive probes.

Argon Gas Flow Through Micro- and Nano-pipettes

Glass micropipettes and nanopipettes are widely used for injection to or aspiration from living cells, and nanopipettes can be also used for nano-fabrication by deposition. Here, we show a non-destructive and contamination-free method of examining the inside of glass micropipettes and nanopipettes by argon gas flow through the pipettes from the tip-top to the tube-end. The exit pressure for each pipette was measured at the entrance pressure of 50 kPa, and the semi-log plot of the vacuum conductance versus pipette inner diameter is compared with the calculation using tapered tube approximation assuming molecular flow. The experimental results were consistent in trend with the calculation, and the difference between the experimental results and the calculation was due to the intermediate gas flow as the pipette inner diameter increases. Therefore, the formula of the calculation was transformed using the Gompertz function to fit the experimental results well. By utilizing the plot of the inner diameter dependence of the exit pressure, the inner diameter of glass pipettes can be estimated from our developed gas flow method.

DNA-decorated multilamellar cholesterol assemblies for nucleic acid detection in the micrometer-scale solid-state nanopore

The large-sized glass nanopipette (GN) allows for an access of large targets and show the benefits of excellent mechanical stability and reproducibility. The nanoscale DNA-decorated multilamellar cholesterol assemblies are introduced into the GN with several nanometers, and could in situ grow into a micrometer-scale aggregates under DNA signal stimulus, developing a simple and sensitive strategy for DNA detection in the micrometer-scale GN. The detection limit of target DNA reaches 2.69 fM in a GN with a diameter of up to ca. 4.76 μm.

Current oscillations from bipolar nanopores for statistical monitoring of hydrogen evolution on a confined electrochemical catalyst.

Taking advantage of bipolar electrochemistry and a glass nanopipette, continuous single bubbles can be controlled which are generated and detached from a nanometer-sized area of confined electrochemical catalysts. The observed current oscillations offer opportunities to rapidly collect data for the statistical analysis of single-bubble generation on and departure from the catalysts.

МНОГОМОДОВЫЙ СКАНИРУЮЩИЙ МИКРОСКОП ИОННОЙ ПРОВОДИМОСТИ С СИСТЕМОЙ ПЬЕЗОИНЕРЦИАЛЬНОГО ПЕРЕМЕЩЕНИЯ

A scanning ion conductance microscope (SICM) has been developed, operating in several modes: DC mode, current modulation mode, and hopping mode. SICM employs a piezoelectric-inertial movement system. The nanoprobes, in the form of glass nanopipettes with an internal radius of r ~ 50 nm, have been created and tested. The current-voltage characteristics I (V) and current dependences on the distance between the probe and the sample I (z) (approach/withdrawal curves) were measured. Images of a polymeric test object with a periodic structure and a biological object (CHO cell) were obtained, their quality was assessed, and the features of the SICM operation in various modes are discussed. The multimode SICM provides non-destructive, non-contact visualization of soft objects in a liquid conducting medium with nanometer spatial resolution in various measuring modes and can be used in biology, cytology, electrochemistry, and medicine when studying inorganic soft objects, biological objects in buffer media, etc.

Specially Resolved Single Living Cell Perfusion and Targeted Fluorescence Labeling Based on Nanopipettes.

Targeted delivery and labeling of single living cells in heterogeneous cell populations are of great importance to understand the molecular biology and physiological functions of individual cells. However, it remains challenging to perfuse fluorescence markers into single living cells with high spatial and temporal resolution without interfering neighboring cells. Here, we report a single cell perfusion and fluorescence labeling strategy based on nanoscale glass nanopipettes. With the nanoscale tip hole of 100 nm, the use of nanopipettes allows special perfusion and high-resolution fluorescence labeling of different subcellular regions in single cells of interest. The dynamic of various fluorescent probes has been studied to exemplify the feasibility of nanopipette-dependent targeted delivery. According to experimental results, the cytoplasm labeling of Sulfo-Cyanine5 and fluorescein isothiocyanate is mainly based on the Brownian movement due to the dyes themselves and does not have a targeting ability, while the nucleus labeling of 4',6-diamidino-2-phenylindole (DAPI) is originated from the adsorption between DAPI and DNA in the nucleus. From the finite element simulation, the precise manipulation of intracellular delivery is realized by controlling the electro-osmotic flow inside the nanopipettes, and the different delivery modes between nontargeting dyes and nucleus-targeting dyes were compared, showcasing the valuable ability of nanopipette-based method for the analysis of specially defined subcellular regions and the potential applications for single cell surgery, subcellular manipulation, and gene delivery.

Applications of nanopipettes in electrochemical analysis

AbstractNowadays, the application of glass nanopipettes in electrochemical analysis has attracted a lot of attention with the increasing demand for sensitive analysis and diagnostic tools for cellular biomolecules. The ultrasmall nanopipettes are becoming a bridge between the macro‐ and nanoworlds as they can transport very small volumes of liquids and materials as well as acting as electrochemical biosensors. In this minireview, we discuss the fabrication and characterization of nanopipettes and introduce our recent studies using nanopipettes for cell electroanalysis. The future application of nanopipettes has also been discussed.

Glass nanopipette sensing of single entities

• Preparation, characterization and modification of glass nanopipette is introduced. • The electrochemical sensing mechanism of glass nanopipette is summarized. • Latest research of glass nanopipette combined with other technologies is introduced. • The advantages of glass nanopipette in single entity detection are introduced. • Challenges and prospects of glass nanopipette in this realm are discussed. Evaluating the laws of matter, life processes, and reaction kinetics from the perspective of the basic single entity elements of material, chemical, and life sciences comprises a fascinating frontier in analytical chemistry. Glass nanopipette sensing aims to detect interactions between single molecules, the physical and chemical properties of single particles, and the important roles of single cells in life systems. Currently, it is a vital branch of analytical chemistry. In this review, we introduce the preparation and characterization of glass nanopipettes, a combined detection method with a vision for a single entity, and their advances in the analysis of single molecules, single particles, and single cells.

Highly Sensitive Glass Nanopipette Sensor Using Composite Probes of DNA-Functionalized Metal-Organic Frameworks.

Pore structure-based analytical techniques have great potential applications for the detection of biological molecules. However, the sophistication of traditional pore sensors is restricted in their applicability of analytical chemistry due to a lack of effective carrier probes. Here, we used porous coordination network-224 (PCN-224) composite probes in conjunction with a glass nanopipette (GN) as a sensing platform. The sensor exhibits a good fluorescence signal and a change in GN's ionic current at the same time. Due to the volume exclusion mechanism coming from PCN-224, the detection limit of target DNA reaches 10 fM in a GN with a diameter of up to ca. 260 nm, outperforming a simple probe. The structure of the composite probe is optimized by the probe's pairing efficiency. Furthermore, the sensor can also discriminate between 1-, 3-, and 5-mismatch DNA sequences and capture the target DNA from a complex mixture. Based on the GN platform, a series of techniques for detecting biomolecules are expected to emerge because of its simplicity, robustness, and universality by incorporating advanced nanoprobes.

Quantification of Asymmetric Ion Transport in Glass Nanopipettes near Charged Substrates

AbstractTaking advantage of asymmetric ion transport in nanopipettes, the surface charges of various substrates have been successfully imaged by scanning ion conductance microscopy (SICM). The predominant electrostatic interaction between charged nanopipettes and substrates thus needs to be better elucidated and quantified, as traditional SICM theory is not adequate to describe the resulting ion current. Herein, we use both experimental and simulation methods to study the asymmetric ion‐transport processes in nanopipettes near charged substrates, and correlate surface charge and geometry parameters to the resulting ionic current. It is found that for negatively charged substrates and nanopipettes, positive feedback responses are displayed at the high conductivity states, and they are sensitive to the applied voltages and surface charges. While at the low conductivity states, comparable negative feedback responses are largely independent of experimental conditions. The elucidated and quantified asymmetric ion transport features would allow more accurate measurements and analysis for approaching and surface charge mapping in SICM applications.

Solid-state nanopores and nanochannels for the detection of biomolecules

Solid-state nanopores and nanochannels are a powerful detection platform for biomolecule sensing. The confined space inside the nanopores and their ability to be functionalized make these materials a uniquely advantageous detection method. Solid-state nanopores avoid the intrinsic fragility of biological nanopores and have excellent robustness and manufacturability. Improvements to the sensitivity, selectivity, controllability, and reproducibility of solid-state nanopores for detection are current areas of growth for the field. This review presents the state-of-the-art technology for solid-state nanopores and nanochannels. Fabrication protocols of various types of nanopores and nanochannels are described, including silicon nitride-based pores, polymer nanochannels, and glass nanopipettes. Moreover, we discuss the strategies for appending and functionalization that confer advanced physical and chemical properties to the solid-state nanopores. Finally, the applications and future potential for solid-state nanopores are presented, including the detection of DNA, drugs, proteins, and viruses.

Nanoscale Electrochemistry Study Using SECCM Option of Park Systems

Scanning electrochemical cell microscopy (SECCM) is a nanoelectrochemical scanning probe technique that creates a confined electrochemical cell (droplet) via meniscus contacting the surface by employing an electrolyte filled micropipette containing quasi-reference counter electrode(QRCE).[1] It is particularly useful to investigate the heterogeneity, or local electrochemical properties of the electrode surfaces by direct mapping the electroactivity with nanoscale resolution.In this study, the electrochemically reversible [Ru (NH3)6]3+/2+ electron transfer process at a highly ordered pyrolytic graphite (HOPG) surface was recorded using Park NX12 AFM system. A single barrel glass nanopipette with Ag/AgCl inserted as QRCE was utilized. Using previous successful experience in commercializing pipette-based electrochemical microscopy,[2] Park Systems’ hardware and software enables localized nanoscopic cyclic voltammetry measurements conducted at each pixel each time the meniscus contacts the surface. Thus, a spatially resolved surface electroactivity mapping of HOPG at various scan rates were created with high-throughput at the micro-/nanoscale. The difference in electrochemical activity measured at the edge plane relative to the basal plane surface was shown.This work demonstrates that Park Systems’ commercial SECCM option is promising for quantitative electroanalysis at the nanoscale, with potential applications in energy storage (battery) studies and corrosion research. [1] Gao, R., Edwards, M. A., Qiu, Y., Barman, K., & White, H. S. (2020). Visualization of Hydrogen Evolution at Individual Platinum Nanoparticles at a Buried Interface. Journal of the American Chemical Society. [2] Shi, W., Goo, D., Jung, G., Pascual, G., Kim, B., & Lee, K. Simultaneous Topographical and Electrochemical Mapping using Scanning Ion Conductance Microscopy–Scanning Electrochemical Microscopy (SICM-SECM). Figure 1