Ionic Current Response Research Articles

A bioinspired heterostructured nanochannel based on dendrimer-gold network and aluminum oxide arrays for sensitive detection of miRNA

BackgroundMicroRNAs, as oncogenes or tumor suppressors, enable to up or down-regulate gene expression during tumorigenesis. The detection of miRNAs with high sensitivity is crucial for the early diagnosis of cancer. Inspired by biological ion channels, artificial nanochannels are considered as an excellent biosensing platform with relatively high sensitivity and stability. The current nanochannel biosensors are mainly based on homogeneous membranes, and their monotonous structure and functionality limit its further development. Therefore, it is necessary to develop a heterostructured nanochannel with high ionic current rectification to achieve highly sensitive miRNA detection. ResultsIn this work, an asymmetric heterostructured nanochannel constructed from dendrimer-gold nanoparticles network and anodic aluminum oxide are designed through an interfacial super-assembly method, which can regulate ion transport and achieve sensitive detection of target miRNA. The symmetry breaking is demonstrated to endow the heterostructured nanochannels with an outstanding ionic current rectification performance. Arising from the change of surface charges in the nanochannels triggered by DNA cascade signal amplification in solution, the proposed heterogeneous nanochannels exhibits excellent DNA-regulated ionic current response. Relying on the nucleic acid's hybridization and configuration transformation, the target miRNA-122 associated with liver cancer can be indirectly quantified with a detection limit of 1 fM and a wide dynamic range from 1 fM to 10 pM. The correlation fitting coefficient R2 of the calibration curve can reach to 0.996. The experimental results show that the method has a good recovery rate (98%–105 %) in synthetic samples. SignificanceThis study reveals how the surface charge density of nanochannels regulate the ionic current response in the heterostructured nanochannels. The designed heterogeneous nanochannels not only possess high ionic current rectification property, but also enable to induce superior transport performance by the variation of surface chemistry. The proposed biosensor is promising for applications in early diagnosis of cancers, life science research, and single-entity electrochemical detection.

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.

Detection of Unfolded Cellular Proteins Using Nanochannel Arrays with Probe‐Functionalized Outer Surfaces

AbstractNanochannel technology has emerged as a powerful tool for label‐free and highly sensitive detection of protein folding/unfolding status. However, utilizing the inner walls of a nanochannel array may cause multiple events even for proteins with the same conformation, posing challenges for accurate identification. Herein, we present a platform to detect unfolded proteins through electrical and optical signals using nanochannel arrays with outer‐surface probes. The detection principle relies on the specific binding between the maleimide groups in outer‐surface probes and the protein cysteine thiols that induce changes in the ionic current and fluorescence intensity responses of the nanochannel array. By taking advantage of this mechanism, the platform has the ability to differentiate folded and unfolded state of proteins based on the exposure of a single cysteine thiol group. The integration of these two signals enhances the reliability and sensitivity of the identification of unfolded protein states and enables the distinction between normal cells and Huntington's disease mutant cells. This study provides an effective approach for the precise analysis of proteins with distinct conformations and holds promise for facilitating the diagnoses of protein conformation‐related diseases.

Detection of Unfolded Cellular Proteins Using Nanochannel Arrays with Probe-Functionalized Outer Surfaces.

Nanochannel technology has emerged as a powerful tool for label-free and highly sensitive detection of protein folding/unfolding status. However, utilizing the inner walls of a nanochannel array may cause multiple events even for proteins with the same conformation, posing challenges for accurate identification. Herein, we present a platform to detect unfolded proteins through electrical and optical signals using nanochannel arrays with outer-surface probes. The detection principle relies on the specific binding between the maleimide groups in outer-surface probes and the protein cysteine thiols that induce changes in the ionic current and fluorescence intensity responses of the nanochannel array. By taking advantage of this mechanism, the platform has the ability to differentiate folded and unfolded state of proteins based on the exposure of a single cysteine thiol group. The integration of these two signals enhances the reliability and sensitivity of the identification of unfolded protein states and enables the distinction between normal cells and Huntington's disease mutant cells. This study provides an effective approach for the precise analysis of proteins with distinct conformations and holds promise for facilitating the diagnoses of protein conformation-related diseases.

Vancomycin sensing using a phenylboronic acid‐modified nanopore pipette

AbstractNanopore sensing measures the changes in charge and physical state around the nanopores as changes in ionic current. In this study, we performed vancomycin (VCM) sensing with nanopores modified via Au−S bonds with dithiobis(4‐butyrylamino‐m‐phenylboronic acid) (DTBA‐PBA), a derivative of phenylboronic acid (PBA) bearing a thiol. First, we modified a 3‐mm‐diameter Au electrode with a DTBA‐PBA self‐assembled monolayer (SAM) to confirm the VCM response of the PBA interface by cyclic voltammetry. DTBA‐PBA was then immobilized in the same manner on a nanopore pipette coated with an Au layer. We measured the VCM concentration from the change in ion current using the nanopore pipette. A VCM concentration‐dependent ionic current response was observed in the range of 0.01–1 mM. Many kinds of pharmaceuticals can bind to PBA; therefore, this method could be used for quick, easy, in situ quantification of not only VCM but also other pharmaceuticals with serious side effects.

Metal-Organic Framework Sub-Nanochannels Formed inside Solid-State Nanopore with Proton Ultra-High Selectivity.

Metal-Organic frameworks (MOFs) have the advantages of high porosity, angstrom-scale pore size, and unique structure. In this work, a kind of MOFs, UiO-66 and its derivatives (including aminated UiO-66-(NH2 )2 and sulfonated UiO-66-(NH-SAG)2 ), were constructed on the inner surface of solid-state nanopores for ultra-selective proton transport. UiO-66 and UiO-66-(NH2 )2 nanocrystal particles were in-situ grown at the orifice of glass nanopores firstly, which were used to investigate the ionic current responses in LiCl and HCl solutions when the monovalent anions (Cl- ) were unchanged. Compared with UiO-66-modifed nanopores, the aminated MOFs modification (UiO-66-(NH2 )2 ) can improve the proton selectivity obviously. However, when the UiO-66-(NH-SAG)2 nanopore is prepared by further post-modification with sulfo-acetic acid, lithium ions can hardly pass through the channel, and the interaction between protons and sulfonic acid groups can promote the transport of protons, thus achieving ultra-high selectivity to protons. This work provides a new way to achieve sub-nanochannels with high selectivity, which can widely be used in ion separation, sensing and energy conversion.

Electrolyte Concentration Dependence on Ionic Current Responses of Au Nanoparticles Irradiated with a Near-Infrared Laser Beam

Electrolyte Concentration Dependence on Ionic Current Responses of Au Nanoparticles Irradiated with a Near-Infrared Laser Beam

Ionic Signal Amplification of DNA in a Nanopore.

Ionic signal amplification is a key challenge for single-molecule analyses by solid-state nanopore sensing. Here, a permittivity gradient approach for amplifying ionic blockade characteristics of DNA in a nanofluidic channel is reported. The transmembrane ionic current response is found to change substantially through modifying the liquid permittivity at one side of a pore with an organic solvent. Imposing positive liquid permittivity gradients with respect to the direction of DNA electrophoresis, this study observes the resistive ionic signals to become larger due to the varying contributions of molecular counterions. On the contrary, negative gradients render adverse effects causing conductive ionic current pulses upon polynucleotide translocations. Most importantly, both the positive and negative gradients are demonstrated to be capable of amplifying the ionic signals by an order of magnitude with a 1.3-fold difference in the transmembrane liquid dielectric constants. This phenomenon allows a novel way to enhance the single-molecule sensitivity of nanopore sensing that may be useful in analyzing secondary structures and genome sequence of DNA by ionic current measurements.

Cover Picture: Electrochemical Detection of Tyrosinase in Cell Lysates at Functionalized Nanochannels via Amplifying of Ionic Current Response (Electroanalysis 6/2022)

Cover Picture: Electrochemical Detection of Tyrosinase in Cell Lysates at Functionalized Nanochannels via Amplifying of Ionic Current Response (Electroanalysis 6/2022)

Identifying Single-Stranded DNA by Tuning the Graphene Nanogap Size: An Ionic Current Approach.

Solid nanopore-based deoxyribonucleic acid (DNA) sequencing has led to low-cost, fast, reliable, controlled, and amplified or label-free and high-resolution recognition and identification of DNA nucleotides. Solid-state materials and biological nanopores have a low signal-to-noise ratio (SNR) and generally are too thick to read at single-nucleotide resolution. The issue with solid-state nanopores is that the DNA strands stick to the nanopore sides and on the surface during the translocation process. The coexistence of DNA nucleotides on the surface and the nanopore sides will complicate the ionic current signals, making nucleotide detection difficult. Therefore, different sized nanogaps can be promising to overcome some of these issues. Using all-atom molecular dynamics (MD) simulations, we have studied the translocation of single-stranded (ss) DNA through solid-state nanogaps embedded in a graphene membrane device. A nucleotide-specific DNA sequencing technique is proposed based on unique differences in the ionic current responses for all the four ssDNA nucleotides (dAMP16, dGMP16, dTMP16, and dCMP16). As the individual homogeneous ssDNA translocate through the nanogaps, characteristic changes are observed in the ionic current. Our results show that ssDNA nucleotides can translocate through the proposed graphene nanogap devices by applying an external electric field. In addition, the sticking issue can be resolved using graphene nanogaps during the ssDNA translocation processes. Therefore, the significant difference in ionic current sensitivity and the translocation event/time yielded by the graphene nanogap-based devices reveal possibilities for utilizing it for ultrafast nanogap-based DNA sequencing.

Numerical Analysis of Identification of Ion Species Using Nanochannels

Recently, ionic current responses in mico- and nanofluidic channels have attracted significant attention from both fundamental and application perspectives in various research fields. For single particle analysis, ionic current responses are often caused by interactions between electrolyte ions and target particles as well as the volume exclusion effect due to the transport of target particles. Therefore, the behavior of ions in narrow confined spaces is required to measure more precisely to obtain detailed information of particles. In this study, we propose a novel method to identify ion species in electrolyte solutions, using the ion selectivity of nanochannels. Applying an external potential on an electrolyte solution for a probe, which is separated from a sample solution by a nanochannel, ion concentrations are separately determined. As a result, it is found that electroosmotic flows have an important role to distinguish ion species, breaking a symmetry of ion transport phenomena that are dominated by diffusion and electrophoretic transport.

Electrochemical Detection of Tyrosinase in Cell Lysates at Functionalized Nanochannels via Amplifying of Ionic Current Response

AbstractTyrosinase is an important enzyme in the metabolism of melanin and catecholamine, as well as an important antigen in autoimmunity of vitiligo. However, it is still a challenge to develop a selective and sensitive sensing approach for monitoring of tyrosinase in cell environment. In this work, a sensing strategy based on ion current rectification induced by nanochannels was established to electrochemically detect inactive tyrosinase. Functionalized alumina nanochannels were decorated by tyramine as an electrochemical platform to selectively determine tyrosinase via chemically specific reaction between phenolic hydroxyl groups of tyramine and tyrosinase. On the other hand, Fe2+ was further bonded with dihydroxyl groups generated by tyrosinase in nanochannels to amplify charge changes, resulting in the enhancement on the detection sensitivity for tyrosinase. Based on this principle, the developed electrochemical sensor showed a good linearity towards tyrosinase from 2 to 50 U/mL with a detection limit down to 0.83 U/mL, which can fulfil the requirement for detection of tyrosinase in cells. Eventually, the present electrochemical sensor was successfully employed in the selective and sensitive determination of tyrosinase in cell lysates. The results suggested that the activity of tyrosinase in B16 cells was 1.7‐fold higher than that of Hela cells.

Application of Micropore Device for Accurate, Easy, and Rapid Discrimination of Saccharomyces pastorianus from Dekkera spp.

Traceability analysis, such as identification and discrimination of yeasts used for fermentation, is important for ensuring manufacturing efficiency and product safety during brewing. However, conventional methods based on morphological and physiological properties have disadvantages such as time consumption and low sensitivity. In this study, the resistive pulse method (RPM) was employed to discriminate between Saccharomyces pastorianus and Dekkera anomala and S. pastorianus and D. bruxellensis by measuring the ionic current response of cells flowing through a microsized pore. The height and shape of the pulse signal were used for the simultaneous measurement of the size, shape, and surface charge of individual cells. Accurate discrimination of S. pastorianus from Dekkera spp. was observed with a recall rate of 96.3 ± 0.8%. Furthermore, budding S. pastorianus was quantitatively detected by evaluating the shape of the waveform of the current ionic blockade. We showed a proof-of-concept demonstration of RPM for the detection of contamination of Dekkera spp. in S. pastorianus and for monitoring the fermentation of S. pastorianus through the quantitative detection of budding cells.

A Practical Electrochemical Nanotool for Facile Quantification of Amino Acids in Single Cell.

Though significant advances are made in the arena of single-cell electroanalysis, quantification of intracellular amino acids of human cells remains unsolved. Exemplified by l-histidine (l-His), this issue is addressed by a practical electrochemical nanotool synergizing the highly accessible nanopipette with commercially available synthetic DNAzyme. The fabricated nanotools are screened before operation of a single-use manner, and the l-His-provoked cleavage of the DNA molecules can be sensibly transduced by the ionic current rectification response, the intrinsic property of nanopipette governed by its interior surface charges. Regional distribution of cytosolic l-His level in human cells is electrochemically quantified for the first time, and time-dependent drug treatment effects are further revealed. This work unveils the possibility of electrochemistry for quantification of cytosolic amino acids of a spatial- and time-based manner and ultimately enables a better understanding of amino acid-involved events in living cells.

Recent advances in protein sequencing

Function or disfunction of proteins depends on the primary structures, and protein sequencing, which provides key information on protein related biological processes and disease, plays important roles in biological, biomedical, clinical research and application. To obtain the precise protein sequences, researchers developed different methods over the past few decades, and these methods include conventional methods and newly methods. The former includes Edman degradation and mass spectrometry (MS), and the latter includes single-molecule detection, nanopore and other lately developed techniques. In the 1960s, the classic Edman degradation was firstly developed for sequencing protein molecules from N-terminus using cyclic chemical reaction. Afterwards, solid-state, and gas-state Edman degradation was further developed that still plays a significant role in the modern technologies. This review discusses the principle and limits of Edman degradation. Moreover, we discussed advantages and shortcomings of MS-based approaches, which are the current standard methods for protein sequencing applications. Single-molecule approaches could bring revolution in proteomics, realizing high sensitivity for the low-abundance protein detection and single-cell proteomics. With the development of the single-molecule nucleic acid sequencing, four kinds of basic groups of DNA/RNA can be effectively detected using label-free or fluorescence labelling strategies. However, it is still a challenge to label and analyze all twenty kinds of amino acid residues. Moreover, sensitive optical detection has been utilized for high throughput protein sequencing using fluorescence labelling. In this approach, selected residues of peptides were labelled, and the C-terminus was anchored onto the glass substrate. N-terminus was degraded through Edman cycles. Finally, the sequence can be analyzed through the wide-field fluorescence signals. This method has potential of large-scale, sensitive, and parallel detection. We have discussed its principle and characteristic features in detail. Nanopore, including biological nanopore and solid-state nanopore, has been emerged as powerful technologies for protein sequencing. Nanopore can provide single-molecule sensing interface and controlled nano-confined space enabling ultimate sensitivity and high spatiotemporal resolution. The mechanism of nanopore-based technologies depends on the interaction of functional group and the nanopore, inducing the current modulations. The information of peptides can be obtained by monitoring the ionic current responses. Arrayed nanopores have potential of high-throughput detection at low-abundance. It is still in early stage of development and some challenges need to be addressed. As “finger-print” signal, Raman spectrum is an ideal candidate for protein sequencing. However, very weak signals can significantly restrict its application, especially at low concentration of target molecule. Surface enhanced Raman spectroscopy (SERS) can enhance the Raman signal to achieve the detection on the scale of a single molecule. Combination of the SERS and nanopore has demonstrated powerful capability of label-free detection of ten kinds of amino acids. Moreover, this method offers a new strategy for protein sequencing. Comparing with the weak Raman signal, fluorescence signals are more accessible, even on the level of single molecule. Several molecular dynamics (MD) simulations have been discussed to show possibility of fluorescence labelled protein sequencing within nanopore. Nevertheless, some drawbacks need to be addressed, especially the high-cost fabrication of nanopore and translocation of proteins through a pore. Specifically, this review also discusses the future challenges as well as summarize recent efforts to break the bottleneck of the current protein sequencing, promoting development of medical treatment, disease diagnosis and related fields.

Effect of Electrolyte Concentration on Cell Sensing by Measuring Ionic Current Waveform through Micropores.

Immunostaining has been widely used in cancer prognosis for the quantitative detection of cancer cells present in the bloodstream. However, conventional detection methods based on the target membrane protein expression exhibit the risk of missing cancer cells owing to variable protein expressions. In this study, the resistive pulse method (RPM) was employed to discriminate between cultured cancer cells (NCI-H1650) and T lymphoblastoid leukemia cells (CCRF-CEM) by measuring the ionic current response of cells flowing through a micro-space. The height and shape of a pulse signal were used for the simultaneous measurement of size, deformability, and surface charge of individual cells. An accurate discrimination of cancer cells could not be obtained using 1.0 × phosphate-buffered saline (PBS) as an electrolyte solution to compare the size measurements by a microscopic observation. However, an accurate discrimination of cancer cells with a discrimination error rate of 4.5 ± 0.5% was achieved using 0.5 × PBS containing 2.77% glucose as the electrolyte solution. The potential application of RPM for the accurate discrimination of cancer cells from leukocytes was demonstrated through the measurement of the individual cell size, deformability, and surface charge in a solution with a low electrolyte concentration.

Coordination of Ligand-Protected Metal Nanoclusters and Glass Nanopipettes: Conversion of a Liquid-Phase Fluorometric Assay into an Enhanced Nanopore Analysis.

We propose a unique concept for transforming the liquid-phase fluorometric assay into an enhanced nanopore analysis, which is based on the analyte binding-mediated changes in the surface chemistry of noble metal nanostructures in a confined space. In a proof-of-concept trial, the bovine serum albumin-protected gold nanoclusters (BSA-Au NCs) were designed as the sensing unit for biothiol determination. Through the specific interaction between biothiols and BSA-Au NCs, the validation system not only performed well in aqueous fluorescent detection but also can be developed into a more selective and sensitive nanopore sensor. In the confined space of the nanopore, the BSA-Au NC film with high density formed, and the addition of biothiols triggered the fluorescence enhancement as well as the ionic current response, hence leading to the construction of the dual-signal-output (fluorescence/ion current signal) system. The fluorescence signal proved that the ionic current change corresponded to the specific recognition process, improving the reliability of our nanopore method. Moreover, the ionic current response from the BSA-Au NC film can be used to quantify cysteine in a broad dynamic range of 0.001-1 pM with a detection limit as low as 1 fM. Such a strategy can be used to detect biothiols in complex biological fluids such as human serum. Therefore, the present work provided a new design strategy for a glass nanopipette sensor inspired by the principles of numerous and diverse fluorometric assays. It also sheds light on how the coupling of electrical and optical signals improves the accuracy, sensitivity, and selectivity of the glass nanopipette platform.

A Supersmall Single-Cell Nanosensor for Intracellular K + Detection

Intracellular potassium ions (K+) play pivotal roles in many physiological processes. Several K+ sensors have been developed for probing cellular K+ fluctuations. Nevertheless, the existing solutio...

Ultrasensitive and Label-Free Detection of Cell Surface Glycan Using Nanochannel-Ionchannel Hybrid Coupled with Electrochemical Detector.

In this work, asymmetric nanochannel-ionchannel of porous anodic alumina (PAA) coupled with electrochemical detector was used for sensitive and label-free detection of cell surface glycan. The amplified ionic current caused by array nanochannels as well as the ionic current rectification (ICR) caused by asymmetric geometry endows PAA with sensitive ionic current response. Functionalized with the special molecular probe, the constructed nanofluidic device can be used for selective recognition and detection of glycan in a real-time and label-free format. In addition, due to the subnanosize of ionchannels, the probe immobilization and glycan recognition is carried out on the outer surface of PAA, avoiding the blockage of PAA nanochannel by samples, which promises the reproducibility and accuracy of the present method toward bioanalysis. Results show that the glycan concentration ranging from 10 fM to 10 nM can be successfully detected with a detection limit of ∼10 aM, which is substantially lower than most previous works. The designed strategy provides a valuable platform for sensitive and label-free detection of cell surface glycan, which acts as a promising candidate in pathological research and cancer diagnosis.

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing