A highly sensitive nanopore platform for measuring RNase A activity.

Ribonuclease A (RNase A) plays significant roles in several physiological and pathological conditions and can be used as a valuable diagnostic biomarker for human diseases such as myocardial infarction and cancer. Hence, it is of great importance to develop a rapid and cost-effective method for the highly sensitive detection of RNase A. The significance of RNase A assay is further enhanced by the growing attention from the biotechnology and pharmaceutical industries to develop RNA-based vaccines and drugs in large part as a result of the successful development of mRNA vaccines in the COVID-19 pandemic. Herein, we report a label-free method for the detection of RNase A by monitoring its proteolytic cleavage of an RNA substrate in a nanopore. The method is ultra-sensitive with the limit of detection reaching as low as 30 fg per milliliter. Furthermore, sensor selectivity and the effects of temperature, incubation time, metal ion, salt concentration on sensor sensitivity were also investigated.

Probe-assisted detection of Fe3+ ions in a multi-functionalized nanopore.

Iron is an essential element that plays critical roles in many biological/metabolic processes, ranging from oxygen transport, mitochondrial respiration, to host defense and cell signaling. Maintaining an appropriate iron level in the body is vital to the human health. Iron deficiency or overload can cause life-threatening conditions. Thus, developing a new, rapid, cost-effective, and easy to use method for iron detection is significant not only for environmental monitoring but also for disease prevention. In this study, we report an innovative Fe3+ detection strategy by using both a ligand probe and an engineered nanopore with two binding sites. In our design, one binding site of the nanopore has a strong interaction with the ligand probe, while the other is more selective toward interfering species. Based on the difference in the number of ligand DTPMPA events in the absence and presence of ferric ions, micromolar concentrations of Fe3+ could be detected within minutes. Our method is selective: micromolar concentrations of Mg2+, Ca2+, Cd2+, Zn2+, Ni2+, Co2+, Mn2+, and Cu2+ would not interfere with the detection of ferric ions. Furthermore, Cu2+, Ni2+, Co2+, Zn2+, and Mn2+ produced current blockage events with quite different signatures from each other, enabling their simultaneous detection. In addition, simulated water and serum samples were successfully analyzed. The nanopore sensing strategy developed in this work should find useful application in the development of stochastic sensors for other substances, especially in situations where multi-analyte concurrent detection is desired.

Nanopore Single-molecule Analysis of Biomarkers: Providing Possible Clues to Disease Diagnosis.

Biomarker detection has attracted increasing interest in recent years due to the minimally or non-invasive sampling process. Single entity analysis of biomarkers is expected to provide real-time and accurate biological information for early disease diagnosis and prognosis, which is critical to the effective disease treatment and is also important in personalized medicine. As an innovative single entity analysis method, nanopore sensing is a pioneering single-molecule detection technique that is widely used in analytical bioanalytical fields. In this review, we overview the recent progress of nanopore biomarker detection as new approaches to disease diagnosis. In highlighted studies, nanopore was focusing on detecting biomarkers of different categories of communicable and noncommunicable diseases, such as pandemic Covid-19, AIDS, cancers, neurologic diseases, etc. Various sensitive and selective nanopore detecting strategies for different types of biomarkers are summarized. In addition, the challenges, opportunities, and direction for future development of nanopore-based biomarker sensors are also discussed.

Nanopore discrimination and sensitive plasma detection of multiple natriuretic peptides: The representative biomarker of human heart failure.

Natriuretic peptides can relieve cardiovascular stress and closely related to heart failure. Besides, these peptides also have preferable interactions of binding to cellular protein receptors, and subsequently mediate various physiology actions. Hence, detection of these circulating biomarkers could be evaluated as a predictor ("Gold standard") for rapid, early diagnosis and risk stratification in heart failure. Herein, we proposed a measurement to discriminate multiple natriuretic peptides via the peptide-protein nanopore interaction. The nanopore single-molecular kinetics revealed that the strength of peptide-protein interactions was in the order of ANP > CNP > BNP, which was demonstrated by the simulated peptide structures using SWISS-MODEL. More importantly, the peptide-protein interaction analyzing also allowed us to measure the peptide linear analogs and structure damage in peptide by single-chemical bond breakup. Finally, we presented an ultra-sensitive detection of plasma natriuretic peptide using asymmetric electrolyte assay, obtaining a detection limit of ∼770 fM for BNP. At approximately, it is 1597 times lower than that of using symmetric assay (∼1.23 nM), 8 times lower than normal human level (∼6 pM), and 13 times lower than the diagnostic values (∼10.09 pM) complied in the guideline of European Society of Cardiology. That said, the designed nanopore sensor is benefit for natriuretic peptides measurement at single molecule level and demonstrates its potential for heart failure diagnosis.

Simultaneous Dual-Site Identification of 5mC/8oG in DNA Triplex Using a Nanopore Sensor.

DNA triplex participates in delivering site-specific epigenetic modifications critical for the regulation of gene expression. Among these marks, 5mC with 8oG functions comprehensively on gene expression. Recently, few research studies have emphasized the necessity of incorporation detection of 5mC with 8oG using one DNA triplex at the same time. Herein, DNA triplex structure was designed and tailored for the site-specific identification of 5mC with 8oG by means of nanopore electroanalysis. The identification was associated with the distinguishable current modulation types caused by DNA unzipping through the nanopore in an electrical field. Results demonstrated that the epigenetic modification proximity to the latch zone or constriction area of the nanopore enables differentiation of modification series at single nucleotide resolution in one DNA triplex, at both physiological and mildly acidic environment. In addition, our nanopore method enables the kinetic and thermodynamic studies to calculate the free energy of modified DNA triplex with applied potentials. Gibbs' energy provided the direct evidence that the DNA triplex with these epigenetic modifications is more stable in acidic environment. Considering modified DNA functions significantly in gene expression, the presented method may provide future opportunities to understand incorporating epigenetic mechanisms of many dysregulated biological processes on the basis of accurate detection.

Chemistry solutions to facilitate nanopore detection and analysis.

Characteristic ionic current modulations will be produced in a single molecule manner during the communication of individual molecules with a nanopore. Hence, the information regarding the length, composition, and structure of a molecule can be extracted from deciphering the electrical message. However, until now, achieving a satisfactory resolution for observation and quantification of a target analyte in a complex system remains a nontrivial task. In this review, we summarize the progress and especially the recent advance in utilizing chemistry solutions to facilitate nanopore detection and analysis. The discussed chemistry solutions are classified into several major categories, including covalent/non-covalent chemistry, redox chemistry, displacement chemistry, back titration chemistry, chelation chemistry, hydrolysis-chemistry, and click chemistry. Considering the significant success of using chemical reaction-assisted nanopore sensing strategies to improve sensor sensitivity & selectivity and to study various topics, other non-chemistry based methodologies can undoubtedly be employed by nanopore sensors to explore new applications in the interdisciplinary area of chemistry, biology, materials, and nanotechnology.

Nanoparticle-assisted detection of nucleic acids in a polymeric nanopore with a large pore size.

Rapid and accurate detection of nucleic acids is of paramount importance in many fields, including medical diagnosis, gene therapy and virus identification. In this work, by taking advantage of two DNA hybridization probes, one of which was immobilized on the surface of gold nanoparticles, while the other was free in solution, detection of short length nucleic acids was successfully achieved using a large size (20 nm tip diameter) polyethylene terephythalate (PET) nanopore. The sensor was sensitive and selective: DNA samples with concentrations at as low as 0.5 nM could be detected within minutes and the number of mismatches can be discerned from the translocation frequency. Furthermore, the nanopore can be repeatedly used many times. Our developed large-size nanopore sensing platform offers the potential for fieldable/point-of-care diagnostic applications.

Simultaneous detection of multiple proteases using a non-array nanopore platform.

Multiplexing methods which are capable of measurement of multiple analytes in a single assay are of great importance in many fields. The conventional strategy for simultaneous detection of multiple species is to construct a sensor array. Herein, we report an innovative multiplex multi-analyte detection platform in a non-array format for protease measurement. By monitoring protease degradation of a single peptide substrate containing two cleavage sites for a disintegrin and metalloproteinase 10 (ADAM10) and a disintegrin and metalloproteinase 10 (ADAM17) in a single nanopore, simultaneous detection and quantification of these two model proteases in mixture samples could satisfactorily be accomplished. Our developed multiplexing sensing platform has the potential to be coupled with the traditional sensor array to further improve the multiplexing capability of the sensor, which may find useful applications in clinical diagnosis and prognosis.

Enzyme Hinders HIV-1 Tat Viral Transport and Real-Time Measured with Nanopores.

HIV-1 Tat protein, an intercellular transporter with a determinant function of delivering "information-rich" molecules in viral multiplication, was tryptic-hydrolyzed and real-time single molecule-monitored in a transmembrane pore. The electrokinetic studies revealed the catalytic and inhibitory effects on enzymatic digestion associated with Ca2+ and Cu2+ ions, respectively, in response to binding interactions with trypsin. Our strategy permits accurate and distinguishable sensing of Ca2+ and Cu2+via an enzyme assay. In addition, considering the closer mimic of the real situation of HIV spread, measurements in the serum and on cells were also investigated. Transmembrane current measurements together with fluorescence microscopy imaging indicated the potential to perturb the Tat transport in the serum environment and on cells. Because the involved Tat proteolysis should prevent the occurrence of viral delivery, the presented method probably enables efficient hindrance to HIV-1 infection, in complementary to current traditional treatments.

Nanopore Stochastic Sensing Based on Non-covalent Interactions.

A variety of species could be detected by using nanopores engineered with various recognition sites based upon non-covalent interactions, including electrostatic, aromatic, and hydrophobic interactions. The existence of these engineered non-covalent bonding sites was supported by the single-channel recording technique. The advantage of the non-covalent interaction-based sensing strategy was that the recognition site of the engineered nanopore was not specific for a particular molecule but instead selective for a class of species (e.g., cationic, anionic, aromatic, and hydrophobic). Since different species produce current modulations with quite different signatures represented by amplitude, residence time, and even characteristic voltage-dependence curve, the non-covalent interaction-based nanopore sensor could not only differentiate individual molecules in the same category but also enable differentiation between species with similar structures or molecular weights. Hence, our developed non-covalent interaction-based nanopore sensing strategy may find useful application in the detection of molecules of medical and/or environmental importance.

Analysis with biological nanopore: On-pore, off-pore strategies and application in biological fluids.

Inspired from ion channels in biology, nanopores have been developed as promising analytical tools. In principle, nanopores provide crucial information from the observation and analysis of ionic current modulations caused by the interaction between target analytes and fluidic pores. In this respect, the biological, chemical and physical parameters of the nanopore regime need to be well-understood and regulated for intermolecular interaction. Because of well-defined molecular structures, biological nanopores consequently are of a focal point, allowing precise interaction analysis at single-molecule level. In this overview, two analytical strategies are summarized and discussed accordingly, upon the challenges arising in case-dependent analysis using biological nanopores. One kind of strategies relies on modification, functionalization and engineering on nanopore confined interface to improve molecular recognition sites (on-pore strategies); The other kind of highlighted strategies concerns to measurement of various chemistry/biochemistry based interactions triggered by employed molecular agents or probes (off-pore strategies). In particularly, a few recent paradigms using these strategies for practical application of accurate analysis of biomarkers in biological fluids are illustrated. To end, the challenging and future outlook of using analytical tools by means of biological nanopores are depicted.

Nanopore detection of metal ions: Current status and future directions.

In this review, we highlight recent research efforts that aimed at developing nanopore sensors for detection of metal ions, which play a crucial role in environmental safety and human health. Protein pores use three stochastic sensing-based strategies for metal ion detection. The first strategy is to construct engineered nanopores with metal ion binding sites, so that the interaction between the target analytes and the nanopore can slow the movement of metal ions in the nano-channel. Second, large molecules such as nucleic acids and especially peptides could be utilized as external selective molecular probes to detect metal ions based on the conformational change of the ligand molecules induced by the metal ion-ligand chelation / coordination interaction. Third, enzymatic reactions can also be used as an alternative to the molecule probe strategy in the situation that a sensitive and selective probe molecule for the target analyte is difficult to obtain. On the other hand, by taking advantage of steady-state analysis, synthetic nanopores mainly use two strategies (modification and modification-free) to detect metals. Given the advantages of high sensitivity & selectivity, and label-free detection, nanopore-based metal ion sensors should find useful application in many fields, including environmental monitoring, medical diagnosis, and so on.

Joint Entropy-Assisted Graphene Oxide-Based Multiplexing Biosensing Platform for Simultaneous Detection of Multiple Proteases.

Due to the limited clinical utility of individual biomarkers, there is growing recognition of the need for combining multiple biomarkers as a panel to improve the accuracy and efficacy of disease diagnosis and prognosis. The conventional method to detect multiple analyte species is to construct a sensor array, which consists of an array of individual selective probes for different species. In this work, by using cancer biomarker matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs) as model analytes and functionalized nanographene oxide (nGO) as a sensing element, we developed a multiplexing fluorescence sensor in a nonarray format for simultaneous measurement of the activities of multiple proteases. The constructed nGO-based biosensor was rapid, sensitive, and selective and was also utilized for the successful profiling of ADAMs/MMPs in simulated serum samples. Furthermore, we showed that joint entropy and programming could be utilized to guide experiment design, especially in terms of the selection of a subset of proteases from the entire MMPs/ADAMs family as an appropriate biomarker panel. Our developed nGO-based multiplex sensing platform should find useful application in early cancer detection and diagnosis.

Chemically functionalized conical PET nanopore for protein detection at the single-molecule level.

Proteins are essential for all living organisms, and perform a wide variety of functions in the cell and human body, including structural, mechanical, biochemical, and signaling. Since proteins can serve as valuable biomarkers for health status and diseases states, and enable personalized medicine, sensitive and rapid detection of proteins is of paramount importance. Herein, we report a chemically functionalized conical shaped poly-(ethylene terephthalate) nanopore (PET nanopore) as a stochastic sensing element for detection of proteins at the single-molecule level. We demonstrate that the PET nanopore sensor is not only sensitive and selective, but also can differentiate proteins rapidly, offering the potential for label-free protein detection and characterization. Our developed PET nanopore sensing strategy not only provides a general platform for exploring fundamental protein dynamics and rapid detection of proteins at the single-molecule level, but also opens new avenues toward advanced deeper understanding of enzymes, development of more efficient biosensing technologies, and constructing novel biomimetic nanopore systems.

Single-molecule Study on the Interactions between Cyclic Nonribosomal Peptides and Protein Nanopore.

Nonribosomal peptides (NRPs) are a type of secondary metabolites mostly originated from microorganisms such as bacteria and fungi. Their proteolytic stability, highly selective bioactivity, and microorganism-specificity have made them an attractive source of drugs for the pharmaceutical industry. Herein, with microcystins (MCs) as a NRP model, we, for the first time, proposed a sensitive method to study the interactions between NRPs and the protein nanopore. Due to the large molecular size (~3 nm diameter) of MCs and their net negative charges, MCs failed to translocate through the α-hemolysin (α-HL) protein channel. Our results demonstrated that the biomolecular interaction of MC-α-HL protein was significantly affected by the applied potential bias. The constant blockage amplitude in the voltage-dependent studies indicated that the current modulation events were dominantly contributed to the bumping interaction between MCs and the α-HL protein under the electrophoretic force. The mean residence time of the bumping events exhibited a two-stage decrease (from 1.90 ms to 1.02 ms, and from 1.02 ms to 0.69 ms) at the threshold voltages of -70 mV and -100 mV, respectively. Using our strategy (i.e., based on their electrophoretic driven interaction with the α-HL protein pore), discrimination of different MC molecules (MC-LR, MC-RR, MC-YR and linear analog) with varied branched residues could be accomplished. This work should provide an insight in developing a rapid and effective method for the identification of cyclic NRPs as valuable biomarkers for fungal infections.

Enzymatic reaction-based nanopore detection of zinc ions.

We report a label-free nanopore sensor for the detection of Zn2+ ions. By taking advantage of the cleavage of a substrate peptide by zinc-dependent enzymes, nanomolar concentrations of Zn2+ ions could be detected within minutes. Furthermore, structurally similar transition metals such as Ni2+, Co2+, Hg2+, Cu2+, and Cd2+ did not interfere with their detection. The enzymatic reaction-based nanopore sensing strategy developed in this work may find potential applications in environmental monitoring and medical diagnosis.

Slowing down DNA translocation velocity using a LiCl salt gradient and nanofiber mesh.

Solid-state nanopores are considered an attractive basis for single-molecule DNA sequencing. At present, one obstacle to be overcome is the improvement of their temporal resolution, with the DNA molecules remaining in the sensing volume of the nanopore for a long period of time. Here, we used a composite system of a concentration gradient of LiCl in solution and a nanofiber mesh to slow the DNA perforation speed. Compared to different alkali metal solutions with the same concentration, LiCl can extend the dwell time to 20 ms, five times longer than NaCl and KCl. Moreover, as the concentration gradient increases, the dwell time can be tuned from dozens of milliseconds to more than 100 ms. When we introduce a nanofiber mesh layer on top of the pore in the asymmetric solution, the DNA molecules get retarded by 162-185 [Formula: see text]s/nt, which is three orders of magnitude slower than the bare nanopore. At the same time, because the molecule absorption region becomes larger at the pore vicinity, the higher molecule capture rate improves the detection efficiency.

Rapid and sensitive detection of the activity of ADAM17 using a graphene oxide-based fluorescence sensor.

A disintegrin and metalloproteinase 17 (ADAM17) has become a novel biomarker and potential therapeutic target for the early detection and treatment of human cancers. In this work, by covalently attaching fluorescently labeled ADAM17 substrate peptide (Pep-FAM) molecules to carboxylated graphene oxide (cGO) and monitoring the cleavage of the peptide substrate by ADAM17, we developed a cGO-Pep-FAM fluorescence sensor for the rapid, sensitive and accurate detection of ADAM17. The sensor was highly sensitive with a detection limit of 17.5 picomolar. Furthermore, the sensor was selective: structure similar proteases such as ADAM9 and MMP-9 would not interfere with ADAM17 detection. In addition, simulated serum samples were successfully analyzed. Our developed cGO-Pep-FAM sensing strategy should find useful applications in disease diagnosis and drug screening.

Salt-Mediated Nanopore Detection of ADAM-17.

ADAM-17 (a disintegrin and metalloproteinase 17) plays an important role in various physiological and pathophysiological processes. Overexpression/underexpression of ADAM-17 could lead to various diseases. In this work, by taking advantage of ionic strength and salt gradient, and monitoring the cleavage of a substrate peptide by ADAM-17 in a nanopore, we developed a label-free sensor for the rapid detection of ADAM-17. The sensor was highly sensitive and selective: picomolar concentrations of ADAM-17 could be detected within minutes, while structure similar proteases such as ADAM-9 and MMP-9 did not interfere with its detection. Our developed nanopore sensing strategy should find useful applications in the development of nanopore sensors for other proteases of biological, pharmaceutical, and medical importance.

Displacement chemistry-based nanopore analysis of nucleic acids in complicated matrices.

To overcome the effect of other components of complicated biological samples on nanopore stochastic sensing, displacement chemical reaction was utilized to selectively extract the target nucleic acid from whole blood. Given its simplicity and high sensitivity for detecting nucleic acids, our developed displacement chemistry-based nanopore sensing strategy offers the potential for fieldable/point-of-care diagnostic applications.