Programmable nanoreactor for stochastic sensing (pnrss)

Abstract

A system and method for characterizing a target analyte is provided. The system comprises a nanopore and a polymer chain, the polymer chain comprising a tethering site and a reaction segment, wherein the polymer chain is tethered through the tethering site such that the polymer chain cannot pass through the nanopore, and wherein the reaction segment comprises at least one sensing module that can interact with a single molecule of the target analyte.

Description

Technical Field

[0001] This invention relates to systems and methods for identifying analytes using nanopores. Background Technology

[0002] Although described and represented at the single-molecule level, chemical reactions are rarely monitored or characterized at this level; rather, they occur holistically. Current instruments, including scanning probe microscopy... 1 Tip / Surface Enhanced Raman Spectroscopy 2 Molecular structure 3 Single-molecule force spectroscopy 4 or biological nanopores 5 Few techniques can resolve the discrete steps of a chemical reaction in a single molecule. However, these existing techniques report different aspects of single-molecule properties. Scanning channel microscopy and molecular knot microscopy focus on the electronic states of the target molecule. Raman spectroscopy focuses on studying the vibrations of chemical bonds. And force spectroscopy measures the elasticity of the molecule.

[0003] Bio-nanopores, including α-hemolysin (α-HL) 6 Mycobacterium smegmatis porin A (MspA) 7 Aerosols 8 、CsgG 9 ClyA (cytolysin A) 10 fragaceatoxin C (FraC) 11 Pleurotolysin (PlyA / B) 12 Outer membrane protein G (OmpG) 13 phi29 connector 14 Others, such as [list of other proteins], are a class of large-channel proteins developed for single-molecule sensing. They can detect nucleic acids during the translocation of analytes through the constriction at the pore. 15 peptides 16 ,protein 17 and small molecules 18 The conformational characteristics. The MinION nanopore sequencer employs a similar measurement scheme. TM Able to directly report nucleic acid sequences 19 By Bayley et al. 5 The pioneering engineered α-HL nanopores possess unique fixed reaction sites within their cavities, enabling them to react with discrete, freely displaced reactants to form monomolecular reactors, thus resolving the binding of individual metal ions. 20-22 However, single-molecule chemical measurements of nanopores using α-HL typically report weak event amplitudes, with measured values ​​of 1–5 pA. 23This hinders its ability to achieve further finer resolution. Undesirable reaction sites uniformly distributed within the cylindrical cavity of the α-HL can also interfere with measurements, requiring excessive effort in pore engineering. 22 Most bio-nanopores exhibit oligomeric symmetry. 6 Therefore, a great deal of effort is required to generate heterogeneous oligomer assemblies to introduce unique reaction sites. 5 This is a niche technology mastered by only a few in the field. Nanoporous single-molecule chemical measurements using engineered homopolymeric porous proteins will inevitably report simultaneous binding from multiple reactants, making them unsuitable for event identification and quantification. 24-27 Modifications within the pore lumen can unpredictably interfere with pore assembly or stability, thus necessitating extensive protein screening. To introduce non-natural reactive sites into the pore lumen, semi-synthetic α-HL was prepared via natural chemical linking (NCL). 28,29 However, the preparation of nanopores via NCL requires extremely complex purification procedures, and its usability is not always guaranteed when engineering different sites or pore types. Other existing techniques utilize internal linkers within the pore cavity, such as cyclodextrins. 30,31 or protein 18,32 To gain new sensing capabilities, these methods require suitable adaptors. The presence of large enzyme adaptors can also result in undesirable resolution for distinguishing molecular analytes with minimal structural differences. To address these issues, technological breakthroughs are needed to enable the complete freedom to place reaction sites of any type, number, or spatial combination at any location within the pore. Summary of the Invention

[0004] A first aspect of the present invention provides a system for characterizing a target analyte, the system comprising:

[0005] Nanopores; and

[0006] Polymer chain, the polymer chain comprising chain-connecting sites and reactive segments,

[0007] The polymer chain is ligated through the ligation site, preventing the polymer chain from passing through the nanopore, and the reaction section includes at least one sensing module that can interact with a single molecule of the target analyte.

[0008] In some implementations, the reaction section includes two or more sensing modules that can interact with two or more different target analytes.

[0009] In some implementations, each sensing module consists of one, two or more sensing parts, and each sensing part can interact with one or two or more binding sites of a single molecule of the target analyte.

[0010] In some embodiments, the sensing portion is selected from the group consisting of any nucleotide base, any amino acid, 1,2,3-triazole, phenylboronic acid (PBA), or any combination thereof.

[0011] In some embodiments, at least one of the sensing modules consists of two adjacent purines selected from the group consisting of guanine and adenine.

[0012] In some embodiments, the reaction section is prepared by any one or any combination of the following methods:

[0013] a. Incorporating one or more monomers containing a sensing component into the reaction section;

[0014] b. Incorporating one or more monomers containing functional groups into the reaction section, and chemically modifying the functional groups into a sensing component; or

[0015] c. Incorporating one or more monomers comprising a first reactive stem into the reaction section, and causing the first reactive stem to react with a second reactive stem, the second reactive stem being connected to the sensing portion.

[0016] In some implementations, the first and second reaction handles are click reaction handles.

[0017] In some embodiments, the first and second reaction handles are selected from the group consisting of azides and alkynes.

[0018] In some embodiments, the polymer chain is linked to a barrier molecule or the nanoporous protein.

[0019] In some embodiments, the barrier molecule is a protein capable of specifically binding to a small molecule compound, the binding site contains the small molecule compound, and the polymer chain is bound to the barrier molecule through the specific binding of the small molecule compound to the protein.

[0020] In some embodiments, the blocking molecule is an antibody to streptavidin or a hapten, and the small molecule compound is biotin or the hapten.

[0021] In some embodiments, the linking site comprises a small molecule that can react with natural amino acids on the surface of the barrier molecule or the nanoporous protein, and the polymer chain is linked to the barrier molecule by the reaction between the small molecule compound and the natural amino acid.

[0022] In some embodiments, a first reactive stem is introduced onto the surface of the barrier molecule or the nanoporous protein, the tethering site includes a second reactive stem, and the polymer chain is tethered to the barrier molecule by a reaction between the first reactive stem and the second reactive stem.

[0023] The polymer chain further includes an extension segment, and the extension segment is configured to allow the reactive segment to be located in a region suitable for measuring blockage. In some embodiments, the polymer chain further includes an extension segment, and the extension segment is configured to allow the reactive segment to be located in a region suitable for measuring blockage.

[0024] In some embodiments, the polymer chain further includes a traction segment, and the traction segment is configured to hold the reactive segment in a region suitable for measuring blockage.

[0025] In some implementations, the traction section comprises any one of the following:

[0026] a. Polymer chains that tend to pass through the nanopore channels in an electric field applied to the nanopore;

[0027] b. Coupling sites that can react with natural amino acids on the surface of the channels of the nanopores;

[0028] c. A second reaction handle that can react with a first reaction handle introduced into the surface of the channel of the nanopore; or

[0029] d. Polymer chains that can pass through the channels of the nanopores and form a three-dimensional structure outside the nanopores with a size larger than the outlet of the nanopores.

[0030] In some implementations, the traction segment is a nucleic acid with a length of 10 nt or longer.

[0031] In some embodiments, the polymer chain is based on nucleic acids, nucleic acid analogs, peptides, polysaccharides, homopolymers, copolymers, or any combination thereof.

[0032] In some implementations, the target analyte is selected from the group consisting of:

[0033] Ions containing metallic elements; preferably ions containing alkaline earth metals or transition metals; more preferably AuCl4. - Mg 2+ Ca2+ Ba 2+ Ni 2+ Cu 2+ Co 2+ Zn 2+ Cd 2+ Ag 2+ or Pb 2+ ;

[0034] Monosaccharides; preferably ribose, fructose or mannose; more preferably D-(-)-ribose, D-fructose or D-(+)-mannose;

[0035] Oligosaccharides; preferably disaccharides or trisaccharides; more preferably 4-O-β-d-galactopyranosyl-d-fructofuranose (lactulose), 6-O-α-D-glucopyranosyl-D-fructofuranose (isomaltulose) or 4-ObD-galactosylsucrose (galactosylsucrose);

[0036] Polysaccharides;

[0037] Glucoside;

[0038] Polyphenols, such as anthocyanins or proanthocyanidins;

[0039] Catecholamines;

[0040] Catecholamine derivatives; preferably adrenaline, noradrenaline, or isoproterenol;

[0041] Polyols; preferably compounds containing two ortho-hydroxy groups, a 1,2-cis-diol or a 1,3-cis-diol moiety; more preferably 3,4-dihydroxymandelic acid, 4-hydroxy-3-methoxymandelic acid (VMA), 3,4-dihydroxyphenylacetic acid, catechol, ethylene glycol, glycerol, L-lactic acid or vitamins (e.g., vitamin C or vitamin B6);

[0042] The compound is in protonated or deprotonated form; preferably, tris is in protonated or deprotonated form.

[0043] Compounds containing a ribose moiety; preferably nucleotides, nucleosides, analogs thereof or their monophosphate derivatives or polyphosphate derivatives thereof; more preferably ribonucleotides, deoxyribonucleotides, galidesvir, ribavirin, favipiravir-RTP, remdesivir or their triphosphate metabolites, cytidine 5'-monophosphate (5'-CMP);

[0044] Hydrogen peroxide;

[0045] Oligopeptides or cyclic peptides;

[0046] Buffer reagent; preferably tris;

[0047] Small molecule drugs; preferably nucleoside analogs; more preferably galidesvir, ribavirin, favipiravir-RTP, remdesivir or their triphosphate metabolites;

[0048] Neurotransmitters; preferably catecholamines or their derivatives;

[0049] Compounds with specific chirality; preferably L-norepinephrine or D-norepinephrine;

[0050] Analytes containing isotopes; preferably catechol-D6 (deuterium-substituted for all hydrogen atoms on the catechol);

[0051] Chemical intermediates;

[0052] Or any combination thereof.

[0053] In some embodiments, the nanopore is a biological nanopore, a solid nanopore, or a DNA nanopore.

[0054] In some embodiments, the protein nanopore is a MspA, α-HL, aerolysin, ClyA, FhuA, FraC, PlyA / B, CsgG Phi 29 linker or its homolog or variant.

[0055] In some implementations, the system comprises two or more nanopores.

[0056] Another aspect of the present invention provides a method for characterizing a target analyte, the method comprising:

[0057] (i) providing a system according to any one of claims 1-22;

[0058] (ii) Apply a voltage between the two sides of the nanopore and allow a polymer chain to enter the nanopore;

[0059] (iii) Allowing target analytes to pass through the nanopores; and

[0060] (iv) Measure the ion current through the nanopore to provide a current pattern, and characterize the target analyte based on the current pattern.

[0061] In some embodiments, the polymer chain of the system includes two or more sensing modules that can interact with two or more different target analytes, and the method is used to characterize the two or more target analytes.

[0062] In some implementations, the method includes:

[0063] (i) providing a system according to any one of claims 1-22;

[0064] (ii) A first voltage is applied between the two sides of the nanopore and a polymer chain is allowed to enter the nanopore;

[0065] (iii) Allowing a first target analyte to pass through the nanopore; and

[0066] (iv) Measure the ion current through the nanopore to provide a current pattern, and characterize the first target analyte based on the current pattern;

[0067] (v) The voltage between the two compartments is converted into a second voltage that is opposite to the direction of the first voltage, thereby causing the polymer chains in the nanopore to exit from the nanopore;

[0068] (vi) Converting the voltage between the two compartments into the first voltage and allowing another polymer chain to enter the nanopore; and

[0069] (vi) Apply steps (iii)-(iv) to a second target analyte that is different from the first target analyte.

[0070] In some implementations, the sensing module is capable of irreversibly interacting with the first target analyte and / or the second target analyte.

[0071] In some implementations, the target analyte is selected from the group consisting of:

[0072] Ions containing metallic elements; preferably ions containing alkaline earth metals or transition metals; more preferably AuCl4. - Mg 2+ Ca 2+ Ba 2+ Ni 2+ Cu 2+ Co 2+ Zn 2+ Cd 2+ Ag 2+ or Pb 2+ ;

[0073] Monosaccharides; preferably ribose, fructose or mannose; more preferably D-(-)-ribose, D-fructose or D-(+)-mannose;

[0074] Oligosaccharides; preferably disaccharides or trisaccharides; more preferably 4-O-β-d-galactopyranosyl-d-fructofuranose (lactulose), 6-O-α-D-glucopyranosyl-D-fructofuranose (isomaltulose) or 4-ObD-galactosylsucrose (galactosylsucrose);

[0075] Polysaccharides;

[0076] Glucoside;

[0077] Polyphenols, such as anthocyanins or proanthocyanidins;

[0078] Catecholamines;

[0079] Catecholamine derivatives; preferably adrenaline, noradrenaline, or isoproterenol;

[0080] Polyols; preferably compounds containing two ortho-hydroxy groups, a 1,2-cis-diol or a 1,3-cis-diol moiety; more preferably 3,4-dihydroxymandelic acid, 4-hydroxy-3-methoxymandelic acid (VMA), 3,4-dihydroxyphenylacetic acid, catechol, ethylene glycol, glycerol, L-lactic acid or vitamins (e.g., vitamin C or vitamin B6);

[0081] Compounds in protonated or deprotonated form; preferably tris in protonated or deprotonated form;

[0082] Compounds containing a ribose moiety; preferably nucleotides, nucleosides, analogs thereof or their monophosphate derivatives or polyphosphate derivatives thereof; more preferably ribonucleotides, deoxyribonucleotides, galidesvir, ribavirin, favipiravir-RTP, remdesivir or their triphosphate metabolites, cytidine 5'-monophosphate (5'-CMP);

[0083] Hydrogen peroxide;

[0084] Oligopeptides or cyclic peptides;

[0085] Buffer reagent; preferably tris;

[0086] Small molecule drugs; preferably nucleoside analogs; more preferably galidesvir, ribavirin, favipiravir-RTP, remdesivir or their triphosphate metabolites;

[0087] Neurotransmitters; preferably catecholamines or their derivatives;

[0088] Compounds with specific chirality; preferably L-norepinephrine or D-norepinephrine;

[0089] Analytes containing isotopes; preferably catechol-D6 (deuterium-substituted for all hydrogen atoms on the catechol);

[0090] Chemical intermediates;

[0091] Or any combination thereof. Attached Figure Description

[0092] Figure 1This section presents a conceptual demonstration of PNRSS. a. PNRSS Chain. The PNRSS chain consists of the aforementioned functional modules. The reaction section is the most critical module, containing one or more reaction sites that form fixed reactants. b. Measurement Setup. During the PNRSS process, MspA nanopores are used to dock the PNRSS chain of streptavidin-based chains. The reaction section (dark yellow) is located precisely at the pore constriction for optimal performance. c. PNRSS for Detecting Ni 2+ Design of a single-molecule reaction between the PNRSS and diguanine reactants. Two adjacent guanines on the PNRSS chain co-bind Ni. 2+ Ions. d. Continuous traces containing different states measured by PNRSS. Initially, the hole is unoccupied (i), reporting the hole current. Then, the PNRSS chain is trapped by the hole, causing the blockage level to drop immediately to (ii). Upon insertion of Ni... 2+ Afterwards, I was observed b and I p The reversible transformation between them proves that the PNRSS chain did not interact with Ni. 2+ The state when combined with (iii) or combined with (iv). e. △I relative to t off Density scatter plot. The local density around each point is color-coded. A histogram of ΔI fitted using Gaussian is located to the right of the scatter plot. Results include 13435 events. f. Concentration dependence. The reciprocal of the interval between events (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off ) relative to [Ni 2+ Plot the graph. Perform measurements as described in the method. Apply PNRSS chain 13G / 14G (Table 1). Add nickel sulfate to the trans side at the desired final concentration.

[0093] Figure 2 PNRSS using non-natural reactive components is shown. a. Introduction of 1,2,3-triazole (TAZ) into the PNRSS chain and its reaction mechanism. The PNRSS chain 14TAK (Table 1) reacts with 3-azidopropylamine, as... Figure 17 As described in [the text]. The generated TAZ is used as a stationary reactant. Ni 2+ It is a mobile reactant that participates in the reversible coordination with TAZ. b. Trace representation. Contains Ni. 2+ Combine the continuous traces of events. In Ni 2+ Characteristic noise fluctuations were observed throughout the process. c. Representative events. Magnified display of the combined events. Ni 2+ Noise fluctuations during the binding process indicate the potential for reactive intermediates to be observed via PNRSS. d. ΔI relative to t offDensity scatter plot. Local density around each point is color-coded. Highly consistent event clusters were observed. Results include 905 events. e. Concentration dependence. The inverse of the interval between events (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off ) relative to [Ni 2+ Plot the graph (Table 6). Perform measurements as described in the method. Add nickel sulfate to the trans side at the desired final concentration.

[0094] Figure 3 The PNRSS using phenylboronic acid is shown. a. Introduction of phenylboronic acid (PBA) and the corresponding reaction mechanism. For example... Figure 24-26 As described in the method, PBA is introduced via CuAAC. b. PBA on 14PBA is used as a stationary reactant. Mobile reactants containing 1,2-diol or 1,3-diol, such as catechol (b), ethylene glycol (c), glycerol (d), L-lactic acid (e), vitamin C (f), or vitamin B6 (g), all reported clear and distinct binding events. h. Resorcinol lacking a compatible reactive group did not report any binding events. Experiments were performed as described in the method. Catechol (500 μM, b), ethylene glycol (18 mM, c), glycerol (12 mM, d), L-lactic acid (5 mM, e), vitamin C (1.6 mM, f), vitamin B6 (50 μM, g), or resorcinol (1 mM, h) were added to the trans side to reach the aforementioned final concentrations. Figures 27-38 The corresponding △I is provided relative to t. off Scatter plot and 1 / τ on and 1 / τ off The results are relative to histograms of different analyte concentrations and times.

[0095] Figure 4 Repeated PNRSS measurements of the irreversible reaction are shown. a. Introduction of PBA and the corresponding reaction mechanism. For example... Figure 24-26 As described above, PBA is introduced via CuAAC. PBA on the PNRSS chain serves as a stationary reactant. Hydrogen peroxide, as a mobile reactant, can reversibly bind to PBA or irreversibly oxidize PBA to phenol. b. Traces of reversible (i)-(ii) and irreversible (iii) reactions containing PBA. c. PNRSS strategy for dealing with irreversible reactions. Upon irreversible oxidation, the stationary PNRSS chain is voltage-popped and reloaded to initiate a new measurement cycle. d. Traces containing repeated PNRSS measurements. Four consecutive measurement cycles are shown. Star labels mark the moments when voltage popping and reloading occur (Video 3).

[0096] Figure 5PNRSS sensing of adrenaline, noradrenaline, and isoproterenol is shown. a. Reaction mechanism. Noradrenaline, adrenaline, and isoproterenol all contain a 1,2-phenylenediol moiety, which can bind PBA. b. Representative events of adrenaline, noradrenaline, or isoproterenol binding. Events are low-pass Butterworth filtered with a cutoff frequency of 100Hz. Figure 50 All events are negative (I) b p cd. A representative trace containing norepinephrine, epinephrine, or isoproterenol binding events. The trace is a Butterworth filter, divided into low-pass (c) and high-pass (d) sections. The cutoff frequency is 100Hz. Figure 50 e. Generate a confusion matrix based on 1455 events input to the SVC model. Figure 51 f. Scatter plot of the standard deviation (SD) of low-pass (Lp) and high-pass (Hp) amplitudes from PNRSS sensor catecholamine mixture events. Figure 52 The results include 150 events. The decision boundaries for dividing the scatter plot into green (norepinephrine), pink (epinephrine), and orange (isoproterenol) color-coded regions were determined using a machine learning algorithm. Figure 51 ).

[0097] Figure 6 The PNRSS sensing of remdesivir and its triphosphate metabolite is shown. a. Sensing mechanism. Both remdesivir and its metabolite contain a ribose moiety that can bind to PBA. b. Representative events of remdesivir and its metabolite. Remdesivir and its metabolite are labeled R and M, respectively. Butterworth low-pass filtering with a cutoff frequency of 100 Hz was applied to the events. Figure 50 Both types of events appear to be positive (I). b >I p The binding of remdesivir results in events with prolonged durations of characteristic noise fluctuations. On the other hand, remdesivir metabolites report transient events with minimal noise. c. Scatter plot of event amplitude standard deviation (SD) relative to event dwell time. The histogram of event amplitude standard deviation (SD) and its Gaussian fit are plotted on the right. Results include 119 events. d. Scatter plot of high-pass (Hp) and low-pass (Lp) amplitude SD. The two analytes are clearly separated in the scatter plot. Results include 126 events. e. Continuous traces containing binding events from remdesivir and remdesivir metabolites. The identifier for each event is invoked based on the event characteristics described in d.

[0098] Figure 7 Ni was shown 2+ ​Binding with diguanine reactants. a. Schematic diagram. (See diagram below.) Figure 1 PNRSS measurements were performed as described in section c. A buffer solution of 1.5 M KCl, 10 mM HEPES, and pH 7.0 was used. A potential of +180 mV was continuously applied. The PNRSS chain 13G / 14G (Table 1) contains two adjacent guanines, which synergistically act as binding Ni. 2+ The ligand. Ni is added to the trans side as a mobile reactant. 2+ a. Achieve the desired final concentration. 2+ Representative traces obtained from concentration. Ni 2+ The concentration was adjusted between 0 and 1 mM and marked on the left side of each corresponding trace, indicating that when Ni 2+ The incidence of events increases with increasing concentration.

[0099] Figure 8 The PNRSS without a fixed reactant is shown. a. Schematic diagram. Measurements were performed as described in the method. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 7.0 was used. A potential of +180 mV was continuously applied. PNRSS chain 14X (Table 1) was applied, where the reaction segment consists of five consecutive base-free sites. Ni 2+ As a mobile reactant. In principle, no base site can bind Ni. 2+ b. Representative traces from the corresponding PNRSS measurements. Ni is added to the trans side. 2+ The final concentration was 0-1 mM. Ni was labeled to the left of each corresponding trace. 2+ The final concentration of Ni was not observed. 2+ Based on the events, the conclusion is that the PNRSS chain 14X did not report any Ni. 2+ Combine the events.

[0100] Figure 9 Ni was shown 2+ Binding with diguanine reactants (simulated). Ni 2+ Optimized structures bound to bisguanine ligands. (dGMP)2-Ni-4wt with (a) low-spin state and (c) high-spin state, and (b) low-spin state and (d) high-spin state, are shown. Water molecules involved in the binding are also shown. Green, gray, blue, red, orange, and white spheres represent Ni, C, N, O, P, and H atoms, respectively.

[0101] Figure 10The PNRSS measurement and data analysis are shown. a. Representative traces of the PNRSS measurement. The animation illustrates the different states of the hole during the measurement process. State (i) represents an unoccupied hole, in which the measured current is the hole current (I0). States (ii) and (iii) represent holes occupied by the PNRSS chain, where the fixed reactants of the PNRSS chain do not bind to (ii) or bind to the mobile reactants in (iii). The measured current in states (ii) or (iii) is defined as I... p Or I b b. Enlarged view of the trace containing the binding events. The continuous binding events, displayed as resistance pulses (gray), are clearly observed. The event amplitude (ΔI) is defined as ΔI = I... b -I p The binding of mobile reactants may produce a positive (I) reaction. b >I p ) or negative (I b p The event, t, generates a positive or negative ΔI value. on ) and event dwell time (t) off The definition of ) is as described on the trace. cd. Average inter-event interval (τ) on (c) and average event dwell time (τ) off The derivation of (d). t on and t off We fit the data using a single exponential function y = a*exp(-x / τ) to obtain the average inter-event interval (τ). on ) and average event dwell time (τ) off e. Derivation of the average event magnitude The result originates from the center position of the Gaussian fit. Figure 1 The measurement results described herein use PNRSS chains 13G / 14G (Table 1) and Ni 2+ Performed. Unless otherwise stated, all analyses of PNRSS measurements in this document were performed according to the definitions stated herein.

[0102] Figure 11 Ni was shown 2+ τ bound to diguanine reactants on and τ off It presents the use of different Ni 2+ The inter-event interval (t) obtained from the concentration on ) and event dwell time (t) off The histograms of the events were obtained. All histograms were fitted using a single exponential function y = a*exp(-x / τ) to derive the average inter-event interval (τ). on ) and average event dwell time (τ) off ​), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chains 13G / 14G (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 7.0. Continuously apply a +180mV potential.

[0103] Figure 12 The binding of other metal ions to diguanine reactants is shown. a. Schematic diagram. PNRSS measurement and Figure 1 Similar to that described in c. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 7.0 was used. A +180 mV potential was continuously applied. Two adjacent guanines on the PNRSS chain (13G / 14G) were used as immobilization reactants (Table 1). Divalent ions such as Zn were also used. 2+ Cd 2+ Co 2+ or Cu 2+ Used as a mobile reactant. (be. when Zn) 2+ (b) Cd 2+ (c) Co 2+ (d) or Cu 2+ (e) Representative traces measured by PNRSS when placed on the trans side. The final concentration of the added divalent ion is indicated to the left of each corresponding trace. Based on the results, Zn 2+ (b) or Cd 2+ (c) The report was not linked to the PNRSS chain. However, Co 2+ (d) or Cu 2+ (e) shows a clear binding event. The above results indicate that Co can also be observed using PNRSS. 2+ or Cu 2+ Binding with diguanine ligand 9 Further research will be conducted in a separate follow-up study.

[0104] Figure 13 The PNRSS measurement using a single adenine reactant is shown. a. Schematic diagram. The measurement was performed as described in the method. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 7.0 was used. A +180 mV potential was continuously applied. PNRSS chain 14A (Table 1) was applied. 14A contains a single adenine and was used as the immobilization reactant. Ni 2+ Used as a mobile reactant. b. Adding different final concentrations of Ni to the trans side. 2+ Representative traces at that time. Ni 2+ The concentration is indicated on the left side of each trace. Ni 2+ Binding to a single adenine produces a sharp, negative event. c.1 / τ on or 1 / τ offCompared to Ni 2+ A graph of concentration. 1 / τ on with Ni 2+ The final concentration is linearly correlated. However, 1 / τ off d. ΔI relative to t off Scatter plot of events. Ni 2+ The concentration was 0.8 mM. All events were extracted from traces recorded continuously over 15 minutes. The scatter plot included 1137 events. Each point was color-coded based on its local point density. A major population of approximately ~39 pA and a minor population of approximately ~56 pA in ΔI were identified. The event histogram for ΔI is appended to the right of the scatter plot. The two populations in ΔI were Gaussian fitted separately and their histograms are superimposed. These observations demonstrate that Ni can be observed using PNRSS. 2+ Coordination interactions between adenine and individual adenine. Different choices of immobilized reactants lead to different binding kinetics. Based on previous studies, adenine has two possible binding sites to bind Ni. 2+ This may help explain the observation of two event groups. 92 .

[0105] Figure 14 Ni was shown 2+ τ bound to a single adenine reactant on and τ off It presents the use of different Ni 2+ Concentration inter-event interval (t) on ) and event dwell time (t) off The histograms of the events were obtained. All histograms were fitted using a single exponential function y = a*exp(-x / τ) to derive the average inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14A (Table 1). Use a buffer solution of 1.5 M KCl, 10 mM HEPES, pH 7.0. Continuously apply a +180 mV potential.

[0106] Figure 15 The PNRSS measurement using a single guanine reactant is shown. a. Schematic diagram. The measurement was performed as described in the method. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 7.0 was used. A +180 mV potential was continuously applied. The PNRSS chain 14G (Table 1) was applied. 14G contains a single guanine and was used as the immobilizing reactant. Ni 2+ Used as a mobile reactant. b. Adding different final concentrations of Ni to the trans side. 2+ Representative traces at that time. Ni 2+The concentrations are marked on the left side of each trace. Ni was observed. 2+ The combined events are spike and negative events. c.1 / τ on or 1 / τ off Compared to Ni 2+ A graph of concentration. 1 / τ on with Ni 2+ The final concentration is linearly correlated. However, 1 / τ off d. ΔI relative to t off Scatter plot of events. Ni 2+ The concentration was 0.8 mM. All events were extracted from traces recorded continuously over 15 minutes. The scatter plot included 1773 events. Each point was color-coded based on its local point density. Individual event clusters were identified, with ~30 pA measured in ΔI. The event histogram of ΔI, superimposed with its Gaussian fit, was appended to the right of the scatter plot. These observations demonstrate that Ni can be observed using PNRSS. 2+ Coordination interactions with guanine alone. Different choices of immobilized reactants lead to different binding kinetics. 92 .

[0107] Figure 16 Ni was shown 2+ τ bound to a single guanine reactant on and τ off It presents the use of different Ni 2+ Concentration inter-event interval (t) on ) and event dwell time (t) off The histograms of the events were obtained. All histograms were fitted using a single exponential function y = a*exp(-x / τ) to derive the average inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14G (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 7.0. Continuously apply a potential of +180mV.

[0108] Figure 17 The chemical synthesis of 14TAZ in the PNRSS chain is shown. a. Animation of TAZ incorporation into the PNRSS chain. b. CuAAC reaction. To generate triazoles on the PNRSS chain, 3-azidopropylamine was reacted with 14TAK (Table 1) via a Huisgen copper(I)-catalyzed azido-alkyne 1,3-dipolar cycloaddition reaction (CuAAC). 93In short, 10 μL of DNA 14TAK (100 μM) solution, 6 μL of 3-azidopropylamine (330 mM) in acetonitrile solution, 1.5 μL of copper sulfate (20 mM), 3 μL of sodium ascorbate (20 mM), and 3.5 μL of Milli-Q water were added to 6 μL of HEPES buffer (100 mM HEPES, pH 7.4), and the mixture was shaken at 600 rpm for 4 h at 25 °C. Then, 6 μL of LEDTA solution (100 mM) was added to the mixture to terminate the reaction. The product DNA was purified using a Micro Bio-Spin 6 Columns (Bio-Rad) instrument. To confirm successful conjugation, the purified product was analyzed by liquid chromatography-mass spectrometry (Xevo G2-XS QTOF MS + Acuqity UPLC I-Class plus, Waters Corporation) equipped with an electrospray ionization (ESI) source. The product DNA, referred to as 14TAZ (Table 1), was directly used for downstream PNRSS measurements. c. Mass spectrometry results for 14TAK: Calculated value: 18271.0, Measured value: 18272.1. d. Mass spectrometry results for 14TAZ: Calculated value: 18371.1, Measured value: 18372.3.

[0109] Figure 18 The single-molecule characteristics of the PNRSS chain 14TAZ are shown. a. Triazole formation and its Ni 2+ Sensing mechanism. The PNRSS chain 14TAK (Table 1) contains a single alkyne (blue arc). 3-azidopropylamine (gray symbol) is reacted with 14TAK via CuAAC (…). Figure 17 ), producing 14TAZ, which contains a single triazole as a stationary reactant (blue + gray). 2+ Used as a mobile reactant, reversibly coordinating with triazoles. 94 bc. Using 14TAK(b) or 14TAZ(c) for I p Measurement. Apply a +180mV potential. When 14TAK(b) or 14TAZ(c) is trapped by the orifice, I is observed. p The significant difference provides single-molecule evidence for successful TAZ formation. This difference is also summarized in the event histograms of 14TAK(d) or 14TAZ(f) when static pore blockage measurements are performed. Table 5 summarizes the average blockage magnitude. Value. For example, PNRSS is performed using 14TAK(e) or 14TAZ(g). Ni 2+ It was used as a mobile reactant and added to the trans side at a final concentration of 1 mM. No Ni was observed using 14TAK(e). 2+The binding event was observed. However, characteristic binding was observed using 14TAZ(g), further confirming that TAZ had been generated on the chain.

[0110] Figure 19 Ni was shown 2+ Binding with triazole. a. Schematic diagram. (e.g.) Figure 2 PNRSS measurements were performed as described in [the document]. A PNRSS chain of 14TAZ (Table 1) containing a single triazole was used as the stationary reactant. Ni 2+ It forms reversible coordination with triazoles and acts as a mobile reactant. b. Using different Ni... 2+ Representative traces obtained from concentration. Ni 2+ The concentration was adjusted between 0 and 1 mM, and marked to the left of each corresponding trace. Ni 2+ Combining to generate negative events (I) b p ). When you 2+ When bound to triazoles, it produces characteristic noise, clearly reporting the occurrence of this specific reaction. When Ni... 2+ As concentration increases, the event occurrence rate increases, providing concrete evidence that the observed events are caused by Ni. 2+ Caused by combination.

[0111] Figure 20 Ni was shown 2+ τ bound to triazole on and τ off It presents the use of different Ni 2+ Concentration inter-event interval (t) on ) and event dwell time (t) off The histograms of the events were obtained. All histograms were fitted using a single exponential function y = a*exp(-x / τ) to derive the average inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14TAZ (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 7.0. Continuously apply a potential of +180mV.

[0112] Figure 21 Co was displayed 2+ Binding with triazole. a. Schematic diagram. Measurements were performed as described in the method. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 7.0 was used. A +180 mV potential was continuously applied. A PNRSS chain of 14TAZ (Table 1) was applied. 14TAZ contains a single triazole and is used as a fixative. Co 2+ Used as a mobile reactant. b. Add Co to the trans side. 2+ ​Representative traces at that time. Co 2+ The concentration was adjusted between 0 and 250 μM, and each was marked to the left of the corresponding trace. Co was observed. 2+ The combined events are spike and negative events. c.1 / τ on or 1 / τ off Compared to Co 2+ A graph of concentration. 1 / τ on With Co 2+ The final concentration is linearly correlated. However, 1 / τ off d. ΔI relative to t off A scatter plot of events. Co 2+ The concentration was 0.2 mM. All events were extracted from traces recorded continuously over 15 minutes. The scatter plot included 3632 events. Each point was color-coded based on its local point density. Individual event clusters were identified, measured to -25 pA in ΔI. The event histogram of ΔI is appended to the right of the scatter plot. The event histogram of ΔI is superimposed with its Gaussian fit and appended to the right of the scatter plot. These observations indicate that Co can be observed using PNRSS. 2+ Coordination interactions between Ni and single triazoles. However, the binding properties are related to Ni. 2+ different

[0113] Figure 22 Co was displayed 2+ τ bound to triazole on and τ off It presents the use of different Co 2+ Concentration inter-event interval (t) on ) and event dwell time (t) off The histograms of the events were obtained. All histograms were fitted using a single exponential function y = a*exp(-x / τ) to derive the average inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14TAZ (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 7.0. Continuously apply a potential of +180mV.

[0114] Figure 23 The diagram shows the sequential addition of Co when measuring with 14TAZ. 2+ and Ni 2+ Measurements were performed as described in the method. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 7.0 was used. A potential of +180 mV was continuously applied. The PNRSS chain was measured over 14 TAZ (Table 1). a. Representative PNRSS trace without added mobile reactant. b. Measured with Co added at a final concentration of 250 μM.2+ A representative PNRSS trace at that time. Only Co was observed. 2+ Based on the event. c. Further add 400 μM Ni to a final concentration. 2+ A representative PNRSS trace was observed at that time. Co was observed. 2+ and Ni 2+ In conjunction with the event. Ni 2+ The unique event noise generated provides clear evidence for event identification.

[0115] Figure 24 The 4-(azidomethyl)phenylboronic acid was shown. 1 H NMR spectrum. 1 H NMR (BRUKER AVANCE III, 400MHz, 298K, DMSO-d6) δ 8.08 (s, 2H), 7.81 (d, J = 8.0Hz, 2H), 7.33 (d, J = 8.0Hz, 2H), 4.44 (s, 2H). 90

[0116] Figure 25 The chemical synthesis of 14PBA on the PNRSS chain is shown. a. Animated diagram of PBA introduction. b. Reaction. To generate PBA on the PNRSS chain, 4-(azidomethyl)phenylboronic acid was reacted with 14TAK (Table 1) via a Huisgen copper(I)-catalyzed azidide-alkyne 1,3-dipolar cycloaddition reaction (CuAAC). 95In short, 10 μL of DNA 14TAK (100 μM), 6 μL of 4-(azidomethyl)phenylboronic acid solution (dissolved in MeCN, 200 mM), 1.5 μL of copper sulfate (20 mM), 3 μL of sodium ascorbate (20 mM), and 3.5 μL of LiMilli-Q water were added to 6 μL of HEPES buffer (100 mM HEPES, pH 7.4), and the mixture was shaken at 600 rpm for 4 h at 25 °C. Then, 6 μL of EDTA solution (100 mM) was added to the mixture to terminate the reaction. The product DNA was purified using Micro Bio-Spin6 Columns (Bio-Rad). To confirm successful conjugation, the purified product was analyzed by liquid chromatography-mass spectrometry (Xevo G2-XS QTOF MS + Acuqity UPLC I-Class plus, Waters Corporation) equipped with an electrospray ionization (ESI) source. This functionalized DNA, designated 14PBA (Table 1), was directly used for downstream PNRSS measurements. c. Mass spectrometry results for 14PBA. For [14PBA], calculated mass: 18448.0. For [14PBA-2H2O], calculated mass: 18412.0, measured mass: 18413.1. During the mass spectrometry measurement, each borate molecule lost 2 H2O molecules, likely due to the strong intramolecular interaction between borate and the DNA phosphate backbone, a phenomenon also reported in the literature. 96 .

[0117] Figure 26 The monomolecular characterization of the PNRSS chain 14PBA is shown. a. Introduction of phenylboronic acid (PBA) and its catechol sensing mechanism. The PNRSS chain 14TAK (Table 1) contains a single alkyne (blue arc). 14PBA is produced by reacting 4-(azidomethyl)phenylboronic acid (grey symbol) with 14TAK via CuAAC, containing a single PBA as a stationary reactant. Detailed synthesis and characterization information for 14PBA is available in [link to table]. Figure 24-25 Provided in [the text]. Catechols that form reversible interactions with PBA are used as mobile reactants. bc. I [the text is incomplete and requires further context to translate accurately]. p Measurement. Apply a +160mV potential. When 14TAK(b) or 14PBA(c) is trapped by the orifice, I is observed. p The significant difference provides single-molecule evidence for the successful PBA conjugation. When 14TAK(d) or 14PBA(f) is measured during static pore blockage measurements, in I... p This difference is also summarized in the event histogram. Table 7 summarizes the average blocking magnitude. Values. For example, PNRSS was performed using 14TAK(e) or 14PBA(g). Catechol was used as a mobile reactant and added to the trans side at a final concentration of 500 μM. No catechol binding events were observed using 14TAK(e). However, characteristic binding was observed using 14PBA(g), further confirming that PBA had been successfully conjugated to the chain.

[0118] Figure 27 The binding of catechol to PBA is shown. a. Schematic diagram. PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, which can bind catechol. 97 As shown in the animation. b. Representative traces containing catechol binding events. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. Catechol was added to the trans side to a final concentration of 0-0.5 mM, and marked to the left of each corresponding trace. Catechol binding produces a positive event (Ig). b >I p The incidence of events increases with increasing catechol concentration. c. Concentration dependence. The reciprocal of the interval between events, 1 / τ. on and the reciprocal of the dwell time 1 / τ off Plot the final concentration of catechols relative to the trans side. 1 / τ on This shows a linear correlation with catechol concentration. 1 / τ off d. ΔI relative to t off A scatter plot of 100 events was generated. The histogram of ΔI was overlaid with its Gaussian fit and plotted to the right of the scatter plot. The catechol concentration was 400 μM. Events were extracted from traces recorded continuously over 15 minutes.

[0119] Figure 28 This shows the τ-type of catechol binding to PBA. on and τ off The event intervals (t) were presented for different catechol concentrations. on ) and event dwell time (t) off Histograms of the events were generated. Catechol was added to the trans side at a final concentration of 0.1–0.5 mM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted with a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0. Continuously apply a +160mV potential.

[0120] Figure 29 The binding of ethylene glycol to PBA is shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, which can bind ethylene glycol. 98 As shown in the animation. b. Representative traces containing ethylene glycol binding events. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. Ethylene glycol was added to the trans side to a final concentration of 0-18 mM, and marked to the left of each corresponding trace. The event occurrence rate increased with increasing ethylene glycol concentration. c. Concentration dependence. The reciprocal of the event interval (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off Plot the final concentration of ethylene glycol relative to 1 / τ. on This demonstrates a linear correlation with ethylene glycol concentration. 1 / τ off d. ΔI relative to t off A scatter plot of 135 events is generated. The histogram of ΔI is overlaid with its Gaussian fit and plotted to the right of the scatter plot. The ethylene glycol concentration is 14 mM. Events are extracted from traces recorded continuously over 15 minutes.

[0121] Figure 30 This shows the τ-type of ethylene glycol binding with PBA. on and τ off The event intervals (t) were presented using different ethylene glycol concentrations. on ) and event dwell time (t) off Histograms of the events were generated. Ethylene glycol was added to the trans side at a final concentration of 2-18 mM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted using a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0. Continuously apply a +160mV potential.

[0122] Figure 31 The binding of glycerol to PBA is shown. a. Schematic diagram. PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, which can bind glycerol. 98As shown in the animation. b. Representative traces containing glycerol binding events. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. Glycerol was added to the trans side to a final concentration of 0-12 mM, and marked to the left of each corresponding trace. The event occurrence rate increased with increasing glycerol concentration. c. Concentration dependence. The reciprocal of the interval between events (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off Plot the relative final concentration of glycerol. 1 / τ on This demonstrates a linear correlation with glycerol concentration. 1 / τ off d. ΔI relative to t off A scatter plot of ΔI is generated. The scatter plot includes 267 events. The histogram of ΔI is overlaid with its Gaussian fit and plotted to the right of the scatter plot. The glycerol concentration is 10 mM. Events are extracted from traces recorded continuously over 15 minutes.

[0123] Figure 32 The τ-binding of glycerol with PBA was shown. on and τ off The event intervals (t) at different glycerol concentrations were presented. on ) and event dwell time (t) off Histograms of the events were generated. Glycerin was added to the trans side at a final concentration of 4-12 mM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted with a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0. Continuously apply a +160mV potential.

[0124] Figure 33 The binding of L-lactic acid to phenylboronic acid (PBA) is shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, which can bind L-lactic acid. 97 As shown in the animation. b. Representative traces containing L-lactate binding events. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. L-lactate was added to the trans side at a final concentration of 0-5 mM, and marked to the left of each corresponding trace. The event occurrence rate increased with increasing L-lactate concentration. c. Concentration dependence. The reciprocal of the event interval (1 / τ) on ) and the reciprocal of the dwell time (1 / τ)off Plot the final concentration of L-lactic acid relative to 1 / τ. on This demonstrates a linear correlation with L-lactic acid concentration. 1 / τ off d. ΔI relative to t off A scatter plot of ΔI is generated. The scatter plot includes 213 events. The histogram of ΔI is overlaid with its Gaussian fit and plotted to the right of the scatter plot. The lactate concentration is 4 mM. Events are extracted from traces recorded continuously over 15 minutes.

[0125] Figure 34 This shows the τ-binding of L-lactic acid with PBA. on and τ off The event intervals (t) were presented using different L-lactic acid concentrations. on ) and event dwell time (t) off Histograms of the events were generated. L-lactic acid was added to the trans side at a final concentration of 1-5 mM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted using a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0. Continuously apply a +160mV potential.

[0126] Figure 35 The binding of vitamin C to PBA is shown. a. Schematic diagram. PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, which can bind vitamin C. 99 As shown in the animation. b. Representative traces containing vitamin C binding events. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. Vitamin C was added to the trans side to a final concentration of 0-2 mM, and the results were marked to the left of each corresponding trace. The event occurrence rate increased with increasing vitamin C concentration. c. Concentration dependence. The reciprocal of the interval between events (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off Plot the relative final concentration of vitamin C. 1 / τ on This demonstrates a linear correlation with vitamin C concentration. 1 / τ off d. ΔI relative to t off A scatter plot of 120 events was generated. The histogram of ΔI, superimposed with its Gaussian fit, was plotted to the right of the scatter plot. The vitamin C concentration was 1.6 mM. Events were extracted from traces recorded continuously over 15 minutes.

[0127] Figure 36 This shows the τ binding of vitamin C to PBA. on and τ off The study presented the inter-event intervals (t) for different vitamin C concentrations. on ) and event dwell time (t) off Histograms of the events were generated. Vitamin C was added to the trans side at a final concentration of 0.4–2.0 mM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted with a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0. Continuously apply a +160mV potential.

[0128] Figure 37 The binding of vitamin B6 to PBA is shown. a. Schematic diagram. PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, which can bind vitamin B6. 100 As shown in the animation. b. Representative traces containing vitamin B6 binding events. A buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0 was used. A +160mV potential was continuously applied. Vitamin B6 was added to the trans side to a final concentration of 0-0.05mM, and marked to the left of each corresponding trace. The event occurrence rate increased with increasing vitamin B6 concentration. c. Concentration dependence. The reciprocal of the interval between events (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off Plot the relative final concentrations of vitamin B6. (1 / τ) on This demonstrates a linear correlation with vitamin B6 concentration. (1 / τ) off d. ΔI remains constant relative to t off A scatter plot of 763 events was generated. The histogram of ΔI, superimposed with its Gaussian fit, was plotted to the right of the scatter plot. The vitamin B6 concentration was 40 μM. Events were extracted from traces recorded continuously over 15 minutes.

[0129] Figure 38 It showed that vitamin B6 binds to PBA. on and τ off The study presented the time intervals between events (t) when using different concentrations of vitamin B6. on ) and event dwell time (t) offHistograms of the events were generated. Vitamin B6 was added to the trans side at a final concentration of 0.01-0.05 mM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted with a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0. Continuously apply a +160mV potential.

[0130] Figure 39 The binding of Tris to PBA is shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, which can bind tris, as shown in the animation. b. Representative traces containing tris binding events. A buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0 was used. A potential of +160mV was continuously applied. Tris was added to the trans side at a final concentration of 0-1.0mM, and marked to the left of each corresponding trace. The event occurrence rate increased with increasing tris concentration. c. Concentration dependence. The reciprocal of the event interval (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off Plot the final concentration relative to tris. (1 / τ) on This demonstrates a linear correlation with tris concentration. (1 / τ) off The event remains constant. Events are extracted from traces recorded continuously for 15 minutes for each condition.

[0131] Figure 40 This shows the τ of tris combined with PBA. on and τ off The inter-event intervals (t) using different tris concentrations are presented. on ) and event dwell time (t) off Histograms of the event intervals were generated. Tris was added to the trans side at final concentrations of 0.2–1.0 mM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted with a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0. Continuously apply a +160mV potential.

[0132] Figure 41 The following shows the observation of chemical intermediates using PNRSS. a. A proposed tris reaction model when bound to PBA. Tris can be protonated or deprotonated depending on the ambient pH. b. Representative PNRSS events containing the chemical intermediate, obtained at pH 8.0. Transitions between states 0, 1, or 2 were observed. However, transitions to other states were never observed. cd. Representative traces obtained at pH 7.0 (c) or pH 8.0 (d). At pH 7.0, tris bound to PBA only produces one type of blocking level I. b1 However, at pH 8.0, tris binds to I... b1 A new blocking level I is generated above. b2 ef. ΔI relative to t formed by events obtained at pH 7.0 (e) or pH 8.0 (f). off The event scatter plot. In the scatter plot, I in ΔI was observed at pH 8.0. b2 A new event population. All the above measurements were performed as described in the method. 1577 events were included in e. 8261 events were included in f. Measurements at higher pH values ​​yielded higher event occurrence rates. 14PBA was used as the PNRSS chain. All the above measurements were performed using a buffer solution of 1.5M KCl and 10mM tris. A +160mV potential was continuously applied. Scatter plots (e, f) were formed from consecutive 15-minute records for each condition.

[0133] Figure 42 This study demonstrates the chemical properties of the irreversible oxidation of PBA. PNRSS measurements are similar to... Figure 4 The procedure was performed as described above. The electrolyte buffer consisted of 1.5 M KCl, 10 mM HEPES, and pH 8.0. PNRSS chain 14PBA was added to the cis side to a final concentration of 10 nM. H₂O₂ and Ni were simultaneously added to the trans side. 2+ Glycerol and glycerol were used at final concentrations of 5.4 mM, 0.2 mM, and 8 mM, respectively. A potential of +160 mV was continuously applied. a. Representative traces obtained during the PNRSS process. The binding of H2O2 or glycerol to PBA produces a positive event, while Ni 2+ The combination produces a negative event. PBA can also be irreversibly oxidized by H2O2 to produce phenol (marked with a red arrow). After that, the combination of H2O2 or glycerol is no longer observed in the trace. b. Enlarged view of the trace segment from a (marked with blue). H2O2 (green triangle), glycerol (orange circle) and Ni 2+ The combinations of (purple squares) are marked on the traces. c. Suggested combination mechanism. d. Enlarged view of the trace segment (gray marker) from a. Only Ni 2+The binding (purple square) can still be observed. e. Suggested mechanism: The disappearance of the H2O2 and glycerol binding event and Ni 2+ The retention of the bond confirms the hypothesis that PBA has been irreversibly oxidized to phenol.

[0134] Figure 43 The binding of norepinephrine to PBA is shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, capable of binding norepinephrine, as shown in the animation. b. Representative traces of norepinephrine binding events. Figure 5 PNRSS measurements were performed. The electrolyte buffer consisted of 1.5 M KCl, 10 mM HEPES, and pH 8.0. A +160 mV potential was continuously applied. Norepinephrine was added to the trans side at a final concentration of 0-180 μM, and the trans side was marked to the left of each corresponding trace. The event occurrence rate increased with increasing norepinephrine concentration. c. Reaction Mechanism 101 d. Concentration dependence. Norepinephrine concentrations were adjusted between 20-180 μM. Recordings were taken continuously for 15 minutes for each condition. Figure 10 The derivation of 1 / τ on and 1 / τ off Values. The mean and standard deviation values ​​are derived from three independent measurements for each condition. The reciprocal of the interval between events (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off Plot the final concentration of norepinephrine relative to 1 / τ. on This demonstrates a linear correlation with norepinephrine concentration. (1 / τ) off (e. ΔI remains constant relative to t) off A scatter plot of ΔI was generated. The scatter plot includes 106 events. The histogram of ΔI is overlaid with its Gaussian fit and plotted on the right side of the scatter plot. The norepinephrine concentration was 140 μM. Events were extracted from traces recorded continuously over 15 minutes.

[0135] Figure 44 The τ-binding of norepinephrine with PBA was shown. on and τ off The event intervals (t) were presented using different norepinephrine concentrations. on ) and event dwell time (t) off Histograms of the events were generated. Norepinephrine was added to the trans side at final concentrations of 20–180 μM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted using a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on ) and average event dwell time (τ) off), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5 MKCl, 10 mM HEPES, pH 8.0. Continuously apply a +160 mV potential.

[0136] Figure 45 The binding of adrenaline to PBA is shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, capable of binding adrenaline, as shown in the animation. b. Representative traces of the adrenaline binding event. Figure 5 PNRSS measurements were performed. The electrolyte buffer consisted of 1.5 M KCl, 10 mM HEPES, and pH 8.0. A +160 mV potential was continuously applied. Epinephrine was added to the trans side at a final concentration of 0-180 μM, and the trans side was marked to the left of each corresponding trace. The event occurrence rate increased with increasing epinephrine concentration. c. Reaction Mechanism 101 d. Concentration dependence. Adrenaline concentration was adjusted between 20-180 μM. Recordings were taken continuously for 15 minutes for each condition. Figure 10 The derivation of 1 / τ on and 1 / τ off Values. The mean and standard deviation values ​​are derived from three independent measurements for each condition. The reciprocal of the interval between events (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off Plot the relative final concentration of adrenaline. (1 / τ) on This demonstrates a linear correlation with adrenaline concentration. (1 / τ) off (e. ΔI remains constant relative to t) off A scatter plot was generated from traces recorded continuously over 15 minutes. The scatter plot includes 109 events. The histogram of ΔI is overlaid with its Gaussian fit and plotted to the right of the scatter plot. The adrenaline concentration was 140 μM. Events were extracted from traces recorded continuously over 15 minutes.

[0137] Figure 46 This shows the τ binding of adrenaline to PBA. on and τ off The event intervals (t) were presented using different adrenaline concentrations. on ) and event dwell time (t) off Histograms of the events were generated. Epinephrine was added to the trans side at final concentrations ranging from 20 to 180 μM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted using a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on ) and average event dwell time (τ) off), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0. Continuously apply a potential of +160mV.

[0138] Figure 47 The binding of isoproterenol to PBA is shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, capable of binding isoproterenol, as shown in the animation. b. Representative traces of isoproterenol binding events. Figure 5 PNRSS measurements were performed. The electrolyte buffer consisted of 1.5 M KCl, 10 mM HEPES, and pH 8.0. A +160 mV potential was continuously applied. Isoproterenol was added to the trans side at a final concentration of 0-180 μM, and the trans side was marked to the left of each corresponding trace. The event occurrence rate increased with increasing isoproterenol concentration. c. Reactivity Mechanism 101 d. Concentration dependence. Isoproterenol concentration was adjusted between 20-180 μM. Recordings were taken continuously for 15 minutes for each condition. Figure 10 The derivation of 1 / τ on and 1 / τ off Values. The mean and standard deviation values ​​are derived from three independent measurements for each condition. The reciprocal of the interval between events (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off Plot the final concentrations relative to isoproterenol. (1 / τ) on This demonstrates a linear correlation with isoadrenaline concentration. (1 / τ) off (e. ΔI remains constant relative to t) off A time-series scatter plot. The scatter plot was generated from traces recorded continuously over 15 minutes. The scatter plot includes 101 events. The histogram of ΔI is overlaid with its Gaussian fit and plotted on the right side of the scatter plot. The isoproterenol concentration was 140 μM. Events were extracted from traces recorded continuously over 15 minutes.

[0139] Figure 48 This demonstrates the τ-binding of isoproterenol with PBA. on and τ off The event intervals (t) were presented using different isoproterenol concentrations. on ) and event dwell time (t) off Histograms of the events were generated. Isoproterenol was added to the trans side at final concentrations of 20–180 μM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted with a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5 MKCl, 10 mM HEPES, pH 8.0. Continuously apply a +160 mV potential.

[0140] Figure 49 The positive and negative events are shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, capable of binding catechol or norepinephrine, as shown in the animation. b. Representative traces of catechol and norepinephrine binding events. Catechol and norepinephrine were added simultaneously to the trans side to final concentrations of 400 μM and 140 μM, respectively. Binding of catechol or norepinephrine to PBA reported positive (I) events, respectively. b,C ,I b,C >I p ) or negative event (I b,N ,I b,N p It also provides visualizations (Video 4). c. Magnified display of different binding events. Representative binding events from catechol (top left) or norepinephrine (top right) are shown. The chemical structures of catechol (bottom left) or norepinephrine (bottom right) when bound to PBA are also shown. d. ΔI relative to t off A scatter plot of events over time. Events were extracted from traces recorded continuously over 15 minutes. The scatter plot includes a total of 119 events. From the scatter plot, the binding events of catechol and norepinephrine produce two distinct groups. The histogram of ΔI is plotted to the right of the scatter plot. The two peaks of ΔI are fitted with Gaussians and superimposed on the histogram.

[0141] Figure 50 ​Frequency division is shown. When probing with PNRSS, the binding of catecholamines (e.g., norepinephrine, epinephrine, and isoproterenol) to PBA produces rich chemical process information, manifested as fluctuations in different frequency domains. The chemical structures of the borate ester complexes formed by the binding of (a) norepinephrine, (e) epinephrine, or (i) isoproterenol to PBA are shown. During PNRSS, the raw traces were acquired at a sampling rate of 25 kHz and low-pass filtered at 1 kHz. The recorded traces were divided into low-pass and high-pass portions by frequency, and filtered by Butterworth. A cutoff frequency of 100 Hz and a filter order of 2 were selected. The raw events formed by the binding of (b) norepinephrine, (f) epinephrine, or (j) isoproterenol to PBA are shown. The low-pass portion of the events formed by the binding of (c) norepinephrine, (g) epinephrine, or (k) isoproterenol to PBA is shown. The high-pass portion of the event generated by the binding of (d) norepinephrine, (h) epinephrine, or (l) isoproterenol to PBA is shown. Specifically, norepinephrine does not exhibit fluctuations in the low-pass portion of the event, but epinephrine and isoproterenol exhibit slight telegraphic transitions. Isoproterenol can be distinguished from epinephrine by recognizing its distinctive noise characteristics in the high-pass portion of the event.

[0142] Figure 51 This demonstrates the machine learning workflow. (Using...) Perform machine learning, It is a commercial AutoML platform developed based on evolutionary algorithms for automated model design. For the learning process (I), PNRSS measurements were performed using norepinephrine, epinephrine, or isoproterenol as the sole analyte, respectively. Figures 43-48 The raw time traces in the .abf file were extracted using the neo module (v0.8.0, https: / / pypi.org / project / neo-python / ) in Python. Events in the traces were extracted using a custom event segmentation program written in Python. The extracted event frequencies were then divided into high-pass and low-pass components using the Butterworth filter integrated into the SciPy module in Python. The cutoff frequency was set to 100Hz, and the filter order was set to 2. The standard deviations of the high-pass and low-pass components were calculated and applied separately to form the feature matrix. The 1455 events in the feature matrix were then input into DarwinML. 102The platform is used for model building. In short, 80%, 10%, and 10% of the events are used as training, validation, and test datasets, respectively. The training and validation sets are used to build and validate the model. A 10x cross-validation method is applied. More than 10 popular models are used, such as SVC (SVM for classification), logistic regression, random forest, XGboost, LightGBM, RidgeClassifier, MLPClassifier, BaggingClassifier, etc. When evaluated using the test set (the remaining 10% of all 1455 events), the SVC model reports a highest accuracy score of 99.6%. The trained SVC model is further validated across all 1455 events, reporting an overall accuracy score of 98.3%. The confusion matrix results are as follows. Figure 5 As shown in f. For the prediction process (II), a PNRSS was performed using a sample mixture. The raw current trace was divided into high-frequency and low-frequency components. SVC was applied to label events ( Figure 5 f). To generate the decision boundaries, grids are generated in the 0-3pA region of Lp SD and the 1-4.5pA region of Hp SD, with an interval of 0.01pA. When inferring from the SVC model, event type regions can be identified using these grid parameters. The boundaries separating these regions are considered the decision boundaries (f). Figure 5 f).

[0143] Figure 52 The sequence of additions of norepinephrine, epinephrine, and isoproterenol is shown. Figure 5 PNRSS measurements were performed. The buffer solution used was 1.5M KCl, 10mM HEPES, pH 8.0. A +160mV potential was continuously applied. Norepinephrine (N), epinephrine (E), and isoproterenol (I) were added sequentially to the trans compartment to final concentrations of 280μM, 280μM, and 180μM, respectively. a. Representative trace obtained with only norepinephrine added. b. Scatter plot of events generated from the 15-minute continuous trace obtained as described in a. c. The scatter plot includes 241 events. Representative trace with further addition of epinephrine. d. Scatter plot of the corresponding events generated from the 15-minute trace obtained as described in c. The scatter plot includes 323 events. e. Representative trace obtained with further addition of isoproterenol. f. Scatter plot of the corresponding events generated from the 15-minute trace obtained under the conditions described in e. The scatter plot includes 388 events. A scatter plot of events is generated based on the standard deviation (SD) values ​​of the low-pass (Lp) and high-pass (Hp) values ​​for each event, such as... Figure 50 As stated above.

[0144] Figure 53The binding of remdesivir to PBA is shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, which can bind remdesivir, as shown in the animation. b. Reaction mechanism. The ribose moiety of remdesivir binds to PBA to form a borate ester. 103 c. Representative traces containing remdesivir binding events. The electrolyte buffer was 1.5 M KCl, 10 mM HEPES, pH 8.0. A +160 mV potential was continuously applied. 10 mM remdesivir dissolved in DMSO was added to the trans side to achieve a final concentration of 0-100 μM, and the corresponding trace was marked to the left of each trace. The event occurrence rate increased with increasing remdesivir concentration. d. Concentration dependence. The reciprocal of the event interval (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off Plotting relative to the final concentration of remdesivir. 1 / τ on This demonstrates a linear correlation with remdesivir concentration. However, 1 / τ off Keep constant. e. △I relative to t off A scatter plot of ΔI is shown. The scatter plot includes 118 events. The histogram of ΔI is overlaid with its Gaussian fit and plotted to the right of the scatter plot. The remdesivir concentration was 80 μM. Events were extracted from traces recorded continuously over 15 minutes.

[0145] Figure 54 This shows the τ binding of remdesivir to PBA. on and τ off The event intervals (t) for different concentrations of remdesivir are presented. on ) and event dwell time (t) off Histograms of the trans side were generated. Remdesivir was added to the trans side at a final concentration of 20-100 μM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted with a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0. Continuously apply a potential of +160mV.

[0146] Figure 55 The binding of remdesivir metabolites to PBA is shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, which can bind remdesivir triphosphate metabolites, as shown in the animation. b. Reaction mechanism. The ribose moiety of remdesivir triphosphate metabolites binds to PBA to form a borate ester.103 c. Representative traces of remdesivir triphosphate binding events. The electrolyte buffer was 1.5 M KCl, 10 mM HEPES, pH 8.0. 10 mM remdesivir triphosphate, dissolved in DMSO, was added to the trans side to a final concentration of 0-600 μM, and labeled to the left of each corresponding trace. A +160 mV potential was continuously applied. The event occurrence rate increased with increasing remdesivir triphosphate concentration. d. Concentration dependence. The reciprocal of the event interval (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) off Plotting the final concentrations of remdesivir triphosphate metabolites relative to 1 / τ. on This demonstrates a linear correlation with the concentration of remdesivir triphosphate metabolites. However, 1 / τ off Keep constant. e. △I relative to t off A scatter plot of ΔI is shown. The scatter plot includes 130 events. The histogram of ΔI is overlaid with its Gaussian fit and plotted on the right side of the scatter plot. The concentration of remdesivir triphosphate metabolite is 500 μM. Events are extracted from traces recorded continuously over 15 minutes.

[0147] Figure 56 This demonstrates the τ binding of remdesivir metabolites to PBA. on and τ off The event intervals (t) for different concentrations of remdesivir metabolites are presented. on ) and event dwell time (t) off Histograms of remdesivir metabolites were plotted. Remdesivir metabolites were added to the trans side at final concentrations of 200–600 μM. The applied concentration was labeled to the left of each corresponding histogram. All histograms were fitted using a single exponential function y = a*exp(-x / τ) to derive the mean inter-event interval (τ). on ) and average event dwell time (τ) off ), and label them on each corresponding histogram. Perform PNRSS measurements as described in the method. Apply PNRSS chain 14PBA (Table 1). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 8.0. Continuously apply a +160mV potential.

[0148] Figure 57 The instructions show the sequential addition of remdesivir metabolite and remdesivir. Figure 6PNRSS measurements were performed. The electrolyte buffer consisted of 1.5 M KCl, 10 mM HEPES, and pH 8.0. A +160 mV potential was continuously applied. Remdesivir metabolite (M) and remdesivir (R) were added sequentially to the trans compartment to final concentrations of 500 μM and 20 μM, respectively. a. Representative trace obtained when only remdesivir metabolite was added. Remdesivir metabolite binding events are marked with a purple M character. b. Event scatter plot of the low-pass (Lp) standard deviation versus high-pass (Hp) standard deviation of the 15-min continuous recording trace, as described in a. The scatter plot includes 100 events. Only remdesivir metabolite binding events (M) were observed in the scatter plot. c. Representative trace obtained when remdesivir was further added. Newly occurring remdesivir binding events are marked with a magenta R character. d. Event scatter plot of the low-pass (Lp) standard deviation versus high-pass (Hp) standard deviation of the 15-min continuous recording trace, as described in c. The scatter plot includes 126 events. Two distinctly separate event clusters were observed in the plot, demonstrating binding to remdesivir metabolites (M) and remdesivir (R), respectively.

[0149] Figure 58 Display of PNRSS using α-HL. a. Measurement setup. During PNRSS, α-hemolysin (α-HL) nanopores were used to accommodate the PNRSS chain of the streptavidin-based chain. b. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, capable of binding isoproterenol, as shown in the animation. c. Representative traces of isoproterenol binding events (purple circles). Even without the addition of isoproterenol, some negative spike noise was observed, indicating a detectable interaction between phenylboronic acid and the amino acid residues within the pore. However, this was not observed when measuring with MspA. The binding of isoproterenol to PBA produces a negative event. PNRSS measurements were performed as described in the Methods section. The electrolyte buffer was 1.5 M KCl, 10 mM HEPES, pH 8.0. A +160 mV potential was continuously applied. Isoproterenol was added to the trans side to a final concentration of 0–30 μM. The applied concentration was marked to the left of each corresponding trace. The event occurrence rate increased with increasing isoproterenol concentration. Illustration: Expanded view combining events. d. Concentration dependence. Isoproterenol concentrations were adjusted between 10–30 μM. Continuous recordings were performed for 15 min for each condition. Figure 10 Obtain the τ from the data on and τ off Values. The mean and standard deviation values ​​are derived from three independent measurements for each condition. The reciprocal of the interval between events (1 / τ) on ) and the reciprocal of the dwell time (1 / τ) offPlot the final concentrations relative to isoproterenol. 1 / τ on This demonstrates a linear correlation with isoproterenol concentration. However, 1 / τ off Keep constant. e. △I relative to t off A scatter plot was generated. The histogram of ΔI was overlaid with its Gaussian fit and plotted to the right of the scatter plot. Individual event populations were identified, with ~-4.6 pA measured in ΔI. The isoproterenol concentration was 30 μM. Events were extracted from traces recorded continuously over 10 minutes. The number of bound events was 100.

[0150] Figure 59 The PNRSS chain without the traction segment is shown. a. Schematic diagram of the 14TAK-NTS PNRSS chain (Table 1). The 14TAK-NTS has no traction segment. b. Representative trace of PNRSS measurement using the 14TAK-NTS of the streptavidin-based chain. Measurements were performed as described in the method. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. Without the traction segment, the 14TAK-NTS of the streptavidin-based chain could not be effectively captured by MspA. Only transient pore blockage was observed. This confirms that the traction segment of the PNRSS chain is essential for introducing the chain into the well by electrophoresis and retaining the chain in the lumen for continuous measurement.

[0151] Figure 60 The binding of catechol to PBA is shown at different voltages. a. Representative traces of catechol binding to PBA when +80mV, +100mV, +120mV, +140mV, or +160mV voltages are applied. The concentration of catechol on the trans side is maintained at 200 μM. The binding of catechol to PBA produces a positive event. The event occurrence rate is generally constant when the applied voltage is adjusted. As the voltage increases, the event amplitude (ΔI = I) increases. b -I p Increase. b.1 / τ on or 1 / τ off A graph relative to the applied voltage. 1 / τ changes as different voltages are applied. on and 1 / τ off c. Average event magnitude A graph relative to the applied voltage. The average event amplitude when a larger voltage is applied. Relatively large. The relationship is exponential with the applied voltage. Events are extracted from traces recorded continuously over 15 minutes. Three independent measurements (N=3) are performed for each condition to generate statistics.

[0152] Figure 61The binding of norepinephrine to PBA at different voltages is shown. a. Representative traces of norepinephrine binding to PBA when applied voltages of +80 mV, +100 mV, +120 mV, +140 mV, or +160 mV. The concentration of norepinephrine on the trans side is maintained at 60 μM. Binding of norepinephrine to PBA produces a negative event. The event occurrence rate increases with increasing voltage. The event amplitude increases with increasing voltage. b. 1 / τ on or 1 / τ off A graph relative to the applied voltage. 1 / τ on It is linearly related to voltage. However, 1 / τ off c. Average event magnitude A graph showing the relationship with the applied voltage. As the voltage increases, The absolute value increases and is linearly correlated with voltage. Events are extracted from traces recorded continuously over 15 minutes. Three independent measurements (N=3) are performed for each condition to generate statistics.

[0153] Figure 62 The binding of norepinephrine to PBA at different salt concentrations is shown. a. Representative traces of norepinephrine-PBA binding events are shown when 0.5M, 1.5M, or 2.5M KCl electrolyte buffer (other components: 10mM HEPES, pH 8.0) are applied. The concentration of norepinephrine on the trans side is maintained at 60 μM. A potential of +160 mV is continuously applied. The binding of norepinephrine to PBA produces a negative event. The event occurrence rate decreases as the KCl concentration increases. However, the event amplitude increases. b. 1 / τ on or 1 / τ off A graph showing the KCl concentration relative to the electrolyte buffer solution. 1 / τ on It shows a linear negative correlation with [KCl], while 1 / τ off c. Average event magnitude The graph relative to [KCl]. As [KCl] increases, The absolute value increases. Events are extracted from traces recorded continuously over 15 minutes. Three independent measurements (N=3) are performed for each condition to generate statistics.

[0154] Figure 63 The binding of norepinephrine to PBA at different temperatures is shown. ae. Representative traces of norepinephrine-PBA binding events are shown when temperatures are set to 5°C, 10°C, 15°C, 20°C, or 25°C. The concentration of norepinephrine on the cis side is maintained at 400 μM. Binding of norepinephrine to PBA produces a negative event. The event occurrence rate and event duration increase with increasing temperature. f.1 / τ onA graph relative to temperature. g.1 / τ off A graph relative to temperature. 1 / τ on and 1 / τ off All of these relationships are exponential with temperature and can be described using the Arrhenius relation. 22 hK b A graph relative to temperature. K b The decrease is observed with increasing temperature. All measurements involving temperature changes were performed using an Orbit Mini micro dual-layer workstation (Nanion Technologies GmbH, Germany) at a sampling rate of 1.25 kHz without further digital filtering. Events were extracted from traces recorded continuously over 15 minutes. Three independent measurements (N=3) were performed for each condition to generate statistical data.

[0155] Figure 64 The PNRSS assay for human urine samples is shown. Measurements were performed as described in the method. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. The PNRSS chain 14 PBA was applied (Table 1). a. Representative PNRSS trace without urine sample addition. A positive spike event occurred randomly before any urine sample was added. b. Representative trace when 20 μL of human urine sample was added to the trans side. c. Representative trace when 50 μL of urine sample was added to the trans side. No additional events were observed from the human urine sample, confirming that the urine sample did not cause any interference to the measurement.

[0156] Figure 65 The PNRSS assay for vitamin B6 in urine is shown. a. Workflow. The assay consists of three steps: urine sample collection (I), premixing of urine at different concentrations of vitamin B6 (II), and PNRSS assay (III). Urine samples were collected from healthy volunteers (Asian, male, 27 years old). For calibration and feasibility testing, vitamin B6 was added to the urine samples to achieve final concentrations of 10, 15, 20, 25, or 30 μM. 50 μL of urine sample containing vitamin B6 was added to the trans side before each PNRSS measurement. bd. Representative traces obtained with different urine samples. Events related to vitamin B6 are marked with purple circles. Inset: Expanded view combining events. Generally, the event rate increases with the addition of higher concentrations of vitamin B6. e.1 / τ on or 1 / τ off A graph relative to the concentration of vitamin B6 in urine. (1 / τ) on This demonstrates a linear correlation between 1 / τ and the concentration of vitamin B6 in urine. off () Remain constant. f. △I relative to t offA scatter plot was generated. The scatter plot included 189 events. The histogram of ΔI was overlaid with its Gaussian fit and plotted to the right of the scatter plot. Individual event clusters were identified, with ΔI measurements of ~7.4 pA. The concentration of vitamin B6 in urine was 40 μM. Events were extracted from traces recorded continuously over 10 minutes.

[0157] Figure 66 PNRSS measurements using the polymer PNRSS chain are shown. a. Schematic diagram. The polymer PNRSS chain 14PBA-Spacer9 consists of oligonucleotides and polymers. The polymer unit is formed by the polymerization of three molecules of ethylene glycol, as shown in the animation. Furthermore, 14PBA-Spacer9 contains a single PBA capable of binding norepinephrine. b. Representative trace without added moving reactant. c. Representative trace with norepinephrine binding event. A buffer solution of 1.5 MKCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. Experiments were performed as described in the method. Norepinephrine was added to the trans side to a final concentration of 0.1 mM. Recordings were taken continuously for 10 min under each condition.

[0158] Figure 67 A conceptual demonstration of fPNRSS is shown. a. Flowchart of immobilized PNRSS (fPNRSS). As described in the method, an amino-modified PNRSS chain 14TAK is reacted with 4-(azidomethyl)phenylboronic acid via CuAAC. The product then binds to an MspA protein containing a cysteine ​​mutation via a Sulfo-SMCC linker. b. Measurement setup. Initially, the pore is unoccupied (i) and the pore current is reported as I0 (i). The PNRSS chain conjugated to the pore is then captured at a voltage of 20 mV, causing the blockage level to immediately decrease to I. p (ii). c. Representative trace without added moving reactant. d. Representative trace containing norepinephrine binding event. Use a buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0. Continuously apply a +160 mV potential. Perform experiments as described in the method. Add norepinephrine to the trans side to a final concentration of 0.2 mM. Record continuously for 10 min for each condition.

[0159] Figure 68The concept of lPNRSS is shown. a. Flowchart of the locked PNRSS (lPNRSS). The lPNRSS chain contains a locking segment that forms a hairpin structure to prevent the lPNRSS chain from escaping from the orifice. Furthermore, the lPNRSS chain 14PBA contains a single PBA capable of binding norepinephrine. b. Measurement setup. Initially, the orifice is unoccupied (i) and the orifice current is reported as I0 (i). Then the lPNRSS chain is captured by the orifice, causing the blockage level to immediately decrease twice (ii and iii) to reach the final blockage level, I0. p (iii). The blocking level (ii) is caused by the opening of the hairpin structure, as shown in the animation. c. Representative traces of norepinephrine binding events. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. The experiment was performed as described in the method. Norepinephrine was added to the trans side to a final concentration of 0.05 mM. Recordings were taken continuously for 10 min under each condition.

[0160] Figure 69 The binding of sugars to PBA is shown. a. Schematic diagram. PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, capable of binding sugars, as shown in the animation. b. Representative traces without added moving reactants. ce. Representative traces of binding events (purple circles) containing D-(-)-ribose (c), D-fructose (d), and D-(+)-mannose (e). The structural formula of the sugar is labeled to the left of each corresponding trace. The spike event in blank (b) is from Tris. Tris is the component used to provide buffering capacity and react with phenylboronic acid (Figures S33-S35). However, the event of Tris binding to PBA (b) is significantly different from the event of sugar binding to PBA (ce). A buffer solution of 1.5 M KCl, 10 mM Tris, pH 8.0 was used. A potential of +140 mV was continuously applied. The experiment was performed as described in the Methods section. Add D-(-)-ribose (4 mM, c), D-fructose (4 mM, d), and D-(+)-mannose (6 mM, e) to the trans side to achieve the above final concentrations. Record data continuously for 10 minutes for each condition.

[0161] Figure 70 The binding of 5'-CMP to PBA is shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, which can bind cytidine 5'-monophosphate (5'-CMP), as shown in the animation. b. Representative trace without the addition of the moving reactant. c. Representative trace containing the 5'-CMP binding event (purple circle). d. ΔI relative to t offA scatter plot was generated. The scatter plot includes 65 events. The histogram of e.ΔI was overlaid with its Gaussian fit. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. Experiments were performed as described in the method. 5'-CMP was added to the trans side to a final concentration of 0.8 mM. Recordings were taken continuously for 10 min for each condition.

[0162] Figure 71 The PNRSS identification of enantiomeric norepinephrine is shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, capable of binding L or D-norepinephrine, as shown in the animation. b. Representative traces obtained when only L-norepinephrine is added. Binding of L-norepinephrine to PBA produces only one type of obstruction level. c. Representative traces when D-norepinephrine is further added. However, D-norepinephrine binding produces a new obstruction level I. b,D Exceeding I b,L d. A scatter plot of events generated from traces continuously recorded over 10 minutes, as described in b. The scatter plot includes 136 events. e. A scatter plot of events generated from traces continuously recorded over 10 minutes, as described in c. The scatter plot includes 199 events. Here, the ability of PNRSS to distinguish enantiomers was validated using L or D-norepinephrine. This system will also be used to study other enantiomeric catecholamines, such as DL-3,4-dihydroxyphenylalanine (DL-DOPA), DL-epinephrine, and DL-isoproterenol, in future work. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. Experiments were performed as described in the method. L or D-norepinephrine was added to the trans side to a final concentration of 0.15 mM. Continuous recording was performed for 10 minutes for each condition.

[0163] Figure 72The binding of catechol-D6 to PBA is shown. a. Schematic diagram. The PNRSS chain 14PBA (Table 1) contains a single PBA at site 14, capable of binding catechol-D6, as shown in the animation. Catechol-D6 is a deuterium compound, and deuterium replaces all hydrogen atoms on the catechol. b. Representative trace without the addition of the moving reactant. c. Representative trace containing the catechol-D6 binding event. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A + 160 mV potential was continuously applied. Experiments were performed as described in the Methods section. Catechol-D6 was added to the trans side to a final concentration of 0.3 mM. Recordings were taken continuously for 10 min under each condition. Catechol-D6 was purchased from Cambridge Isotope Laboratories, Inc. (USA).

[0164] Figure 73 Polysaccharide sensing using PNRSS is shown. Phenylboronic acid (PBA) was introduced for the detection of sugars containing vicinal diols. Representative polysaccharides containing fructose, such as 4-O-β-d-galactopyranosyl-d-fructose (lactulose, a), 6-O-α-D-glucopyranosyl-D-fructose (isomaltulose, b), and 4-ObD-galactosylsucrose (galactosylsucrose, c), all reported clear and distinct binding events. Potential vicinal diols used for binding are marked in red. Of the three polysaccharides, galactosylsucrose had the lowest affinity for PBA. This is because the 1,2-cis-diol in the fructose group, known to have a high affinity for PBA, was disrupted to form a glycosidic bond. Experiments were performed as described in the Methods section. Lactulose (a), isomaltulose (b), and galactosylsucrose (c) were added to the trans compartment at a final concentration of 8 mM for each analyte.

[0165] Figure 74 The binding of 3,4-dihydroxymandelic acid (3,4-dihydroxymandelic acid) to PBA is shown. a. Schematic diagram. PNRSS chain 14PBA contains a single PBA at site 14, capable of binding 3,4-dihydroxymandelic acid, as shown in the animation. b. Representative traces of the 3,4-dihydroxymandelic acid binding event. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A potential of +160 mV was continuously applied. 3,4-dihydroxymandelic acid was added to the trans side at final concentrations of 0 and 0.4 mM, and marked to the left of the corresponding traces. The binding of 3,4-dihydroxymandelic acid produces a positive event (I0). b >I p c. △I relative to t off Scatter plot of ΔI. d. Histogram of ΔI, superimposed with its Gaussian fit result. 3,4-Dihydroxymandelic acid concentration was 0.4 mM. Events were extracted from traces recorded continuously over 15 minutes. ΔI = 26.6349 pA.

[0166] Figure 75 The binding of 4-hydroxy-3-methoxymandelic acid (VMA) to PBA is shown. a. Schematic diagram. The PNRSS chain 14PBA contains a single PBA at site 14, capable of binding VMA, as shown in the animation. b. Representative traces containing the VMA binding event. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. VMA was added to the trans side at final concentrations of 0 and 2 mM, marked to the left of the corresponding traces. VMA binding produces a positive event (I0). b >I p c. △I relative to t off d. Scatter plot of ΔI. Histogram of ΔI, superimposed with its Gaussian fitting result. VMA concentration is 2 mM. Events were extracted from traces recorded continuously over 15 minutes. ΔI = 26.4662 pA.

[0167] Figure 76 The binding of 3,4-dihydroxyphenylacetic acid (3,4-dihydroxyphenylacetic acid) to PBA is shown. a. Schematic diagram. The PNRSS chain 14PBA contains a single PBA at site 14, capable of binding 3,4-dihydroxyphenylacetic acid, as shown in the animation. b. Representative traces of the 3,4-dihydroxyphenylacetic acid binding event. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used. A +160 mV potential was continuously applied. 3,4-dihydroxyphenylacetic acid was added to the trans side at final concentrations of 0 and 0.5 mM, and marked to the left of the corresponding traces. The binding of 3,4-dihydroxyphenylacetic acid produces a positive event (I0). b >I p c. △I relative to t off Scatter plot of ΔI. d. Histogram of ΔI, superimposed with its Gaussian fit result. 3,4-Dihydroxyphenylacetic acid concentration was 0.5 mM. Events were extracted from traces recorded continuously over 15 minutes. ΔI = 25.1196 pA. Detailed Implementation

[0168] The embodiments described herein can be more readily understood by referring to the following detailed description, examples, and claims, as well as the descriptions preceding and following them. It should be understood that the embodiments described herein are not limited to any particular use, method, and / or product. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting.

[0169] Furthermore, the following description is provided as effective teaching on the various embodiments in their best, currently known aspects. Those skilled in the art will recognize that many changes can be made to the described aspects while still obtaining the beneficial results of this disclosure. It will also be apparent that some of the desired benefits of the invention can be obtained by selecting some features of the various embodiments without utilizing others. Therefore, those skilled in the art will recognize that many modifications and adjustments to the various embodiments described herein are possible, and in some cases may even be desirable and part of this disclosure. Thus, the following description is provided as an illustration of the principles of the embodiments described herein and not as a limitation thereof.

[0170] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are described hereafter. All publications mentioned herein disclose and describe methods and / or materials in connection with reference to those publications.

[0171] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of the error of the system or method used to determine that value. For example, the term "about" may refer to a range equal to a particular value plus or minus twenty percent (+ / - 20%). In any embodiment discussed in the context of numerical values ​​used in conjunction with the term "about," it is particularly considered that the term "about" may be omitted.

[0172] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “analyte” includes one analyte and multiple different analytes, and reference to “the molecule” includes reference to one or more molecules. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a basis for the use of exclusive terms such as “unique,” ​​“only,” etc., in relation to the descriptive or “negative” use of claim elements.

[0173] The terms “comprise,” “include,” “contain,” “have,” and variations thereof, such as “comprising,” “comprises,” and “comprised,” are intended not to exclude further additions, components, integers, or steps. These terms also encompass the meaning of “consist of” or “consisting of.”

[0174] The term “and / or” refers to any one, any several, or all of the elements connected by the term.

[0175] It should be understood that the methods of the present invention can be performed in vivo, in vitro, or ex vivo. The methods of the present invention may not be used for disease treatment purposes, and / or for disease diagnosis purposes.

[0176] As used herein, the term "modified" refers to an altered state or structure of the molecules of the present invention. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally, for example, by introducing molecules or groups through covalent linkage.

[0177] The terms "alkynyl" and "alkynyl group" are used interchangeably and refer to -C≡C-.

[0178] The terms "azide" and "azido" are used interchangeably and both refer to -N3.

[0179] Single-molecule chemical reactions caused by the rapid formation or breaking of chemical bonds are difficult to observe even with state-of-the-art instruments. Bio-nanopores can be designed as single-molecule reactors to detect the binding of single-atom ions or the transient appearance of chemical intermediates. However, this type of pore engineering is technically challenging, which greatly limits its further development. We propose a general strategy, “programmable nano-reactors for stochastic sensing” (PNRSS), through which various single-molecule reactions involving hydrogen peroxide, metal ions, ethylene glycol, glycerol, lactic acid, vitamins, catecholamines, or nucleoside analogs can be directly observed. PNRSS provides fine sensing resolution that can be further enhanced by artificial intelligence algorithms. Remdesivir, a nucleoside analog and investigational antiviral drug for the treatment of COVID-19, can be distinguished from its active triphosphate form using PNRSS, demonstrating its potential application in pharmacokinetics or drug screening.

[0180] Single-molecule chemical reactions caused by the rapid formation or breaking of chemical bonds are difficult to observe even with state-of-the-art instruments. Bio-nanopores can be designed as single-molecule reactors to detect the binding of single-atom ions or the transient appearance of chemical intermediates. However, this type of pore engineering is technically challenging, which greatly limits its further development. We propose a general strategy, “programmable nano-reactors for stochastic sensing” (PNRSS), through which various single-molecule reactions involving hydrogen peroxide, metal ions, ethylene glycol, glycerol, lactic acid, vitamins, catecholamines, or nucleoside analogs can be directly observed. PNRSS provides fine sensing resolution that can be further enhanced by artificial intelligence algorithms. Remdesivir, a nucleoside analog and investigational antiviral drug for the treatment of COVID-19, can be distinguished from its active triphosphate form using PNRSS, demonstrating its potential application in pharmacokinetics or drug screening.

[0181] We propose a general strategy, the “Programmable Nanoreactor for Random Sensing” (PNRSS), to democratize nanopore-based single-molecule chemistry research. PNRSS involves the synergistic synthesis of polymer chains and nanopores (defined as PNRSS chains and PNRSS pores, respectively). The PNRSS chain itself consists of functional modules defined as tie sites, extension segments, reaction segments, and traction segments. Figure 1 a). Tie sites are used to attach one end of the strand to a ligand (e.g., streptavidin). The PNRSS strand of the streptavidin ligand is passed through the electrophoretic well and remains fully extended in the PNRSS well. Figure 1 b). This setup has previously been used to distinguish different nucleic acid sequences. 15,33,34 However, it has never been used for single-molecule chemical measurements in nanopores. This setup significantly reduces the technical barriers to pore engineering. The design of the PNRSS chain is completely flexible, and the corresponding synthesis can be performed using low-cost commercial services. The length of the extended segments of the PNRSS chain is optimized to an accuracy of [insert accuracy here]. The reaction segment is positioned at the pore constriction point to achieve optimal performance. One or more reaction sites within the reaction segment form a stationary reactant that directly participates in the reaction under study. The traction segment maintains the electrophoretic forces on the chain. The mobile reactant bound to the stationary reactant is placed in the measurement environment and plays a role when bound to the stationary reactant. The PNRSS chain can consist of any synthetic polymer, such as nucleic acids, peptides, polysaccharides, or combinations thereof, but for the study of a wider range of single-molecule reactions, the composition of the PNRSS chain should be arbitrarily programmable. DNA is the most studied synthetic polymer and can be easily and economically synthesized, chemically modified, enzymatically treated, purified, characterized, and stored.35 This method is not limited to DNA, but it is an ideal component of the PNRSS chain.

[0182] PNRSS pores should possess sharp and narrow confinement for high spatial resolution, rigid and reproducible structures for high measurement consistency, and chemically inert cavities to minimize undesirable reactions. Recent reports on engineered Mycobacterium smegma porin A (MspA) in single-molecule chemistry applications have demonstrated its structural advantages, with a significantly expanded event amplitude (~55 pA). 27 However, this paper applies the MspA (method) to demonstrate all PNRSS measurements.

[0183] In summary, single-molecule chemical reactions caused by the rapid formation or breaking of chemical bonds are difficult to observe even with state-of-the-art instruments. Bio-nanopores can be designed as single-molecule reactors capable of detecting the binding of single-atom ions or the transient appearance of chemical intermediates. However, this type of pore engineering is technically challenging, which greatly limits its further development. We propose a general strategy, “Programmable Nanoreactors for Random Sensing” (PNRSS), through which various single-molecule reactions involving hydrogen peroxide, metal ions, ethylene glycol, glycerol, lactic acid, vitamins, catecholamines, or nucleoside analogs can be directly observed. PNRSS provides fine sensing resolution that can be further enhanced by artificial intelligence algorithms. Remdesivir, a nucleoside analog and investigational antiviral drug for the treatment of COVID-19, can be distinguished from its active triphosphate form using PNRSS, demonstrating the application of PNRSS in pharmacokinetics or drug screening.

[0184] Nanopores

[0185] As used herein, the term "nanopore" generally refers to a pore, channel, or tube with a diameter on the nanometer scale that extends through a membrane. Nanopores can have a characteristic width or diameter on the order of 0.1 nanometers (nm) to approximately 1000 nm.

[0186] The nanopores of the present invention can be of any form and can be biological nanopores or synthetic nanopores. As known to those skilled in the art, the nanopores of the present invention can be, for example, solid nanopores, protein nanopores, hybrid solid-protein nanopores, or DNA origami nanopores.

[0187] Examples of protein nanopores include α-hemolysin (α-HL), Mycobacterium smegmatis porin A (MspA), aerolysin, curli production assembly / transport component (CsgG), outer membrane porin F (OmpF), cytolysin A (ClyA), ferric hydroxamate uptake component A (FhuA), fragaceatoxin C (FraC), pleurodactylysin A (PlyA) / pleurodactylysin B (PlyB), curli production assembly / transport component (CsgG), and Phi29 linker protein. Protein nanopores can be naturally occurring wild-type protein nanopores or homologs or variants of wild-type protein nanopores.

[0188] The sequences of wild-type protein nanopores are available in GenBank at https: / / www.ncbi.nlm.nih.gov / . For example, wild-type MspA can have the following amino acid sequence:

[0189] GLDNELSLVDGQDRTLTVQQWDTFLNGVFPLDRNRLTREWFHSGRAKYIVAGPGADEFEGTLELGYQIGFPWSLGVGINFSYTTPNILIDDGDITAPPFGLNSVITPNLFPGVSISADLGNGPGIQEVATFSVDVSGAEGGVAVSNAHGTVTGAAGGVLLRPFARLIASTGDSVTTYGEPWNMN (SEQ ID NO: 1).

[0190] As those skilled in the art know, protein nanopores typically include constricted regions, which are the narrowest parts of the nanopore channel. Protein nanopores may also include vestibules located at one end of the nanopore channel, which are also part of the nanopore channel but have a larger diameter than the constricted regions.

[0191] Some protein nanopores may contain two or more monomers that associate with each other to form a channel, wherein each monomer may be identical or different. Any monomer forming the protein nanopore may be selected from wild-type proteins, or their homologs or variants. In some embodiments, all monomers in the protein nanopore are identical.

[0192] As defined herein, the term "homologous" is a gene or its protein product that has a similar structure and function to another gene or its protein product. A homolog may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with its counterpart. The term "homologous" is sometimes used to refer to a relationship between genes or their protein products that diverged due to a speciation event (see "orthologous homologs"), or to refer to a relationship between genes or their protein products that diverged due to a gene replication event (see "paralogous homologs"). The term "orthologous homolog" refers to a gene or its protein product that evolved from a common evolutionary origin in different species. The term "paralogous homolog" refers to a gene that is related by replication within the genome.

[0193] Variants may have one or more mutations (e.g., one or more additions, substitutions, and / or deletions of amino acids) compared to their wild-type mutations, and retain the ability to form channels.

[0194] Those skilled in the art will readily understand how to determine the identity of two polypeptides. For example, identity can be calculated after aligning two sequences to achieve the highest possible level of identity. For instance, to determine the "percentage of identity" between two amino acid sequences or two nucleic acids, the sequences are arranged for optimal comparison purposes (e.g., vacancies can be introduced into the sequence of the first amino acid or nucleic acid sequence to achieve optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences can be a function of the number of identical positions shared by the two sequences (i.e., percentage of identity = number of identical positions / total number of positions (e.g., overlapping positions) × 100). In one embodiment, the two sequences are of the same length. Sequence identity can be determined in a variety of different ways and using various algorithms. To determine sequence identity, various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) can be used to align sequences. These methods and programs are available on the World Wide Web, including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , and mafft.cbrc.jp / alignment / software / . See, for example, Altschul et al. (1990), J.Mol.Bioi. 215:403-10.

[0195] In some embodiments, mutations (e.g., the addition, substitution, and / or deletion of one or more amino acids) can occur at any site, such as on the pore surface of the protein nanopore, at the edge of the periplasmic ring, or on the outer side. In some embodiments, for protein nanopores with a conical cavity, mutations can occur in the constricted region and / or vestibule of the protein nanopore.

[0196] In some embodiments, the variant of the protein nanopore may contain at least one additional positively charged amino acid, at least one additional negatively charged amino acid, at least one less positively charged amino acid, or at least one less negatively charged amino acid in its pore cavity, compared to its parent protein.

[0197] In some embodiments, one or more positively charged amino acids in the pore of the protein nanopore are replaced by negatively charged amino acids, and each negatively charged amino acid may be the same or different; or one or more negatively charged amino acids in the pore of the protein nanopore are replaced by positively charged amino acids, and each positively charged amino acid may be the same or different.

[0198] As an example of a variant, a variant of MspA may contain (i) a mutation such that amino acids at positions 90, 91, and 93 contain a neutrally charged amino acid, and (ii) one or more mutations at positions 88, 105, 108, 118, 126, 134, 138, or 139; preferably, compared to wild-type MspA, variant MspA may contain the D90N / D91N / D93N or D93N / D91N / D90N / D118R / D134R / E139K mutations. D90N / D91N / D93N or D93N / D91N / D90N / D118R / D134R / E139K means that the mutant contains all six listed mutants; more preferably, the variant of MspA may have only the D90N / D91N / D93N (M1 MspA) or D93N / D91N / D90N / D118R / D134R / E139K (M2 MspA) mutations compared to wild-type MspA. The numbers used herein identify the site of the mutagenesis, where the first amino acid immediately following the start codon is defined as 1.

[0199] In this invention, the protein nanopore can be a recombinant protein.

[0200] Examples of solid-state nanopores include nanopores made of solid materials such as SiNx, graphene, glass, and quartz.

[0201] Examples of hybrid nanopores include protein nanopores disposed in a solid membrane or solid nanopores having protein nanopores embedded therein.

[0202] Nanopores can be modified, for example, chemically modified. Nanopores can be chemically modified in any way and at any location, such as on the surface of the pore cavity. Protein nanopores can be chemically modified by linking molecules to one or more amino acids (e.g., cysteine ​​or lysine). Suitable methods for such modifications are well known in the art. Nanopores can be chemically modified by linking any molecule. For example, nanopores can be chemically modified by linking a reaction handle. Protein nanopores can be chemically modified by linking an adaptor (e.g., cyclodextrin) that affects the physical or chemical properties of the nanopore.

[0203] Preferably, the protein nanopores used in this invention do not spontaneously gate even at 150mV-200mV or higher. "Gating" refers to a spontaneous change in the conductivity through a protein channel, which is typically temporary (e.g., lasting from 1-10 milliseconds to up to 1 second). For some protein nanopores, the likelihood of gating increases with the application of higher voltages. Typically, during gating, the conductivity of the protein decreases, and conductivity may therefore permanently cease (i.e., the channel may permanently close), a process that is irreversible. Optionally, gating refers to the spontaneous change in the conductivity through the protein channel to less than 75% of its open-state current.

[0204] The methods for preparing nanopores are well known to those skilled in the art. For example, protein nanopores can be prepared by prokaryotic expression and are easily purified by chromatography, while solid nanopores can be prepared by etching methods using focused ion beams and high-energy electron beams.

[0205] Polymer chain (PNRSS chain)

[0206] As used herein, a PNRSS chain is typically a polymer chain containing one or more sensing modules; therefore, in this invention, a PNRSS chain is also referred to as a polymer chain. In this invention, the PNRSS chain enters the channel of a nanopore. Preferably, the PNRSS chain extends within the channel of the nanopore. After the target analyte enters the channel of the nanopore, the interaction between the sensing module and the target analyte causes nanopore blockage, which is measurable, for example, as a change in ion current. The analyte can be characterized by measuring the blockage caused by the interaction. Characterization of different analytes can be achieved by using different sensing modules, thereby making the PNRSS chain programmable.

[0207] The polymer chains of the present invention can be driven into nanopores and extended in the channels of nanopores in any way, for example by a voltage across the nanopores.

[0208] The polymer chain of the present invention includes at least a chain-linking site and a reactive segment. The polymer chain may be charged, for example, positively or negatively charged.

[0209] Tethering sites are used to tether (or constrain) polymer chains, preventing them from migrating through the nanopores. Tethering sites can be located at one end of the polymer chain. The polymer chain is tethered so that the reactive segment is located in a region suitable for measuring blockage. The polymer chain can be tethered to any suitable substrate via tethering sites in any suitable manner.

[0210] For example, but not limited to, the substrate can be a barrier molecule. The barrier molecule can have a size that prevents it from passing through (preferably entering) the nanopore. In other words, the size of the barrier molecule, or at least a portion thereof, is larger than the opening of the nanopore. The barrier molecule can have a three-dimensional structure, which determines its size. The barrier molecule can be any molecule that meets the above size requirements, such as a protein molecule.

[0211] Another example of a substrate is the nanopore itself, such as a protein nanopore. Polymer chains can be chained to any suitable location within the protein nanopore, such as any amino acid outside the protein nanopore channel (e.g., the edge or outer side of the periplasmic ring of the protein nanopore).

[0212] The polymer chain can be attached to the substrate by any suitable means known to those skilled in the art.

[0213] For example, a polymer chain can be tied to a barrier molecule via a high binding affinity between a protein and a compound. The barrier molecule can be a protein capable of specifically binding to a compound (e.g., a small molecule), and the tying site contains that compound. The polymer chain can also be tied to the barrier molecule via a high binding affinity between the protein and the compound. For example, the barrier molecule can be an antibody to a hapten, and the tying site can contain the hapten. For example, the barrier molecule can be streptoacidin, and the tying site can contain biotin. As another example, the barrier molecule can be an anti-digoxigenin antibody, and the tying site can contain digoxigenin.

[0214] As another example, the barrier molecule can be any protein containing natural amino acids that can react with small molecules, or the nanoporous protein can contain natural amino acids that can react with small molecules, and the ligation site can contain the small molecule. The natural amino acid can be located on the surface of the barrier molecule or the nanoporous protein. The natural amino acid can be located on the edge or outer surface of the periplasmic ring of the nanoporous protein, or on the surface near the channel opening of the nanoporous protein. Polymer chains can be chained to barrier molecules or nanoporous proteins through reactions between natural amino acids and small molecules that can react with natural amino acids. These reactions include, but are not limited to, the Michael addition reaction between the thiol group of cysteine ​​and maleimide or its derivatives (Nair, DP et al., 2013, The Thiol-Michael Addition Click Reaction: A Powerful and Widely Used Tool in Materials Chemistry. Chemistry of Materials, 26(1), 724–744), the covalent bonding between iodoacetamide or its derivatives and the thiol group of cysteine ​​(Tyagarajan, K. et al., 2003, Thiol-reactive dyes for fluorescence labeling of proteomic samples. ELECTROPHORESIS, 24(14), 2348–2358), and the thiol-ene reaction between the thiol group of cysteine ​​and the vinyl group of the compound (Dondoni, A., 2008, The Emergence of Thiol-Ene Coupling as a Click Process for Materials and Bioorganic). Chemistry. Angewandte Chemie International Edition, 47(47), 8995–8997); Thiol-thiol reaction between the thiol group of cysteine ​​and the thiol of the compound (Gilbert, HF, 1995, [2] Thiol / disulfide exchange equilibria and disulfide bond stability. Biothiols Part A Monothiols and Dithiols, Protein Thiols, and Thiyl Radicals, 8–28); Lysine and trimethylammonium nitrobenzene (Sutton, DAReaction between 1-fluoro-2-nitro-4-trimethylammoniobenzene iodide, a protein-solubilizing reagent. Biochemical Journal, 130(2), 589–595) or aryl halides (Lautrette, G. et al., 2016, Nitrogen Arylation for Macrocyclization of Unprotected Peptides. Journal of the American Chemical Society, 138(27), 8340–8343); reaction between methionine and oxapropidine or its derivatives (Lin, Shixian, et al., 2017, Redox-based reagents for chemoselectivemethionine bioconjugation, Science, 355(6325), 597-602). Natural amino acids that react with small molecule compounds include, but are not limited to, cysteine, lysine and / or methionine. Small molecule compounds that react with natural amino acids include, but are not limited to, maleimide or its derivatives, iodoacetamide or its derivatives, small molecules containing vinyl or thiols, trimethylnitrofluorophenylamine, aryl halides, and / or oxaprodiidine or its derivatives. Preferably, in the barrier molecule or nanoporous protein, the natural amino acid groups that can react with the small molecule compounds are free. Preferably, in the barrier molecule or nanoporous protein, the natural amino acids and / or the natural amino acid groups that can react with the small molecule compounds are exposed on the surface of the barrier molecule or nanoporous protein.

[0215] For example, D56 of MspA can be mutated to cysteine, and the linking site can contain maleimide or its derivative, iodoacetamide or its derivative, or a small molecule containing vinyl or thiol, and the polymer chain is linked to the cysteine ​​at position 56 of MspA.

[0216] As another example, the barrier molecule can be any protein with an introduced first reactive handle, or the first reactive handle can be introduced into a nanoporous protein. For example, a non-natural amino acid containing the first reactive handle can be incorporated into the barrier molecule or nanoporous protein, for example, during the artificial synthesis of the protein or through chemical modification of the protein. Thus, the barrier molecule or nanoporous protein contains an exposed first reactive handle, a tethering site contains a second reactive handle that can react with the first reactive handle, and a polymer chain is tethered to the barrier molecule or nanoporous protein through a reaction between the first and second reactive handles. The natural amino acid can be located on the surface of the barrier molecule or nanoporous protein. The first reactive handle can be located on the surface of the barrier molecule, or on the edge or outer side of the periplasmic ring of the nanoporous protein, or on the surface near the channel opening of the nanoporous protein.

[0217] As used herein, the term "reactor handle" means a chemical molecule, chemical part, or chemical group that is exposed and can react with another reactor handle. A reactor handle pair consists of a first reactor handle and a second reactor handle, wherein the first reactor handle can react with the second reactor handle. Reactor handle pairs are known to those skilled in the art. Reactor handle pairs that can be used in this invention include, but are not limited to, click reactor handles.Examples of reaction handle pairs include, but are not limited to, azides and alkynes that can react with each other via copper(i)-catalyzed alkyne-azide cycloaddition (CuAAC); azides and difluorocyclooctylenes that can react with each other via copper-free alkyne-azide cycloaddition; azides and phosphine that can react via staudinger linkage; thiols and alkenes that can react with each other via radical addition; thiols and maleimides that can react with each other via Michael addition; and amines and parafluorines that can react with each other via nucleophilic substitution (Becer, Hoogenboom and Schubert, Click Chemistry beyond Metal-Catalyzed Cycloaddition, Angewandte Chemie International Edition, 2009, 48:490-4908; Rostovtsev, VV et al., 2002, Astepwise Huisgen cycloaddition process: Copper(i)-catalyzed regioselective “ligation” of azides and terminal). alkynes. Angew. Chem., Int. Ed. 41, 2596–2599; Torne, CW et al., 2002, Peptidotriazoles on solid phase: [1,2,3]-Triazoles by regiospecificcopper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes toazides.J.Org.Chem.67,3057–3064; Agard, NJ et al., 2004, A strainpromoted[3+2]azide-alkyne cycloaddition for covalent modification of blomolecules inliving systems.J.Am.Chem.Soc.126,15046–15047; Kohn, M. and Breinbauer, R., 2004, The Staudinger ligation: A gift to chemical (biology. Angew. Chem., Int. Ed. 43, 3106–3116). Either of the reaction handles can be used as the first reaction handle or the second reaction handle.

[0218] By designing the substrate and ligation sites, the polymer chains of the present invention can be ligated in a variety of flexible ways, not limited to any of the examples described above.

[0219] The terms “polypeptide” and “protein” are used interchangeably and refer to a polymer of amino acids of any length, which may include naturally occurring and non-naturally occurring amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone.

[0220] The reaction section may contain one or more monomers (e.g., two or more, three or more, four or more, or five or more) and one or more sensing modules (also referred to as stationary reactants in this invention). Each sensing module can be viewed as a sensing site or reaction site that interacts with a single molecule of the target analyte (also referred to as a mobile reactant in this invention). When the reaction section contains two or more monomers, the two or more monomers polymerize to form a polymer chain. For example, the reaction section may be positively or negatively charged.

[0221] As used herein, the term "sensing module" refers to a chemical part that can interact with a single molecule of a target analyte. A sensing module may consist of one or more (e.g., two or more) sensing parts.

[0222] As used herein, the term "part" refers to a chemical molecule or any portion of a chemical molecule, such as a functional group. As used herein, the term "sensing part" refers to a chemical molecule or a portion of a chemical molecule that interacts with one or two or more binding sites of a single molecule of a target analyte. The sensing part may be contained in the side chain of a monomeric unit of a reaction segment. As used herein, the term "side chain" refers to a chemical group attached to the core portion of the molecule, referred to as the "main chain" or backbone.

[0223] As used herein, the term "interaction" can refer to a reaction or binding between a sensing module or sensing element and a target analyte, which can be reversible or irreversible. The interaction between the sensing module and the target analyte can cause a measurable change in the ion current passing through the nanopore.

[0224] A sensing module can consist of a sensing part that can interact with a single molecule of the target analyte independently; this sensing part is called the non-cooperative sensing part.

[0225] A sensing module can also consist of two or more sensing parts, wherein the two or more sensing parts interact together with a single molecule of the target analyte, and each sensing part interacts with one or two or more binding sites of the single molecule. The interaction of two or more sensing parts together with a single molecule of the target analyte is called a co-sensing part. Co-sensing parts can be contained within adjacent monomer units. A single molecule of some target analytes may contain two or more binding sites, through which the sensing parts interact with the target analyte. The two or more binding sites in a molecule can be the same or different from each other, for example, having the same or different groups or the same or different bonds. Two or more co-sensing parts in a sensing module can each interact with two or more binding sites in a molecule. The two or more co-sensing parts in a sensing module can be the same or different and can be designed according to the binding sites of the target analyte. Sensing modules composed of co-sensing parts can more easily and advantageously capture analyte molecules.

[0226] For example, if the reaction segment is a nucleic acid, two adjacent purines selected from the group consisting of guanine and adenine can form a sensing module to capture divalent metal ions (e.g., Ni). 2+ Co 2+ or Cu 2+ Zn 2+ Cd 2+ A single purine molecule can have two adjacent purines that are the same or different.

[0227] The total number of sensing modules within the reaction section can be from 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Two or more sensing modules within the reaction section can be identical or different from each other. In some embodiments, all sensing modules within the reaction section are identical. In some embodiments, some sensing modules within the reaction section are identical. In some embodiments, all sensing modules within the reaction section are different from each other.

[0228] The reaction section may contain two or more sensing modules that can interact with different target analytes to facilitate the simultaneous or sequential characterization of different target molecules.

[0229] For example, two or more identical or different sensing modules, or combinations thereof, can be used, provided that the sensing modules can interact with different target analytes. In this case, the different target analytes can interact with two or more sensing modules respectively, thereby being characterized simultaneously.

[0230] For example, two or more different sensing modules can be used, each capable of interacting with a specific target analyte and having no cross-reactivity with other target analytes to be characterized. In this case, different analytes can not only be characterized simultaneously but also separately in successive rounds. For example, a first target analyte in a first sample can be characterized first, and then a second target analyte in a second sample can be characterized sequentially without altering the PNRSS chain. As an example, the reaction section can include a first sensing module interacting with the first target analyte and a second sensing module interacting with the second target analyte, and the first and second sensing modules are distinct, wherein the first sensing module does not interact with the second target analyte and the second sensing module does not interact with the first target analyte. In the first round of measurements, the first target molecule occupies the first sensing module and is characterized, and in the second round of measurements, although the first sensing module is occupied, the second sensing module remains available, allowing the second target molecule to interact with and be characterized.

[0231] Two or more monomer units containing sensing portions may be arranged adjacent to each other. Optionally, two or more monomer units containing sensing portions may be separated by one or more monomer units without sensing portions. A reaction segment may consist of one or more monomer units containing sensing portions, such as one monomer unit containing a sensing portion or two or more monomer units containing sensing portions arranged adjacent to each other. A reaction segment may also include one or more monomer units containing sensing portions and additional monomer units without sensing molecules (e.g., 1-3 monomer units located on either side of any monomer unit containing a sensing portion, or 1-3 monomer units located between any two monomer units containing sensing molecules).

[0232] The size (e.g., diameter or width) of the reaction section with the sensing module should be set such that the reaction section can enter and be contained in the channel of the nanopore, and that the blockage caused by the interaction between the sensing module and the target analyte can be measured.

[0233] While the narrowest regions of nanopores exhibit the highest sensitivity, it should be understood that measurable blockage can occur in any region of the channel. The quality and resolution of the blockage signal are related to the diameter of a particular region of the channel and the size of the blockage present in that region. For the designed reaction segment, those skilled in the art should know which region is suitable to accommodate it, for example, based on the diameter of the nanopore channel, the size of the reaction segment of the polymer chain, and the size of the target analyte. For example, the larger the reaction segment of the polymer chain and the target analyte, the more suitable the channel region with a larger diameter; the smaller the reaction segment of the polymer chain and the target analyte, the more suitable the channel region with a smaller diameter. For example, for some protein analytes with three-dimensional structures, the vestibule of a protein nanopore with a conical cavity may be suitable. For example, for target analytes with small dimensions, a nanopore with a cylindrical cavity or a constricted region of a protein nanopore with a conical cavity may be suitable. Those skilled in the art can determine which region of the nanopore channel is suitable for obtaining a measurable blockage signal caused by the interaction between the reaction segment of the polymer chain and the target analyte. Therefore, those skilled in the art can determine the position of the reaction segment on the polymer chain and / or the length of the reaction segment to position the reaction segment appropriately.

[0234] The sensing module can be designed to interact with a specific target analyte, and the reaction section can be designed to contain the sensing module. Suitable monomeric units can be designed to contain the sensing portions constituting the sensing module. Methods for preparing the designed reaction section having one or more sensing modules should be well known to those skilled in the art. The designed reaction section having one or more sensors can be prepared in any suitable manner, such as by chemical synthesis.

[0235] For example, a reaction segment can be synthesized using monomers containing a sensing portion, such that the sensing portion is incorporated into the reaction segment. In the preparation of the reaction segment, one or more monomers containing a sensing portion are incorporated into the reaction segment. Such monomers include, but are not limited to, natural nucleotides (e.g., guanine nucleotides, adenine nucleotides, thymine nucleotides, cytosine nucleotides, or uracil nucleotides) and amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). For example, nucleotides or nucleotide analogs containing guanine, adenine, thymine, cytosine, or uracil can be used as monomers for synthesizing the reaction segment, and guanine, adenine, thymine, cytosine, or uracil can be used as the sensing portion or sensing module. In some embodiments, the synthesized reaction segment comprises two adjacent purine nucleotides selected from the group consisting of guanine nucleotides and adenine nucleotides. The two adjacent purines constitute the sensing module.

[0236] As another example, the monomers used for synthesis may contain functional groups, and these functional groups may be further modified to form sensing moieties, thereby incorporating the sensing moieties into the reaction section. In the preparation of the reaction section, one or more monomers containing functional groups are incorporated into the reaction section. Such monomers include, but are not limited to, 5-ethynyl-dU-CE phosphorous amide. For example, monomers containing alkynes or azides (e.g., 5-ethynyl-dU-CE phosphorous amide) are used in the synthesis, and the alkynes or azides may be further modified by Huisgen copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) to form 1,2,3-triazoles, which can be used as sensing moieties or sensing modules.

[0237] As another example, the monomer used for synthesis may include a first reactive stem, which may further react with a second reactive stem connected to the sensing portion, thereby incorporating the sensing portion into the reaction section. In the preparation of the reaction section, one or more monomers containing a first reactive stem are incorporated into the reaction section. The term "reactive stem" is defined as above. For example, a monomer containing an alkyne or azide (e.g., 5-ethynyl-dU-CE phosphorous amide) is used for synthesis, wherein the alkyne or azide serving as the first reactive stem may further react with a second reactive stem (azide or alkyne) connected to the sensing portion, such as a second reactive stem connected to PBA.

[0238] The above methods can be used in combination. As an example of the combination of the above methods, the monomer used for synthesis may contain functional groups, and the functional groups may be further modified to form a first reaction handle, and the first reaction handle may further react with a second reaction handle connected to the sensing part, thereby incorporating the sensing part into the reaction section.

[0239] It should be understood that the above-described method for preparing the reaction section with the sensing component is merely exemplary and is not intended to limit the scope of the invention. The sensing component can be incorporated into the reaction section in any suitable manner.

[0240] The sensing module can be designed to match the target molecule to be characterized. Different target analytes can be characterized simply by changing the sensing module on the PNRSS chain. For example, target analytes can include, but are not limited to:

[0241] Ions containing a metallic element, which can be cations or anions, polyatomic ions or monatomic ions, and can contain alkaline earth metals or transition metals, such as AuCl4. - Mg 2+ Ca 2+ Ba 2+ Ni 2+ Cu 2+ Co2+ Zn 2+ Cd 2+ Ag 2+ Pb 2+ wait.

[0242] Sugars, such as monosaccharides, oligosaccharides, or polysaccharides, wherein monosaccharides may be selected from ribose, fructose, and mannose, for example, D-(-)-ribose, D-fructose, and D-(+)-mannose; oligosaccharides may be selected from disaccharides and trisaccharides; wherein examples of disaccharides may include 4-O-β-d-galactopyranosyl-d-fructofuranose (lactulose) or 6-O-α-D-glucopyranosyl-D-fructofuranose (isomaltulose); and examples of trisaccharides may include 4-ObD-galactosylsucrose (galactosylsucrose).

[0243] Glucoside;

[0244] Polyphenols, such as anthocyanins or proanthocyanidins;

[0245] Catecholamines or catecholamine derivatives, such as adrenaline, noradrenaline, or isoproterenol;

[0246] Polyols, such as compounds containing two ortho-hydroxyl groups, 1,2-cis-diol or 1,3-cis-diol moieties, for example, 3,4-dihydroxymandelic acid, 4-hydroxy-3-methoxymandelic acid (VMA), 3,4-dihydroxyphenylacetic acid, catechol, ethylene glycol, glycerol, L-lactic acid or vitamins (e.g. vitamin C or vitamin B6);

[0247] Compounds in protonated or deprotonated form, such as protonated or deprotonated tris;

[0248] Compounds containing a ribose moiety, such as nucleotides, nucleosides, their analogs or monophosphate derivatives or polyphosphate derivatives thereof, such as ribonucleotides or deoxyribonucleotides, galidesvir, ribavirin, favipiravir-RTP, remdesivir or their triphosphate metabolites, such as cytidine 5'-monophosphate (5'-CMP);

[0249] Hydrogen peroxide;

[0250] Oligopeptides or cyclic peptides;

[0251] Buffer reagents, such as tris;

[0252] Small molecule drugs, such as nucleoside analogues, such as galidesvir, ribavirin, favipiravir-RTP, remdesivir or their triphosphate metabolites;

[0253] Neurotransmitters, such as catecholamines or their derivatives;

[0254] Compounds with specific chirality, such as L-norepinephrine or D-norepinephrine;

[0255] Analytes containing isotopes, such as catechol-D6 (deuterium-substituted for all hydrogen atoms in catechol);

[0256] Chemical intermediates;

[0257] Or any combination thereof.

[0258] For example, guanine, adenine, a sensing module consisting of two adjacent purines selected from the group consisting of guanine and adenine, or 1,2,3-triazole can be used as sensing modules to interact with metal-containing ions used as target analytes. The metal-containing ions can be cations or anions. They can be polyatomic or monatomic ions. They can be alkaline earth metal or transition metal ions, such as AuCl4. - Mg 2+ Ca 2+ Ba 2+ Ni 2+ Cu 2+ Co 2+ Zn 2+ Cd 2+ Ag 2+ Pb 2+ wait.

[0259] For example, the PBA can be used as a sensing module to interact with the following target analytes:

[0260] Sugars include monosaccharides, oligosaccharides, or polysaccharides, wherein monosaccharides may be selected from ribose, fructose, and mannose, such as D-(-)-ribose, D-fructose, and D-(+)-mannose; oligosaccharides may be selected from disaccharides and trisaccharides; examples of disaccharides may include 4-O-β-d-galactopyranosyl-d-fructofuranose (lactulose) or 6-O-α-D-glucopyranosyl-D-fructofuranose (isomaltulose); examples of trisaccharides may include 4-ObD-galactosylsucrose (galactosylsucrose);

[0261] Glucoside;

[0262] Polyphenols, such as anthocyanins or proanthocyanidins;

[0263] Catecholamines or catecholamine derivatives, such as adrenaline, noradrenaline, or isoproterenol;

[0264] Polyols, such as compounds containing two ortho-hydroxyl groups, 1,2-cis-diol or 1,3-cis-diol moieties, for example, 3,4-dihydroxymandelic acid, 4-hydroxy-3-methoxymandelic acid (VMA), 3,4-dihydroxyphenylacetic acid, catechol, ethylene glycol, glycerol, L-lactic acid or vitamins (e.g. vitamin C or vitamin B6);

[0265] Compounds in protonated or deprotonated form, such as protonated or deprotonated tris;

[0266] Compounds containing a ribose moiety, such as nucleotides, nucleosides, their analogs or monophosphate derivatives or polyphosphate derivatives thereof, such as ribonucleotides or deoxyribonucleotides, galidesvir, ribavirin, favipiravir-RTP, remdesivir or their triphosphate metabolites, such as cytidine 5'-monophosphate (5'-CMP);

[0267] Hydrogen peroxide;

[0268] Oligopeptides or cyclic peptides;

[0269] Buffer reagents, such as tris;

[0270] Small molecule drugs, such as nucleoside analogues, such as galidesvir, ribavirin, favipiravir-RTP, remdesivir or their triphosphate metabolites;

[0271] Neurotransmitters, such as catecholamines or their derivatives;

[0272] Compounds with specific chirality, such as L-norepinephrine or D-norepinephrine;

[0273] Analytes containing isotopes, such as catechol-D6 (deuterium-substituted for all hydrogen atoms in catechol);

[0274] Chemical intermediates;

[0275] Or any combination thereof.

[0276] In some embodiments, the polymer chain may further include an extension segment between the linker site and the reactive segment, although the extension segment is not required.

[0277] If the length of the PNRSS chain, consisting only of the reaction segment and ligation site, is sufficient to position the reaction segment in a region suitable for measuring the blockage caused by the interaction between the sensing module and the target analyte after the PNRSS chain has been driven into the nanopore channel, then an extension segment may not be necessary. However, if the length of the PNRSS chain, consisting only of the reaction segment and ligation site, is insufficient to position the reaction segment in a region suitable for measuring the blockage caused by the interaction between the sensing module and the target analyte after the PNRSS chain has been driven into the nanopore channel, an extension segment can be used to adjust the position of the reaction segment within the channel, positioning it appropriately within the nanopore channel so that the blockage caused by the interaction between the reaction segment and the target analyte can be measured. In a preferred embodiment, the extension segment is used to position the reaction segment in the narrowest region of the nanopore channel. Therefore, the length of the extension segment depends on the specific needs. The extension segment may contain one or more monomeric units. For example, the extension segment may contain oligonucleotides. The length of the oligonucleotide can be 1 nt or more, 2 nt or more, 3 nt or more, 4 nt or more, 5 nt or more, 6 nt or more, 7 nt or more, 8 nt or more, 9 nt or more, 10 nt or more, 11 nt or more, 12 nt or more, 13 nt or more, 14 nt or more, or 15 nt or more.

[0278] In some implementations, the polymer chain may further include a traction segment, although the traction segment is not required.

[0279] The traction segment can be located on the side of the reaction segment opposite to the tethering site; that is, the traction segment and the tethering site are not on the same side of the reaction segment. For example, the traction segment can be positively or negatively charged.

[0280] The traction segment can be used to hold polymer chains (especially the reactive segment) within the channels of a nanopore. The traction segment keeps the reactive segment within the nanopore channel in a region suitable for measurement of blockages caused by the interaction between the target analyte and the sensing module. The traction segment prevents reverse movement of the polymer chains. The traction segment prevents the polymer chains from exiting the nanopore entrance. The traction segment helps stabilize the reactive segment within the channel region suitable for measurement of blockages caused by the interaction between the reactive segment and the target analyte.

[0281] "Reverse" refers to the direction opposite to the direction in which the polymer chains move into the nanopores.

[0282] "Exiting the nanopore entrance" means that the polymer chain leaves the nanopore from the opening through which it entered.

[0283] A traction segment is not necessary. Measurements can be achieved even without a traction segment to hold the reaction segment within the nanopore, as long as the polymer chains can temporarily enter and extend into the nanopore.

[0284] There are no restrictions on the form of the traction section, as long as it can function in any way.

[0285] For example, the traction segment can be designed as a polymer chain that, under the influence of electrophoretic force or electroosmosis in an electric field applied to the nanopore, tends to move to the other side of the nanopore channel (or tends to pass through the nanopore channel), thereby pulling the reaction segment. For example, the traction segment can contain oligonucleotides. The length of the oligonucleotide can be 10nt or more, 11nt or more, 12nt or more, 13nt or more, 14nt or more, 15nt or more, 16nt or more, 17nt or more, 18nt or more, 19nt or more, 20nt or more, 21nt or more, 22nt or more, 23nt or more, 24nt or more, 25nt or more, 26nt or more, 27nt or more, 28nt or more, 29nt or more, 30nt or more, 31nt or more, 32nt or more, 33nt or more, 34nt or more, 35nt or more, 36nt or more, 37nt or more, 38nt or more, 39nt or more, 40nt or more, 41nt or more, 42nt or more, 43nt or more, 44nt or more, 45nt or more, 50nt or more, or 55nt or more.

[0286] "The other side of the channel" or "the other side of the nanopore" refers to the side opposite the opening through which the polymer chain enters the nanopore.

[0287] As another example, the traction segment can be a coupling site used to chain the reactive segment to the surface of the nanoporous channel. For instance, the traction segment can be a small molecule capable of reacting with native amino acids on the surface of the nanoporous channel. The reactive segment can be chained to the surface of the nanoporous channel via a reaction chain between the native amino acid and the small molecule. Examples of small molecules capable of reacting with native amino acids are as described above.

[0288] As another example, a first reactive stem can be introduced into the surface of a nanoporous channel, and a traction segment can contain a second reactive stem. For instance, a non-natural amino acid containing the first reactive stem can be incorporated into a nanoporous protein, for example, during the artificial synthesis of the protein or through chemical modification of the protein, so that the introduced first reactive stem is located on the surface of the nanoporous channel. The reaction segment can be chained to the surface of the nanoporous channel via the reaction system between the first and second reactive stems. The reactive stems are defined as above.

[0289] As another example, the traction segment can be designed as a polymer chain, and at least a portion of the traction segment can be driven through the nanopore in an electric field applied to it. The portion of the traction segment that passes through the nanopore can form a three-dimensional structure outside the nanopore. The three-dimensional structure can have a size larger than the outlet of the nanopore and can prevent said portion of the traction segment from retracting back into the nanopore. The length of the traction segment can be designed to ensure that the portion capable of forming the three-dimensional structure can pass through the nanopore and reach the outside of the nanopore. In some embodiments, the traction segment can contain nucleic acid. In some embodiments, the three-dimensional structure can be a hairpin structure. In some embodiments, the three-dimensional structure can be a hairpin structure formed by a nucleic acid sequence.

[0290] As used herein, the term "polymer" refers to a molecule comprising two or more monomers linked together by covalent bonds. The term "polymer" includes homopolymers, copolymers, and biopolymers such as nucleic acids or peptides.

[0291] As used herein, the terms “polymer strand,” “polymer chain,” or “polymer” are used interchangeably and can be linear or branched.

[0292] The terms “monomer,” “unit,” “monomer unit,” and “structural unit” are used interchangeably in this invention and refer to the constituent units of a polymer. A polymer chain or the two or more monomers contained within a polymer chain may be the same or different, or some of them may be the same.

[0293] The monomer units of the polymer chains of the present invention are not limited and may include nucleotides or analogs thereof, debased monomers, amino acids or analogs thereof, monosaccharides, monomer units that can polymerize to form homopolymers (e.g., ethylene glycol, which can polymerize to form PEG), monomer units that can polymerize to form copolymers, or any combination thereof. The monomer units that can polymerize to form homopolymers or copolymers may be small molecule compounds.

[0294] As used herein, the terms "small molecule" and "small molecule compound" are used interchangeably and refer to low molecular weight compounds, for example, <900 Daltons, or with a size on the order of 1 nm. In this invention, "small molecule" may mean small molecules other than nucleotides, amino acids, and / or monosaccharides.

[0295] The term "nucleotide analogue" generally refers to a non-naturally occurring nucleotide that has a modified nucleotide base moiety, a modified pentose moiety, and / or a modified phosphate moiety compared to naturally occurring nucleotides (A, T, C, or G). Examples of nucleotide analogues include, but are not limited to, monomeric units of arabinonucleotide (ANA), bridged nucleotide (BNA), cyclohexenyl nucleotide (CeNA), 2'-fluoroarabinonucleotide (FANA), glycol nucleotide (GNA), hexononucleotide (HNA), locked nucleotide (LNA), morpholine, peptide nucleotide (PNA), or threononucleotide (TNA).

[0296] The term "amino acid analog" refers to a compound that is structurally similar to a naturally occurring amino acid, wherein the C-terminal carboxyl group, N-terminal amino group, or side chain functional group has been chemically modified. Amino acid analogs include, but are not limited to, β-amino acids and amino acids in which the amino or carboxyl group is substituted with a similar reactive group (e.g., a primary amine is substituted with a secondary or tertiary amine, or the carboxyl group is substituted with an ester). Aspartic acid-(β-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine.

[0297] The polymer chains of the present invention can be based on (in other words, substantially composed of) nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, homopolymers (e.g., polyethylene, PEG), copolymers, or any combination thereof. That is, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the monomer units of the polymer chains can be polymerized to form nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, homopolymers (e.g., polyethylene, PEG), copolymers, or any combination thereof.

[0298] As used herein, “nucleic acid analogue” refers to a compound that is structurally similar to naturally occurring RNA and DNA. Nucleic acids are chains of nucleotides consisting of three parts: a phosphate backbone, a pentose sugar (ribose or deoxyribose), and one of four nucleobases. Analogs can have alterations to any of these. Nucleic acid analogues can be distinguished from naturally occurring DNA or RNA by changes to their molecular backbone. Examples of nucleic acid analogues include, but are not limited to, arabinoarabinonucleotide (ANA), bridged nucleic acid (BNA), cyclohexenylnucleotide (CeNA), 2'-fluoroarabinonucleotide (FANA), glycol-based nucleic acid (GNA), hexononucleotide (HNA), locked nucleic acid (LNA), morpholino-peptide nucleic acid (PNA), and threononucleotide (TNA).

[0299] Each part of the polymer chain of the present invention (e.g., reactive segment, extension segment, and / or traction segment) may be independently based on (in other words, substantially composed of) nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, homopolymers (e.g., polyethylene, PEG), copolymers, or any combination thereof. That is, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the monomer units of each of these segments (e.g., reactive segment, extension segment, and / or traction segment) may be polymerized to form nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, homopolymers (e.g., polyethylene, PEG), copolymers, or any combination thereof.

[0300] As used herein, the term "nucleic acid analog" refers to a compound that is structurally similar to naturally occurring RNA and DNA and is composed of nucleotide analogs. Nucleic acid analogs include, but are not limited to, arabinonucleotide (ANA), bridging nucleic acid (BNA), cyclohexenylnucleotide (CeNA), 2'-fluoroarabinonucleotide (FANA), glycol-based nucleic acid (GNA), hexononucleotide (HNA), locked nucleic acid (LNA), morpholino-peptide nucleic acid (PNA), or threononucleotide (TNA).

[0301] As used herein, the term "peptide" may include naturally occurring amino acids and / or amino acid analogs.

[0302] In view of the foregoing, each part of the polymer chain can be designed, including tie sites, extension segments, reactive segments, and / or traction segments. Each part of the polymer chain can be implemented in any suitable manner and is not limited to the examples described above.

[0303] Systems and methods for characterizing target analytes

[0304] Nanopores can be placed within a membrane separating a first conductive liquid medium from a second conductive liquid medium, which can be referred to as a nanopore system. The channels of the nanopores are the only pathways connecting the first and second conductive liquid media. Typically, the target analyte is added to at least one of the first and second conductive liquid media. The membrane can be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane formed from a polymer material. The thickness of the membrane through which the nanopores extend can range from 1 nm to approximately 10 μm.

[0305] The fabrication of nanoporous systems is well known. For example, in protein nanoporous systems, when a porin (e.g., MspA) is placed in either a first conductive liquid medium or a second conductive liquid medium separated by a membrane (e.g., a lipid bilayer), the protein can spontaneously insert into the membrane to form nanopores.

[0306] The polymer chain (PNRSS chain) can be placed on either side of the nanopore, i.e., the first conductive liquid medium or the second conductive liquid medium. The target analyte can also be placed on either side of the nanopore, i.e., the first conductive liquid medium or the second conductive liquid medium. In some embodiments, the polymer chain and the target analyte are placed on the same side or different sides of the nanopore.

[0307] When a potential difference (also known as a voltage or electric field) is applied between the first and second conductive liquid media (i.e., an electric field or voltage is applied across the nanopore), a channel ion current is generated through the nanopore, and the polymer chains can be driven from the conductive liquid media into the nanopore and extend, for example, under the action of electrophoretic force and / or electroosmotic flow. The potential difference may be not less than 20mV, not less than 40mV, not less than 60mV, not less than 80mV, not less than 100mV, not less than 120mV, not less than 140mV, not less than 160mV, not less than 180mV, or not less than 200mV; or in the range of about 20mV to 200mV, in the range of about 40mV to 180mV, in the range of about 600mV to 180mV, in the range of about 80mV to 180mV, in the range of about 100mV to 180mV, in the range of about 120mV to 180mV, in the range of about 140mV to 180mV, or in the range of about 160mV to 180mV.

[0308] In some embodiments, the potential difference between the first and second conductive liquid media may change or remain constant. Methods and apparatus for applying an electric field to a nanopore are known to those skilled in the art. For example, an electric field can be applied to a nanopore using a pair of electrodes. As understood, the range of voltages that can be used may depend on the type of nanopore system and the analyte used.

[0309] Nanoporous systems combined with polymer chains can be used to characterize (or identify) target analytes. First, the polymer chain is driven into and remains within the channels of the nanopore. Then, the target analyte is driven into the nanopore and interacts with a sensing module on the polymer chain. This interaction causes blockage, which is measured to characterize the target analyte. The system for characterizing the target analyte may further include the target analyte itself. Optionally, in this system, the target analyte may have already interacted with the sensing module, or it may not have yet interacted with the sensing module.

[0310] Target analytes can be driven into nanopores by electrophoretic forces or concentration gradients (diffusion effects). The analyte interacts with the sensing module present in the nanopore channel, and this interaction leads to the blockage of the ion current, which is measurable, for example, by measuring the current after the analyte enters the nanopore and comparing it with the current when the polymer chain enters the nanopore but the analyte does not. The blockage of the ion current may be related to the identity of the analyte, the interaction between the analyte and the reagent (such as the sensing module), and the binding kinetics of the analyte.

[0311] Typically, "blockage of ion current" can also be referred to as "blocking current," which can be demonstrated by changes in ion current that are clearly distinguishable from noise fluctuations and are usually related to analyte molecules present within the nanopore. The intensity of the blockage, or the change in current, will depend on the properties of the analyte. More specifically, "blockage" can refer to a range where the ion current drops to approximately 5-100% lower than the unblocked current level, remains there for a period of time, and then spontaneously returns to the unblocked level. For example, the blocking current level can be approximately, at least, or at most approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lower than the unblocked current level. Blockage can be referred to as a blocking event or event.

[0312] Measurements can be taken at any suitable temperature, such as -4°C to 100°C, 4°C to 50°C, 5°C to 25°C, or room temperature.

[0313] After the measurement is completed, a reverse voltage can be applied to drive the polymer chain to move in the opposite direction and exit the nanopore. Then, the voltage direction can be reversed again to drive another unoccupied polymer chain from the sensing module into the channel of the nanopore for the next measurement. Therefore, the method of the present invention can reuse the same system for multiple measurements.

[0314] The measurement of current through a nanopore is well known in the art and can be performed using optical or electrical signals. For example, one or more measuring electrodes can be used to measure the current through the nanopore. These can be, for example, patch-clamp amplifiers or data acquisition devices.

[0315] "Liquid media" includes aqueous, organic-aqueous, and organic-only liquid media. Organic media include, for example, methanol, ethanol, dimethyl sulfoxide, and mixtures thereof. Liquids that can be used in the methods described herein are well known in the art. Descriptions and examples of such media (including conductive liquid media) are provided in U.S. Patent No. 7,189,503, which is incorporated herein by reference in its entirety. Salts, detergents, or buffers may be added to such media. Such reagents can be used to alter the pH or ionic strength of the liquid media. In some embodiments, the salt may contain KCl and / or the salt concentration may be 0.5 M to 2.5 M. In some embodiments, the KCl concentration is 1.5 M. Buffers may be HEPES or Tris, etc. The pH of the first conductive liquid media and / or the second conductive liquid media may be 1.0 to 13.0, preferably 6.0 to 8.0, preferably 7.0 to 7.4, depending on the desired charge properties of the target analyte. In some embodiments, the first conductive liquid media and / or the second conductive liquid media do not contain Tris.

[0316] As used in this article, current patterns and current traces are used interchangeably, referring to the change of ion current over time. A current pattern can contain one or more types of blocking events, and can contain one or more individual blocking events of the same type. The distribution, frequency, amplitude, and other characteristics of the blocking events can be obtained from the current pattern.

[0317] In this invention, the nanopores exist in different states during measurement. State I represents an unoccupied nanopore, where the current measured is the opening current (I0). States II and III represent nanopores occupied by PNRSS chains, where the sensing portion of the PNRSS chain either does not interact with the target molecule (II) or interacts with the target molecule (III). The current measured in states (II) or (III) is defined as the first blocking current (I0). p ) or second blocking current (I b )

[0318] As used herein, an "event" refers to the blockage of a nanopore by the target analyte (i.e., the ion current drops to a level approximately 5-100% lower than the first blocking current level, remains at that level for a period of time, and then spontaneously returns to the first blocking current level), or the current change caused by the blockage of the target analyte. As used herein, the first blocking current level refers to the current level measured when the nanopore is occupied by the PNRSS chain, wherein the sensing module of the PNRSS chain does not interact with the target analyte molecule. Those skilled in the art will know how to determine the occurrence of an event.

[0319] Various characteristic parameters can be obtained from the current mode. These characteristic parameters include, but are not limited to, the orifice current (I0) and the amplitude of the first blocking current (I0). p), second blocking current amplitude (I) b ), Event amplitude (ΔI, defined as ΔI = I) b -I p ), duration between events (t) on ), event dwell time (t) off ), average event magnitude One or more of these characteristic parameters can be used to characterize the analyte.

[0320] Characterization of a target analyte may include, but is not limited to, determining the identity of the target analyte, determining whether the target analyte is a specific substance, determining the presence or absence of the target analyte, determining the interaction between the target analyte and a reagent (e.g., the reagent may be a sensing module, and the system and method of the present invention can be used to determine the interaction between the target analyte and the sensing part), or measuring the binding kinetics of the target analyte and the reagent (e.g., the reagent may be a sensing module, and the system and method of the present invention can be used to determine the binding kinetics between the target analyte and the sensing part). Identity may include, but is not limited to, what the analyte is, the structure of the analyte, the protonated or deprotonated state of the analyte, the chirality of the analyte, etc.

[0321] For example, to identify the target analyte, the tested current pattern can be compared with a reference current pattern to determine the target analyte's identity.

[0322] For example, to determine whether a target analyte and a reagent interact, the reagent can be included as a sensing module in the reaction segment of the polymer chain, and the occurrence of an event represents an interaction between the target analyte and the reagent.

[0323] As used in this article, test current mode refers to the current mode obtained by using the analyte being tested (i.e., the target analyte).

[0324] A reference current mode is a current mode used as a reference to determine at least one characteristic of the target analyte. Different reference current modes can be used depending on the purpose of the detection. For example, a reference current mode can be a current mode obtained using a known analyte under the same conditions as the measured current mode. It can be determined whether the measured analyte is the same as or different from the reference analyte.

[0325] In some implementations, the characterization of the target analyte based on the test current pattern can be achieved by using machine learning algorithms.

[0326] In some implementations, the test current mode can be filtered to obtain a high-pass and / or low-pass filter, and the test current mode can be provided from the high-pass and / or low-pass filter. In some implementations, the cutoff frequency of the high-pass and / or low-pass filter is approximately 100 Hz.

[0327] The systems and methods of this invention can be used to characterize single-molecule target analytes. The systems and methods of this invention can characterize a wide range of analytes, provided the size of the analyte allows it to enter the channels of a nanopore. If the analyte can interact with a moiety, that moiety can be used as a sensing module to characterize the analyte using the systems and methods of this invention.

[0328] The systems and methods of the present invention can be used to characterize a variety of different target analytes, for example, by using a polymer chain comprising a plurality (e.g., two or more) sensing modules that can interact with a plurality (e.g., two or more) different target analytes. In some embodiments, a variety of different target analytes can be driven simultaneously into the channels of a nanopore and interact with a plurality of sensing modules, respectively. The resulting multiple interactions can be measured simultaneously and distinguished from each other based on their respective current modes. In some embodiments, a variety of different target analytes can be driven into the channels of a nanopore in different rounds for measurement. For example, the plurality of sensing modules are different from each other, and each sensing module can specifically interact with one or more specific analytes. Such sensing module design can be used to characterize a variety of different target analytes, wherein each target analyte can only interact with one of the plurality of sensing modules. As an illustration, a first target analyte can be driven into the channels of a nanopore and interact with a first sensing module, and a first interaction between the first target analyte and the first sensing module can be measured; then, a second target analyte can be driven into the channels of a nanopore and interact with a second sensing module, and a second interaction between the second target analyte and the second sensing module can be measured. Repeat the above steps for other target analytes until all target analytes have been characterized or all sensing modules have been occupied.

[0329] In the system and method of the present invention, multiple nanopores can be used simultaneously, which can improve the detection limit of the analyte. Preferably, the multiple nanopores are identical.

[0330] This invention also relates to the following aspects:

[0331] Aspect 1. A system for identifying target analytes, the system comprising:

[0332] (1) Nanopores;

[0333] (2) A polymer chain consisting of a linking site, an extension segment, a reaction segment, and a traction segment in sequence;

[0334] The polymer chain is coupled to the chain molecule at the chain linking site, and the size of the chain molecule is larger than the nanopore cavity, preventing it from entering the nanopore cavity;

[0335] The extension segment has a suitable length so that when the polymer chain is loaded into the nanopore, the reaction segment is located at the narrowest part of the nanopore;

[0336] The reaction section contains a first active group that can bind to the target analyte.

[0337] Aspect 2. The system according to aspect 1, wherein a first active group is contained in the polymer backbone.

[0338] Aspect 3. The system according to aspect 2, wherein the polymer backbone comprises one or more polymer monomer derivatives and the first active group is contained in one or more polymer monomer derivatives.

[0339] Aspect 4. The system according to Aspect 1, wherein the first active group is connected to the polymer backbone directly or through one or more connectors.

[0340] Aspect 5. A system for identifying target analytes, the system comprising:

[0341] (1) Nanopores;

[0342] (2) A polymer chain consisting of a linking site, an extension segment, a reaction segment, and a traction segment in sequence;

[0343] The polymer chain is coupled to the chain molecule at the chain linking site, and the size of the chain molecule is larger than the nanopore cavity, preventing it from entering the nanopore cavity;

[0344] The extension segment has a suitable length so that when the polymer chain is loaded into the nanopore, the reaction segment is located at the narrowest part of the nanopore;

[0345] The reaction section contains a second active group, which can directly bind to a compound containing a first active group or bind through one or more linkers; wherein the first active group can bind to the target analyte.

[0346] Aspect 6. The system according to aspect 5, wherein the second active group is contained in the polymer backbone.

[0347] Aspect 7. The system according to aspect 6, wherein the polymer backbone comprises one or more polymer monomer derivatives, and the first active group is contained in one or more polymer monomer derivatives.

[0348] Aspect 8. The system according to aspect 5, wherein the second active group is directly connected to the polymer backbone or connected through one or more connectors.

[0349] Aspect 9. The system according to any one of the preceding aspects, wherein the system further comprises two compartments separated by an interface; wherein each of the two compartments contains a liquid medium and nanopores are located at the interface.

[0350] Aspect 10. The system according to any one of the preceding aspects, wherein the polymer backbone is a nucleic acid, peptide, polysaccharide or any combination thereof.

[0351] Aspect 11. The system according to any one of the preceding aspects, wherein the polymer backbone is DNA, RNA, or a hybrid of DNA and RNA.

[0352] Aspect 12. The system according to any one of the preceding aspects, wherein the reaction section comprises guanine, adenine, or any combination thereof.

[0353] Aspect 13. The system according to any one of the preceding aspects, wherein the reaction section comprises adjacent purines selected from the group consisting of guanine and adenine, preferably two adjacent purines selected from the group consisting of guanine and adenine.

[0354] Aspect 14. The system according to any one of the preceding aspects, wherein the first active group is 1,2,3-triazole.

[0355] Aspect 15. The system according to any one of the preceding aspects, wherein the 1,2,3-triazole is generated by conjugating the azide with the alkyne via a Huisgen copper (I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction.

[0356] Aspect 16. The system according to any one of the preceding aspects, wherein the alkyne is contained in the polymer backbone, or the compound containing the alkyne is directly connected to the polymer backbone or connected through one or more joints.

[0357] Aspect 17. The system according to any one of the preceding aspects, wherein the polymer backbone comprises 5-ethynyl-dU-CE phosphoramide.

[0358] Aspect 18. The system according to any one of the preceding aspects, wherein the azide is 3-azidopropylamine.

[0359] Aspect 19. The system according to any one of the preceding aspects, wherein the first active group is phenylboronic acid.

[0360] Aspect 20. The system according to any one of the preceding aspects, wherein phenylboronic acid is generated by conjugating 4-(azidomethyl)phenylboronic acid with an alkyne via CuAAC.

[0361] Aspect 21. The system according to any one of the preceding aspects, wherein the second active group is an alkyne.

[0362] Aspect 22. The system according to any one of the preceding aspects, wherein the alkyne is contained in the polymer backbone, or the compound containing the alkyne is directly connected to the polymer backbone or connected through one or more joints.

[0363] Aspect 23. The system according to any one of the preceding aspects, wherein the polymer backbone comprises 5-ethynyl-dU-CE phosphoramide.

[0364] Aspect 24. The system according to any one of the preceding aspects, wherein the target analyte is selected from the group consisting of: metal ions, sugars, catecholamines, catecholamine derivatives, compounds containing a 1,2-cis-diol or 1,3-cis-diol moiety, polyols (e.g., catechol), ethylene glycol, glycerol, L-lactic acid, vitamins (e.g., vitamin C or vitamin B6), buffer reagents (e.g., protonated or deprotonated forms of tris), adrenaline, norepinephrine, isoproterenol, antiviral drugs (e.g., remdesivir or its triphosphate metabolites), hydrogen peroxide, polysaccharides, or cyclic peptides.

[0365] Aspect 25. The system according to any one of the preceding aspects, wherein the metal ion is a transition metal ion, preferably Ni. 2+ Zn 2+ Cd 2+ Co 2+ or Cu 2+ .

[0366] Aspect 26. The system according to any one of the preceding aspects, wherein the target analyte is a polyol and the liquid medium is free of Tris buffer.

[0367] Aspect 27. The system according to any one of the preceding aspects, wherein the chain molecule is streptavidin.

[0368] Aspect 28. The system according to any one of the preceding aspects, wherein the ligation site is modified with 5' biotin-TEG.

[0369] Aspect 29. The system according to any one of the preceding aspects, wherein the second active group is capable of binding with a variety of compounds containing different first active groups.

[0370] Aspect 30. The system according to any one of the preceding aspects, wherein the target analyte is a single molecule, a single-atom ion, or a chemical intermediate.

[0371] Aspect 31. The system according to any one of the preceding aspects, wherein the target analyte is a single-atom ion or a chemical intermediate.

[0372] Aspect 32. The system according to any one of the preceding aspects, wherein the nanopore is a protein nanopore, a solid nanopore, or a DNA nanopore.

[0373] Aspect 33. The system according to any one of the preceding aspects, wherein the nanopore has a conical or cylindrical cavity.

[0374] Aspect 34. The system according to any one of the preceding aspects, wherein the protein nanopore is MspA, an M2MspA mutant (D93N / D91N / D90N / D118R / D134R / E139K), α-HL, aerolysin, ClyA, FraC, PlyA / B, or a Phi 29 linker.

[0375] Aspect 35. The system according to any one of the preceding aspects, wherein the nanopores are made of a solid material (e.g., SiNx, graphene, glass, quartz or DNA framework).

[0376] Aspect 36. A method for identifying a target analyte, the method comprising:

[0377] Provides nanopores;

[0378] A polymer chain is provided, which is sequentially composed of a binding site, an extension segment, a reactive segment, and a traction segment; wherein the polymer chain is coupled to a binding molecule at the binding site, and the size of the binding molecule is larger than the nanopore cavity, preventing it from entering the nanopore cavity; wherein the extension segment has a suitable length such that when the polymer chain is loaded into the nanopore, the reactive segment is located at the narrowest point of the nanopore; and wherein the reactive segment has an active group capable of binding to the target analyte.

[0379] This allows polymer chains to enter the nanopores and positions the reaction segment at the narrowest point of the nanopores.

[0380] Enabling the target analyte to pass through nanopores; and

[0381] The change in ion current passing through the nanopore during the displacement process is measured to identify the target analyte.

[0382] Aspect 37. The method according to aspect 36, wherein the first active group is contained in the polymer backbone.

[0383] Aspect 38. The method according to aspect 37, wherein the polymer backbone comprises one or more polymer monomer derivatives and the first active group is contained in one or more polymer monomer derivatives.

[0384] Aspect 39. The method according to aspect 36, wherein the first active group is directly connected to the polymer backbone or connected through one or more connectors.

[0385] Aspect 40. A method for identifying a target analyte, the method comprising:

[0386] Provides nanopores;

[0387] A polymer chain is provided, consisting of a linking site, an extension segment, a reactive segment, and a traction segment in sequence. The polymer chain is coupled to a linking molecule at the linking site, the linking molecule being larger than the nanopore cavity, preventing it from entering the nanopore cavity. The extension segment has a suitable length such that the reactive segment is located at the narrowest point of the nanopore when the polymer chain is loaded into the nanopore. The reactive segment contains a second active group, which can directly bind to a compound containing a first active group or bind through one or more linkers. Furthermore, the first active group can bind to a target analyte.

[0388] The second active group is directly bonded to a compound containing the first active group or combined with it through one or more linkers;

[0389] This allows polymer chains to enter the nanopores and positions the reaction segment at the narrowest point of the nanopores.

[0390] Enabling the target analyte to pass through nanopores; and

[0391] The change in ion current passing through the nanopore during the displacement process is measured to identify the target analyte.

[0392] Aspect 41. The method according to aspect 40, wherein the second active group is contained in the polymer backbone.

[0393] Aspect 42. The method according to aspect 41, wherein the polymer backbone comprises one or more polymer monomer derivatives and the first active group is contained in one or more polymer monomer derivatives.

[0394] Aspect 43. The method according to aspect 40, wherein the second active group is directly connected to the polymer backbone or connected through one or more connectors.

[0395] Aspect 44. A method for identifying multiple target analytes, the method comprising:

[0396] (1) Two compartments are provided, separated by an interface; each of the two compartments contains a liquid medium and the interface has nanopores;

[0397] (2) A polymer chain is provided, which is composed of a linking site, an extension segment, a reaction segment and a traction segment in sequence; wherein the polymer chain is coupled to a linking molecule at the linking site, and the size of the linking molecule is larger than the nanopore cavity, so that it cannot enter the nanopore cavity; wherein the extension segment has a suitable length, such that when the polymer chain is loaded into the nanopore, the reaction segment is located at the narrowest part of the nanopore; and wherein the reaction segment has an active group that can irreversibly bind to the first target analyte;

[0398] (3) Apply a first voltage between the two compartments to allow the polymer chains to enter the nanopore and position the reaction section at the narrowest point of the nanopore;

[0399] (4) Displace the first target analyte through the nanopore; and

[0400] (5) The change in ion current through the nanopore during the displacement process is measured to identify the first target analyte;

[0401] (6) Apply a second voltage opposite to the direction of the first voltage between the two compartments, thereby causing the polymer chains that are irreversibly bound to the first target analyte to leave the nanopore;

[0402] (7) Repeat (3)-(5) to reload another polymer chain into the nanopore and identify another target analyte.

[0403] Aspect 45. The method according to any one of the preceding aspects, wherein the method further comprises providing two compartments separated by an interface; wherein each of the two compartments contains a liquid medium and nanopores are located in the interface.

[0404] Aspect 46. The method according to any one of the preceding aspects, wherein the polymer backbone is a nucleic acid, peptide, polysaccharide or any combination thereof.

[0405] Aspect 47. The method according to any one of the preceding aspects, wherein the polymer backbone is DNA, RNA, or a hybrid of DNA and RNA.

[0406] Aspect 48. The method according to any one of the preceding aspects, wherein the reaction section comprises guanine, adenine, or any combination thereof.

[0407] Aspect 49. The method according to any one of the preceding aspects, wherein the reaction section comprises adjacent purines selected from the group consisting of guanine and adenine, preferably two adjacent purines selected from the group consisting of guanine and adenine.

[0408] Aspect 50. The method according to any one of the preceding aspects, wherein the first active group is 1,2,3-triazole.

[0409] Aspect 51. The method according to any one of the preceding aspects, wherein the 1,2,3-triazole is generated by conjugating the azide with the alkyne via a Huisgen copper (I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction.

[0410] Aspect 52. The method according to any one of the preceding aspects, wherein the alkyne is contained in the polymer backbone, or the compound containing the alkyne is directly connected to the polymer backbone or connected through one or more joints.

[0411] Aspect 53. The method according to any one of the preceding aspects, wherein the polymer backbone comprises 5-ethynyl-dU-CE phosphoramide.

[0412] Aspect 54. The method according to any one of the preceding aspects, wherein the azide is 3-azidopropylamine.

[0413] Aspect 55. The method according to any one of the preceding aspects, wherein the first active group is phenylboronic acid.

[0414] Aspect 56. The method according to any one of the preceding aspects, wherein phenylboronic acid is generated by conjugating 4-(azidomethyl)phenylboronic acid to an alkyne via CuAAC.

[0415] Aspect 56. The method according to any one of the preceding aspects, wherein the second active group is an alkyne.

[0416] Aspect 58. The method according to any one of the preceding aspects, wherein the alkyne is contained in the polymer backbone, or the compound containing the alkyne is directly connected to the polymer backbone or connected through one or more joints.

[0417] Aspect 59. The method according to any one of the preceding aspects, wherein the polymer backbone comprises 5-ethynyl-dU-CE phosphoramide.

[0418] Aspect 60. The method according to any one of the preceding aspects, wherein the target analyte is selected from the group consisting of: metal ions, sugars, catecholamines, catecholamine derivatives, compounds containing a 1,2-cis-diol or 1,3-cis-diol moiety, polyols (e.g., catechol), ethylene glycol, glycerol, L-lactic acid, vitamins (e.g., vitamin C or vitamin B6), buffer reagents (e.g., protonated or deprotonated forms of tris), adrenaline, norepinephrine, isoproterenol, antiviral drugs (e.g., remdesivir or remdesivir triphosphate metabolites), hydrogen peroxide, polysaccharides, or cyclic peptides.

[0419] Aspect 61. The method according to any one of the preceding aspects, wherein the metal ion is a transition metal ion, preferably Ni. 2+ Zn2+ Cd 2+ Co 2+ or Cu 2+ .

[0420] Aspect 62. The method according to any one of the preceding aspects, wherein the target analyte is a polyol and the liquid medium is free of Tris buffer.

[0421] Aspect 63. The method according to any one of the preceding aspects, wherein the chain molecule is streptavidin.

[0422] Aspect 64. The method according to any one of the preceding aspects, wherein the ligation site is modified with 5' biotin-TEG.

[0423] Aspect 65. The method according to any one of the preceding aspects, wherein the second active group is capable of binding with a variety of compounds containing different first active groups.

[0424] Aspect 66. The method according to any one of the preceding aspects, wherein the target analyte is a single molecule, a single-atom ion, or a chemical intermediate.

[0425] Aspect 67. The method according to any one of the preceding aspects, wherein the target analyte is a single-atom ion or a chemical intermediate.

[0426] Aspect 68. The method according to any one of the preceding aspects, wherein the nanopore is a protein nanopore, a solid nanopore, or a DNA nanopore.

[0427] Aspect 69. The method according to any one of the preceding aspects, wherein the nanopore has a conical or cylindrical cavity.

[0428] Aspect 70. The method according to any one of the preceding aspects, wherein the protein nanopore is MspA, an M2MspA mutant (D93N / D91N / D90N / D118R / D134R / E139K), α-HL, aerolysin, ClyA, FraC, PlyA / B, or a Phi 29 linker.

[0429] Aspect 71. The method according to any one of the preceding aspects, wherein the nanopores are made of a solid material (e.g., SiNx, graphene, glass, quartz or DNA framework).

[0430] Aspect 72. The method according to any one of the preceding aspects is used for screening drugs.

[0431] 73. Use of the system according to any one of aspects 1-35 in screening drugs.

[0432] Example 1 uses PNRSS with natural DNA bases

[0433] Natural nucleic acid bases, such as guanine, adenine, or any combination thereof, can act as ligands that can bind metal ions. 36,37 The first PNRSS chain 13G / 14G (Table 1) has two adjacent guanines that synergistically bind to Ni. 2+ ion( Figure 1 c). To avoid interference from other DNA bases, these guanines are surrounded by base-free residues that cannot bind metal ions. PNRSS measurements are performed as described in the method. The 13G / 14G strand is added to the cis chamber to a final concentration of 10 nM. A single well is inserted and a +180 mV potential is continuously applied, initially reporting the opening current I0. Subsequently, the PNRSS strand is captured by electrophoresis and the quiescent residual current I is reported. p (Table 2). Then, Ni was added to the trans chamber. 2+ As a mobile reactant, the final concentration was 1 mM. This immediately led to further blocking events, reaching what is defined as I... b The level of occurrence. The consecutive occurrence of events is observed as I. p and I b Telegraphic conversion between ( Figure 1 d, Video 1). trans-side Ni 2+ The concentration is adjusted between 0-1 mM. Figure 7 ), and then the event occurrence rate is relatively high [Ni 2+ The increase is obvious. However, from another PNRSS chain 14X ( Figure 8 These events were not observed, confirming that they originated from binding to diguanine ligands. 37 This phenomenon was also theoretically simulated, revealing that N(7) and O(6) atoms in Ni 2+ Plays a key role in coordination ( Figure 9 (Table 3, Methods).

[0434] The core parameters for quantitatively describing any PNRSS event are summarized in Figure 10 In the context of the rule, the dwell time t is defined. off and the interval t between events on The blocking amplitude ΔI is defined as I b -I p Using 13G / 14G and Ni 2+ The measured ΔI relative to t off The scatter plot of events shows a single group of events, where ΔI = ~ -60pA ( Figure 1 e) is significantly larger than events produced by monatomic ions, where α-HL is used. 23 At the time of observation, the amplitude was approximately 2 pA. The average dwell time τ off and the average event interval τ on Each by Figure 10 The data was derived from this. The time histogram is summarized in... Figure 11 From this, the corresponding τ can be derived. on and τ off Value. For example... Figure 1 As shown in f, the reciprocal of the average interval between events (1 / τ) on ) and [Ni 2+ It is proportional to τ, which is consistent with the single-step bimolecular model, where 1 / τ on =k on [Ni 2+ However, the average residence time τ off This indicates that [Ni] 2+ The dependence of 1 / τ is negligible, which is consistent with the single-molecule dissociation model, where 1 / τ off =k off Therefore, k on It was determined to be 1 / τ on Compared to [Ni 2+ The slope of the fitted line, and k off From 1 / τ off The average value is determined.

[0435] Other divalent ions, such as Zn, were also tested using 13G / 14G. 2+ Cd 2+ Co 2+ or Cu 2+ This indicates that with Co 2+ and Cu 2 + The binding preference is higher than that of Zn. 2+ or Cd 2+ ( Figure 12 Ni 2+ Co 2+ or Cu 2+ The binding events also differed significantly from one another, indicating that single-ion differentiation can be achieved using PNRSS. Different PNRSS chains, such as 14A, with a single adenine as the immobilized reactant, or 14G, with guanine (Table 1), also provide Ni 2+ Combined events ( Figure 13-16 However, the binding characteristics here differ from those observed using 13G / 14G (Table 4), indicating that different chemical processes are occurring as monitored using PNRSS. Although previous studies have used NMR or infrared spectroscopy for overall analysis... 36,37 However, to our knowledge, no direct single-molecule observation of the binding of metal ions to DNA bases and the corresponding quantitative binding kinetics have been previously reported.

[0436] Example 2 uses PNRSS of 1,2,3-triazole

[0437] Beyond the proof of concept described above, a wider selection of artificial, functional DNA phosphoramidides provides relatively unrestricted freedom for the design and synthesis of PNRSS strands, and radically expands the versatility and complexity of downstream PNRSS measurements. 38 5-Ethynyl-dU-CE phosphoramidide (Glen Research, USA) is a thymine derivative containing an alkyne, used to synthesize the PNRSS chain 14TAK (Table 1, Methods). 14TAK contains a single alkyne at position 14, and any azide can be conjugated to this alkyne via a Huisgen copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction (a widely used click chemistry reaction). 39 3-Azidepropylamine is one of the simplest azides; it reacts with 14TAK (Table 1). Figure 17 ). By mass spectrometry ( Figure 17 ) and single-channel recording ( Figure 18 The product was characterized (Table 5). Successful conjugation was confirmed, producing a new PNRSS chain, named 14TAZ (Table 1). 1,2,3-triazole (TAZ) was generated at position 14 of 14TAZ via the CuAAC reaction. Figure 2 a).

[0438] It has been previously reported that the N(2) or N(3) atoms of TAZ can act as lone pair electron donors and coordination sites for binding metal ions. 40 As evidence of unimolecular behavior, PNRSS measurements were performed, with 14 TAZ of TAZ used as the stationary reactant. Ni was added to the trans side. 2+ Used as a mobile reactant, with a final concentration of 1 mM. Under a continuously applied potential of +180 mV, binding events with high characteristic noise signals were observed continuously. Figure 2 b-2c, Video 2). In ΔI relative to t off The scatter plot reports a single distribution of events. Figure 2 d). By using Ni in the trans side 2+ Increasing the concentration from 0 mM to 1 mM significantly increased the event occurrence rate. Figures 19-20 The reciprocal of the average interval between events (1 / τ) on ) and Ni 2+ Concentration is directly proportional ( Figure 2 e). However, the average residence time τ off This indicates that [Ni] 2+ The dependency on [] is negligible. Co is also used. 2+ Perform similar measurements, where the combined event is displayed as a transient resistance pulse ( Figure 21-22 The average length of stay reported is τ. offIt takes 1.32ms, much shorter than using Ni. 2+ The observed time was 220 ms (Table 6). Rate constant k on and k off From respectively Figure 2 The results in e and 21c. The equilibrium binding constant K. b Calculated as K b =k on / k off Ni 2+ The binding affinity for TAZ is significantly stronger than that for Co. 2+ (Table 6). When simultaneously checking Ni... 2+ and Co 2+ At that time, their combination events are explicitly identified ( Figure 23 Therefore, the PNRSS chain 14TAK demonstrates its versatility as a template for introducing any azide. When reacted with 3-azidopropylamine, the resulting TAZ itself can serve as a stationary reactant, Ni 2+ or Co 2+ It will combine with the reactant.

[0439] Example 3: PNRSS using phenylboronic acid

[0440] However, in most cases involving CuAAC, TAZ is considered a connector to which other functional modules can be attached. 41 Benzylboronic acid (PBA) is a core component of sugar or catecholamine sensors. 42 It reacts with compounds containing 1,2-cis-diol or 1,3-cis-diol moieties to form five- or six-membered borate esters ( Figure 3 a) 43-45 To introduce PBA into the PNRSS chain, 4-(azidomethyl)phenylboronic acid was reacted with the PNRSS chain via CuAAC and 14TAK (method, Figure 24-25 The product was analyzed by mass spectrometry (MS / MS). Figure 25 ) and single-channel recording ( Figure 26 Further characterization (Table 7) confirmed successful coupling and the generation of a new PNRSS chain, termed 14PBA (Table 1). For PNRSS, the PBA at position 14 of 14PBA was used as a stationary reactant. A potential of +160 mV was continuously applied. Polyols (e.g., catechols) 44 ( Figure 3 b, Figure 27-28 ), ethylene glycol 45 ( Figure 3 c, Figures 29-30 ),glycerin 45 ( Figure 3 d, Figures 31-32 L-lactic acid 44 ( Figure 3 e, Figures 33-34 Vitamin C 46 ( Figure 3 f, Figures 35-36 or vitamin B6 47 ( Figure 3 g, Figures 37-38 These were used as mobile reactants and added individually to the trans side at desired concentrations. Despite differences in binding properties and kinetics (Tables 8-9), all the above reactants reported detectable binding to PBA. Conversely, no binding events were observed when using 14TAK as a probe, confirming that these events were a result of binding to PBA. Figure 26 e). Resorcinol, whose reaction structure is incompatible with PBA, failed to produce any binding events. Figure 3 h) further confirmed the reaction mechanism. Figure 3 All observed responses discussed in b-3g reported positive events (I b >I p This is counterintuitive, because binding molecules occupying more space in nanopores are expected to produce negative events (I). b <I p The proposed mechanism is that the binding of any of the above analytes to PBA leads to the formation of anionic borate esters, as reported in previous literature. 48,49 The resulting negative charge can typically enhance the ion flow through the pore. Evidence demonstrating that introducing a negative charge at the pore constriction enhances pore conductivity is that wild-type MspA, which has more negative charge at the pore constriction than M2 MspA, exhibits significantly higher conductivity in the open-pore state than M2 MspA. 7 On the other hand, the overall size of the bound analyte may contribute to reducing the ion flux. Therefore, the overall contribution of the bound analyte to the blocking amplitude may be positive, especially when the analyte under study is a small molecule. Systematic studies using quantum chemical and molecular dynamics simulations can be conducted to further quantify this phenomenon, but these should be performed in separate follow-up studies.

[0441] The core advantage of nanopore-based single-molecule chemistry lies in its ability to detect the transient appearance of chemical intermediates at a resolution of μs. 50 Tris(hydroxymethyl)-aminoethane (tris) is a widely used buffer reagent with a pKa of ~8.3 for its conjugate acid. 51When dissolved in aqueous solutions with a pH close to its pKa, tris exists in both protonated and deprotonated forms in considerable proportions. In either form, the tris molecule, as a polyol, can react with PBA, and chemical intermediates arising from its protonation or deprotonation can be observed. Although tris is readily available and widely used, the binding of tris to PBA appears to have been largely unstudied to date. To demonstrate the direct observation of chemical intermediates using PNRSS, PBA and tris were used as stationary and mobile reactants, respectively. Figures 39-40 At pH 7.0, the binding of tris to PBA produces a positive event, reaching I. b1 Level. However, at pH 8.0, except for I... b1 In addition, other binding levels (I) were observed. b2 When combined with PBA, I was also observed. p I b1 and I b2 Dynamic conversion between them, where I b1 and I b2 tris(representing protonation and deprotonation respectively) Figure 41 A deprotonated state with a higher negative charge reports a higher blocking state (I). b2 >I b1 This observation is consistent with our previous hypothesis that more negative charge generally enhances the ion flow in this measurement setup. This simple example demonstrates that direct observation of chemical intermediates can provide insight into transient chemical processes. When properly designed, the presence of chemical intermediates can also help distinguish compounds with subtle differences. The above demonstration also suggests that the use of Tris buffer should be avoided in any PBA-based polyol sensing assays using PNRSS to prevent interference.

[0442] Example 4: Repeated observation of irreversible reactions

[0443] Based on previously reported single-molecule chemical measurements using nanopores 52 In nanopores, only a single reaction site is permanently immobilized within the pore, meaning that any irreversible chemical reaction at that site immediately terminates the generation of any new chemical process information. Although rarely discussed, this technical limitation restricts nanopore single-molecule chemistry research to reversible reactions. However, with PNRSS, the chain containing the immobilized reactant is chemically separated from the pore. Even if an irreversible reaction occurs, the entire PNRSS chain can be voltage-popped and reloaded, thus restarting a new measurement cycle.

[0444] To demonstrate this, hydrogen peroxide (H₂O₂), a strong oxidizing agent capable of irreversibly oxidizing PBA to phenol, was used. 53 It is used as a mobile reactant. Figure 4a) PBA is used as a stationary reactant. During the PNRSS process, H₂O₂ initially undergoes a reversible reaction with PBA, producing a forward spike event, possibly due to the intermediate state prior to phenol formation. Figure 4 aii), as proposed in the literature 54,55 However, boron atoms have not yet been removed at this stage. Later, these spikes abruptly disappear, giving a smooth baseline. Figure 4 b). In this state, the chain can still interact with Ni. 2+ The reaction indicates that the 1,2,3-triazole linker still exists. However, it can no longer react with any polyol or H2O2, indicating that the borate group has been lost due to irreversible oxidation to phenol. Figure 42 However, a new measurement cycle can be restarted using a voltage scheme. Figure 4 c), therefore, irreversible unimolecular reactions can now be repeatedly monitored, acknowledging this unique property of PNRSS. Figure 4 d, Video 3).

[0445] Example 5: Differences between adrenaline, noradrenaline, and isoproterenol

[0446] In addition to measurements combining kinetics, the rich information from chemical reactions monitored using PNRSS can also be used to identify single molecules. Norepinephrine, epinephrine, and isoproterenol are catecholamine derivatives. 56 Norepinephrine and epinephrine are both natural hormones and neurotransmitters and are used medically.57 Isoproterenol is a sympathomimetic β-adrenergic agonist.58 Specifically, norepinephrine primarily acts on α-receptors and maintains blood pressure, while epinephrine specifically and less intensely stimulates both α and β-receptors and relaxes the respiratory tract and regulates blood flow, heart rate, and glycogen metabolism. Isoproterenol is mainly used to treat bradycardia, cardiac conduction block, and asthma. These functional differences stem from subtle variations in their chemical structures. However, they both contain a 1,2-phenylenediol moiety that reacts with PBA (…). Figure 5 a).

[0447] The PNRSS of these compounds was measured using 14PBA. Norepinephrine was added to the trans side (…). Figures 43-44 ), adrenaline ( Figures 45-46 ) or isoproterenol ( Figures 47-48 The desired final concentration was reached. Under a continuously applied potential of +160 mV, all three compounds reported negative binding events (Ig). b <I p This is consistent with reporting positive events (I) b >I pThe catechols in these samples differed significantly. It is speculated that norepinephrine, epinephrine, and isoproterenol are all cations, which may compensate for the enhanced ion flow resulting from the production of anionic borate esters. This difference was even more pronounced when catechols and norepinephrine were detected simultaneously. Figure 49 (Video 4). Although their binding affinities are similar (Table 10), binding events induced by norepinephrine, epinephrine, or isoproterenol can be distinguished by their unique binding characteristics, including ΔI, τ off Or noise level. The acquired trace is first divided into low-pass and high-pass sections according to frequency, and then filtered by a Butterworth filter with a cutoff frequency of 100Hz. Figure 50 The low-pass portion of events induced by norepinephrine binding reported a smaller ΔI. No further fluctuations were observed in the binding state. Conversely, events induced by adrenaline or isoproterenol binding resulted in a larger ΔI, and secondary telegraphic fluctuations were also observed. Figure 5 b). However, events induced by adrenaline or isoproterenol can be more clearly separated from their high-pass components, with isoproterenol binding producing higher-amplitude high-frequency noise. These differences in binding characteristics are more clearly demonstrated in simultaneous sensing measurements. Figure 5 c and 5d, video 5). The standard deviation (SD) values ​​of the low-pass and high-pass components effectively distinguish events induced by different catecholamine bindings. For automatic event recognition, these two parameters were used to construct a machine learning algorithm (method, Figure 51 In short, this algorithm is based on the Support Vector Classification (SVC) model, a machine learning method used for data classification. 59 A total of 1455 events were obtained using adrenaline, noradrenaline, or isoproterenol as single analytes to determine the model. Based on the confusion matrix results, the accuracy of event reporting induced by isoproterenol was an impressive 99.9%. The accuracy of event reporting induced by adrenaline or noradrenaline was 95.5% or 98.6%, respectively. Figure 5 e). When sensing simultaneously, events are extracted from a continuous recording trace of 15 minutes in length, and the low-pass and high-pass standard deviation values ​​are presented. Figure 5 f). The three event groups, arising from the binding of norepinephrine, epinephrine, or isoproterenol, were clearly distinguished ( Figure 52The decision boundary map generated by the machine learning algorithm was placed above the scatter plot to aid event recognition. The demonstration of the three catecholamines provides concrete evidence that the wealth of information generated by chemical reactions can facilitate single-molecule recognition. Although the reaction between PBA and catecholamines is well known, this is the first single-molecule chemical study on this topic, facilitated by PNRSS. Unlike previous reports on nanopore neurotransmitter sensing, where adrenaline and noradrenaline binding to engineered α-HL reported only weak amplitudes of 0.9 and 1.1 pA, respectively, our PNRSS method, which applied different chemical reactions and a conical pore, produced richer sensing information and larger event amplitudes (~21-32 pA), clearly distinguishing the three catecholamines (Table 11). Machine learning-assisted frequency division analysis further enhanced the sensing performance. Unlike the fluorescent probe design strategies used in holistic studies, which require the chemical synthesis of complex probe structures to distinguish chemically similar catecholamines 63 and 64, the PNRSS strategy requires only a single PBA to distinguish the three catecholamines. This system has also been used to investigate other catecholamines, such as dopamine and L-3,4-dihydroxyphenylalanine (L-DOPA), which are precursors to norepinephrine and epinephrine 65. However, these results will be reported separately in subsequent studies.

[0448] Example 6: Differentiation between remdesivir and remdesivir triphosphate metabolites

[0449] In the development of antiviral drugs, a variety of nucleoside analogue-based compounds have been synthesized, screened, and clinically tested. 66 Remdesivir is a specific nucleoside analogue and investigational antiviral drug. 67 It has been reported that it can effectively treat the disease caused by the 2019 coronavirus (COVID-19), the pandemic that is causing the current global crisis. 68-70 Remdesivir is a prodrug that is metabolized in cells into its active triphosphate form, which works by blocking RNA-dependent RNA polymerase (RdRp), thereby preventing further viral replication. 71 .

[0450] Remdesivir and its triphosphate metabolites both contain a ribose moiety, which reacts with PBA. 72 ( Figure 6 a). The corresponding experiment was designed to place 14PBA on the cisses side. Remdesivir ( Figures 53-54 ) or remdesivir triphosphate ( Figures 55-56Remdesivir and remdesivir triphosphate were treated separately as mobile reactants and added to the trans side at the desired concentrations. A potential of +160 mV was continuously applied. During the PNRSS process, both remdesivir and remdesivir triphosphate reported positive events. However, events from remdesivir were long-lasting and exhibited dramatic fluctuations, while events from remdesivir triphosphate were much shorter-lasting and the observed level fluctuations were negligible. Figure 6 (b, Table 12-13). When the SD relative to the event dwell time is plotted at the blockage level, these different event characteristics are clearly shown. Figure 6 c). The scatter plot of the standard deviations of the high-pass and low-pass filters also illustrates the difference in events, clearly separating the two groups ( Figure 6 d, Figure 57 ). Figure 6 e shows a continuous trace with the addition of two reagents, where the difference between the two event types can be clearly identified (Video 6).

[0451] Despite conflicting conclusions regarding the efficacy of remdesivir in treating COVID-19. 73,74 However, the above demonstration expands the types of analytes that PNRSS can study. Although chemical reactions between PBA and nucleoside analogues have been previously investigated. 72 However, the binding between remdesivir and its derivatives and PBA has not been investigated to date. Their single-molecule binding kinetics were determined in a confined space at the nanoscale via PNRSS (Table 13). Direct differentiation of remdesivir and its metabolites was also achieved. While this paper does not demonstrate the binding of other nucleoside analogues, such as Galidesvir... 75 Ribavirin 76 Or Favipiravir-RTP 77 In principle, similar PNRSS assays could be used for identification. These findings could inspire pharmacokinetic or drug screening applications and may be useful in the current pandemic.

[0452] Example 7

[0453] PNRSS chains can be composed of any synthetic polymer, such as nucleic acids, peptides, polysaccharides, or combinations thereof, but for the purpose of studying a wider range of single-molecule reactions, the composition of the PNRSS chain should be arbitrarily programmable. As shown in the figure above, we designed a PNRSS chain composed of oligonucleotides and polymers (…). Figure 66 a). The polymer unit is formed by the polymerization of three molecules of ethylene glycol. Furthermore, the chain contains a single PBA capable of binding norepinephrine. In this case, norepinephrine reports a negative binding event at +160 mV ( Figure 66 (b and c). In summary, in PNRSS technology, the PNRSS chain can be composed of polymers.

[0454] In PNRSS measurements, the PNRSS chains of streptavidin-based chains pass through electrophoresis into the pores and remain fully extended within the PNRSS pores. However, as... Figure 67 As shown, we demonstrate an alternative PNRSS method without avidin, called immobilized PNRSS (fPNRSS). In this case, we designed a PNRSS chain conjugated to the MspA protein, thus eliminating the need for a PNRSS chain in solution. This PNRSS chain can be electrophoretically inserted into the well and remains fully extended in the PNRSS well at +20 mV. Norepinephrine reports a negative binding event at +160 mV. In summary, the fPNRSS chain can be permanently conjugated to the well to further improve the resolution and consistency of PNRSS.

[0455] like Figure 68 As shown, we present another PNRSS method called locked PNRSS (lPNRSS). The lPNRSS chain contains locking segments that can form a hairpin structure through hydrogen bonding interactions to prevent the lPNRSS chain from escaping from the aperture. Figure 68 (a and b). Furthermore, the lPNRSS chain 14PBA contains a single PBA capable of binding norepinephrine. In this case, norepinephrine reports a negative binding event at +160 mV ( Figure 68 c). In summary, based on the PNRSS method, we set locking segments on the PNRSS chain to form a hairpin structure to prevent the PNRSS chain from randomly escaping from the nanopore. The lPNRSS technique can extend measurement time and improve consistency.

[0456] like Figure 69 As shown, we demonstrate the ability of the PNRSS technique to sense sugars. Three monosaccharides are shown here. It is important to note that some background signals are caused by the Tris buffer. The Tris buffer used at the time could react with phenylboronic acid, introducing background. However, the events of Tris binding to PBA are distinctly different from the events of sugar binding to PBA. In summary, the PNRSS technique, primarily introduced by phenylboronic acid, can directly sense multiple sugars.

[0457] like Figure 70 As shown, we demonstrate the ability of PNRSS technology to sense nucleotides. It's important to note that we used 5'-monophosphate (5'-CMP) as an example here. However, theoretically, PNRSS technology can be used to directly observe nucleosides, nucleotides, and nucleoside analogs with diol structures. In summary, PNRSS technology, primarily introduced through phenylboronic acid, can directly sense a variety of nucleosides, nucleotides, and nucleoside analogs.

[0458] like Figure 71As shown, we demonstrate the ability of PNRSS technology to distinguish molecular chirality. Using the enantiomer norepinephrine as an example, we show the signals from the sequential addition of L- and D-norepinephrine. In summary, PNRSS technology, primarily introduced via phenylboronic acid, can directly distinguish the molecular chirality of catecholamines.

[0459] like Figure 72 As shown, we demonstrate the ability of PNRSS technology to sense isotopic catechols, where hydrogen atoms in the molecule have been replaced by deuterium isotopes. In summary, PNRSS technology can directly sense a variety of isotopic molecules.

[0460] like Figure 73 As shown, we demonstrate the ability of the PNRSS technology to sense polysaccharides. Three polysaccharides are shown here, including disaccharides and trisaccharides. Clear and well-defined binding events are reported for all. In summary, the PNRSS technology, primarily introduced via phenylboronic acid, can directly sense a variety of polysaccharides.

[0461] like Figure 74 As shown, we demonstrate the ability of PNRSS technology to sense molecules containing two reactive groups. 3,4-Dihydroxymandelic acid has two diol structures, which can react with phenylboronic acid in two ways.

[0462] like Figure 75 As shown, we demonstrate the ability of PNRSS technology to sense 4-hydroxy-3-methoxymandelic acid (VMA). VMA has a diol structure that can react with phenylboronic acid.

[0463] like Figure 76 As shown, we demonstrate the ability of PNRSS technology to sense 3,4-dihydroxyphenylacetic acid. 3,4-Dihydroxyphenylacetic acid has a diol structure that can react with phenylboronic acid.

[0464] discuss

[0465] To date, MspA has been used as the pore for PNRSS analysis of 20 analytes. In principle, any nanopore in which a polymer can be chained and fully extended is suitable for PNRSS. However, its performance will depend on the overall structure of the pore. To verify the versatility of PNRSS in working with other channel proteins, a feasibility test was conducted using wild-type α-hemolysin (WTα-HL) as the PNRSS pore and 14PBA (Table 1) as the PNRSS chain. Isoproterenol was used as the mobile reactant (…). Figure 58 Experimentally, isoproterenol binding events were successfully observed, exhibiting a negative (I) reaction. b p ​The resistance pulse confirmed our hypothesis that PNRSS is universal when used with other channel proteins. However, the reported event amplitude (~-4.6 pA) was much smaller than that generated by MspA (~-32.2 pA, Table 11), even though other measurement conditions remained the same. This experimentally demonstrates that MspA, with its overall conical geometry, generates a more concentrated electric field at the pore constriction, thus producing a larger event amplitude. Therefore, it is the best choice for distinguishing PNRSS pores from analytes with similar chemical structures. PNRSS chains without a traction segment (14TAK-NTS, Table 1) were also tested, and no successful PNRSS chain capture was reported. Figure 59 Although the traction segment helps maintain electrophoretic force during measurement, it should be understood that other methods can be used to maintain the PNRSS chain, or that instantaneous measurements can be performed.

[0466] To maintain consistency throughout the text, measurement conditions were generally the same, using a 1.5M KCl, 10mM HEPES buffer solution and applying a high voltage (e.g., +180mV or +160mV). Catechol ( Figure 60 ) or norepinephrine ( Figure 61 As representative electroneutrally neutral or electroneutrally positive analytes, PNRSS measurements were performed at voltage gradients between +80 mV and +160 mV. For both analytes, higher potentials generally resulted in larger reported event amplitudes, suggesting that higher applied potentials yield higher sensing resolution and are therefore more advantageous. Although a potential of +20 mV was sufficient to hold the PNRSS chains within the pores and backflow of the PNRSS chains was rarely observed, at least +60 mV was required to generate sufficiently large event amplitudes for detection. For both analytes, event residence time was generally independent of the applied potential amplitude. However, positively charged norepinephrine reported a significantly higher event occurrence rate when larger potentials were applied, suggesting that electrophoretic forces are crucial when moving the charged reactant. Figure 61 Conversely, for catechol, the event occurrence rate is less modulated by the applied potential, demonstrating that the contribution of electroosmotic flow is negligible. Figure 60 PNRSS can also be performed at different salt concentrations, and generally higher salt concentrations are more conducive to generating larger event amplitudes by producing a larger ion flow through the pore. Figure 62 ).

[0467] The rate of unimolecular chemical reactions can also be controlled by temperature. In the experiment, norepinephrine was used as the model analyte, and phenylboronic acid was used as the stationary reactant. PNRSS was performed on an Orbit Mini nanopore reader (Nanion Technologies GmbH, Germany) with a built-in temperature control module. Figure 63Clearly, the on / off rates and binding affinity are all temperature-regulated. The reaction rate is generally exponentially related to the set temperature, conforming to the rules described by the Arrhenius equation. 78 The trans compartment of the Orbit Mini chip is too small to hold mobile analytes; therefore, in this measurement, norepinephrine was added to the CIS, along with... Figure 43 The events occurred differently in both settings. However, the events were detectable in both settings, confirming that rapid diffusion of small molecule analytes also contributes to the occurrence of the events.

[0468] In this paper, the limit of detection is specifically defined as the lowest final analyte concentration in the measurement chamber acquired during at least five events in a 10-minute continuous measurement process (Table 14). Due to the large volume of the measurement chamber and the small size of the individual nanopore sensors, the detection efficiency is generally not optimal for the current setup. Therefore, in the absence of any sample enrichment, when the target analyte is present in physiological samples at low concentrations, insufficient quantitative events are not expected to be observed. This is the case for adrenaline, noradrenaline, and isoproterenol, whose physiological concentrations in serum are approximately in the nM range. 79 However, the detection limit reported here is approximately 1 μM. From an engineering perspective, this can be improved by introducing thousands of independent parallel nanopore sensors and an extremely flat flow cell, similar to the setup demonstrated in the MinION sequencer. 19 On the other hand, some analytes tested in this paper may be present in high concentrations in natural samples and could potentially be used directly for detection even under the current setup. Vitamin B6 reports an easily identifiable event pattern during the PNRSS process, with a detection limit of ~400 nM. Therefore, we designed an assay method to simulate the detection of vitamin B6 in real human urine samples. In the experiments, adding real human urine samples to the trans side did not report any interfering events. Figure 64 However, urine samples from individuals with added vitamin B6 reported corresponding events, suitable for direct quantification based on the reported calibration curve. Figure 65 These results indicate that PNRSS assays can be performed using real biological samples, similar to other state-of-the-art nanopore assays. 80,81 Unique event patterns help identify events from heterogeneous solutions.

[0469] limit

[0470] However, the above demonstration is far from perfect. For rapid demonstration, all PNRSS were performed with the pores and chains chemically separated. While this sensing mode is advantageous for reproducible measurements of irreversible reactions, the PNRSS chains can be permanently conjugated to the pores to further improve the resolution and consistency of the PNRSS. For example, triazoles generated by the CuAAC reaction may undergo undesirable chemical reactions with transient metal ions (…). Figure 2 However, this interference can be minimized by adding a chelating agent (such as ethylenediaminetetraacetic acid (EDTA)) or by choosing alternative conjugation chemistry. 82 The results in this paper may be enlightening for the preparation of compounds. However, due to the small scale of this single-molecule reactor, PNRSS is currently designed as a sensing method rather than a preparative method. K was measured using PNRSS. b The values ​​were also compared with those reported in the literature (Table 15). While it is difficult to report the results of all chemical reactions in this paper, and it is also difficult to obtain results under exactly the same conditions, a general consistency of the results can be observed. However, we would like to emphasize that the purpose of developing PNRSS is to apply existing knowledge of the chemical interactions between reactants to achieve direct chemical sensing of small molecule analytes, and the results in this paper strongly support this.

[0471] in conclusion

[0472] In summary, this paper reports for the first time PNRSS as a convenient molecular toolkit for studying single-molecule chemical processes using nanopores. An artistic video demonstration (Video 7) is included to better illustrate the concept of PNRSS. The specific aim is to overcome the technical bottleneck of introducing any number or type of reactive groups into any location within a nanopore cavity, a challenge even for the most advanced in the field. However, with PNRSS, this difficulty is transformed into the synthesis of functional DNA oligomers, a routine task performed daily in countless biochemistry labs or offered as a low-cost service by various commercial vendors. Unlike previous setups, PNRSS also enables the reproducible monitoring of irreversible reactions, further expanding the range of chemical reactions previously difficult to study via nanopores. We report a total of 20 single-molecule chemical reactions involving hydrogen peroxide, buffer reagents, transition metal ions, glycerol, lactic acid, vitamins, catecholamine derivatives, or antiviral drugs. The reported event patterns are highly diverse, relating to analyte size, charge, and conformation, and can be used for single-molecule recognition. While limited by the length of this paper, these reactions demonstrate the core aspects of the PNRSS technique and its feasibility and versatility. To the best of our knowledge, this is the largest number of nanopore-based single-molecule chemical reactions reported in a single publication. Most of them have never been studied as single molecules before (Table 16). Given the conical structure of MspA, which effectively concentrates ion flow at its narrow pore confinement, all the single-molecule chemical reactions discussed in this paper demonstrate fully resolved event magnitudes. For PBA, the direct recognition of epinephrine, noradrenaline, and isoproterenol also suggests its direct biomedical applications. The single-molecule differentiation of remdesivir and its triphosphate metabolites by PNRSS may inspire pharmacokinetic measurements of various nucleoside analogue drugs.

[0473] In future studies, multiple immobilized reactants may be used on the same chain for PNRSS, thus increasing the complexity of the sensing. Despite using MspA and α-HL... 6 It has been demonstrated, but other biological nanopores (e.g., aerolysine) 8 or CsgG 9 In principle, it is also compatible with PNRSS, as long as the chain polymer containing the designed reaction sites can fully extend within the pores. ClyA, with its large opening, is another example. 10 fragaceatoxin C (FraC) 11 Pleurotolysin A / B 12 or phi29 connector 14Nanopores can also be used to probe chemical reactions involving larger or more complex mobile reactants, such as polysaccharides, or cyclic peptides. However, these proposed plans to apply PNRSS in conjunction with large channel proteins have not yet been implemented, but may inspire other colleagues in the field.

[0474] Author's Contribution

[0475] SH and WDJ conceived this project. WDJ and CZH performed the measurements. YMG and JM conducted molecular simulations. GRQ designed the machine learning algorithm. YQW, YLSHYJC, and SYZ prepared MspA nanopores. WDJ and XYD performed measurements using α-HL. WDJ and LYW performed measurements at different temperatures. PKZ set up the instrument. SH and WDJ wrote this paper. YQW and WDJ prepared supplementary videos. SH and HYC supervised the project.

[0476] Data and code availability declaration

[0477] If you have a reasonable request, you may request all the data and code provided in this work from the relevant authors.

[0478] Statement of Competitive Interests

[0479] SH and WDJ have filed patent applications describing PNRSS technology and its applications. GRQ is the founder of IntelligenceQubic Technology Co., Ltd., a company that develops artificial intelligence software interfaces. The authors claim no other competing interests.

[0480] Acknowledgments

[0481] The author thanks Prof. Hagan Bayley (Oxford University) for his valuable suggestions during the manuscript preparation process. The author also thanks Prof. Shaolin Zhu, Prof. Congqing Zhu, Prof. Jie Li, and Prof. Ran Xie from Nanjing University, and Mrs. Yiou Ma, Prof. Daoqiang Zhang, and Xiaoyu Guan from Nanjing University of Aeronautics and Astronautics for their inspiring discussions. SH thanks his grandmother, Mrs. Jiqing Xia, for her encouragement during the completion of this project. Sadly, Mrs. Xia passed away before the manuscript was submitted due to her advanced age.

[0482] This project was supported by the National Natural Science Foundation of China (No. 31972917, No. 91753108, No. 21675083), the Fundamental Research Funds for the Central Universities (No. 020514380257, No. 020514380261), the State Key Laboratory of Life Analytical Chemistry (No. 5431ZZXM1902, No. 5431ZZXM1804), the Natural Science Foundation of Jiangsu Province, and the Jiangsu Provincial High-Level Entrepreneurial and Innovative Talent Introduction Program (Individual and Group Programs). It was also supported by the Nanjing University Science and Technology Innovation Fund Project and the Nanjing University Excellent Research Program (Project No. ZYJH004).

[0483] Material

[0484] Pentane, hexadecane, ethylenediaminetetraacetic acid (EDTA), and Genapor X-80 were obtained from Sigma-Aldrich. 1,2-Diphydanoyl-sn-glycerol-3-phosphocholine (DPhPC) was supplied by Avanti Polar Lipids. Potassium chloride (KCl, 99.9%), sodium hydroxide (NaOH, 99.9%), cobalt sulfate heptahydrate (CoSO4·7H2O, 99.99%), nickel sulfate hexahydrate (NiSO4·6H2O, 99.9%), copper sulfate pentahydrate (CuSO4·5H2O, 99.9%), zinc sulfate heptahydrate (ZnSO4·7H2O, 99.995%), cadmium sulfate 8 / 3 hydrate (CdSO4·8 / 3H2O, 99.99%), ethylene glycol (99.9%), and glycerol (99%) were also present. 0.7%, L-lactic acid (98%), pyridoxine (vitamin B6) (98%), 30% hydrogen peroxide solution (H2O2, GR), DL-norepinephrine hydrochloride (97%), DL-adrenaline hydrochloride (98%), DL-isoproterenol hydrochloride (99%), 3-azidopropylamine (95%), anhydrous sodium sulfate (Na2SO4, 99%), dimethyl sulfoxide (DMSO, 99.9%) and dimethyl sulfoxide-d6 (DMSO-d6, D. 99.9% + 0.03% TMS) were from Aladdin (China). Methylboronic acid (97%), catechol (99.5%), resorcinol (AR) and acetonitrile (MeCN, 99.9%) were from Macklin (China). Hydrochloric acid (HCl), acetone (Me2CO, 99.5%), and dichloromethane (DCM, 99.5%) were from Sinopharm (China). Sodium ascorbate (vitamin C) (99%) and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES, 99%) were purchased from Shanghai Yuanye Bio-Technology (China). Pinalol 4-(azidomethyl)phenylboronic acid (95%) was from Alfa Aesar (USA). Remdesivir (99.74%) and remdesivir metabolites (99.87%) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

[0485] Escherichia coli strain BL21(DE3) was obtained from Biomed (China). Streptavidin was obtained from New England Biolabs. Dioxane-free isopropyl-β-D-thiopyranoside (IPTG), kanamycin sulfate, and tris-(hydroxy-methyl)aminomethane (Tris) were obtained from Solarbio Biotechnology (China). Luria-Bertani broth and Luria-Bertani agar were obtained from Hopebio (China). Precision Plus Protein™ Dual Color Standards and TGX™ FastCast™ Acrylamide Kit (12%) were purchased from Bio-Rad.

[0486] The monomeric DNA phosphoramidite 5-ethynyl-dU-CE phosphoramidite was purchased from Glen Research (USA), and the alkyne-containing oligonucleotide 14TAK (Table 1) was synthesized by Shanghai Generay Biotech Co., Ltd. All other DNA oligonucleotides were synthesized by Genscript (New Jersey, USA). The complete sequences are listed in Table 1.

[0487] method

[0488] 1. Nanopore preparation

[0489] The gene encoding the monomeric M2 MspA mutant (D93N / D91N / D90N / D118R / D134R / E139K) was synthesized and inserted into the pet-30a(+) vector. M2 MspA was expressed in *E. coli* BL21(DE3) as previously reported and purified using nickel affinity chromatography (GE Akta Pure, GE Healthcare). 1 Purified M2 MspA spontaneously oligomerized into an axisymmetric octamer form, which was used for all PNRSS measurements in this paper. The octamer M2 MspA is the only nanopore used in this work. For simplicity, it will be referred to as MspA throughout the text unless otherwise stated.

[0490] The gene encoding the monomeric wild-type α-hemolysin (WTα-HL) was synthesized and inserted into the pet-30a(+) vector. The preparation of the heptamer WTα-HL was performed strictly according to previously reported procedures. 83 .

[0491] The plasmid DNA encoding M2 MspA and WTα-HL has been shared in the Molecular Cloud Plasmid Library (https: / / www.molecularcloud.org / s / shuo-huang,GenScript,New Jersey), access codes MC_0101191 and MC_0068416. Citations are required when publishing articles using this plasmid.

[0492] 2. Nanopore Measurement and Data Analysis

[0493] Nanopore measurements were performed in a custom-designed measurement chamber. A self-assembled lipid bilayer formed of 1,2-diphydanyl-sn-glycerol-3-phosphocholine (DPhPC) divided the chamber into cis and trans compartments. Each compartment was filled with 500 μL of electrolyte buffer at pH 7.0 or 8.0, consisting of 1.5 M KCl, 10 mM HEPES. All measurements using PNRSS chains 14X, 13G / 14G, 14A, 14G, and 14TAZ (Table 1) were performed at pH 7.0. All measurements using PNRSS chain 14PBA (Table 1) were performed at pH 8.0 when tris was not used as a mobile reactant. For all measurements using tris, the pH was adjusted to 7.0 or 8.0. A pair of Ag / AgCl electrodes were placed on the cis and trans sides of the chamber, respectively, in contact with the aqueous buffer on each side. The Ag / AgCl electrodes were electrically connected to a patch-clamp amplifier to form a closed circuit. By convention, the electrodes in the CIS compartment are electrically grounded, while the opposite electrodes are the working electrodes.

[0494] To obtain the average blocking level Static orifice blockage measurement using published methods 84 In short, with the insertion of a single MspA, a PNRSS chain was added to the cis side to a final concentration of 20 nM. Voltage schemes of +180 mV or +160 mV (0.9 s) and -100 mV (0.3 s) were repeatedly applied, and I was measured when +180 mV or +160 mV potential was applied. p At least 500 I samples were collected during each experiment. p Events. These events follow a Gaussian distribution. Three independent measurements are performed to obtain... The mean and standard deviation.

[0495] To perform PNRSS, the required PNRSS chain is added to the cis side to a final concentration of 10 nM. A positive potential is continuously applied. At I... p Above this level, the PNRSS event is identified as a further orifice blockage event. Figure 10 ).

[0496] All electrophysiological recordings were performed using an Axopatch 200B patch-clamp amplifier. Acquired traces were low-pass filtered using a Digidata 1550B analog-to-digital converter (Molecular Devices, UK) at a sampling rate of 25 kHz and a corner frequency of 1 kHz. MspAs were added to the cis side for spontaneous single-well insertion. After inserting a single well into the membrane, the electrolyte buffer on the cis side was exchanged to prevent further well insertion. All PNRSS measurements were performed at room temperature (21 ± 2 °C). Nanopore events were extracted using the single-channel search function of Clampfit 10.7 (Molecular Devices, UK). Further analysis was performed in Origin 2019. All color-coded scatter plots were generated using ggplot2, an R package for data visualization.

[0497] 3. Streptavidin DNA conjugation

[0498] To form a streptavidin-based DNA complex, DNA oligomers modified with 5' biotin-TEG (Table 1) were incubated with streptavidin in an equimolar ratio at room temperature (rt) for 10 min. During PNRSS measurements, the formed streptavidin-based DNA complex was added to the cis side at the desired final concentration.

[0499] 4. Optimize the theoretical calculations based on the combined settings.

[0500] All theoretical calculations were performed using the Gaussian 16 software package. 85 Geometric optimization was performed using density functional theory (DFT) and the M06 functional. 86,87 The C, H, O, N, and P atoms are represented by the 6-31+G(d) basis set, while the Ni atom is represented by the LANL2DZ basis set and the associated effective core potential. 88 Low spin (E LS ) state and high spin (E) HS The relative energy (ΔE) between systems in the following states is calculated as follows:

[0501] ΔE=E LS –E HS (1)

[0502] Ni with low-spin (LS) and high-spin (HS) states was investigated. 2+ Possible combination patterns, such as Figure 9 As shown. To conduct further research, Ni was calculated. 2+Two modes of clusters with 4 or 5 H2O molecules were presented. The calculated relative energies ΔE between different spin states are shown in Table 3. The HS states of (dGMP)2-Ni-4wt and (dGMP)2-Ni-5wt are -44.13 and -36.85 kcal / mol lower than the LS states, respectively, indicating that the HS states are energy-favorable. The geometry of the HS states shows an octahedral structure with 6 coordination atoms, and the distance between Ni and N(O) atoms is... However, the octahedral geometry exhibits some degree of deformation in the LS state. The hydrogen-bonded interactions of OH…O and OH…N with Ni... 2+ It plays an important role in the combination of clusters.

[0503] The binding energy (E) was calculated. b This study investigates the binding capacity of different modes. The binding energy is determined by separately calculating the total energy (E) of the complex system and the energy of H₂O (E). wt ), Ni 2+ Ions (E) Ni ) and two deoxyguanosine monophosphates (E dGMP / dGMP The energy difference between the sums of individual energies is obtained, and the calculation formula is as follows:

[0504] E b =E–xE wt –E Ni –E dGMP / dGMP (2)

[0505] Where x is the number of H2O molecules. Ni 2+ The ion can bind with four H₂O molecules and two guanine molecules, with a binding energy of -45.33 kcal / mol. For (dGMP)₂-Ni⁻⁵wt, Ni 2+ An ion can bind one guanine molecule and five H₂O molecules, E b The concentration is -46.07 kcal / mol, and one of the H2O molecules can bind guanine through hydrogen bond interactions of the N(7) atom.

[0506] 5. Introducing functionalized azides into the PNRSS chain

[0507] The 14TAK DNA strand containing alkynes (Table 1) was used as a universal PNRSS strand template to introduce any functional azide. Functional azides, such as 3-azidopropylamine (… Figure 17-18 ) or 4-(azidomethyl)phenylboronic acid ( Figure 24-26 Chemical conjugation was carried out via a Huisgen copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction.

[0508] 6. Procedure for producing 4-(azidomethyl)phenylboronic acid

[0509]

[0510] 4-(azidomethyl)phenylboronic acid pinacol ester (158 mg, 0.6 mmol) and methylboronic acid (360 mg, 6 mmol) were added to a 10 mL reaction tube and dissolved in acetone (2 mL). After further adding 0.1 M NaOH (2 mL), the resulting solution was stirred at room temperature for 12 h. Then, 4 mL of dichloromethane was added to the solution. The resulting mixture was poured into a separatory funnel to remove the organic layer. The pH of the solution was adjusted to 7 by titration with 0.1 M HCl. Then, 4 mL of dichloromethane was added to the aqueous layer to extract the target product. The organic layer was washed with water to remove residual salts. The organic phase was further dried with solid Na₂SO₄. The organic solvent was removed by rotary evaporation to collect 4-(azidomethyl)phenylboronic acid as a white powder (74.2 mg, 68% yield). 89 .pass 1 Further characterization of the product by ¹H NMR spectroscopy confirmed success. 90 (Figure S18).

[0511] Table 1 | Sequence background of all PNRSS chains in this study.

[0512]

[0513] footnote:

[0514] 1. The reaction segment is marked in bold in each sequence.

[0515] 2. Natural DNA bases or combinations thereof (e.g., A, G, or GG) can be used as fixation reactants.

[0516] 3.X represents a baseless site, which cannot bind to any of the mobile reactants tested in this study.

[0517] 4.5' Biotin TEG serves as a tying point, forming a tight bond with the streptavidin barrier.

[0518]

[0519] 5. (TAK) is an alkyne-modified thymine analog (5-ethynyldeoxyuridine, Glen Research, US) used as a universal linker for introducing functional azides. The chemical structure of (TAK) is shown below:

[0520]

[0521] 6. (TAZ) represents 1,2,3-triazole. The chemical structures of nucleotides containing TAZ are provided below. Detailed procedures for their chemical synthesis and characterization are described in the Methods and... Figure 17-18 Provided by China.

[0522]

[0523] 7. (PBA) represents phenylboronic acid. The chemical structures of nucleotides containing TBA are provided below. Detailed procedures for their chemical synthesis and characterization are described in the Methods and... Figure 25-26 Provided by China.

[0524]

[0525] Table 2 | Blocking of 14X, 14A, 14G or 13 / 14G Statistical data. Static pore blockage measurements were performed as described in the method. A buffer solution of 1.5 M KCl, 10 mM HEPES, and pH 7.0 was used. Each PNRSS chain was added to the cis side to a final concentration of 20 nM. I was measured when a +180 mV bias was applied. p ( Figure 10 500 events were collected during each measurement. Three independent measurements were performed for each condition (N=3) to generate statistical data.

[0526]

[0527]

[0528] Table 3 | Relative energies of (dGMP)2-Ni-4wt and (dGMP)2-Ni-5wt.

[0529]

[0530] Table 4|Ni 2+ Combined with 14A, 14G or 13G / 14G and τ off Statistical data. PNRSS measurements are conducted according to... Figure 13 (14A) Figure 15 (14G) and Figure 1 Perform as described in (13G / 14G). Use a buffer solution of 1.5M KCl, 10mM HEPES, pH 7.0. Continuously apply an A+180mV potential. 2+ Combined with 14A, it produces two event groups ( Figure 13-14 ), so that they can be obtained separately and τ off Ni 2+ respectively with 14G ( Figure 15-16 ) or 13G / 14G ( Figure 1 Combine and report individual event groups. Each measurement should include at least 1000 events. Perform three independent measurements (N=3) for each condition to generate statistics.

[0531]

[0532] Table 5 | 14TAK and 14TAZ Statistical data. Static pore blockage measurements were performed as described in the method. A buffer solution of 1.5 M KCl, 10 mM HEPES, pH 7.0 was used. Each PNRSS chain was added to the cis side to a final concentration of 20 nM. I was measured when a +180 mV bias was applied. p ( Figure 10 500 events are obtained from each measurement. Three independent measurements (N=3) are performed for each condition to generate statistics.

[0533]

[0534] Table 6|Ni 2+ or Co 2+ Combined with TAZ τ off and K b Statistical data. PNRSS measurements are conducted according to... Figure 19 (Ni 2+ )and Figure 21 (Co 2+ Perform as described in [reference needed]. Use a buffer solution of 1.5 M KCl, 10 mM HEPES, pH 7.0. Continuously apply a +180 mV potential. Each measurement should include at least 1000 events. Perform three independent measurements (N=3) for each condition to generate statistical data.

[0535]

[0536] Table 7 | PNRSS chains 14TAK and 14PBA Statistical data. Static pore blockage measurements were performed as described in the method. A buffer solution of 1.5 M KCl, 10 mM HEPES, and pH 8.0 was used. I was measured when a bias voltage of +160 mV was applied. p 500 events are obtained from each measurement. Three independent measurements are performed for each condition (N=3) to generate statistics.

[0537]

[0538] Table 8 | Combination of phenylboronic acid with diol and τ off Statistical data. For example... Figure 3PNRSS measurements were performed as described above. A buffer solution of 1.5 M KCl, 10 mM HEPES, and pH 8.0 was used. Each type of mobile reactant was added to the trans side at final concentrations of 400 μM (catechol), 14 mM (ethylene glycol), 10 mM (glycerol), 4 mM (L-lactic acid), 1.6 mM (vitamin C), and 40 μM (vitamin B6). A +160 mV bias was continuously applied during the measurement. and τ off Definitions and data sources, such as Figure 10 As shown. For each measurement, and τ off Values ​​are derived from events recorded within a 15-minute continuous trace. Three independent measurements (N=3) are performed for each condition to generate statistical data.

[0539]

[0540] Table 9 | Kinetic constants for the formation of the complex between PBA and diol. According to... Figures 27-38 PNRSS measurements were performed as described in [the document]. A buffer solution of 1.5 M KCl, 10 mM HEPES, and pH 8.0 was used. A potential of +160 mV was continuously applied. For each measurement, the kinetic constant was derived from events within a 15-minute continuously recorded trace. Three independent measurements were performed for each condition (N=3) to generate statistical data.

[0541]

[0542] Table 10 | Kinetic constants of the interaction between catecholamines and PBA. According to... Figures 43-48 PNRSS measurements were performed as described in [the document]. A buffer solution of 1.5 M KCl, 10 mM HEPES, and pH 8.0 was used. A potential of +160 mV was continuously applied. For each measurement, the kinetic constant was derived from events within a 15-minute continuously recorded trace. Three independent measurements were performed for each condition (N=3) to generate statistical data.

[0543]

[0544]

[0545] Table 11 | ΔI and τ of catecholamine events off Statistical data. According to Figure 43 , Figure 45 and Figure 47PNRSS measurements were performed according to the description in [the document]. A buffer solution of 1.5 M KCl, 10 mM HEPES, and pH 8.0 was used. Each mobile reactant was added to the trans side to a final concentration of 140 μM. A bias of +160 mV was continuously applied during the measurement. For each measurement, [the following parameters were applied]. and τ off The values ​​are derived from events generated by a 5-minute continuous trace. Three independent measurements (N=3) are performed for each condition to generate statistics.

[0546]

[0547] Table 12 | ΔI and τ for Remdesivir and Remdesivir Metabolite Events off Statistical data. For example... Figure 53 and Figure 55 The PNRSS measurement was performed. In separate measurements, remdesivir or a remdesivir metabolite was added to the trans side at a final concentration of 80 μM or 500 μM. A +160 mV bias was continuously applied during the measurement. For each measurement, and τ off The values ​​are derived from events generated by a 5-minute continuous trace. Three independent measurements (N=3) are performed for each condition to form statistical data.

[0548]

[0549] Table 13 | Kinetic constants of remdesivir and its metabolites binding to PBA. (e.g.) Figure 53 and Figure 55 PNRSS measurements were performed. A buffer solution of 1.5 M KCl, 10 mM HEPES, and pH 8.0 was used. A potential of +160 mV was continuously applied. For each measurement, the kinetic constant was derived from events within a 15-minute continuously recorded trace. Three independent measurements (N=3) were performed for each condition to generate statistical data.

[0550]

[0551] Table 14 | Limit of Detection. In this paper, the limit of detection is defined as the lowest concentration of analyte required to detect at least 5 events within 10 min of the measurement. All PNRSS measurements were performed as described in the Methods section. For all measurements using 14A, 14G, 13G / 14G, or 14TAZ, a buffer solution of 1.5 M KCl, 10 mM HEPES, pH 7.0 was used, with a continuous +180 mV potential applied. For all measurements using 14PBA, a buffer solution of 1.5 M KCl, 10 mM HEPES, pH 8.0 was used, with a continuous +160 mV potential applied.

[0552]

[0553]

[0554] Table 15 | The previously reported binding constant K between PNRSS and PNRSS b (M -1 (Summary)

[0555]

[0556]

[0557] Table 16 | Summary of PNRSS measurements in this paper.

[0558]

[0559]

[0560]

[0561]

[0562] Video 1 | Ni 2+ It binds to diguanine reactants. For example... Figure 1 PNRSS measurements were performed. The electrolyte buffer used was 1.5 M KCl, 10 mM HEPES, pH 7.0. PNRSS chains 13G / 14G (Table 1) were added to the cis side to a final concentration of 10 nM. The diguanine on the 13G / 14G chain was used as a fixative. Ni was added to the trans side. 2+ Used as a mobile reactant, with a final concentration of 1 mM. A potential of +180 mV was continuously applied. Initially (first ~0.1 s), the pores were unoccupied, and an opening current (~575 pA) was reported. Subsequently, the PNRSS chain was captured, and the current was observed to drop immediately to ~170 pA. Further binding events occurred successively (0.2–5 s), with ΔI measured at approximately -60 pA. These events are due to Ni 2+ Reversible binding with diguanine reactants leads to, such as Figure 1 As shown in c-1d.

[0563] Video 2 | Ni 2+ Combined with TAZ. For example... Figure 2 PNRSS measurements were performed as described above. The electrolyte buffer used was 1.5 M KCl, 10 mM HEPES, pH 7.0. PNRSS chain 14TAZ (Table 1) was added to the cis side to a final concentration of 10 nM. TAZ was used as a fixative. Ni was added to the trans side. 2+Used as a mobile reactant, with a final concentration of 1 mM. In this video, the PNRSS chain 14TAZ has been trapped in the pores. Ni 2+ The reversible binding with TAZ resulted in events that were subsequently observed as further blockages. When Ni 2+ When combined, all events report characteristic noise fluctuations.

[0564] Video 3 | Repeated measurements of irreversible reactions. (e.g.) Figure 4 PNRSS measurements were performed as described above. The electrolyte buffer used was 1.5 M KCl, 10 mM HEPES, pH 8.0. PNRSS chains of 14PBA (Table 1) were added to the cis side to a final concentration of 10 nM. Phenylboronic acid (PBA) on the 14PBA was used as a stationary reactant. Hydrogen peroxide was added to the trans side as a mobile reactant to a final concentration of 5.4 mM. Hydrogen peroxide can reversibly bind to PBA or irreversibly oxidize it to phenol. Figure 4 (a, 4b) Initially, reversible binding of hydrogen peroxide to PBA was observed sequentially, reporting a spike, positive event (2–80 s). Irreversible oxidation of PBA deactivates the chemical reactivity of the stationary reactants, producing an event-free portion of the trace. By sequentially switching the applied potential to -100 mV (chain ejection) and +160 mV (chain reloading), the deactivated PNRSS chain is ejected and reloaded with another reactive chain. This specific measurement mode of PNRSS allows for repeated measurements of irreversible reactions.

[0565] Video 4 | Positive and negative events obtained using PBA. For example... Figure 10 PNRSS measurements were performed. The electrolyte buffer used was 1.5 M KCl, 10 mM HEPES, pH 8.0. PNRSS chain 14PBA (Table S1) was added to the cis side to a final concentration of 10 nM. Phenylboronic acid (PBA) on 14PBA was used as a stationary reactant. Catechol or norepinephrine was added to the trans side as a mobile reactant, with a final concentration of 280 μM for each analyte. In this video, the PNRSS chain 14PBA is captured in the well, and the reported I... p Values ​​are ~100 pA. Binding to catechol or norepinephrine respectively reports a positive (I) result. b >I p ) or negative (I b p The binding of catechol or norepinephrine on the traces is labeled with C or N, respectively.

[0566] Video 5 | PNRSS sensing of adrenaline, noradrenaline, and isoproterenol. (e.g., ...) Figure 5 ​PNRSS measurements were performed. The electrolyte buffer used was 1.5 M KCl, 10 mM HEPES, pH 8.0. PNRSS chain 14PBA (Table 1) was added to the cis side to a final concentration of 10 nM. Phenylated boric acid (PBA) on 14PBA was used as a stationary reactant. Norepinephrine, epinephrine, and isoproterenol were added to the trans side as mobile reactants to final concentrations of 280 μM, 280 μM, and 180 μM, respectively. A +160 mV potential was continuously applied. In this video, the PNRSS chain 14PBA is captured by the well. Continuous binding of norepinephrine, epinephrine, or isoproterenol to PBA was observed. The raw trace was divided into low-pass (lp, top trace) and high-pass (hp, bottom trace) portions according to frequency. A Butterworth filter was applied for frequency division. The cutoff frequency was set to 100 Hz. Figure 50 Event recognition through machine learning algorithms ( Figure 51 The identified events are labeled as N (norepinephrine), E (epinephrine), or I (isoproterenol).

[0567] Video 6 | PNRSS sensing of remdesivir and its metabolites. (e.g.) Figure 6 PNRSS measurements were performed as shown. The electrolyte buffer used was 1.5 M KCl, 10 mM HEPES, pH 8.0. PNRSS chain 14PBA (Table 1) was added to the cis side to a final concentration of 10 nM. Phenylboronic acid (PBA) on 14PBA was used as a stationary reactant. Remdesivir and its metabolites were added to the trans side as mobile reactants to final concentrations of 20 μM or 500 μM, respectively. A potential of A+160 mV was continuously applied. At the start of this video, the PNRSS chain 14PBA had been captured in the well. The binding of remdesivir or its metabolites to PBA both produced positive events (…). Figure 6 b). Based on their different binding properties ( Figure 6 bd), identify remdesivir or its metabolites, and label them with R or M on the trace, respectively.

[0568] Video 7 | An artistic showcase of PNRSS. The core concept of PNRSS is to lower the technical barriers to protein engineering to prepare heterogeneous oligomeric nanoporous nanoreactors. With PNRSS, reaction components are used as individual modules in a toolkit. New applications are achieved through countless combinations of these modules, without the need for repeated engineering of protein nanopores.

[0569] References

[0570] 1.Huang,S.et al.Identifying single bases in a DNA oligomer withelectron tunnelling.Nature Nanotechnology 5,868-873(2010).

[0571] 2.Zrimsek,A.B.et al.Single-Molecule Chemistry with Surface-and Tip-Enhanced Raman Spectroscopy.Chemical Reviews 117,7583-7613(2017).

[0572] 3.Jia,C.et al.Covalently bonded single-molecule junctions with stableand reversible photoswitched conductivity.Science 352,1443-1445(2016).

[0573] 4.Huang,W.et al.Maleimide–thiol adducts stabilized throughstretching.Nature Chemistry 11,310-319(2019).

[0574] 5.Braha,O.et al.Designed protein pores as components forbiosensors.Chemistry&Biology 4,497-505(1997).

[0575] 6.Song,L.et al.Structure of Staphylococcalα-Hemolysin,a HeptamericTransmembrane Pore.Science 274,1859-1866(1996).

[0576] 7.Butler,T.Z.,Pavlenok,M.,Derrington,I.M.,Niederweis,M.&Gundlach,J.H.Single-molecule DNA detection with an engineered MspA protein nanopore.PNatl Acad Sci USA 105,20647-20652(2008).

[0577] 8.Cao,C.et al.Discrimination of oligonucleotides of different lengthswith a wild-type aerolysin nanopore.Nature Nanotechnology 11,713-718(2016).

[0578] 9.Bock,A.V.,Dill,D.B.&Edwards,H.T.LACTIC ACID IN THE BLOOD OF RESTINGMAN.The Journal of clinical investigation 11,775-788(1932).

[0579] 10.Soskine,M.et al.An Engineered ClyA Nanopore Detects Folded TargetProteins by Selective External Association and Pore Entry.Nano Letters 12,4895-4900(2012).

[0580] 11.Wloka,C.,Mutter,N.L.,Soskine,M.&Maglia,G.Alpha-HelicalFragaceatoxin C Nanopore Engineered for Double-Stranded and Single-StrandedNucleic Acid Analysis.Angewandte Chemie International Edition 55,12494-12498(2016).

[0581] 12.Huang,G.et al.Electro-Osmotic Vortices Promote the Capture ofFolded Proteins by PlyAB Nanopores.Nano Letters 20,3819-3827(2020).

[0582] 13.Fahie,M.A.,Yang,B.,Pham,B.&Chen,M.Tuning the Selectivity andSensitivity of an OmpG Nanopore Sensor by Adjusting Ligand Tether Length.ACSSensors 1,614-622(2016).

[0583] 14.Wendell,D.et al.Translocation of double-stranded DNA throughmembrane-adapted phi29 motor protein nanopores.Nature Nanotechnology 4,765-772(2009).

[0584] 15.Stoddart,D.,Heron,A.J.,Mikhailova,E.,Maglia,G.&Bayley,H.Single-nucleotide discrimination in immobilized DNA oligonucleotides with abiological nanopore.Proceedings of the National Academy of Sciences 106,7702-7707(2009).

[0585] 16.Stefureac,R.,Long,Y.T.,Kraatz,H.B.,Howard,P.&Lee,J.S.Transport ofalpha-helical peptides through alpha-hemolysin and aerolysinpores.Biochemistry 45,9172-9179(2006).

[0586] 17.Van Meervelt,V.et al.Real-Time Conformational Changes andControlled Orientation of Native Proteins Inside a ProteinNanoreactor.Journal of the American Chemical Society 139,18640-18646(2017).

[0587] 18.Galenkamp,N.S.,Biesemans,A.&Maglia,G.Directional conformerexchange in dihydrofolate reductase revealed by single-molecule nanoporerecordings.Nature Chemistry 12,481-488(2020).

[0588] 19.Quick,J.et al.Real-time,portable genome sequencing for Ebolasurveillance.Nature 530,228-232(2016).

[0589] 20.Roozbahani,G.M.,Chen,X.,Zhang,Y.,Wang,L.&Guan,X.Nanopore Detectionof Metal Ions:Current Status and Future Directions.Small Methods 4,2000266(2020).

[0590] 21.Kasianowicz,J.J.,Burden,D.L.,Han,L.C.,Cheley,S.&Bayley,H.Genetically Engineered Metal Ion Binding Sites on the Outside of a Channel's Transmembraneβ-Barrel.Biophysical Journal 76,837-845(1999).

[0591] 22.Choi,L.-S.,Mach,T.&Bayley,H.Rates and Stoichiometries of Metal IonProbes of Cysteine Residues within Ion Channels.Biophysical Journal 105,356-384(2013).

[0592] 23.Braha,O.et al.Simultaneous stochastic sensing of divalent metalions.Nature Biotechnology 18,1005-1007(2000).

[0593] 24.Borsley,S.&Cockroft,S.L.In Situ Synthetic Functionalization of aTransmembrane Protein Nanopore.ACS Nano 12,786-794(2018).

[0594] 25.Zhou,B.,Wang,Y.-Q.,Cao,C.,Li,D.-W.&Long,Y.-T.Monitoring disulfidebonds making and breaking in biological nanopore at single moleculelevel.Science China Chemistry 61,1385-1388(2018).

[0595] 26.Haugland,M.M.,Borsley,S.,Cairns-Gibson,D.F.,Elmi,A.&Cockroft,S.L.Synthetically Diversified Protein Nanopores:Resolving Click ReactionMechanisms.ACS Nano 13,4101-4110(2019).

[0596] 27.Cao,J.et al.Giant single molecule chemistry events observed from atetrachloroaurate(iii)embedded Mycobacterium smegmatis porin Ananopore.Nature Communications 10,5668(2019).

[0597] 28.Lee,J.&Bayley,H.Semisynthetic protein nanoreactor for single-molecule chemistry.P Natl Acad Sci USA 112,13768-13773(2015).

[0598] 29.Lee,J.et al.Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry.Acs Nano 10,8843-8850(2016).

[0599] 30.Bétermier,F.et al.Single-sulfur atom discrimination ofpolysulfides with a protein nanopore for improved batteries.CommunicationsMaterials 1,59(2020).

[0600] 31.Clarke,J.et al.Continuous base identification for single-moleculenanopore DNA sequencing.Nature Nanotechnology 4,265-270(2009).

[0601] 32.Zernia,S.,van der Heide,N.J.,Galenkamp,N.S.,Gouridis,G.&Maglia,G.Current Blockades of Proteins inside Nanopores for Real-Time MetabolomeAnalysis.ACS Nano 14,2296-2307(2020).

[0602] 33.Wallace,E.V.B.et al.Identification of epigenetic DNA modificationswith a protein nanopore.Chemical Communications 46,8195-8197(2010).

[0603] 34.Manrao,E.A.,Derrington,I.M.,Pavlenok,M.,Niederweis,M.&Gundlach,J.H.Nucleotide Discrimination with DNA Immobilized in the MspA Nanopore.PlosOne 6(2011).

[0604] 35.Hogrefe,R.I.,Midthune,B.&Lebedev,A.Current Challenges in NucleicAcid Synthesis.Israel Journal of Chemistry 53,326-349(2013).

[0605] 36.Huq,F.&Peter,M.C.R.Interaction between NiCl2,and nucleobases,nucleosides and nucleotides.Journal of Inorganic Biochemistry 78,217-226(2000).

[0606] 37.Masoud,M.S.,Soayed,A.A.&Ali,A.E.Complexing properties of nucleic-acid constituents adenine and guanine complexes.Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy 60,1907-1915(2004).

[0607] 38.El-Sagheer,A.H.&Brown,T.Click chemistry with DNA.Chemical SocietyReviews 39,1388-1405(2010).

[0608] 39.Kolb,H.C.,Finn,M.G.&Sharpless,K.B.Click Chemistry:Diverse ChemicalFunction from a Few Good Reactions.Angewandte Chemie International Edition40,2004-2021(2001).

[0609] 40.Struthers,H.,Spingler,B.,Mindt,T.L.&Schibli,R.“Click-to-Chelate”:Design and Incorporation of Triazole-Containing Metal-Chelating Systems intoBiomolecules of Diagnostic and Therapeutic Interest.Chemistry–A EuropeanJournal 14,6173-6183(2008).

[0610] 41.Meng,G.et al.Modular click chemistry libraries for functionalscreens using a diazotizing reagent.Nature 574,86-89(2019).

[0611] 42.Wu,X.et al.Selective sensing of saccharides using simple boronicacids and their aggregates.Chemical Society Reviews 42,8032-8048(2013).

[0612] 43.Pizer,R.&Babcock,L.Mechanism of the complexation of boron acidswith catechol and substituted catechols.Inorganic Chemistry 16,1677-1681(1977).

[0613] 44.Babcock,L.&Pizer,R.Dynamics of boron acid complexationreactions.Formation of 1:1 boron acid-ligand complexes.Inorganic Chemistry19,56-61(1980).

[0614] 45.Pizer,R.&Tihal,C.Equilibria and reaction mechanism of thecomplexation of methylboronic acid withpolyols.Inorganic Chemistry 31,3243-3247(1992).

[0615] 46. D.A.&Zümreoglu-Karan,B.Complexation of boric acid with vitaminC.New Journal of Chemistry 33,1874-1881(2009).

[0616] 47. D.A.,Zumreoglu-Karan,B.,Sahin,O.& O.Boric acidcomplexes with thiamine (vitamin B1)and pyridoxine(vitamin B6).InorganicaChimica Acta 413,77-83(2014).

[0617] 48.Lorand,J.P.&Edwards,J.O.Polyol Complexes and Structure of theBenzeneboronate Ion.The Journal of Organic Chemistry 24,769-774(1959).

[0618] 49.Bull,S.D.et al.Exploiting the Reversible Covalent Bonding ofBoronic Acids:Recognition,Sensing,and Assembly.Accounts of Chemical Research46,312-326(2013).

[0619] 50.Ramsay,W.J.,Bell,N.A.W.,Qing,Y.&Bayley,H.Single-MoleculeObservation of the Intermediates in a Catalytic Cycle.Journal of the AmericanChemical Society 140,17538-17546(2018).

[0620] 51.Good,N.E.et al.Hydrogen Ion Buffers for BiologicalResearch.Biochemistry 5,467-477(1966).

[0621] 52.Ramsay,W.J.&Bayley,H.Single-Molecule Determination of the Isomersof d-Glucose and d-Fructose that Bind to Boronic Acids.Angewandte ChemieInternational Edition 57,2841-2845(2018).

[0622] 53.Kuivila,H.G.Electrophilic Displacement Reactions.III.Kinetics ofthe Reaction between Hydrogen Peroxide and Benzeneboronic Acid.Journal of theAmerican Chemical Society 76,870-874(1954).

[0623] 54.Hao,L.,Ding,G.,Deming,D.A.&Zhang,Q.Recent Advances in GreenSynthesis of Functionalized Phenols from Aromatic Boronic Compounds.EuropeanJournal of Organic Chemistry 2019,7307-7321(2019).

[0624] 55.Dutta,A.,Ali,A.A.&Sarma,D.Versatile catalysis of“natural extract”:oxidation of sulfides and alcohols and ipso-hydroxylation of arylboronicacids.Journal of the Iranian Chemical Society 16,2379-2388(2019).

[0625] 56.Reiner,S.,Ambrosio,M.,Hoffmann,C.&Lohse,M.J.Differential Signalingof the Endogenous Agonists at the beta(2)-Adrenergic Receptor.Journal ofBiological Chemistry 285,36188-36198(2010).

[0626] 57.Moore,R.Y.&Bloom,F.E.Central Catecholamine Neuron Systems:Anatomyand Physiology of the Norepinephrine and Epinephrine Systems.Annual Review ofNeuroscience 2,113-168(1979).

[0627] 58.Nichtova,Z.,Novotova,M.,Kralova,E.&Stankovicova,T.Morphologicaland functional characteristics of models of experimental myocardial injuryinduced by isoproterenol.General Physiology and Biophysics 31,141-151(2012).

[0628] 59.Cortes,C.&Vapnik,V.Support-vector networks.Machine Learning 20,273-297(1995).

[0629] 60.Ptak,T., P.,Dobosz,A.,Rydzewska,A.&Prokopowicz,M.Potentiometric and NMR complexation studies of phenylboronic acid PBA andits aminophosphonate analog with selected catecholamines.Journal of MolecularStructure 1040,59-64(2013).

[0630] 61.Zhang,S.,Tang,Y.,Chen,Y.,Zhang,J.&Wei,Y.Boronic acid-modifiedpolyhedral oligomeric silsesquioxanes on polydopamine-coated magnetizedgraphene oxide for selective and high-capacity extraction of thecatecholamines epinephrine,dopamine and isoprenaline.Microchimica Acta 187,77(2020).

[0631] 62.Boersma,A.J.,Brain,K.L.&Bayley,H.Real-Time Stochastic Detection ofMultiple Neurotransmitters with a Protein Nanopore.ACS Nano 6,5304-5308(2012).

[0632] 63.Secor,K.E.&Glass,T.E.Selective Amine Recognition:Development of aChemosensor for Dopamine and Norepinephrine.Organic Letters 6,3727-3730(2004).

[0633] 64.Zhang,L.,Liu,X.A.,Gillis,K.D.&Glass,T.E.A High-AffinityFluorescent Sensor for Catecholamine:Application to Monitoring NorepinephrineExocytosis.Angewandte Chemie International Edition 58,7611-7614(2019).

[0634] 65.Flatmark Catecholamine biosynthesis and physiological regulationin neuroendocrine cells.Acta Physiologica Scandinavica 168,1-17(2000).

[0635] 66.Seley-Radtke,K.L.&Yates,M.K.The evolution of nucleoside analogueantivirals:A review for chemists and non-chemists.Part 1:Early structuralmodifications to the nucleoside scaffold.Antiviral Research 154,66-86(2018).

[0636] 67.Warren,T.K.et al.Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys.Nature 531,381-385(2016).

[0637] 68.Holshue,M.L.et al.First Case of 2019 Novel Coronavirus in theUnited States.New England Journal of Medicine 382,929-936(2020).

[0638] 69.McMahon,J.H.,Udy,A.&Peleg,A.Y.Remdesivir for the Treatment ofCovid-19-Preliminary Report.New England Journal of Medicine 383,992-993(2020).

[0639] 70.Wang,M.et al.Remdesivir and chloroquine effectively inhibit therecently emerged novel coronavirus (2019-nCoV)in vitro.Cell Research 30,269-271(2020).

[0640] 71.Yin,W.et al.Structural basis for inhibition of the RNA-dependentRNA polymerase from SARS-CoV-2 by remdesivir.Science 368,1499-1504(2020).

[0641] 72.Martin,A.R.,Vasseur,J.-J.&Smietana,M.Boron and nucleic acidchemistries:merging the best of both worlds.Chemical Society Reviews 42,5684-5713(2013).

[0642] 73.Beigel,J.H.et al.Remdesivir for the Treatment of Covid-19—FinalReport.New England Journal of Medicine(2020).

[0643] 74.Pan,H.et al.Repurposed antiviral drugs for COVID-19–interim WHOSOLIDARITY trial results.medRxiv,2020.2010.2015.20209817(2020).

[0644] 75.Warren,T.K.et al.Protection against filovirus diseases by a novelbroad-spectrum nucleoside analogue BCX4430.Nature 508,402-405(2014).

[0645] 76.Oxford,J.S.Inhibition of the Replication of Influenza A and BViruses by a Nucleoside Analogue (Ribavirin).Journal of General Virology 28,409-414(1975).

[0646] 77.Furuta,Y.et al.Favipiravir(T-705),a novel viral RNA polymeraseinhibitor.Antiviral Research 100,446-454(2013).

[0647] 78.Peleg,M.,Normand,M.D.&Corradini,M.G.The Arrhenius EquationRevisited.Critical Reviews in Food Science and Nutrition 52,830-851(2012).

[0648] 79.Ross,G.A.et al.Plasma and 24 H-Urinary CatecholamineConcentrations in Normal and Patient Populations.Annals of ClinicalBiochemistry 30,38-44(1993).

[0649] 80.Fahie,M.A.,Yang,B.,Mullis,M.,Holden,M.A.&Chen,M.SelectiveDetection of Protein Homologues in Serum Using an OmpG Nanopore.AnalyticalChemistry 87,11143-11149(2015).

[0650] 81.Galenkamp,N.S.,Soskine,M.,Hermans,J.,Wloka,C.&Maglia,G.Directelectrical quantification of glucose and asparagine from bodily fluids usingnanopores.Nature Communications 9,4085(2018).

[0651] 82.Patterson,D.M.,Nazarova,L.A.&Prescher,J.A.Finding the Right(Bioorthogonal)Chemistry.ACS Chemical Biology 9,592-605(2014).

[0652] 83.Wang,Y.et al.Osmosis-Driven Motion-Type Modulation of BiologicalNanopores for Parallel Optical Nucleic Acid Sensing.ACS Applied Materials&Interfaces 10,7788-7797(2018).

[0653] 84.Stoddart,D.,Heron,A.J.,Mikhailova,E.,Maglia,G.&Bayley,H.Single-nucleotide discrimination in immobilized DNA oligonucleotides with abiological nanopore.Proceedings of the National Academy of Sciences 106,7702-7707(2009).

[0654] 85.Frisch,M.J.et al.(Wallingford,CT;2016).

[0655] 86.Zhao,Y.&Truhlar,D.G.The M06 suite of density functionals for maingroup thermochemistry,thermochemical kinetics,noncovalent interactions,excited states,and transition elements:two new functionals and systematictesting of four M06-class functionals and 12 other functionals.TheoreticalChemistry Accounts 120,215-241(2008).

[0656] 87.Zhao,Y.&Truhlar,D.G.Density Functionals with Broad Applicabilityin Chemistry.Accounts of Chemical Research 41,157-167(2008).

[0657] 88.Wadt,W.R.&Hay,P.J.Ab initio effective core potentials formolecular calculations.Potentials for main group elements Na to Bi.TheJournal of Chemical Physics 82,284-298(1985).

[0658] 89.Hinkes,S.P.A.&Klein,C.D.P.Virtues of Volatility:A FacileTransesterification Approach to Boronic Acids.Organic Letters 21,3048-3052(2019).

[0659] 90.Zhou,J.,Stapleton,P.,Haider,S.&Healy,J.Boronic acid inhibitors ofthe class Aβ-lactamase KPC-2.Bioorganic&Medicinal Chemistry 26,2921-2927(2018).

[0660] 91.Masoud,M.S.,Soayed,A.A.&Ali,A.E.Complexing properties of nucleic-acid constituents adenine and guanine complexes.Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy 60,1907-1915(2004).

[0661] 92.Huq,F.&Peter,M.C.R.Interaction between NiCl2,and nucleobases,nucleosides and nucleotides.Journal of Inorganic Biochemistry 78,217-226(2000).

[0662] 93.Mai,K.,Lin,J.,Zhuang,B.,Li,X.&Zhang,L.-M.Cationic dendronizationof amylose via click chemistry for complexation and transfection of plasmidDNA.International Journal of Biological Macromolecules 79,209-216(2015).

[0663] 94.Struthers,H.,Spingler,B.,Mindt,T.L.&Schibli,R.“Click-to-Chelate”:Design and Incorporation of Triazole-Containing Metal-Chelating Systems intoBiomolecules of Diagnostic and Therapeutic Interest.Chemistry–A EuropeanJournal 14,6173-6183(2008).

[0664] 95.Steinmeyer,J.&Wagenknecht,H.-A.Synthesis of DNA Modified withBoronic Acid:Compatibility to Copper(i)-Catalyzed Azide–AlkyneCycloaddition.Bioconjugate Chemistry 29,431-436(2018).

[0665] 96.Dai,C.et al.The first chemical synthesis of boronic acid-modifiedDNA through a copper-free click reaction.Chemical Communications 47,3598-3600(2011).

[0666] 97.Pizer,R.&Babcock,L.Mechanism of the complexation of boron acidswith catechol and substituted catechols.Inorganic Chemistry 16,1677-1681(1977).

[0667] 98.Pizer,R.&Tihal,C.Equilibria and reaction mechanism of thecomplexation of methylboronic acid with polyols.Inorganic Chemistry 31,3243-3247(1992).

[0668] 99. D.A.&Zümreoglu-Karan,B.Complexation of boric acid with vitaminC.New Journal of Chemistry 33,1874-1881(2009).

[0669] 100. D.A.,Zumreoglu-Karan,B.,Sahin,O.& O.Boric acidcomplexes with thiamine (vitamin B1)and pyridoxine(vitamin B6).InorganicaChimica Acta 413,77-83(2014).

[0670] 101.Zhang,S.,Tang,Y.,Chen,Y.,Zhang,J.&Wei,Y.Boronic acid-modifiedpolyhedral oligomeric silsesquioxanes on polydopamine-coated magnetizedgraphene oxide for selective and high-capacity extraction of thecatecholamines epinephrine,dopamine and isoprenaline.Microchimica Acta 187,77(2020).

[0671] 102.Qi,F.et al.arXiv:1901.08013(2018).

[0672] 103.Martin,A.R.,Vasseur,J.-J.&Smietana,M.Boron and nucleic acidchemistries:merging the best of both worlds.Chemical Society Reviews 42,5684-5713(2013).

[0673] 104.Peleg,M.,Normand,M.D.&Corradini,M.G.The Arrhenius EquationRevisited.Critical Reviews in Food Science and Nutrition 52,830-851(2012).

Claims

1. A system for characterizing a target analyte, the system comprising: Nanopores; and Polymer chain, the polymer chain comprising chain-connecting sites and reactive segments, The polymer chain is tethered through the tethering site such that the polymer chain cannot pass through the nanopore, and the reaction section includes at least one sensing module that can interact with a single molecule of the target analyte entering the nanopore. The interaction between the sensing module and the target analyte can cause blockage, and the polymer chain can enter and be contained in the channels of the nanopores so that the reaction section is located in a region suitable for measuring the blockage caused by the interaction between the sensing module and the target analyte.

2. The system of claim 1, wherein the reaction section comprises two or more sensing modules, the sensing modules being capable of interacting with two or more different target analytes.

3. The system of claim 1, wherein each sensing module comprises one, two or more sensing parts, and each sensing part can interact with one or two or more binding sites of a single molecule of the target analyte.

4. The system of claim 3, wherein the sensing portion is selected from the group consisting of any nucleotide base, any amino acid, 1,2,3-triazole, phenylboronic acid (PBA), or any combination thereof.

5. The system of claim 3, wherein at least one of the sensing modules comprises two adjacent purines selected from the group consisting of guanine and adenine.

6. The system according to claim 1, wherein the reaction section is prepared by any one or any combination of the following methods: a. Incorporating one or more monomers containing a sensing component into the reaction section; b. Incorporating one or more monomers containing functional groups into the reaction section, and chemically modifying the functional groups into a sensing component; or c. Incorporating one or more monomers comprising a first reactive stem into the reaction section, and causing the first reactive stem to react with a second reactive stem, the second reactive stem being connected to the sensing portion.

7. The system of claim 6, wherein the first reaction handle and the second reaction handle are click reaction handles.

8. The system according to claim 7, wherein the first reaction handle and the second reaction handle are selected from the group consisting of azides and alkynes.

9. The system according to any one of claims 1-8, wherein the polymer chain is linked to a barrier molecule or a nanoporous protein.

10. The system of claim 9, wherein the blocking molecule is a protein capable of specifically binding to a small molecule compound, the binding site contains the small molecule compound, and the polymer chain is bound to the blocking molecule by the specific binding of the small molecule compound to the protein.

11. The system of claim 10, wherein the blocking molecule is an antibody to streptavidin or a hapten, and the small molecule compound is biotin or the hapten.

12. The system of claim 9, wherein the ligation site comprises a small molecule capable of reacting with a natural amino acid on the surface of the barrier molecule or the nanoporous protein, and the polymer chain is ligated to the barrier molecule by a reaction between the small molecule compound and the natural amino acid.

13. The system of claim 9, wherein a first reactive stem is introduced onto the surface of the barrier molecule or the nanoporous protein, the tethering site includes a second reactive stem, and the polymer chain is tethered to the barrier molecule by a reaction between the first reactive stem and the second reactive stem.

14. The system according to any one of claims 1-8, wherein the polymer chain further comprises an extension segment, and the extension segment is configured to allow the reactive segment to be located in a region suitable for measuring blockage.

15. The system according to any one of claims 1-8, wherein the polymer chain further comprises a traction segment, and the traction segment is configured to hold the reactive segment in a region suitable for measuring blockage.

16. The system of claim 15, wherein the traction section comprises any one of the following: a. Polymer chains that tend to pass through the nanopore channels in an electric field applied to the nanopore; b. Coupling sites that can react with native amino acids on the surface of the channels of the nanopores; c. A second reaction handle, which can react with a first reaction handle introduced into the surface of the channel of the nanopore; or d. Polymer chains that can pass through the channels of the nanopores and form a three-dimensional structure outside the nanopores with a size larger than the outlet of the nanopores.

17. The system of claim 15, wherein the traction segment is a nucleic acid with a length of 10 nt or longer.

18. The system according to any one of claims 1-8, wherein the polymer chain is based on nucleic acid, nucleic acid analog, polypeptide, polysaccharide, homopolymer, copolymer or any combination thereof.

19. The system according to any one of claims 1-8, wherein the target analyte is selected from the group consisting of: Ions containing metallic elements; Monosaccharides; Oligosaccharides; Polysaccharides; Glucoside; Polyphenols; Catecholamines; Catecholamine derivatives; Polyols; Protonated or deprotonated compounds; Compounds containing ribose; Hydrogen peroxide; Oligopeptides or cyclic peptides; Buffer reagent; Small molecule drugs; Neurotransmitters; Compounds with specific chirality; Analytes containing isotopes; Chemical intermediates; Or any combination thereof.

20. The system of claim 19, wherein The ions containing metallic elements are ions containing alkaline earth metals or transition metals. The monosaccharide is ribose, fructose, or mannose; The oligosaccharide is a disaccharide or a trisaccharide; The polyphenols are anthocyanins or proanthocyanidins; The catecholamine derivative is adrenaline, noradrenaline, or isoproterenol. The polyol is a compound containing two ortho-hydroxy groups, a 1,2-cis-diol or a 1,3-cis-diol moiety; The protonated or deprotonated form of the compound is a protonated or deprotonated form of tris; The compound containing the ribose moiety is a nucleotide, nucleoside, analogue thereof, or a monophosphate derivative thereof or a polyphosphate derivative thereof; The buffer reagent is tris; The small molecule drug is a nucleoside analog drug; The neurotransmitter is catecholamine or its derivative; The compound with a specific chirality is L-norepinephrine or D-norepinephrine; The isotopic analyte is catechol-D6 (deuterium-substituted catechol with all hydrogen atoms replaced).

21. The system of claim 19, wherein The ion containing the metal element is AuCl4. - Mg 2+ Ca 2+ Ba 2+ Ni 2+ Cu 2+ Co 2+ Zn 2+ Cd 2+ Ag 2+ or Pb 2 + ; The monosaccharide is D-(-)-ribose, D-fructose, or D-(+)-mannose; The oligosaccharide is 4-O-β-d-galactopyranosyl-d-fructofuranose (lactulose), 6-O-α-D-glucopyranosyl-D-fructofuranose (isomaltulose), or 4-ObD-galactosylsucrose (galactosylsucrose). The polyol is 3,4-dihydroxymandelic acid, 4-hydroxy-3-methoxymandelic acid (VMA), 3,4-dihydroxyphenylacetic acid, catechol, ethylene glycol, glycerol, L-lactic acid or vitamin; The compounds containing the ribose moiety are ribonucleotides, deoxyribonucleotides, galidesvir, ribavirin, favipiravir-RTP, remdesivir or its triphosphate metabolites, and cytidine 5'-monophosphate (5'-CMP). The small molecule drug is galidesvir, ribavirin, favipiravir-RTP, remdesivir, or its triphosphate metabolites.

22. The system of claim 19, wherein The polyol is vitamin C or vitamin B6.

23. The system according to any one of claims 1-8, wherein the nanopore is a biological nanopore, a protein nanopore, a solid nanopore, or a DNA nanopore.

24. The system of claim 23, wherein the protein nanopore is MspA, α-HL, aerosol, ClyA, FhuA, FraC, PlyA / B, CsgG Phi 29 linker or its homolog or variant.

25. The system according to any one of claims 1-8, wherein the system comprises two or more nanopores.

26. A method for characterizing a target analyte, the method comprising: (i) Providing a system according to any one of claims 1-25; (ii) Applying a voltage between the two sides of the nanopore allows a polymer chain to enter the nanopore; (iii) Allowing the target analyte to pass through the nanopores and interact with the sensing module of the reaction section; and (iv) Measure the blocking ion current through the nanopore caused by the interaction between the sensing module and the target analyte to provide a current pattern, and characterize the target analyte based on the current pattern.

27. The method of claim 26, wherein the polymer chain of the system comprises two or more sensing modules that can interact with two or more different target analytes, and wherein the method is used to characterize two or more target analytes.

28. The method of claim 26, wherein the method comprises: (i) Providing a system according to any one of claims 1-25; (ii) A first voltage is applied between the two sides of the nanopore to allow a polymer chain to enter the nanopore; (iii) Allowing the first target analyte to pass through the nanopore and interact with the sensing module of the reaction section; and (iv) Measure the blocking ion current through the nanopore caused by the interaction between the sensing module and the target analyte to provide a current pattern, and characterize the first target analyte based on the current pattern; (v) The voltage between the two compartments is converted into a second voltage opposite to the direction of the first voltage, thereby causing the polymer chains in the nanopore to exit from the nanopore; (vi) Convert the voltage between the two compartments into the first voltage and allow another polymer chain to enter the nanopore; and (vi) Apply steps (iii)-(iv) to a second target analyte that is different from the first target analyte.

29. The method of claim 28, wherein the sensing module is capable of irreversibly interacting with the first target analyte and / or the second target analyte.

30. The method according to any one of claims 26-29, wherein the target analyte is selected from the group consisting of: Ions containing metallic elements; Monosaccharides; Oligosaccharides; Polysaccharides; Glucoside; Polyphenols Catecholamines; Catecholamine derivatives; Polyols; Compounds in protonated or deprotonated form; Compounds containing ribose; Hydrogen peroxide; Oligopeptides or cyclic peptides; Buffer reagent; Small molecule drugs; Neurotransmitters; Compounds with specific chirality; Analytes containing isotopes; Chemical intermediates; Or any combination thereof.

31. The method of claim 30, wherein Ions containing metallic elements are ions containing alkaline earth metals or transition metals. The monosaccharide is ribose, fructose, or mannose; The oligosaccharide is a disaccharide or a trisaccharide; The polyphenols are anthocyanins or proanthocyanidins; The catecholamine derivative is adrenaline, noradrenaline, or isoproterenol. The polyol is a compound containing two ortho-hydroxy groups, a 1,2-cis-diol or a 1,3-cis-diol moiety; The protonated or deprotonated form of the compound is a protonated or deprotonated form of tris; The compound containing the ribose moiety is a nucleotide, nucleoside, analogue thereof, or a monophosphate derivative thereof or a polyphosphate derivative thereof; The buffer reagent is tris; The small molecule drug is a nucleoside analog drug; The neurotransmitter is catecholamine or its derivative; The compound with a specific chirality is L-norepinephrine or D-norepinephrine; The isotopic analyte is catechol-D6 (deuterium-substituted catechol with all hydrogen atoms replaced).

32. The method of claim 30, wherein The ion containing the metallic element is AuCl4. - Mg 2+ Ca 2+ Ba 2+ Ni 2+ Cu 2+ Co 2+ Zn 2+ Cd 2+ Ag 2+ or Pb 2+ ; The monosaccharide is D-(-)-ribose, D-fructose, or D-(+)-mannose; The oligosaccharide is 4-O-β-d-galactopyranosyl-d-fructofuranose (lactulose), 6-O-α-D-glucopyranosyl-D-fructofuranose (isomaltulose), or 4-ObD-galactosylsucrose (galactosylsucrose). The polyol is 3,4-dihydroxymandelic acid, 4-hydroxy-3-methoxymandelic acid (VMA), 3,4-dihydroxyphenylacetic acid, catechol, ethylene glycol, glycerol, L-lactic acid or vitamin; The compounds containing the ribose moiety are ribonucleotides, deoxyribonucleotides, galidesvir, ribavirin, favipiravir-RTP, remdesivir or its triphosphate metabolites, and cytidine 5'-monophosphate (5'-CMP). The small molecule drug is galidesvir, ribavirin, favipiravir-RTP, remdesivir, or its triphosphate metabolites.

33. The method of claim 30, wherein The polyol is vitamin C or vitamin B6.

Images