WO2025063893A2

WO2025063893A2 - A nanopore system and method for determining properties of nanoparticles

Info

Publication number: WO2025063893A2
Application number: PCT/SG2024/050600
Authority: WO
Inventors: Slaven Garaj; Patrick Seamus Doyle; Kun Li; Arjav Shah; Swati SHIKHA
Original assignee: Singapore Mit Alliance For Research And Technology Centre; National University of Singapore; Massachusetts Institute of Technology
Current assignee: Singapore Mit Alliance For Research And Technology Centre; National University of Singapore; Massachusetts Institute of Technology
Priority date: 2023-09-20
Filing date: 2024-09-20
Publication date: 2025-03-27
Anticipated expiration: 2026-03-20
Also published as: WO2025063893A9; WO2025063893A3

Abstract

A method and system for determining mechanical properties of nanoparticles. The method comprises the steps of providing an electrically conducting fluid comprising the nanoparticles dispersed therein in a fluidic cell configured with a membrane having a nanopore extending through a thickness of the membrane; applying a voltage across the fluidic cell for effecting a translocation event of at least one of the nanoparticles through the nanopore; monitoring a current across the fluid cell for a period starting before the translocation event and ending after the translocation event to measure a current blocking signal; and determining the mechanical properties of the nanoparticles from the measured current blocking signal; wherein a size of the nanoparticles is larger than a pore size of the nanopore; and wherein an aspect ratio of thickness of the membrane to the pore size of the nanopore is smaller than 1.

Description

A NANOPORE SYSTEM AND METHOD FOR DETERMINING PROPERTIES OF NANOPARTICLES

Field of Invention

The present invention relates to the field of biosensor. In particular, the invention relates to a nanopore system and method suitable for determining mechanical properties of nanoparticles.

Background

Any mention and/or discussion of prior art throughout tire specification should not be considered, in any way, as an admission that this prior art is well known or forms part of common general knowledge in the field.

Mechanical properties of nanoparticles such as viruses, vesicles, and DNA origami particles can be used to provide information on their structure, such as vesicle lamellar structure, viral capsid thickness, internal cargo content and osmotic pressures. Additionally, key functions and features of the nanoparticles can also be elucidated by determining mechanical properties of the nanoparticles. For instance, viral DNA packaging and ejection strategies depend on the mechanical properties of the viral capsid, which exhibit drastic changes in stiffness before and after viral maturation, and in different environments. The malignancy of cancer cells can also be determined based on the stiffness of nanometer-sized vesicles known as exosomes that are secreted from the cancer cells. Particularly, an increase in stiffness of the exosomes from the combined contribution of the bending modulus and osmotic pressure of the exosomes is correlated with an increase in malignancy of the parent cancer cells. The mechanical properties of nanoparticles used in cancer drug delivery are also crucial for therapeutic efficacy, whereby soft nanoparticles with excellent deformability are associated with less macrophage uptake and removal by biological filtration systems, resulting in longer blood circulation time and higher tumor accumulation. As such, mechanical characterization of nanoparticles such as viruses or exosomes can be a useful tool for diagnostics and fingerprinting.

Atomic force microscopy (AFM) and nanoindentation are conventional techniques for characterizing the mechanical properties of nanoparticles. However, the shortcomings for both techniques include the influence of the substrate on the sample cannot be eliminated despite being in a liquid environment. Furthermore, low throughput and complicated procedures associated with AFM and nanoindentation limits their use in clinical applications. To achieve high-throughput and ultra-fast measurement on nanoparticlcs, nano-channels are preferably used. Nano-channels rely on object deformation to determine mechanical properties of nanoparticles, which can be achieved using two deformationgenerating methods, namely spatial restriction and electrodeformation. Both methods involve the use of nano-channels having aspect ratios (Length/diameter) of more than 5, and channel lengths that generally exceed 50 nm. Nonetheless, the large contact area between nanoparticles and inner surface of the nano-channel involving spatial restriction results in significant friction and adhesion, while size variation within the nano-channel reduces accuracy of deformation measurement, calculation and subsequent modelling. Although the use of electrodeformation can avoid contact between nanoparticles and the nano-channel’s inner surface, fluctuations in deformation measurements are large while the magnitude and stability of object deformation are low.

To overcome tire disadvantages of nano-channels as mentioned above, various methods and systems involving the use of nanopores are developed. For example, Patent Cooperation Treaty Publication Number WO 2014/165372 Al discloses the use of solid- state nanopore for differentiation of biomolecules and analysis of their internal content. Particularly, the translocation of biomolecules across the nanopores which are larger than the biomolcculc under investigation provides a signal that can be resolved to determine structural characteristics such as presence or absence of nucleic acid and the order or sequence of nucleic acids within the biomolecule. Chinese Patent Publication Number CN 113686235 A discloses a method of estimating morphological characteristics of protein using nanopore biomolecular sensing. It is disclosed that morphology estimation is achieved by analyzing the relationship between relative blocking current and orientation angle of the protein translocating across a nanopore which is larger than the protein under investigation. Additionally, Patent Cooperation Treaty Publication Number WO 2005/017025 A2 discloses the use of a nanopore to determine conformation of a polymer based on time-dependent changes in transport properties of the nanopore such as capacitance, optical property or chemical structure when the polymer traverses through the nanopore, which is larger than the polymer under investigation. Nonetheless, the abovementioned use of nanopores that are larger than the nanoparticles under investigation is limited in terms of the type of nanoparticles that can be analysed, and the properties that can be determined. Embodiments of the present invention seek to address one or more of the abovementioned problems.

Summary

According to a first aspect of the present invention, a method for determining mechanical properties of nanoparticles is provided, the method comprising the steps of: providing an electrically conducting fluid comprising the nanoparticles dispersed therein in a fluidic cell configured with a membrane having a nanopore extending through a thickness of the membrane; applying a voltage across the fluidic cell for effecting a translocation event of at least one of the nanoparticles through the nanopore; monitoring a current across the fluid cell for a period starting before the translocation event and ending after the translocation event to measure a current blocking signal; and determining the mechanical properties of the nanoparticles from the measured current blocking signal; wherein a size of the nanoparticles is larger than a pore size of the nanopore; and wherein an aspect ratio of thickness of the membrane to the pore size of the nanoporc is smaller than 1.

According to a second aspect of the present invention, a nanopore system for determining mechanical properties of nanoparticlcs is provided, comprising: a fluid cell configured for receiving an electrically conducting fluid comprising the nanoparticles dispersed therein, wherein the fluidic cell is configured with a membrane having a nanopore extending through a thickness of the membrane; a source for applying a voltage across the fluidic cell for effecting a translocation event of at least one of the nanoparticles through the nanopore; a monitor configured for monitoring a current across the fluid cell for a period starting before the translocation event and ending after the translocation event to measure a current blocking signal for determining the mechanical properties of the nanoparticles from the measured current blocking signal; wherein a pore size of the nanoporc is chosen to be smaller than a size of the nanoparticlcs; and wherein an aspect ratio of thickness of tire membrane to the pore size of the nanopore is smaller than 1. Brief Description of the Drawings

This invention will be described by way of non-limiting embodiments of the present invention, with reference to the accompanying drawings, in which:

FIG. 1 illustrates the mechanism by which properties of nanoparticles are determined according to an embodiment of the present invention;

FIG. 2 illustrates a typical translocation event characteristic of an icosahcdral DNA origami particle translocating through a 15-nm-diameter SiNx nanopore in IM KC1 environment under an applied bias voltage of substantially 150 mV;

FIGs. 3A to 3F illustrate current blocking signals of translocation events for DNA origami particles using the creep mode through an 8-nm-diamctcr SiNx nanoporc at substantially 150-mV bias;

FIG. 4 illustrates the scatter plot of squeezing-translocation-induced conductance change (AG) versus event duration of DNA origami particles translocating using the creep mode through an 8-nm-diamctcr SiNx nanoporc at substantially 150-mV bias;

FIGs. 5A to 5F illustrate current blocking signals of translocation events of DNA origami particles using the plop mode through an 8-nm-diameter SiNx nanopore at substantially 600-mV bias;

FIG. 6 illustrates the scatter plot of AG versus event duration of DNA origami particles translocating using the plop mode through an 8-nm-diameter SiNx nanopore at substantially 600-mV bias;

FIGs. 7A to 7F illustrate current blocking signals of translocation events of DNA origami particles using a transition between creep and plop modes through an 8-nm-diamctcr SiNx nanopore at substantially 300-mV bias;

FIG. 8 illustrates the scatter plot of AG versus event duration of DNA origami particles translocating using a transition between creep and plop modes through an 8-nm-diameter SiNx nanoporc at substantially 300-mV bias;

FIGs. 9A to F illustrate current blocking signals of translocation events of DNA origami particles using the creep mode through a 15-nm-diameter SiNx nanopore at substantially 100-mV bias;

FIGs. 10A to H illustrate current blocking signals of translocation events of liposomes having (10A) unilamellar, (10C) multilamellar, (10E) single-core multivesicular and (10G) double-core multivesicular structures using the plop mode through a 100-nm-diameter SiNx nanopore at substantially 150-mV bias, and Cryo-EM images of the liposomes having (10B) unilamellar, (10D) multilamellar, (10E) single-core multivesicular, and ( 1 OH) double-core multivesicular structures;

FIGs. HA to 11C illustrate soft nanoparticles being extruded using nanopores having different thickness according to an embodiment of the present invention;

FIG. 12 illustrates the mechanism by which applied voltage and pressure are used in combination to squeeze and translocate a virus through the nanoporc of the present invention;

FIGs. 13A and 13B illustrate the (10A) network layout of machine learning and (10B) confusion matrix for deep neural network classification that are used to distinguish the structure and rigidity of nanoparticlcs of different species having a similar size;

FIGs. 14A to 14F illustrate current blocking signals of squeezing translocation events of Adeno-associated virus (AAV) through a 30-nm-diameter SiNx nanopore at substantially 350-mV bias;

FIG. 15 illustrates the scatter plot of AG versus event duration of Adeno-associated virus translocating through a 30-nm-diameter SiNx nanopore at substantially 350-mV bias;

FIGs. 16A to 16F illustrate current blocking signals of translocation events of liposomes using the creep mode through a 100-nm-diameter SiNx nanopore at substantially 300-mV bias;

FIG. 17 illustrates the scatter plot of AG versus event duration of liposomes translocating using the creep mode through a 100-nm-diameter SiNx nanopore at substantially 300-mV bias;

FIGs. 18A to 18F illustrate current blocking signal of translocation events of polymersomes using the creep mode through a 100-nm-diameter SiNx nanopore at substantially 150-mV bias;

FIG. 19 illustrates the scatter plot of AG versus event duration of polymersomes translocating using the creep mode through a 100-nm-diameter SiNx nanopore at substantially 150-mV bias;

FIGs. 20A and 20B illustrate DNA origami particles having (10A) icosahedron and (10B) pentagonal bipyramidal structures;

FIG. 21 shows the transmission electron microscopy (TEM) image of nanopore A of the present invention; FIGs. 22A to 22E show the scatter plot of AG versus event duration of icosahedron type

DNA origami particles translocating through the nanopore as illustrated in Figure 21 at about 60 - 200 mV bias;

FIGs. 23A to 23E illustrate the ECD distribution of icosahedron type DNA origami particles translocating through the nanopore as illustrated in Figure 21 at about 60 - 200 mV bias;

FIGs. 24A to 24E illustrate the scatter plot of AG versus event duration of pentagonal bipyramidal type DNA origami particles translocating through the nanopore as illustrated in Figure 21 at about 60 - 200 mV bias;

FIGs. 25A to 25E illustrate the ECD distribution of pentagonal bipyramidal type DNA origami particles translocating through the nanopore as illustrated in Figure 21 at about 60

- 200 mV bias;

FIG. 26 shows the TEM image of nanopore B of the present invention;

FIGs. 27A to 27F illustrate the scatter plot of AG versus event duration of icosahedron type DNA origami particles translocating through the nanopore as illustrated in Figure 26 at about 100 - 350 mV bias;

FIGs. 28A to 28F illustrate the ECD distribution of icosahedron type DNA origami particles translocating ihrough the nanopore as illustrated in Figure 26 at about 100 - 350 mV bias;

FIGs. 29A to 29D illustrate the scatter plot of AG versus event duration of pentagonal bipyramidal type DNA origami particles translocating through the nanopore as illustrated in Figure 26 at about 70 - 200 mV bias;

FIGs. 30A to 30D illustrate the ECD distribution of pentagonal bipyramidal type DNA origami particles translocating through the nanopore as illustrated in Figure 26 at about 70

- 200 mV bias;

FIG. 31 shows the TEM image of nanopore C of the present invention;

FIGs. 32A to 32D illustrate the scatter plot of AG versus event duration of icosahedron type DNA origami particles translocating through the nanoporc as illustrated in Figure 31 at about 200 - 600 mV bias;

FIGs. 33A to 33D illustrate the ECD distribution of icosahedron type DNA origami particles translocating through the nanopore as illustrated in Figure 1 at about 200 - 600 mV bias; FIGs. 34A to 34F illustrate the scatter plot of AG versus event duration of pentagonal bipyramidal type DNA origami particles translocating through the nanopore as illustrated in Figure 31 at about 150 - 600 mV bias;

FIGs. 35A to 35F illustrate the ECD distribution of pentagonal bipyramidal type DNA origami particles translocating through the nanopore as illustrated in Figure 31 at about 150 - 600 mV bias;

FIGs. 36A to 36F show the CryoEM images of vesicles having compositions of (A) 40%DSPC-40%Cholesterol-20%DGPS, (B) 50%DSPC-40%Cholesterol-10%DGPS, (C) 80%DSPC-10%Cholesterol-10%DOPS, (D) 100%DSPC, (E) and (F) PEO(700)-b- PBD(1200);

FIG. 37 illustrates the characterization results of dynamic light scattering (DLS) for vesicles having compositions of 40%DSPC-40%Cholesterol-20%DOPS, 50%DSPC- 40%Cholesterol-10%DOPS, 80%DSPC-10%Cholesterol-10%DOPS, 100%DSPC, and PEG(700)-b-PBD(1200);

FIGs. 38A and 38B illustrate the scatter plot of AG versus event duration of polymersomes (PEG(700)-b-PBD(1200)) translocating through a nanopore having a diameter of substantially 100 nm at (38A) 150 mV and (38B) 200 mV bias;

FIGs. 39A and 39B illustrate the ECD distribution of polymersomes (PEO(700)-b- PBD(1200)) translocating through a nanopore having a diameter of substantially 100 nm at (39A) 150 mV and (39B) 200 mV bias;

FIGs. 40A to 40D illustrate the scatter plot of AG versus event duration of liposomes having a composition ratio of DSPC:Cholesterol:DOPS = 50:40:10 that are translocating through a nanoporc having a diameter of substantially 100 nm at about 250 - 400 mV bias;

FIGs. 41A to 41D illustrate the ECD distribution of liposomes having a composition ratio of DSPC:Cholesterol:DOPS = 50:40:10 that are translocating through a nanopore having a diameter of substantially 100 nm at about 250 - 400 mV bias;

FIGs. 42A to 42D illustrate the scatter plot of AG versus event duration of liposomes having a composition ratio of DSPC:Cholesterol:DOPS = 80:10:10 that are translocating through a nanopore having a diameter of substantially 100 nm at about 200 - 350 mV bias;

FIGs. 43A to 43D illustrate the ECD distribution of liposomes having a composition ratio of DSPC:Cholcstcrol:DOPS = 80:10:10 that arc translocating through a nanoporc having a diameter of substantially 100 nm at about 200 - 350 mV bias; FIGs. 44A to 44E illustrate the scatter plot of AG versus event duration of Adeno- associated virus translocating through a nanopore having a diameter of substantially 30 nm at about 200 - 400 mV bias;

FIGs. 45A to 45E illustrate the ECD distribution of Adcno-associatcd virus translocating through a nanopore having a diameter of substantially 30 nm at about 200 - 400 mV bias; and

FIG. 46 illustrates another embodiment of the present invention having a three-layer nanopore structure for measuring the plastic deformation of nanoparticles.

FIGs. 47A to 47K show (47A-47C) schematic diagrams of nanopore elastometry working principle, schematic representations and CryoEM images of biological nanostructures (47D and 47H), DNA origami icosahedral sphere (hereafter referred to as DNA ball, 47E and 471), DNA origami star-shaped particle (hereafter referred to as DNA coin) liposome (47F and 47J), and polymersome (47G and 47K), ionic current trace of (47L) DNA ball and (47M) DNA coin squeezing through a 15-nm-diameter SiNx nanopore at different voltages, and (47N) liposome and (47) polymersome squeezing through a 100-nm-diamctcr SiNx nanopore.

FIGs. 48A to 48H show the scatter of squeezing translocation events of (48A-48D) DNA ball and (48E to 48H) liposome at a series of different voltages. The horizontal axis represents the duration of the events, while the vertical axis represents the variation in nanopore conductivity, defined as the current change divided by voltage.

FIGs. 49A to 49G show (49A) the translocation speed versus bias voltage for DNA ball and DNA coin squeezing through a 15-nm-diameter SiNx nanopore at a series of different voltages, the transition of these two regimes occurs at the onset voltage Von. (49B) probability distribution histogram of the duration of translocation events for DNA ball at a bias voltage of 150mV, which is fitted using the First Passage Time theory, extracting the average speed v and distribution width o, (49C to 49F) schematics of the funnelling model (dashed lines) and funnelling length (arrows) of the particles with different geometries or orientations docking at the nanopore, (49G) the predicted translocation velocity verses the applied bias in the 1-D funnelling model.

FIGs. 50A to 50H show (50A-50-D) TEM images of SiNx nanopores with diameters ranging from 23 nm down to 8 nm, the correlation between translocation speed v and bias voltage for (50E) DNA ball and (50F) DNA coin at different voltages and pore sizes, (50G) and (50H) represent the correlation between duration and distribution width with bias voltage for the two types of DNA particles, respectively.

FIGs. 51A to 51E show the proportion of poration events to the total number of events is as a function of the applied voltage, the diameter of nanopores is 100 nm, and the thickness of SiNx membrane is 12 nm, the buffer is 0.5 M KC1 solution, at an applied voltage of 350 mV, liposomes of 10 mol% cholesterol concentration generate (5 IB) a typical single-peak event and (5 ID) a typical poration event, (51A_the proportion of Poration events to the total number of events increases with increasing applied voltage, with a more significant growth trend observed for liposomes with lower cholesterol concentration, the total number of events is the sum of single-peak events and poration events, (51C and 5 IE) are schematic diagrams of the liposome poration process.

Detailed Description

One or more specific and alternative embodiments of the present invention will now be described with reference to the attached drawings. It shall be apparent to one skilled in the art, however, that this invention may be practised without such specific details. Some of the details may not be described at length so as not to obscure the invention. For ease of reference, common reference numerals or series of numerals will be used throughout the figures when referring to the same or similar features common to the figures.

Embodiments of the present invention can provide a nanopore system that can control translocation speed of nanoparticles traversing the nanopore so as to determine different mechanical or structural properties of the nanoparticles. Particularly, varying ranges of driving force are applied to produce different squeezing modes, including a mode referred to as Randomly- Activated Regime, also sometimes referred to as creep mode herein and a mode referred to as Frictional Regime, also sometimes referred to as plop mode herein. The Randomly-Activated Regime and the Frictional Regime are used to determine the frame structure and inner structure of nanoparticles respectively, hi some experiments, the transition between those modes can be used to determine stiffness or strength of nanoparticles, also sometimes referred to herein as transitional mode.

Embodiments of the present invention can further provide a nanopore system that can be configured with a nanopore that is smaller than the nanoparticle under investigation. For example, For DNA particles (size 33-38 nm), pore diameters of 8-23 nm are used, for liposome particles (diameter 1 10-160 nm), pore diameters of 100 nm are used, for viruses (diameter 35 nm), pore diameters of 26-30 nm are used. In addition, embodiments of the present invention can provide a nanopore system that can be configured with a nanopore having different range of thickness to determine different mechanical properties of nanoparticles. Particularly, nanopores having a thickness of substantially 0.5 to 20 nm are used to reduce friction and adhesion between nanopore and nanoparticles, thereby increasing precision of size and shape of nanoparticle deformation through the nanopore. Additionally, nanopores having a thickness of substantially 20 to 50 nm can be used to increase resistance of the nanopore against nanoparticles to prolong translocation time and determine bending strength of the nanoparticles. In addition, embodiments of the present invention can provide a nanoporc system with a nanoporc that has an aspect ratio, here the ratio of pore length (i.e. thickness of membrane) to pore size (i.e. pore diameter) of less than 1.

In an example embodiment of the present invention, the nanopore system further comprises an amplifier connected to one of the electrodes to receive and amplify the current blocking signal.

In an example embodiment of the present invention, the nanopore system is configured so as to control translocation speed of the nanoparticlcs across the nanoporc by the applied voltage and, optionally, by a fluid pressure gradient.

Further, according to an example embodiment of the present invention, the nanopore system is enclosed within a Faraday cage.

In an example embodiment of the present invention discloses that the membrane comprises silicon nitride (SiNx), silicon oxide, aluminium oxide, hafnium oxide, 2D materials including graphene, fluorinated graphene, molybdenum disulfide, tungsten disulfide, hexagonal boron nitride and MXenes (e.g., Ti2CTx and Ti3C2Tx) or a combination of two or more thereof.

An example embodiment of the present invention discloses that the membrane has a thickness of about 0.5 to 50 nm.

An example embodiment of the present invention discloses that the nanopore has a diameter of about 8 to 100 nm.

An example embodiment of the present invention discloses that the nanoporc system further comprises sealing members between the membrane and the fluid storages to prevent leakage of the fluid. According to an example embodiment of the present invention, the sealing members are rubber gaskets are polydimethyl siloxane (PDMS) gaskets.

According to an example embodiment of the present invention, the electrodes arc configured to provide an applied voltage of about 0.1 to 5 V across the fluidic cell.

According to an example embodiment of the present invention, the electrodes are Ag/AgCl electrodes.

An example embodiment of the present invention discloses that the nanoparticles are viruses, DNA origami particles, liposomes or polymersomes.

An example embodiment of the present invention discloses that the salt is potassium chloride (KC1) having a concentration of about 1 M.

An example embodiment of the present invention discloses that a pressure gradient between the fluid storages ranges from about 0.1 kPa to about 800 kPa may be used to assist in controlling the translocation speed.

An example embodiment of the present invention discloses that the current blocking signal provides information on mechanical properties of tire nanoparticles.

FIG. 1 shows an illustration of the mechanism by which the nanopore system (1 ) according to an example embodiment of the present invention is used to determine properties of nanoparticles (2). The nanopore system (1) comprises a fluidic cell (3) configured with a membrane (4) having a nanopore (5), and a pair of electrodes (6) is configured to apply voltage across the fluidic cell (3). The diameter of the nanopore used may vary between substantially 8 to 100 nm according to application. It is noted that the data presented herein is based on single nanopores, but arrays of nanopores may be used in different embodiments. The electrodes (6) in this example embodiment are configured to provide an applied voltage of about 0.1 to 5 V across the fluidic cell (3), and the electrodes (6) are Ag/AgCl electrodes.

The nanopore system (1 ) further comprises at least two fluid storages (7) in fluid communication to the nanopore (5), each of the storages (7a, b) is configured with an inlet (8) to receive a fluid and an outlet (9a, b) to deposit the fluid on the fluidic cell (3), wherein tire fluid comprises am electrically conducting fluid such as physiological saline or other saline solutions. For example, a syringe may be used to introduce the fluid into the fluid storages through the inlets. In one embodiment, the salt is potassium chloride (KC1) having a concentration of about 0.1 M to about 4 M. Preferably, the salt is potassium chloride (KC1) having a concentration of about 1 M. It is noted that other electrically conducting fluids such as NaCl, LiCl may be used in different example embodiments. The role of the electrically conducting fluid is to conduct electricity for generating the measurement signal

(c.g. ionic current blockade) according to example embodiments.

In a preferred embodiment of the present invention, one of the fluid storages (e.g. 7a) receives a fluid comprising the electrically conducting fluid and nanoparticles (2), while the other fluid storage (e.g. 7b) receives a fluid comprising the electrically conducting fluid only. Preferably, the nanopore system (1) further comprises sealing members (14) between the membrane (4) of the fluidic cell (3) and the fluid storages (7) to prevent leakage of the fluid. Preferably, the sealing members (14) are rubber gaskets are polydimethylsiloxane (PDMS) gaskets.

Upon operation of the nanopore system (1), in one embodiment the applied voltage from the electrodes (6) exerts a force that pushes the charged nanoparticles (2) to deform and pass through the nanopore (5) having a relatively smaller dimension. Preferably, the nanopore (5) has an aspect ratio (i.e. here the ratio of the thickness of the membrane to the pore diameter) of less than 1. Preferably, the nanopore (5) has a diameter of about 8 to 100 nm. FIG. 2 illustrates a typical translocation event characteristic of a nanoparticle (2), particularly an icosahedral DNA origami particle translocating through a 15-nm-diameter SiNx nanopore (5) in IM KC1 environment under an applied bias voltage of substantially 150 mV. The charge event deficit (ECD) is determined as the time integral of a current blocking signal when the nanoparticle (2) translocates through the nanopore (5). The current blockade mat be characterized by a mean of the difference in current AI_mcan or the maximum difference in current Almax.

Referring again to FIG. 1, preferably, the nanopore system (1) further comprises an amplifier (12) connected to one of the electrodes (6) to receive and amplify the current blocking signal. The nanopore system (1) comprises a detector (10) that receives the amplified current blocking signal from the amplifier (12) to monitor translocation behavior of nanoparticles (2), and determine properties of the nanoparticles (2). Preferably, the current blocking signal provides information on mechanical properties of the nanoparticles (2). Preferably, the nanoparticles (2) are viruses, DNA origami particles, liposomes or polymersomes. Preferably, the nanopore system (1) is enclosed within a Faraday cage (13), thereby reducing noise from external sources such as electromagnetic fields.

In a preferred embodiment of the present invention, the fluid storages (7) are fluidically linked via tire nanopore (5) and airtight upon operation of the nanopore system (1), while one of the fluid storages (7a) comprises a pressure pump (11) to provide fluidic pressure to the fluid thereby creating a pressure gradient between the fluid storages (7a, b). The pressure gradient works in association with the voltage across the fluidic cell (3) to squeeze the nanoparticles (2) through the nanopore (5) and a translocation of the nanoparticles (2) is detected as a current blocking signal. Preferably, the pressure gradient between the fluid storages (7a, b) ranges from about 0.1 kPa to about 800 kPa. Preferably, the nanopore system (1) is configured with multiple squeezing modes determined by the fluidic pressure and the applied voltage so as to control translocation speed of the nanoparticles (2) across the nanopore (5). Advantageously, the different squeezing modes can be used to extract different mechanical or structural information about the nanoparticlcs (2), wherein the creep mode and plop mode can be used to determine the frame structure and inner structure of nanoparticles (2) respectively, according to an example embodiment, while the transition between creep and plop modes can be used to determine stiffness or strength of nanoparticles (2), according to an example embodiment, as will be described in more detail below. In some embodiments, the nanopore system (1) may employ the combined use of applied voltage and pressure gradient to apply varying ranges of driving force without compromising signal-to-noisc ratio to produce the different squeezing modes, for example for virus-related data (e.g. AAV).

FIGS. 3, 5 and 7 illustrate current blocking signals of translocation events for DNA origami particles through an 8-nm-diameter SiNx nanopore (5) using creep mode, plop mode and transition between creep and plop modes, respectively. Voltage bias of substantially 150-mV, 600-mV and 300-mV were used for creep mode, plop mode and transition between creep and plop modes, respectively. The mean current blockade from each squeezing mode is divided by their respective voltage bias to obtain squeezingtranslocation-induced conductance change (AG), which is then used to plot against event duration of nanoparticles (2) translocating across the nanopore (5) to generate scatter plots as shown in FIGS. 4, 6 and 8 for creep mode, plop mode and transition between creep and plop modes, respectively.

FIG. 9 further illustrates current blocking signals of translocation events of DNA origami particles using the creep mode through a 15-nm-diameter SiNx nanopore (5) at substantially 100-mV bias. The current blockade in this example embodiment has three steps as indicated by dashed lines in FIG. 9, which are used to tomographically scan the frame structure of the DNA origami particles. Additionally, FIG. 10 illustrates current blocking signals of translocation events of liposomes having (A) unilamellar, (C) multilamellar, (E) single-core multivesicular and (G) double-core multivesicular structures using the plop mode through a 100-nm-diamctcr SiNx nanoporc at substantially 150-mV bias, according to various example embodiment. The results obtained from each translocation event corresponded with the Cryo-EM images of the liposomes having (B) unilamellar, (D) multilamellar, (F) single-core multivesicular, and (H) double-core multivesicular structures.

In a preferred embodiment of the present invention, the membrane (4) comprises silicon nitride (SiNx), silicon oxide, aluminium oxide, hafnium oxide, 2D materials including graphene, fluorinated graphene, molybdenum disulfide, tungsten disulfide, hexagonal boron nitride and MXenes (e.g., Ti2CTx and Ti3C2Tx) or a combination of two or more thereof. Preferably, the membrane (4) has a thickness of about 0.5 to 50 nm. Advantageously, the thickness of the membrane can be configured to provide a nanopores with a range of thicknesses for different membranes to determine different properties of nanoparticles. For example, FIG. 11 illustrates how nanoparticles (2) are extruded using nanopores having different thickness to obtain different information regarding the properties of the nanoparticles (2). Particularly, in FIG. 11A and 11B nanopores (5a, b) having a thickness of substantially 0.5 to 20 nm, respectively (not to scale), are used to reduce the contact area between nanopore (5) and nanoparticles (2), thereby reducing friction and adhesion between the same. This provides an increase in precision of size and shape of the nanoparticlc (2) deformation through the nanoporc (5), and further enables tomography of mechanical properties to determine structural details of the nanoparticles (2). Additionally, in FIG. 11C nanopores (5c) can be configured to have a thickness of > 20 to 50 nm, which increases resistance of the nanopore (5c) against nanoparticles (2). This prolongs the translocation time of nanoparticlcs (2) across the nanoporc (5c), which is useful for determining bending strength of the nanoparticles. All nanoparticle sizes are larger than the pore diameter, and the pore diameter is larger than the pore length, according to example embodiments.

FIG. 12 illustrates the mechanism by which applied voltage and, optionally, pressure are used in combination to squeeze and translocate a nanoparticle (2), for example, a virus through the nanopore (5) of the present invention. The nanoparticle (2) translocates through the nanopore (5) via electrophoretic force induced by the applied voltage from the electrodes (6), while fluidic pressure provided by the pressure pump (11) creates a pressure induced flow against the nanoparticle’s (2) movement so as to control its translocation speed through the nanopore (5).

The current blocking signal obtained is detected and analyzed by the detector (10) when a nanoparticle (2) translocates through the nanopore (5) according to an example embodiment can, for example, be used to distinguish the structure and rigidity of nanoparticles (2) of different species having a similar size. In an example embodiment, the detector (10) further comprises a processor having a machine learning algorithm to determine the properties of the nanoparticles (2) based on the current blocking signals received, whereby all determinations or predictions are further validated using a confusion matrix. FIG. 13 illustrates a network layout 1300 of machine learning and confusion matrix for deep neural network classification with two hidden layers containing 100 neurons each (>90% accuracy) that are used to distinguish two different nanoparticles (2) which are designated as species 1 and species 2, based on their raw resistive pulses obtained during translocation across the nanopore (5), according to an example embodiment. The nanopore system (1) according to an example embodiment is able to determine that species 1 has an icosahedral structure, while species 2 has a pentagonal structure.

The nanopore system (1) according to an example embodiment can also be used to determine the mechanical properties of nanoparticles (2) such as Adeno- associated virus, liposomes and polymersomes. FIGS. 14, 16 and 18 illustrates current blocking signals of translocation events for Adcno-associatcd virus, liposomes and polymersomes respectively, wherein Adeno-associated virus involves the use of a 30-nm-diameter SiNx nanopore (5) with substantially 350 mV bias; liposomes involve the use of a 100-nm-diameter SiNx nanoporc (5) with substantially 300-mV bias; and polymersomes involve the use of a 100- nm-diameter SiNx nanopore (5) with substantially 150-mV bias. The membranes uses have a thickness of 10-12 nm. The pore diameters are: for DNA particles (particle size 33-38 nm), pore diameters of 8-23 nm; for liposome particles (particle size 110-160 nm), pore diameters of 100 nm; and for viruses (particle size 350 nm), pore diameters of 26-30 nm. The mean current blockade for each type of nanoparticle (2) is divided by their respective voltage bias to obtain AG, which is used to plot against event duration of nanoparticles (2) translocating across the nanopore (5) to generate scatter plots as shown in FIGS. 15, 17 and 19 for Adeno-associated virus, liposomes and polymersomes respectively. FIG. 20 illustrates two DNA origami particles having icosahedron (FIG. 20A) and pentagonal bipyramidal (FIG. 20B) structures with diameters of about 33 and 38 nm, respectively. The DNA origami particles are subjected to nanopore system (1) according to an example embodiment with three different nanoporcs (5) designated as nanoporcs A, B and C as shown in FIGS. 21, 26 and 31, respectively. Both types of DNA origami particles were translocated through nanopore A using different voltage bias of substantially 60 mV, 80 mV, 100 mV, 150 mV and 200 mV, whereby current blocking signals obtained are used to generate the scatter plots of AG versus event duration of icosahedron type and pentagonal bipyramidal type DNA origami particles as shown in FIGS. 22 and 24 respectively. The ECD distributions for icosahedron type and pentagonal bipyramidal type DNA origami particles translocating through nanopore A are as shown in FIGS. 23 and 25 respectively, which differs between both types of DNA origami particles at different voltage bias. It is noted that ECD is not affected by filtering effects, but it cannot extract the translocation speed, as one of the important parameters for further characterizing mechanical properties.

Both types of DNA origami particles are also subjected to translocation through nanopore B using different voltage bias of substantially 100 mV, 150 mV, 200 mV, 250 mV, 300 mV and 350 mV, whereby current blocking signals obtained are used to generate the scatter plots of AG versus event duration of icosahedron type and pentagonal bipyramidal type DNA origami particles as shown in FIGS. 27 and 29 respectively. Similar to what was observed for nanopore A, there is a difference in ECD distributions obtained at different voltage bias between icosahedron type and pentagonal bipyramidal type DNA origami particles translocating through nanoporc B as shown in FIGS. 28 and 30 respectively. The translocation of icosahedron type and pentagonal bipyramidal type DNA origami particles through nanopore C is carried out using voltage bias of substantially 200 mV, 400 mV, 500 mV and 600 mV, whereby current blocking signals obtained are used to generate the scatter plots of AG versus event duration of icosahedron type and pentagonal bipyramidal type DNA origami particles as shown in FIGS. 32 and 34 respectively. The ECD distributions for icosahedron type and pentagonal bipyramidal type DNA origami particles translocating through nanopore C are as shown in FIGS. 33 and 35 respectively, which differs between both types of DNA origami particles at different voltage bias.

FIG. 36 shows CryoEM images of vesicles having different compositions, namely liposomes comprising distearoyl phosphatidylcholine (DSPC), cholesterol, anionic dioleoylphosphatidylserine, and a combination of two or more thereof, and polymersomes comprising polyethylene oxide (PEO) having a number average molecular weight (Mn) of 700, and polybutadicnc (PBD) having a Mn of 1200. Dynamic light scattering (DLS) is used to characterize the aforementioned vesicles as shown in FIG. 37, which include liposomes comprising 40% of DSPC, 40% of cholesterol and 20% of DOPS; liposomes comprising 50% of DSPC, 40% of cholesterol and 10% of DOPS; liposomes comprising 80% of DSPC, 10% of cholesterol and 10% of DOPS; liposomes comprising 100% of DSPC; and polymersomes PEO(700)-b-PBD(1200).

The properties of polymersomes of PEO(700)-b-PBD(1200) are determined using the nanopore system (1) according to an example embodiment. The polymersomes of PEO(700)-b-PBD(1200) are translocated through a nanopore (5) having a diameter of 100 nm using voltage bias of substantially 150 mV and 200 mV, whereby current blocking signals obtained are used to generate the scatter plots of AG versus event duration of the polymersomes as shown in FIG. 38. The ECD distributions for polymersomes of PEG(700)-b-PBD(1200) are as shown in FIG. 39, which differs when translocating through the nanopore at different voltage bias of substantially 150 mV and 200 mV. As will be appreciated by a person skilled in the art, liposome particles have a wide size distribution, and it is in practice difficult to ensure that the pore diameter is smaller than the smallest particles. On the other hand, liposome particles are not as compressible as DNA particles, so to prevent large particles from completely clogging the pores, a moderately sized pore diameter was chosen in the experiments, which diameter is still smaller than the diameter of 80% of the liposome particles under investigation.

The properties of two different liposomes designated as liposome A and liposome B are also determined using the nanopore system (1) according to an example embodiment. Liposome A comprises 50% of DSPC, 40% of cholesterol and 10% of DOPS, while liposome B comprises 80% of DSPC, 10% of cholesterol and 10% of DOPS. FIG. 37, shows DLS characterization results, i.e. liposomes incorporating 40 mol% cholesterol and 10% of DOPS exhibited diameters averaging 161 ± 43 nm. Conversely, liposomes formulated with 10 mol% cholesterol and 10% of DOPS demonstrated a reduced diameter of 139 ± 39 nm. Polymersomes displayed a diameter measuring 123 ± 40 nm. Liposome A is translocated through a nanopore (5) having a diameter of 100 nm using voltage bias of substantially 250 mV, 300 mV, 350 mV and 400 mV, whereby current blocking signals obtained are used to generate the scatter plots of AG versus event duration of liposome A as shown in FIG. 40. The effect of different voltage bias on the translocation behavior of liposome A is as illustrated by the different ECD distributions obtained in FIG. 41. Liposome B is translocated through a nanopore (5) having a diameter of 100 nm using voltage bias of substantially 200 mV, 250 mV, 300 mV and 350 mV, whereby current blocking signals obtained are used to generate the scatter plots of AG versus event duration of liposome B as shown in FIG. 42. Similar to what was observed for liposome A, tire use of different voltage bias affected the translocation behaviour of liposome B, as illustrated by the different ECD distributions obtained in FIG. 43.

The nanopore system (1) according to and example embodiment is further used to determine properties of Adeno-associated virus (size/diameter of about 35 nm). The Adeno-associated virus is translocated through a nanopore (5) having a diameter of 30 nm using different voltage bias of substantially 200 mV, 250 mV, 300 mV, 350 mV and 400 mV, whereby current blocking signals obtained are used to generate the scatter plots of AG versus event duration of the virus as shown in FIG. 44. The effect of different voltage bias on the translocation behavior of Adeno-associated virus is as illustrated by the different ECD distributions obtained in FIG. 45.

Another nanopore system according to an example embodiment of the present invention is as illustrated in FIG. 46, wherein the nanopore system (46) is provided with a fluidic cell (47) having a three-layer nanopore structure for measuring plastic deformation of nanoparticles. Particularly, the nanopores (48, 49) at the top and bottom layer are larger in diameter, here larger than the size of the nanoparticle (50), so as to measure the size of nanoparticle (50) before and after translocating through the three-layer nanopore structure of the nanoporc system (46) for measuring mechanical properties of the nanoparticlc (50). The middle layer is configured with a nanopore (51) having a smaller diameter compared to the top and bottom layer, here smaller than the size of the nanoparticle (50), thereby providing a function of squeezing the nanoparticle to measure its plastic deformation. Electrodes (52, 53) are used to apply a voltage across the fluidic cell (47) to effect the translocations while measuring the current using a current meter (54).

FROM MANUSCRIPT

Further results and discussion according to example embodiments.

On a 12 nm- thick SiNx membrane, individual nanopores were fabricated using focused electron beam or Helium ion beam sputtering techniques, with a diameter range of 8-100 nm. Following meticulous surface cleaning, here the nanopore chips were soaked in piranha solution (a mixture of 3 parts of concentrated sulfuric acid and 1 part of 35 wt.% hydrogen peroxide solution) at 1 15°C for 1 hour to decompose most organic matter and hydroxylate surface (by adding -OH groups) which made the chips highly hydrophilic, these nanopore chips were sandwiched between a pair of gaskets forming a fluidic cell configured with the membrane having the individual nanopores and disposed between two fluid reservoirs, as described above with reference to FIG. 1. The solution within the fluidic cell comprised 0.2-1.0 M KC1, 50 mM Tris, and pH ^; ” 8. The nanopore was employed to interconnect two fluidic reservoirs, and an electric bias (50 - 600 mV) was applied to drive negatively charged particles through the nanopore. When the particle size exceeded that of the pore, the particles were squeezed through. FIG. 47A depicts the theoretical initial stage where particles 4700 are depicted to approach the entrance of the nanopore 4702 in a membrane 4704 under the influence of the electric field. Subsequently, in FIG. 47B, upon encountering the nanopore 4702, the particle 4700 undergoes deformation, with certain portions penetrating deeper into the pore. Finally, as illustrated in FIG. 47C, the particles 4700 continue to traverse through the nanopore 4700, eventually passing through it entirely, while undergoing mechanical deformation.

DNA-NPs were prepared using scaffolded DNA assembly methods [1]. Two different geometric structures (icosahedral DNA ball FIG. 47D, star-shaped DNA coin FIG. 47E) with edge lengths of 52-bp and 84-bp were utilized. The diameters of these particles were measured via dynamic light scattering (DLS) as 38 nm and 33 nm, respectively. The size and geometry of the DNA-nanoparticles (NPs) were confirmed using cryo-electron microscopy (cryo-EM), as depicted in FIGs. 47H and 471. These materials are negatively charged due to the solvent-exposed negative oxygen ions on the phosphate backbone of DNA. DNA-NPs were chosen as a model system due to their highly uniform size and geometry, nearly eliminating the influence of sample heterogeneity on translocation events. The materials for polymersomes (FIG. 47G) and liposomes (FIG. 47F) were Poly(ethylene oxide)-b-poly(butadiene) diblock copolymer (PEO(700)-b-PBD(1200)) and DSPC- Cholesterol-DOPS, respectively. Different mechanical properties of membranes of the lipid were achieved by adjusting the proportions of lipid molecules (50mol%DSPC- 40mol%Cholcstcrol-10mol%DOPS and 80mol%DSPC-10mol%Cholcstcrol-

10mol%DOPS), where membranes with higher percentages of cholesterol exhibited lower stiffness. Liposomes and polymersomes were prepared by repeated extrusion of polymer/lipid films through a 30 nm filter more than 30 times. Based on the DLS characterization results, liposomes incorporating 40 mol% cholesterol exhibited diameters averaging 161 ± 43 nm and a potential of -36 ± 7 mV. Conversely, liposomes formulated with 10 mol% cholesterol demonstrated a reduced diameter of 139 ± 39 nm alongside a C, potential of -21 ± 9 mV. Polymersomes displayed a diameter measuring 123 ± 40 nm with a potential of -7 ± 9 mV. Their shapes and internal membrane structures are depicted in cryo-EM images (FIGs. 47J and 47K).

Before introducing the sample particles, ion transport within the nanopore generated a stable baseline current as shown in FIGs. 471 to 470. In contrast, modulation of the current as blockade signals occurs when particles translocate through the nanopore. For DNA-NPs, a high-concentration (I M KC1, 50 mM Tris, and 10 mM EDTA) buffer was utilized, and the signal amplitude reflected the extent to which the nanopore volume was occupied. At low voltages (100 mV), distinct step-like features were observed as shown in FIGs. 47L and 47M. DNA balls exhibited three distinct blocking levels, whereas DNA coins displayed two levels. As the voltage increased to 150 mV, the total duration of blocking events significantly shortened, and the step-like features became less distinct. When the voltage increased to 200 mV, the duration time of translocation through the pore further decreased, resulting in rapid and smooth passage, generating spike-like blockade signals.

FIGs. 47N and 470 demonstrate more intricate patterns of blockage signals of liposomes and polymersomes. This is attributed to the wide size distribution and diverse internal membrane structures of liposomes and polymersomes. During the analysis, two common event shapes were noted: spike-shaped events (FIG. 51B) and events with pronounced fluctuations in current over duration, the latter referred to as poration events (FIG. 51D). Both liposomes and polymersomes displayed more spike-shaped blocking signals at lower voltages, originating from two hypothesized mechanisms: either the passage of particles smaller than the pore size, or collision of particles with the pore wall upon reaching the pore entrance, followed by return to the bulk region. With increasing voltage, signals with longer durations emerged, exhibiting noticeable oscillatory with amplitudes significantly exceeding the baseline noise level.

The average current blockade amplitude within the duration of each event was extracted and normalized by the applied voltage to obtain the voltage-independent conductance change (AG) in the nanopore. Tn the scatter plots in FIGs. 48A to 48H, the horizontal axis represents the duration, and the vertical axis represents AG. Statistical analysis revealed that under 150 mV bias, DNA coins passing through an 8 nm diameter nanopore (FIGs. 48A to 48D) exhibited a wide range of durations, with little variation in AG. As voltage increased, the distribution of AG remained nearly unaffected by voltage, but the duration of translocation significantly decreased, with a narrower distribution range. For liposomes with 10 mol% cholesterol (FIGs. 48E to 48H), the duration of pore events exhibited a trend of narrowing distribution with increasing voltage, and the distribution shifted towards shorter durations.

In FIG. 49B, the distribution of duration for DNA balls passing through a 15-nm-diamctcr nanopore at 200 mV voltage is presented, exhibiting an asymmetric shape with a long tail extending towards longer durations. First-passage time distributions obtained from the onedimensional Fokker-Planck equation were used to extract the translocation velocity of particles [2],

Here, F(r) is the probability density distribution function of event duration (as depicted by the fitted curve in FIG. 49B), L represents the trajectory length of the particle (simplified as the pore length Lp_Ore plus the particle diameter

L

Du is the diffusion coefficient along the axis direction, r is translocation duration and v is the drift velocity. FIG. 49A illustrates the drift velocity of DNA balls and DNA coins passing through a 15 nm diameter nanopore under 50 - 350 mV voltage drive. Both types of particles exhibit two distinct regimes: at low voltage, the velocity is low and depends super-linearly on the voltage, a regime that is interpreted as limited by thermally activated creeping and squeezing of the particles through the pore entrance. This regime, which exhibits a wide distribution of event durations, is referred to as “randomly activated” herein, at higher voltage, in contrast, the velocity scales linearly with voltage, which is consistent with a friction-dominated regime. In this regime, the electric field provides sufficient energy to overcome the energy barrier, driving ballistic transport of particles through the pore. At this point, pore translocation velocity depends on the balance between driving force and frictional force. The slope of the curve is defined herein as the constrained mobility, p_c.

To gain further insight on the particles’ mechanical properties, specifically geometric and elastic properties, from these experiments, a 1-D funnel model is proposed that divides the translocation dynamics into two parts: deforming into the pore and traveling through the pore. Tn order to enter the pore, a particle larger than the pore diameter must compress in the surface directions (x, y), raising its free energy by a deformation cost A, which relates to the particle’s elastic modulus and the ratio between pore size and particle diameter. In the absence of potential difference, this energy barrier must be overcome by thermal fluctuations alone, which leads to a crossing rate following an Arrhenius law, vocexp(A/kBT) where ks is the thermal energy. In the presence of a voltage, the particle is subject to a force y along the z direction perpendicular to the surface, which tilts the energy landscape towards entering the pore. To quantify the degree of cooperativity between this perpendicular force and the parallel compression required to enter the pore, the funnelling length, I , is introduced which indicates how much a particle moves into the pore as it squeezes to enter it. In this simplified ID model, tire deformation energy is a linear-byparts function of the position z of the particle, such that the energy cost A is distributed over a length If This parameter represents how much the parallel force component is projected perpendicularly by the particle’s geometry, facilitating compression and passage through the pore: when !,=() (e.g. for the square particle in FIG. 49C) there is no cooperativity between parallel and perpendicular force, and the energy barrier to entering the pore is independent of the bias. In contrast, a particle with If comparable to its size (FIG. 49D) smoothly funnels into the pore, and the energy barrier is reduced when the electrostatic potential difference tilts the energy: an electrostatic bias \|/_c~A//y is sufficient to suppress the energy barrier, marking the transition between activated and frictional regimes. Beyond the particles’ shape, the funnel length might also be influenced by its Poisson ratio. FIGs. 49E and 49F show the funnel shapes for elliptical and circular particles entering the nanoporc, respectively, illustrating that the Poisson ratio would distinguish DNA ball and DNA coin geometries. In the proposed model, the particle undergoes Langevin dynamics in the tilted energy landscape composed of the elastic and electrostatic potential energies, and thus incorporates three key parameters: p (the particle’s mobility within the pore), A (the deformation energy barrier), and If (the funnelling length), along with controllable parameters such as the electrostatic drift \|/, and the pore length L. FIG. 49G illustrates the theoretical prediction curve for translocation velocity versus applied bias, indicating that p_c depends on the geometric factor L/(L+ If), and the y-axis intercept provides direct access to the energy barrier A. FIGs. 50A to 50D present TEM imaging of a series of nanopores used in the experiment with diameters ranging from 8 nm to 23 nm. For nanopores of different sizes, it was observed that the translocation speeds of DNA balls and DNA coins exhibited similar trends. Specifically, particles passing through pores of different diameters experienced a plateau of the random-activated regime, followed by a linear growth phase of the frictional regime. As the voltage increased, the width of the translocation duration distribution also decreased significantly as shown in FIGs. 50G to 50H. However, the critical points and p_c vary depending on the pore size. The position of the critical voltage, Von, directly reflects the value of A, which was found to exhibit an exponential relationship with the pore diameter. The p_c can decode the geometric characteristics, L/(L+ If), of particles. As shown in FIG. 52A, the offset of the critical voltage, V_on, exhibits an exponential relationship with the pore diameter for the two types of DNA particles. With reference to FIG. 52B, which illustrates the correlation between constraint mobility and pore diameter, where the constraint mobility is defined as the slope of the linear relationship between translocation speed v and bias in the plastic regime, the constraint mobility reflects the combination of two main resistances that particles experience after collapsing: frictional resistance from the pore surface and viscous resistance from the solution.

Analysis of liposome events is relatively complex mainly due to the complexity of the current signal shapes. Therefore, a comprehensive code for identifying different types of current signal events was developed and these events were categorized as typical spikeshaped events and poration events, as shown in FIGs. 51B and 51D. An interesting phenomenon is observed in FIG. 51A where the proportion of poration events shows an exponential growth as the voltage increases. Further comparison of liposomes with different material compositions revealed that the occurrence of poration events is primarily influenced by the material's breakdown characteristics rather than surface charge or material rigidity. Liposomes with 10 mol% cholesterol exhibit higher rigidity compared to those with 40 mol% cholesterol, yet the former has a higher incidence of poration events under the same voltage. The breakdown potential of planar lipid bilayers increases from 0.6 V to 1.3 V with the increase in cholesterol concentration from 0 to 50 mol% [3]. It was found that membranes more prone to breakdown tend to have a higher proportion of poration events under the same voltage. Tire breakdown potential of Polymersome (PEO-b- PBD) is around 9 V [4J, hence exhibiting the lowest poration event rate. The areal strain at rupture a_c (^ 15-20%) of Polymersome [5, 6] is much greater than those sustainable by any lipid membrane (typically only tolerate 4-6%) [7, 8]. Therefore, Polymersomes can deform by increasing surface area to translocate due to their superior material elasticity, resulting in spike-shaped events.

Nanopore fabrication according to an example embodiment

The fabrication of nanopores was conducted on Norcada chips (NTDB-B105V122), which have a 12±2nm thickness, square (10x10pm), low-stress SiNx membrane window centered within a 200pm-thick silicon frame. To address various pore size requirements, two distinct fabrication techniques were employed. Firstly, a TEM (JEOL 2010F) operating at 200 kV was employed for nanopores within the diameter range of 8 - 23 nm. By focusing a tightly controlled electron beam on the membrane, material sputtering occurred, leading to the creation of nanopores. The nanopore size was finely tuned by adjusting the electron beam intensity. Additionally, a Helium Ion Microscope (HIM), Zeiss Orion Nanofab was employed for 100-nm-diameter nanopores. Operating at an acceleration voltage of 30 kV with an aperture of 10 pm, the spot size was varied around 3 to achieve an ion current ranging from 2.1 to 2.3 pA. The shape properties were standardized circular with a size of 100 nm. Scan pixel spacing for both X and Y were set to Inm, ensuring precise control over the fabrication process. To etch nanoporcs, a dose of 2c+18 ions/cm2 of helium ion beam was applied.

Lipid/ polymer nanoparticle fabrication

Membrane compositions for Polymersomes and liposomes were selected using Poly(ethylene oxide)-b-poly(butadiene) diblock copolymer (PEO (700)-b-PBD (1200)) as the polymer source. For liposomes, the lipid components included DSPC (1,2-distearoyl- sn-glycero-3-phosphocholine), Cholesterol, and DOPS (l,2-dioleoyl-sn-glycero-3- phospho-L- serine (sodium salt)). Lipid/polymer solutions were prepared by adding 200 pL chloroform. Thin films were formed by drying polymer/lipid-chloroform solutions with a gentle nitrogen stream, rotating to coat the tube walls. Tubes were sealed with parafilm, punctured, and vacuum desiccated for 4 hours to remove chloroform and maintain membrane properties. To hydrate thin films, a clean magnetic stir bar was introduced, and KC1 buffer (typically at a concentration 0.2 M or 0.5 M) was added. Stirring occurred at 200-300 rpm, and after several hours, gentle vortexing ensured complete hydration. Hydration proceeded overnight at room temperature. Vesicles were then extruded using the Avanti® Mini-Extruder to reduce their size. Polymersomes underwent 31 extrusions, while liposomes underwent 41 extrusions. Post-extrusion, the solution exhibited decreased turbidity.

DNA origami nanoparticlc fabrication

The DNA origami structures were designed using DAEDALUS, with custom ssDNA scaffolds fabricated using bacteriophage systems as previously reported. The sequences of the scaffolds and staples for the DNA nanoparticles can be found in section XX of the SI. The self-assembly mixture was prepared in nuclease-free water supplemented with TAE and 12 mM MgC12. 30 nM scaffold and 150 nM staples prepared in a self-assemble mixture and subjected to the following annealing protocols: for the DNA ball, 65 °C for 15 min, 61°C for 90 min, 60°C for 90 min, 25°C for 5 min; for the DNA coin, 95°C for 5 min, 80-75°C at 1°C per 5 min, 75-30°C at 1°C per 15 min, and 30-25°C at 1°C per 10 min. After assembly, the nanoparticles were purified into PBS using Amicon Ultra centrifugal filters (100 kDa, 2000 x g, 3x) and were stored at 4 °C prior to use. DNA nanoparticles were characterized with dynamic light scattering (50 nM in PBS).

Cryo-EM

The Cryo-EM grid preparation process commenced by applying DNA-NPs (1 nM), liposome (2.5 mg/ml) and polymcrsomc (2.5 mg/ml) samples at a concentration of (lipid) onto Tedpella ultrathin carbon film on Lacey carbon support film (400 mesh, copper) grids. These grids were then glow-discharged for 20 seconds to clean and enhance hydrophilicity. Subsequently, blotting was performed using an FEI Vitrobot Mark IV with a blot time of 1 s, blot force of 1, and wait time of 1 s, all carried out at 4°C with 100% humidity. The samples were then plunge-frozen in liquid ethane. For Cryo-EM imaging, an FEI Tecnai Arctica 200 kV cryo-TEM equipped with a Falcon 3EC camera was utilized, operating at a magnification of 53,000x with an exposure time of 1 s.

Nanopore squeezing according to an example embodiment

After cleaning the nanopore chips in piranha solution for one hour, they were carefully enclosed between two PDMS gaskets to prevent any potential leakage, and subsequently placed between two reservoirs (compare also description above with reference to FIG. 1. Ag/AgCl electrodes were then carefully inserted into both reservoirs and linked to a current amplifier (Axon Axopatch 200B alongside the Digidata 1440B data acquisition system) to apply a consistent voltage difference, while concurrently recording the resulting ionic current. To enhance electromagnetic shielding from the surrounding environment, the entire apparatus was housed within a grounded Faraday cage. For the DNA-NPs samples, a dilution to a concentration of 1 nM was prepared in a solution containing 1 M KC1, 50 mM Tris, and 10 mM EDTA, adjusted to a pH of 8.3. The liposome samples of 250 pg/ml lipid concentration were prepared in 0.5 M KC1 and 50 mM Tris buffer with pH = 8.4. The polymersome samples of 250 ug/ml polymer concentration were prepared in 0.2 M KC1 and 50 mM Tris buffer with pH = 7.9. The sample solution was then introduced into the reservoir on the cis side reservoir. Voltage variations ranging from 50mV to 600mV were applied across the nanopore. Notably, during translocation events, the electrical current passing through the pore deviated from the baseline current (i.e., in the absence of particles), facilitating nanomechanical property measurements. Signal conditioning was achieved using a fourth-order Bessel filter set at a bandwidth of 100 kHz, followed by digitization at a sampling rate of 500 kHz. Subsequent analyses pertaining to event durations and amplitudes were conducted utilizing the transalyzer package in conjunction with custom MATLAB scripts.

FIG. 53 shows a flowchart 5300 illustrating a method for determining mechanical properties of nanoparticles. At step 5302, an electrically conducting fluid comprising the nanoparticles dispersed therein is provided in a fluidic cell configured with a membrane having a nanoporc extending through a thickness of the membrane. At step 5304, a voltage is applied across the fluidic cell for effecting a translocation event of at least one of the nanoparticles through the nanopore. At step 5306, a current across the fluid cell is monitored for a period starting before the translocation event and ending after the translocation event to measure a current blocking signal. At step 5308, the mechanical properties of the nanoparticles are determined from the measured current blocking signal; wherein a size of the nanoparticles is larger than a pore size of the nanopore; and wherein an aspect ratio of thickness of the membrane to the pore size of the nanopore is smaller than 1.

The mechanical properties may comprise one or more of a group consisting of frame structure of the nanoparticlcs, inner structure of the nanoparticlcs, stiffness of the nanoparticles, strength of the nanoparticles, and poration events of the nanoparticles.

The method may comprise applying a pressure gradient across the fluidic cell for assisting in the effecting of the translocation event.

The method may comprise varying the applied voltage of respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, and determining the mechanical properties of the nanoparticles from the measured plurality of current blocking signals.

The method may comprise varying the pore size of the nanoporcs of respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, and determining the mechanical properties of the nanoparticles from the measured plurality of current blocking signals.

The method may comprise varying the thickness of the membrane of respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, and determining the mechanical properties of the nanoparticles from the measured plurality of current blocking signals.

The method may comprise amplifying the current blocking signal.

The method may comprise using a Faraday cage around the fluidic cell.

The membrane may have a thickness of about 0.5 to 50 nm.

The nanopore may have a diameter of about 8 to 100 nm.

The applied voltage may be in a range from about 0.1 to 5 V.

The nanoparticles may comprise one or more of a group consisting of viruses, DNA origami particles, liposomes and polymersomes.

In one embodiment, a nanopore system for determining mechanical properties of nanoparticles is provided, comprising a fluid cell configured for receiving an electrically conducting fluid comprising the nanoparticlcs dispersed therein, wherein the fluidic cell is configured with a membrane having a nanopore extending through a thickness of the membrane; a source for applying a voltage across the fluidic cell for effecting a translocation event of at least one of the nanoparticles through the nanopore; a monitor configured for monitoring a current across the fluid cell for a period starting before the translocation event and ending after the translocation event to measure a current blocking signal for determining the mechanical properties of the nanoparticles from the measured current blocking signal; wherein a pore size of the nanoporc is chosen to be smaller than a size of the nanoparticles; and wherein an aspect ratio of thickness of the membrane to the pore size of the nanopore is smaller than 1.

The mechanical properties comprise one or more of a group consisting of frame structure of the nanoparticles, inner structure of the nanoparticles, stiffness of the nanoparticles, strength of the nanoparticles, and poration events of the nanoparticles. The nanopore system may comprise a pump for applying a pressure gradient across the fluidic cell for assisting in the effecting of the translocation event.

The source may be configured for varying the applied voltage of respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, for determining the mechanical properties of the nanoparticles from the measured plurality of current blocking signals.

The fluid cell may be configurable with membranes having different pore sizes for respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, for determining the mechanical properties of the nanoparticlcs from the measured plurality of current blocking signals.

The fluid cell may be configurable with membranes having different thicknesses for respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, for determining the mechanical properties of the nanoparticles from the measured plurality of current blocking signals.

The nanopore system may comprise an amplifier for amplifying the current blocking signal.

The nanoporc system may comprise a Faraday cage around the fluidic cell.

The membrane may have a thickness of about 0.5 to 50 nm.

The nanopore may have a diameter of about 8 to 100 nm.

The applied voltage may be in a range from about 0.1 to 5 V.

The membrane may comprise one or more of a group consisting of silicon nitride (SiNx), silicon oxide, aluminium oxide, hafnium oxide, 2D materials comprising graphene, fluorinated graphene, molybdenum disulfide, tungsten disulfide, hexagonal boron nitride and MXenes (comprising Ti2CTx and Ti3C2Tx).

The nanoporc system may comprise electrodes for applying the voltage.

Aspects of the systems and methods described herein such as, but not limited to, the data capture and data analysis for determination/classification of properties of the nanoparticles may be implemented on computing dcvicc(s), including cloud-based computing dcvicc(s) and/or Internet-of-Things computing device(s), for example as functionality programmed into any of a variety of circuitry, including programmable logic devices (PLDs), such as field programmable gate arrays (FPGAs), programmable array logic (PAL) devices, electrically programmable logic and memory devices and standard cell-based devices, as well as application specific integrated circuits (ASICs). Some other possibilities for implementing aspects of the system include: microcontrollers with memory (such as electronically erasable programmable read only memory (EEPROM)), embedded microprocessors, firmware, software, etc. Furthermore, aspects of the system may be embodied in microprocessors having software-based circuit emulation, discrete logic (sequential and combinatorial), custom devices, fuzzy (neural) logic, quantum devices, and hybrids of any of the above device types. Of course the underlying device technologies may be provided in a variety of component types, e.g., metal-oxide semiconductor field-effect transistor (MOSFET) technologies like complementary metal-oxide semiconductor (CMOS), bipolar technologies like emitter-coupled logic (ECL), polymer technologies (e.g., Silicon-conjugated polymer and metal-conjugated polymer-metal structures), mixed analog and digital, etc.

The various functions or processes disclosed herein may be described as data and/or instructions embodied in various computer-readable media, in terms of their behavioral, register transfer, logic component, transistor, layout geometries, and/or other characteristics. Computer-readable media in which such formatted data and/or instructions may be embodied include, but are not limited to, non-volatile storage media in various forms (e.g., optical, magnetic or semiconductor storage media) and carrier waves that may be used to transfer such formatted data and/or instructions through wireless, optical, or wired signaling media or any combination thereof. When received into any of a variety of circuitry (e.g. a computer), such data and/or instruction may be processed by a processing entity (e.g., one or more processors).

It will be appreciated by a person skilled in the art that numerous variations and/or modifications may be made to the present invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments arc, therefore, to be considered in all respects to be illustrative and not restrictive. Also, the invention includes any combination of features described for different embodiments, including in the summary section, even if the feature or combination of features is not explicitly specified in the claims or the detailed description of the present embodiments.

In general, in the following claims, the terms used should not be construed to Emit the systems and methods to the specific embodiments disclosed in the specification and the claims, but should be construed to include all processing systems that operate under the claims. Accordingly, the systems and methods are not limited by the disclosure, but instead the scope of the systems and methods is to be determined entirely by the claims.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in a sense of "including, but not limited to." Words using the singular or plural number also include the plural or singular number respectively. Additionally, the words "herein," "hereunder," "above," "below," and words of similar import refer to this application as a whole and not to any particular portions of this application. When the word "or" is used in reference to a list of two or more items, that word covers all of the following interpretations of the word: any of the items in the list, all of the items in the list and any combination of the items in the list.

References

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[3] P. Kramar, D. Miklavcic, Bioelectrochemistry 2022, 144, 108004.

[4] D. Branton, D. W. Deamer, A. Marziali, H. Bayley, S. A. Benner, T. Butler, M. Di Ventra, S. Garaj, A. Hibbs, X. Huang, S. B. Jovanovich, P. S. Krstic, S. Lindsay, X. S. Ling, C. H. Mastrangelo, A. Meller, J. S. Oliver, Y. V. Pershin, J. M. Ramsey, R. Riehn, G. V. Soni, V. Tabard-Cossa, M. Wanunu, M. Wiggin, J. A. Schloss, Nat. Biotechnol. 2008, 26, 1146.

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Claims

CLAIMS:

1. A method for determining mechanical properties of nanoparticles, the method comprising the steps of: providing an electrically conducting fluid comprising the nanoparticles dispersed therein in a fluidic cell configured with a membrane having a nanopore extending through a thickness of the membrane; applying a voltage across the fluidic cell for effecting a translocation event of at least one of the nanoparticles through the nanopore; monitoring a current across the fluid cell for a period starting before the translocation event and ending after the translocation event to measure a current blocking signal; and determining the mechanical properties of the nanoparticles from the measured current blocking signal; wherein a size of the nanoparticles is larger than a pore size of the nanopore; and wherein an aspect ratio of thickness of the membrane to the pore size of the nanopore is smaller than 1.

2. The method of claim 1 , wherein the mechanical properties comprise one or more of a group consisting of frame structure of the nanoparticles, inner structure of the nanoparticles, stiffness of the nanoparticles, strength of the nanoparticles, and poration events of the nanoparticles.

3. The method of claims 1 or 2, comprising applying a pressure gradient across the fluidic cell for assisting in the effecting of the translocation event.

4. The method of any one of the preceding claims, comprising varying the applied voltage of respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, and determining the mechanical properties of the nanoparticles from the measured plurality of current blocking signals.

5. The method of any one of the preceding claims, comprising varying the pore size of the nanopores of respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, and determining the mechanical properties of the nanoparticles from the measured plurality of current blocking signals.

6. The method of any one of the preceding claims, comprising varying the thickness of the membrane of respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, and determining the mechanical properties of the nanoparticles from the measured plurality of current blocking signals.

7. The method of any one of the preceding claims, comprising amplifying the current blocking signal.

8. The method of any one of the preceding claims, comprising using a Faraday cage around the fluidic cell.

9. The method of any one of the preceding claims, wherein the membrane has a thickness of about 0.5 to 50 nm.

10. The method of any one of the preceding claims, wherein the nanopore has a diameter of about 8 to 100 nm.

11. The method of any one of the preceding claims, wherein the applied voltage is in a range from about 0.1 to 5 V.

12. The method of any one of the preceding claims, wherein the nanoparticles comprise one or more of a group consisting of viruses, DNA origami particles, liposomes and polymersomes.

13. A nanopore system for determining mechanical properties of nanoparticles, comprising: a fluid cell configured for receiving an electrically conducting fluid comprising the nanoparticlcs dispersed therein, wherein the fluidic cell is configured with a membrane having a nanopore extending through a thickness of the membrane; a source for applying a voltage across the fluidic cell for effecting a translocation event of at least one of the nanoparticlcs through the nanoporc; a monitor configured for monitoring a current across the fluid cell for a period starting before the translocation event and ending after the translocation event to measure a current blocking signal for determining the mechanical properties of the nanoparticles from the measured current blocking signal; wherein a pore size of the nanopore is chosen to be smaller than a size of the nanoparticles; and wherein an aspect ratio of thickness of the membrane to the pore size of the nanoporc is smaller than 1.

14. The nanopore system of claim 13, wherein the mechanical properties comprise one or more of a group consisting of frame structure of the nanoparticlcs, inner structure of the nanoparticles, stiffness of the nanoparticles, strength of the nanoparticles, and poration events of the nanoparticles.

15. The nanopore system of claims 13 or 14, comprising a pump for applying a pressure gradient across the fluidic cell for assisting in the effecting of the translocation event.

16. The nanopore system of any one of claims 13 to 15, wherein the source is configured for varying the applied voltage of respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, for determining the mechanical properties of the nanoparticles from the measured plurality of current blocking signals.

17. The nanopore system of any one of claims 13 to 16, wherein the fluid cell is configurable with membranes having different pore sizes for respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, for determining the mechanical properties of the nanoparticles from the measured plurality of current blocking signals.

18. The nanopore system of any one of claims 13 to 17, wherein the fluid cell is configurable with membranes having different thicknesses for respective ones of a plurality of translation events for measuring a plurality of corresponding current blocking signals, for determining the mechanical properties of the nanoparticlcs from the measured plurality of current blocking signals.

19. The nanopore system of any one of claims 1 to 18, comprising an amplifier for amplifying the current blocking signal.

20. The nanopore system of any one of claims 13 to 19, comprising a Faraday cage around the fluidic cell.

21. The nanopore system of any one of claims 13 to 20, wherein the membrane has a thickness of about 0.5 to 50 nm.

22. The nanopore system of any one of claims 13 to 21, wherein the nanopore has a diameter of about 8 to 100 nm.

23. The nanoporc system of any one of claims 13 to 22, wherein the applied voltage is in a range from about 0.1 to 5 V.

24. The nanopore system of any one of claims 13 to 23, wherein the nanoparticles comprise one or more of a group consisting of viruses, DNA origami particles, liposomes and polymersomes.

25. The nanopore system of any one of claims 13 to 24, wherein the membrane comprises one or more of a group consisting of silicon nitride (SiNx), silicon oxide, aluminium oxide, hafnium oxide, 2D materials comprising graphene, fluorinated graphene, molybdenum disulfide, tungsten disulfide, hexagonal boron nitride and MXenes (comprising Ti2CTx and Ti3C2Tx).

26. The nanopore system of any one of claims 13 to 25, comprising electrodes for applying the voltage.