Systems and methods for inertial-dynamic capture and sensing of single molecules

Abstract

The invention relates to a nanopore sensing device for inertia dynamic migration and single molecule sensing. Some embodiments include: a centrifuge rotor; a centrifuge tube; a single or a plurality of flow cell modules; comprising a single or a plurality of nanopores; a signal detection module; a signal amplifier module; a control module; and a wireless communication module. The centrifugal force field is adjusted through power, meanwhile, the balance state of electrophoretic force and electroosmosis force in the nanopores is kept by adjusting the pH value of electrolyte in the nanopores or exciting surface charges on the silicon nanopores through visible light, and accurate adjustment of molecular migration parameters such as speed, direction and molecular selectivity is provided. To optimize temporal and spatial resolutions of molecular sensing with high S / N ratio signal readout.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Patent Application No. 18 / 477,768, filed on September 29, 2023, the entire content of which, including any tables, figures, or drawings, is incorporated herein by reference. Technical field

[0003] The present invention relates to high - sensitivity measurement devices. More specifically, the present invention relates to improved nanopore sensing systems and methods. Background art

[0004] When a molecule drifts through a nanopore (also known as a nano - pore) under the drive of an applied potential difference, the ion channel is temporarily blocked, and a resulting sensing signal (in the form of current, voltage, resistance, conductance) is generated [ACS Chem. Biol. 2012, 7, 1935 - 1949; Phys. Chem. Chem. Phys., 2022, 24, 19948 - 19955]. Nanopore sensing is a technique achieved by measuring the sensing signal with electrodes on the nanopore [Nat. Nanotech. 12, 360 - 367 (2017); Nat. Nanotech. 17, 708 - 713 (2022); Nat. Nanotech. 17, 976 - 983 (2022); Nature Reviews Materials 2020, 5(12), 931 - 951]. This technique can be integrated into portable sensing devices with electronic equipment [K. Chuah, et al. Nature Communications 2019, 10, 2109]. In fact, the so - called nanopore sequencing technology has made significant contributions to many branches of life science in the past two decades [N. S. Galenkamp, et al. Nature Communications, 9, 4085 (2018); Bayley, H. Nanopore sequencing: from imagination to reality. Clin. Chem. 61, 25–31 (2015).]

[0005] In principle, nanopores with appropriate structural dimensions can resolve the size and conformation of the molecule under discussion [Phys. Life rev. 9, 125 - 158 (2012)]. Electromigration of individual molecules is commonly employed in nanopores, and the migration speed is determined by the electrophoretic force and viscous drag of the molecules in the solution and the pore. However, it is difficult to control the migration speed of molecules in nanopores, resulting in short residence times of sensing signals and low conformational sensitivity. The high migration speed and low conformational sensitivity for signal readings limit the accuracy of nanopore molecular recognition [Adv. Mater. 2018, 30, 1704680; Venkatesan, B. M. & Bashir, R. Nanopore sensor for nucleic acid analysis. Nat. Nanotechnol. 6, 615–624 (2011)].

[0006] Although it has been successfully demonstrated that the speed of protein motors can be controlled using biological nanopores, it remains challenging to achieve a stable feeding rate of protein motors and a high conductivity drop as in solid-state nanopores [Fragasso, ACS nano, 2020, Brinkerhoff, Science 2021]. On the other hand, nanopositioners have been used to achieve controlled migration in glass nanopores. However, this method requires the confinement of molecules, thereby preventing their complete migration [Leitao NatNanotec 2023]. Summary of the Invention

[0007] Embodiments of the present invention provide systems and methods for driving nanoscale objects through nanopores by centrifugation, such that the molecules under study undergo inertia - dynamics - controlled migration and a controlled residence time in the nanopores, with highly conformation - sensitive signal readout. The utilization of the inertial force generated by centrifugation effectively separates the single - molecule migration process from experimental parameters (such as ionic strength and bias voltage) and the signal detection process, which uses the same pair of electrodes to apply a bias voltage across the nanopore. In certain embodiments, by adjusting the pH value of the electrolyte in the nanopore or using surface charges excited by light on silicon nanopores, the electrophoretic force and electroosmotic force are effectively balanced, while the electric field still serves as an independent sensing method covering the nanopore.

[0008] Although electrodynamic translocation usually results in high and uncontrolled translocation speeds of individual molecules in nanopores, low conformational sensitivity, non-uniform conductance signals, and short residence times ranging from microseconds to milliseconds [Tang, L. et al. Nat. Commun. 12, 913 (2021).], inertial dynamic translocation can effectively control the speed and direction of single-molecule translocation, thereby enabling uniform sensing readouts with high conformational sensitivity and residence times of up to hundreds of milliseconds, and being able to programmably and selectively sense individual molecules from complexes composed of multiple molecules. Additionally, inertial dynamic translocation can help achieve reversible sensing and selective translocation of single molecules, enabling repetitive and addressable molecular sensing with high spatial and temporal resolution. Therefore, the sensing signal can be optimized by independently controlling the centrifugal force in the nanopore, including its signal-to-noise ratio and residence time, to achieve highly distinguishable single-molecule fingerprints with improved detection limits.

[0009] Embodiments provide an in-tube nanopore sensing device similar to a centrifuge tube, which can be conveniently placed in a centrifuge to implement an inertial-dynamic nanopore sensing system. Such an in-tube nanopore device basically includes a nanopore for sensing or multiple nanopores for multiplex sensing, a flow cell module for loading samples into each nanopore, a signal detection module for each nanopore, a signal amplification module for each nanopore, one or more control modules, and one or more wireless communication modules that wirelessly transmit sensing signals and enable real-time sensing of individual molecules.

[0010] According to an embodiment of the present invention, the inertial-dynamic nanopore sensing system of the present invention further includes one or more data processing modules for recording and analyzing the signals of detected individual molecules. Brief Description of the Drawings

[0011] Figure 1A is a schematic diagram of an in-tube nanopore sensing device in a centrifuge according to an embodiment of the present invention, which device has a nanopore module including a single nanopore in a flow cell module and a signal detection module that measures signals (e.g., in the form of current, voltage, resistance, and / or conductance) using an applied bias voltage.

[0012] Figure 1B is a detailed schematic diagram of a detection module for a single nanopore in a single flow cell module according to an embodiment of the present invention.

[0013] Figure 1C is a schematic diagram of an in-tube nanopore sensing device in a centrifuge according to an embodiment of the present invention, which device has a nanopore module including multiple nanopores in multiple flow cell modules, wherein each nanopore has a signal detection module that applies a bias voltage and measures signals.

[0014] Figure 1D It is a detailed schematic diagram of multiple detection modules for multiple nanopores in multiple flow cell modules according to an embodiment of the present invention.

[0015] Figure 1E It is a schematic diagram of a signal amplification module, a control module, and a wireless communication module according to an embodiment of the present invention.

[0016] Figure 2A It is a schematic diagram of the force analysis of the molecule to be measured at different pH values according to an embodiment of the present invention. The external forces applied to the target molecule (i.e., bovine serum albumin or BSA) are mainly the electrophoresis effect f EP , the electroosmotic effect f EO and the centrifugal force f c . Adjusting the pH value can convert the electrophoresis effect f EP and the electroosmotic effect f EO into each other, so that the electrophoresis effect and the electroosmotic effect reach equilibrium, that is, f EP =f EO .

[0017] Figure 2B It is a schematic diagram of the force analysis of the molecule to be measured when irradiating the nanopore with different optical powers according to an embodiment of the present invention. The external forces applied to the target molecule (i.e., antibody) are mainly determined by f EP , f EO , f c . By adjusting the optical power, the balance between the electrophoresis effect and the electroosmotic effect can be achieved, that is, f EP =f EO .

[0018] Figure 2C It is a series of pulsed sensing signals received through wireless communication according to an embodiment of the present invention. The first current drop and duration (I 1 , t 1 ) related to molecule capture in stage (ii), and the second current drop and duration (I 2 , t 2 ) generated due to molecule migration in stage (iii). The amplitude of the current block I is defined as I 1 +I 2 , and the residence time t d is calculated as t 1 +t 2 .

[0019] Figure 2D It is a schematic diagram of different stages of the movement of molecules through the nanopore according to an embodiment of the present invention.

[0020] Figures 3A to 3E It is precise regulation according to an embodiment of the present invention, including speed ( Figure 3B ), direction ( Figure 3C) Molecular selectivity ( Figure 3D ) and reversible single-molecule sensing ( Figure 3E ) of the parameters of molecular migration. The external forces acting on the target molecule are mainly the electrophoresis effect f EP , electroosmotic effect f EO and centrifugal force f c . Figure 3D In, f CO , f anti and f GFP are the electrophoresis effects acting on three example molecules of green fluorescent protein (GFP)-antibody conjugate, antibody and GFP respectively.

[0021] Figures 4A to 4G are the sensing signals of six example molecules measured at different rotation speeds at their respective pH values in the equilibrium state according to an embodiment of the present invention. Figure 4A shows in detail Figure 4B to detail Figure 4G The relationship between them, including common size (10nm), current (600pA) and time (10ms) scales. β represents the ratio of the length to the diameter of the molecule (i.e., r / R), where r and R are the polar semi-axis and equatorial semi-axis of the molecule respectively. The time ratio (α) is calculated as t 1 / t 2 . Figures 4B to 4G is magnified to show details.

[0022] Figure 5A shows a series of residence time histograms of Au@PEG detected by the device according to an embodiment of the present invention at different rotation speeds.

[0023] Figure 5B is a diagram showing the relationship between the residence time and rotation speed of Au@PEG at different bias voltages according to an embodiment of the present invention.

[0024] Figure 5C is a diagram showing the sensing signal amplitude of Au@PEG at different rotation speeds and bias voltage values according to an embodiment of the present invention.

[0025] Figure 5D shows a series of residence time histograms of EpCAM detected by the device according to an embodiment of the present invention at different rotation speeds.

[0026] Figure 5E is a diagram showing the relationship between the residence time and rotation speed of EpCAM at different bias voltages according to an embodiment of the present invention.

[0027] Figure 5F is a diagram showing the sensing signal amplitude of EpCAM at different rotation speeds and biases according to an embodiment of the present invention.

[0028] Figure 6A Shows the molecular mass and shape distribution according to an embodiment of the present invention. Six molecules can be classified into three categories according to the weight factor zeta (ζ) of their shape and mass, i.e., K' / m, where ζ includes ζ 1 (amylase, Fab), ζ 2 (streptavidin, EpCAM, and BSA), and ζ 3 (Au@PEG), and ζ 1 > ζ 2 > ζ 3 . Where K' is the shape factor determined solely by the ratio β of the molecular length to the diameter, and m is the molecular weight.

[0029] Figure 6B Is a diagram showing the residence time measured at a rotational speed of 2000 rpm according to an embodiment of the present invention.

[0030] Figure 6C Is a diagram showing the residence time measured at a rotational speed of 4000 rpm according to an embodiment of the present invention.

[0031] Figure 6D Is a diagram showing the time ratio measured at a rotational speed of 2000 rpm according to an embodiment of the present invention.

[0032] Figure 6E Is a diagram showing the time ratio measured at a rotational speed of 4000 rpm according to an embodiment of the present invention.

[0033] Figure 7A Is a diagram showing the sensing signal of the antibody EpCAM IgG according to an embodiment of the present invention.

[0034] Figure 7B Is a diagram showing the sensing signal of the antibody-antigen complex EpCAM IgG-EpCAM according to an embodiment of the present invention.

[0035] Figure 7C Shows the longitudinal monitoring of the dissociation of the antibody-antigen complex according to an embodiment of the present invention.

[0036] Figure 7D Is a diagram showing the sensing signal trajectory of Au@PEG nanoparticles in the bimolecular aggregation state according to an embodiment of the present invention.

[0037] Figure 7E Is a diagram showing the sensing signal trajectory of Au@PEG nanoparticles in the trimolecular aggregation state according to an embodiment of the present invention.

[0038] Figure 7F Shows the longitudinal sensing of the aggregation of Au@PEG nanoparticles according to an embodiment of the present invention.

[0039] Figures 8A to 8C Shows the sensing signals of molecules (i.e., PEG 10000 chains) under centrifugal forces applied in different directions. When the centrifugal force is perpendicular to the nanopore ( Figure 8B ), the centrifugal force is perpendicular to the etched sidewall ( Figure 8C ). Figure 8A Shows the details Figure 8B and Figure 8C relationship between, including the scale of amperage (100 pA) and time (0.15 s).

[0040] Figures 9A to 9D Respectively show the selective sensing signals of green fluorescent protein (GFP)-antibody conjugates ( Figure 9B ), antibodies ( Figure 9C ), and GFP ( Figure 9D ) measured under illumination at specific powers of 60 mW, 75 mW, and 110 mW. Figures 9B to 9D Are the equilibrium states of the conjugate, antibody, and GFP, respectively, where the electrokinetic force tends to 0. Figure 9A Shows the selective migration events controlled by adjusting the illumination power.

[0041] Figure 10 Shows the reversible sensing signal of the antibody measured with a reversible capture process: the nanopore provides a baseline current (i); 60 kDa PEG electroosmosis docks onto the nanopore (ii); the centrifugal force promotes the transfer of the molecule (i.e., the antibody) into or out of the nanopore by reversibly changing the direction of the force between forward and reverse (iii, iv, and v). f c is the centrifugal force applied to the antibody, and f eo-peg is the electrophoretic effect applied to the EPG.

[0042] It should be understood that the examples and factors described in the figures are for illustrative purposes only, and the embodiments are not limited to sensing these molecules with specific factors. Those skilled in the art will readily recognize a wide range of application fields, including but not limited to "small molecules", "chain molecules", and "molecular complexes". Detailed Description

[0043] Embodiments provide a nanopore sensing device for single-molecule inertial-kinetic migration and sensing. Some embodiments include: a centrifuge rotor; a centrifuge tube; one or more flow cell modules; a nanopore module including one or more nanopores; a signal detection module; a signal amplifier module; a control module; and a wireless communication module. By kinetically regulating the centrifugal force field while maintaining the balance of electrophoretic and electroosmotic forces in the nanopore by adjusting the pH value of the electrolyte in the nanopore or by using visible light to excite the surface charges on the silicon nanopore, precise regulation of molecular migration parameters such as velocity, direction, and molecular selectivity is provided to optimize the temporal and spatial resolution of molecular sensing with high S / N ratio signal readout. Thus, the conformational sensitivity of chain-like and granular molecules has been determined by programmable migration parameters. Using the provided inertial-force actuated in-tube nanopore device, discrimination of molecular conformation and longitudinal monitoring of morphological changes have been demonstrated by quantifying the residence time and measuring features in the sensing signal trajectory. In addition, programmable migration and selective recognition of individual molecules from complexes composed of multiple molecules have improved the quantification of the single-molecule shape factor.

[0044] To achieve more precise molecular sensing and more accurate molecular actuation, embodiments of the present invention provide a new single-molecule sensing device that incorporates an inertial-force kinetic actuation single-molecule migration method into an in-tube nanopore system, as Figures 1A to 1E shown in the exemplary and non-limiting examples in. Embodiments include a centrifuge tube having a single nanopore within a single flow cell module or multiple nanopores within parallel multiple flow cell modules (e.g., see Figures 1A to 1D ). Each signal detection module can apply a voltage bias to the nanopore and measure the sensing signal (e.g., see Figure 1E ). In some embodiments, the measured signal is first amplified by a signal amplifier module to a sensitivity of at least -1.081 V / nA, which is cascaded to a differential circuit with a circuit noise as low as 0.4 pA (e.g., root mean square at a sampling rate of 50 kHz). Then, the amplified sensing signal is digitized by the control module at a sampling rate of at least 50 kHz before being wirelessly transmitted to an external receiver at a baud rate of 115200 bits per second through the in-tube wireless communication module.

[0045] Embodiments also measure multi-molecule migration signals. Novel silicon nanopore fabrication is also a component of some embodiments. Embodiments of the inertial-kinetic molecular migration method can be applied to a variety of nanopores without external limitations on the nanopore type.

[0046] As Figures 2A to 2DAs shown, in some embodiments, the sensor provides an extended molecular migration time in milliseconds within the nanopore, enabling accurate molecular fingerprinting by measuring the sensing signal received with microsecond resolution via wireless communication. The embodiments can precisely describe the molecular structure by reading the sensing signal. Additionally, the provided nanopore device and / or method provides an adjustable residence time on demand by kinetically modulating the centrifugal force while maintaining the electrophoretic and electroosmotic forces in balance within the nanopore.

[0047] This balance can be achieved by adjusting the pH value of the analyte medium or by utilizing light to modulate the surface charge excited on the silicon nanopore (e.g., see Figure 2A and Figure 2B ). Inertial-kinetic molecular migration provides a series of precise regulations of molecular migration parameters, enabling the device to provide feedback on molecular conformation, with a long residence time and a high signal-to-noise ratio for single-shot readout (e.g., see Figures 3A to 3E ). The single-molecule fingerprinting ability of the provided nanopore device and / or method can sense the mass, shape, and configuration of different molecules and nanoparticles by quantifying the residence time and detecting characteristic sensing signals, including but not limited to bovine serum albumin (BSA), gold nanoparticles coated with polyethylene glycol (Au@PEG), epithelial cell adhesion molecule (EpCAM), EpCAM-antibody complex, streptavidin, and α-amylase.

[0048] Embodiments of the present invention will now be described with reference to exemplary embodiments in conjunction with the accompanying drawings and their various advantages.

[0049] Figures 1A to 1C One embodiment of the present invention shown in

[0050] provides a nanopore sensing device, comprising:

[0051] a centrifuge rotor 1;

[0052] a single or multiple flow cell modules 3 and 5;

[0053] a nanopore module 4 comprising a single or multiple nanopores;

[0054] a signal detection module 6;

[0055] a signal amplifier module 7;

[0056] a control module 8; and

[0057] a wireless communication module 9.

[0058] Figures 1A to 1EA nanopore sensing device according to an embodiment of the present invention is shown. To achieve the controllable migration of molecules, a nanopore module 4 is integrated in the upper and lower flow cells 3 and 5 of the flow cell module to provide inertia-kinetic regulated molecular migration, i.e., inertia-kinetic migration, in a laboratory-scale centrifuge composed of a rotor 1 and a tube 2. The traditional molecular electrokinetic migration behavior is dominated by the electrophoretic and electroosmotic velocities in the nanopore. After introducing a constant bias potential on both sides of the nanopore module 4 through the signal detection module 6, the inertia-kinetic migration of molecules is mainly regulated by the centrifugal force and the superimposed balance force generated by the electrophoretic and electroosmotic effects in the nanopore module 4.

[0059] Inside the tube, several in-tube modules are sequentially connected, including a signal amplifier module 7, an analog-to-digital converter (ADC) and a microcontroller of the control module 8, and a wireless communication module 9, which are used to amplify, detect and wirelessly transmit the sensing signal. In some embodiments, the microcontroller can integrate the ADC to avoid additional wiring noise and other interference sources or signal attenuation. The preamplifier circuit board 7 sets the sensitivity of the circuit, which corresponds to the signal amplitude of proteins and nanoparticles generated in the nanopore. The noise-free cascode and low-noise processing based on the differential amplifier circuit make the preamplifier circuit board 7 an excellent low-level signal detector. The sampling rate of the control module 8 is set (for example, set to 50 kHz) to maintain the conformational sensitivity of the sensing signal with a long dwell time (>1 ms). Here, the conformational sensitivity refers to the shape and statistical characteristics of the signal pulse, which enables the analysis of the types of biomolecules in the solution [ACS Nano, 8, 6, 6425-6430, 2014; Nature Nanotechnology, 16, 2021, 1244-1250].

[0060] When the nanopore module 4 is centrifuged, the centrifugal force can effectively capture individual molecules by overcoming Brownian motion and the potential barrier (ΔU) generated due to molecule-pore interactions and migrate individual molecules through the nanopore. Overcoming ΔU means that biomolecules need to overcome the chemical pore-particle interaction to enter the pore [ACS Nano 2020, 14, 15816-15828]. The measured sensing signal clearly describes three different stages related to the inertia-kinetic migration in the nanopore module 4, including (i) molecules outside the sensing region, (ii) molecules inside the sensing region, and (iii) molecules passing through the nanopore, as Figures 2C to 2D shown. The molecular motion in stages (i) and (ii) is controlled by the centrifugal force and Brownian motion, while the centrifugal force and potential barrier play a dominant role in the molecular behavior in stage (iii).

[0061] Materials and Methods

[0062] All patents, patent applications, provisional applications, and publications referenced or cited herein are hereby incorporated by reference in their entireties, including all figures, to the extent that they are not inconsistent with the explicit teachings of this specification.

[0063] The following are examples of procedures for practicing the present invention. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight and all solvent mixture ratios are by volume.

[0064] Example 1 - Verification of the Inertial-Kinetic-Migration Mechanism

[0065] To confirm the fingerprint resolution detection function of the nanopore sensing device according to an embodiment of the present invention, a device within a tube containing a 15 nm nanopore was used to sense six representative molecules, each having a unique mass and shape, including EpCAM, BSA, antigen-binding fragment (Fab), streptavidin, Au@PEG, and α-amylase. For each molecule, a low molecule concentration of approximately 0.1 nM was used to minimize molecular interactions, and the molecules were simultaneously injected into the flow cell 3 of the device within the tube. Each molecular movement was reflected in the recorded current blockade signal, including the current baseline associated with stage (i), the first current drop and duration (t1) associated with molecular capture in stage (ii), and the second current drop and duration (t2) due to molecular migration in stage (iii). The residence time t d was calculated as t 1 +t 2 . Then, molecular migration sensing was performed at different rotation speeds. The captured current blockade signal pulses indicated that the residence time and time ratio were related to the molecular weight m, molecular shape K', and rotation speed ω, demonstrating distinguishable migration characteristics of individual molecules, as Figures 4A to 4G shown.

[0066] The device was used to test Au@PEG and EpCAM at different bias voltages and rotation speeds, as Figures 5A to 5F shown. For example, a 100 kDa biomolecule was subjected to a centrifugal force of 2.3×10 -6 pN at 2000 rpm and 9.2×10 -6 pN at 4000 rpm. The histogram of the Au@PEG residence time ( Figure 5A ) indicated that both its mean value and standard deviation decreased with an increase in the rotation speed ω during the inertial-kinetic-regulated molecular migration event, and were independent of the bias voltage level ( Figure 5B ). It was found that the signal amplitude ( Figure 5C ) was independent of the rotation speed and increased with an increase in the bias voltage. As for EpCAM ( Figures 5D - 5F) Molecular translocation shows similar characteristics in terms of the rotational speed ω and the relationship with the bias voltage. The residence times of Au@PEG and EpCAM differ by more than an order of magnitude because of their different molecular weights (or masses), even though they have similar sizes of about 5 nanometers. For molecular sensing, the residence time and amplitude of the signal pulse can be advantageously optimized by adjusting the rotational speed and the bias voltage.

[0067] To verify the ability of the nanopore sensing device according to an embodiment of the present invention to characterize molecular mass and shape, the molecules to be measured are first classified into three categories according to the weight factor ζ of shape and mass, as Figure 6A shown. The detection data ( Figures 6B - 6C ) of the nanopore sensing device shows that the residence time decays exponentially with the molecular weight, and the higher the centrifugal rotational speed, the faster the residence time decays. At the same time, since the ζ 1 class of molecules has a larger shape and mass weight factor, its decay rate is slower than that of the ζ 2 class of molecules, as Figures 6B - 6C shown. As for the correlation between α and K', the time ratio decays exponentially with the shape factor, and the results of the ζ 1 class of molecules show a slower decay rate than those of the ζ 2 class of molecules, as Figures 6D - 6E shown.

[0068] During the molecular capture process, the molecular motion is dominated by the competition between the centrifugal force and Brownian diffusion, so it can be described by the Langevin equation.

[0069]

[0070] where v (β) is the molecular velocity, f e (β) = f c is the external force applied to the molecule, and g(t) is the Gaussian noise term generated by the random collision force.

[0071] In addition, based on the Einstein - Smoluchowski relationship, the diffusion coefficient D(β) of the molecule can be described as follows:

[0072]

[0073] where k is the Boltzmann constant, T is the environmental temperature, and f(β) is the viscous resistance coefficient; the molecular diffusion coefficient is highly correlated with the molecular shape K'. Therefore, the capture time t 1 can be calculated using the formula 12πμRK'L / fc, where μ represents the dynamic viscosity coefficient of the molecule, R represents the equatorial semi - axis of the molecule, L represents the sensing length, and f c represents the centrifugal force applied to the molecule. As for the motion of the molecule during the nanopore translocation process (for example, see Figure 2D iii), the translocation time t2 (The time until desorption) is a random variable that depends on the dissociation rate of the bulk.

[0074] Therefore, t 2 can be calculated using a form similar to Eyring, where h represents a force-related factor, t represents the mean of the exponential distribution, f 0 represents the viscous force opposite to the molecular motion caused by f drag1 represents the viscous force opposite to the molecular motion caused by f c k represents the Boltzmann constant, and T represents the ambient temperature. Therefore, the time ratio α and the residence time t d can be calculated using the following equation:

[0075]

[0076]

[0077] Here, m represents the molecular weight, ζ represents the weight factor of shape and mass, ρ represents the distance between the target and the centrifuge rotation axis, and ω represents the rotational speed.

[0078] Example 2 - Longitudinal monitoring results of complex sensing

[0079] To verify the longitudinal monitoring function of the nanopore sensing device according to an embodiment of the present invention, the device is used to detect morphological changes caused by molecular interactions, such as the dissociation of the EpCAM IgG (antibody) and EpCAM (antigen) complex. By comparing the characteristic signal traces of the antibody molecule and the antibody-antigen complex, it is found that the signal of the complex has a ratio of the second peak amplitude to the first peak amplitude (Ip2 / Ip1 ≥ 1.5) greater than that of the antibody (Ip2 / Ip1 ≤ 1.0), as Figures 7A to 7B shown. The characteristics in the signal trace provide a novel and advantageous method for evaluating the binding affinity of the antibody-antigen complex. The dissociation sensing experiment was repeated every 20 minutes for a total of 4 times, indicating that the EpCAM IgG-EpCAM complex gradually dissociates over time at equilibrium, and it was observed that the ratio of the complex to the antibody in the solution gradually decreases over time, as Figure 7C shown.

[0080] The embodiments of the nanopore sensing device provided by the present invention can also be used for longitudinal detection of morphological changes of molecules during molecular aggregation and polymerization processes. To prove this, the initial Au@PEG solution was first treated with ultrasound, and then the aggregation sensing experiment was repeated every 10 minutes for a total of 4 times. The characteristic sensing signals of Au@PEG in single nanoparticles, bimolecular aggregates, and trimolecular aggregates showed single peaks, double peaks, and triple peaks, respectively, as Figures 7D to 7EAs shown. By utilizing these features of signal readout (e.g., calculating the number of peaks of each molecular migration), it was found that the proportion of bimolecular and trimolecular aggregates in the Au@PEG nanoparticle solution gradually increased over time and finally exceeded the proportion of single nanoparticles, as Figure 7F shown. Here, 4.8 nm spherical nanoparticles Au@PEG were synthesized by a kinetically controlled seed growth method. First, 3.5 nm gold seeds were synthesized by injecting chloroauric acid into a mixed solution of sodium citrate and tannic acid at 70 °C. Subsequently, the size of the gold seeds was increased to 4.8 nm by diluting the seed solution and injecting aliquots of the gold precursor [Chem. Mater. 28, 1066 - 1075 (2016)].

[0081] Example 3 - Programmable Migration Direction

[0082] To verify the function of the nanopore sensing device according to the embodiments of the present invention for controlling the migration direction of molecules, the device was used to detect chain molecules, such as PEG chains, and achieved an extension of the molecular residence time by hundreds of times. 10 kDa PEG was tested while applying centrifugal forces in different directions, as Figures 8A to 8C shown. When the direction of the centrifugal force was adjusted from perpendicular to the nanopore to perpendicular to the etched sidewall, the residence time of the sensing signal was extended (from <100 msec to >200 msec), and the signal-to-noise ratio was optimized (from <1.5 to >4.5). Compared with traditional nanopore devices such as protein nanopores, the inertial dynamics method showed more than an order of magnitude improvement in weight resolution (i.e., 0.1 kDa) when measuring PEG.

[0083] Example 4 - Selective Molecular Migration

[0084] To confirm the ability of the nanopore sensing device according to the embodiments of the present invention to selectively migrate and characterize molecules, the device was used to detect a mixture of GFP - antibody conjugates, antibodies, and GFP. By adjusting the illumination power to 60 mW (GFP - antibody conjugate), 75 mW (antibody), and 110 mW (GFP) respectively, an equilibrium state of these three molecules was achieved (see Figure 9A to Fig. 9D). Here, the electrokinetic forces applied to the molecules tended to an equilibrium state. By comparing the fingerprint signals obtained for the conjugate, antibody, and GFP in different equilibrium states, it was found that most molecular inertial dynamic migrations occurred at the unique equilibrium states of different molecules. Therefore, the selectivity of molecular migration was achieved by adjusting the intensity of the electroosmotic effect of the nanopore through illumination intensity.

[0085] Example 5 - Reversible Single - Molecule Sensing

[0086] To verify the reversible monitoring function of the nanopore sensing device according to an embodiment of the present invention, the device is used to detect signal changes associated with different stages of the reversible movement of molecules in and out of the nanopore module 4, including (i) applying a voltage across the nanopore module 4 to establish a baseline current. (ii) A charged and permeable nanostructure (i.e., 60 kDa PEG of 15 nm nanopore) is driven electrophoretically to the nanopore 4 to dock at the entrance of the nanopore 4, thereby closing the nanocavity. (iii) A single molecule (i.e., antibody) can be captured in the closed nanopore 4 by forward centrifugal force while the antibody is in equilibrium. (iv) When the centrifugal force becomes reverse, the single molecule can be driven out of the nanopore 4 again. (v) The direction of the centrifugal force is further adjusted to be forward so that the single molecule is recaptured in the nanopore 4. Therefore, after the nanopore 4 is covered with a charged and permeable nanostructure, reversible single-molecule sensing is achieved by adjusting the direction of the centrifugal force.

[0087] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and those skilled in the art can make various modifications or changes accordingly, which should be included within the spirit and scope of this application and the scope of the appended claims. Additionally, any element or limitation of any invention or its embodiment disclosed herein can be combined with any and / or all other elements or limitations (individually or in any combination) disclosed herein or of any other invention or its embodiment, and all such combinations are within the scope of the present invention, but not limited thereto.

[0088] Exemplary embodiments

[0089] Example 1. A nanopore sensing device, comprising:

[0090] A centrifuge rotor (1);

[0091] A centrifuge tube (2); and

[0092] A nanopore module, comprising a single or multiple nanopores (4) located within the centrifuge tube (2).

[0093] Example 2. The nanopore sensing device according to Example 1, comprising:

[0094] One or more flow cell modules (3) and (5), separated by the nanopore (4); and

[0095] A signal detection module (6) spanning the nanopore (4).

[0096] Example 3. The nanopore sensing device according to Example 2, comprising:

[0097] A signal amplifier module (7);

[0098] A control module (8); and

[0099] Wireless communication module (9).

[0100] Example 4. The nanopore sensing device according to Example 3, wherein the signal amplifier module (7), the control module (8), and the wireless communication module (9) are respectively installed inside the centrifuge tube (2) and are configured and adapted to rotate with the centrifuge tube (2).

[0101] Example 5. The nanopore sensing device according to Example 4, wherein the centrifuge rotor (1) is configured and adapted to provide different rotational speeds and directions to the centrifuge tube (2).

[0102] Example 6. The nanopore sensing device according to Example 4, wherein the signal detection module (6) across the nanopore module (4) is configured and adapted to provide different voltage potentials.

[0103] Example 7. The nanopore sensing device according to Example 6, wherein the signal amplifier module (7) is configured and adapted to detect the sensing signal flowing through the detection module (6) across the nanopore module (4) over time.

[0104] Example 8. The nanopore sensing device according to Example 7, wherein the control module (8) is configured and adapted to form a digital representation of the signal detected by the signal amplifier module (7).

[0105] Example 9. The nanopore sensing device according to Example 1, wherein the nanopore structure has a wide range of applications.

[0106] Example 10. The nanopore sensing device according to Example 9, wherein the nanopore has an adjustable pore size.

[0107] Example 11. The nanopore sensing device according to Example 10, wherein the adjustable pore size is adjustable in the nanometer range.

[0108] Example 12. A method for inertial-kinetic migration and sensing of single molecules, comprising:

[0109] Providing a nanopore sensing device, the nanopore sensing device including a nanopore, a driving circuit, a sensing circuit, and a communication device, which are respectively located inside a centrifuge tube;

[0110] Applying a potential across the nanopore through the driving circuit;

[0111] Applying a centrifugal force across the nanopore through the operation of the centrifuge tube;

[0112] Driving a molecule to be measured through the nanopore by inertial-kinetic migration; and

[0113] Capturing a digital representation of the sensing signal passing through the nanopore over time through the sensing circuit.

[0114] Example 13. The method according to Example 12, comprising:

[0115] Transmitting a digital representation of the sensing signal through a communication device; and

[0116] Receiving the digital representation of the sensing signal at a location outside the centrifuge tube.

[0117] Example 14. The method according to Example 12, comprising:

[0118] Extracting features from the digital representation of the sensing signal.

[0119] Example 15. The method according to Example 14, wherein the features are selected from the group comprising: the amplitude of the signal pulse amplitude; the dwell time of the signal pulse; local peaks in one of the above; the mean, median or standard deviation of any series of the above; any of the above decay times; the variation of one of the above; the difference between two of the above; and any of the above counts.

[0120] Example 16. The method according to Example 14, wherein the features include dwell time or pulse amplitude.

[0121] Example 17. The method according to Example 14, wherein the features include the ratio of the second peak amplitude to the first peak amplitude.

[0122] Example 18. The method according to Example 14, wherein the features include the count of the number of peaks migrated by each molecule.

[0123] Example 19. A nanopore sensing device, comprising:

[0124] A centrifuge rotor (1);

[0125] A centrifuge tube (2);

[0126] A nanopore module, comprising a single or multiple nanopores (4) located within the centrifuge tube (2);

[0127] One or more flow cell modules (3) and (5), separated by the nanopore module (4);

[0128] A signal detection module (6), spanning the nanopore (4);

[0129] A signal amplifier module (7);

[0130] A control module (8); and

[0131] A wireless communication module (9);

[0132] Among them, the signal amplifier module (7), the control module (8), and the wireless communication module (9) are respectively installed in the centrifuge tube (2) and are configured and adapted to rotate with the centrifuge tube (2);

[0133] Among them, the centrifuge rotor (1) is configured and adapted to provide a rotational speed between 1,000 and 4,000 revolutions per minute, i.e., rpm, to the centrifuge tube (2);

[0134] Among them, the nanometer pore structure has a wide range of applications;

[0135] Among them, the nanopore has an adjustable pore size; and

[0136] Among them, the adjustable pore size is adjustable within the nanometer range.

[0137] Example 20. The nanopore sensing device according to Example 19, wherein the signal detection module (6) spanning the nanopore module (4) is configured and adapted to provide a voltage potential between 0.3 volts, i.e., V, and 0.6 V;

[0138] Among them, the preamplifier circuit board (7) is configured and adapted to detect the sensing signal flowing through the signal detection module (6) spanning the nanopore module (4) over time; and

[0139] Among them, the microcontroller with the control module (8) is configured and adapted to form a digital representation of the sensing signal detected by the signal amplifier module (7).

Claims

1. A nanopore sensing device, comprising: a centrifuge rotor (1); a centrifuge tube (2); and a nanopore module, including single or multiple nanopores (4) located within the centrifuge tube (2).

2. The nanopore sensing device according to claim 1, comprising: single or multiple flow cell modules (3) and (5), separated by the nanopore (4); and a signal detection module (6) spanning the nanopore (4).

3. The nanopore sensing device according to claim 2, comprising: a signal amplifier module (7); a control module (8); and a wireless communication module (9).

4. The nanopore sensing device according to claim 3, wherein the signal amplifier module (7), the control module (8), and the wireless communication module (9) are respectively installed within the centrifuge tube (2) and are configured and adapted to rotate with the centrifuge tube (2).

5. The nanopore sensing device according to claim 4, wherein the centrifuge rotor (1) is configured and adapted to provide different rotational speeds and directions to the centrifuge tube (2).

6. The nanopore sensing device according to claim 4, wherein the signal detection module (6) spanning the nanopore module (4) is configured and adapted to provide different voltage potentials.

7. The nanopore sensing device according to claim 6, wherein the signal amplifier module (7) is configured and adapted to detect the sensing signal flowing through the detection module (6) spanning the nanopore module (4) over time.

8. The nanopore sensing device according to claim 7, wherein the control module (8) is configured and adapted to form a digital representation of the signal detected by the signal amplifier module (7).

9. The nanopore sensing device according to claim 1, wherein the nanopore structure has a wide range of applications.

10. The nanopore sensing device according to claim 9, wherein the nanopore has an adjustable pore size.

11. The nanopore sensing device according to claim 10, wherein the adjustable pore size can be adjusted within the nanometer range.

12. A method for inertial - kinetic migration and sensing of single molecules, comprising: providing a nanopore sensing device, the nanopore sensing device including a nanopore, a drive circuit, a sensing circuit, and a communication device, which are respectively located within a centrifuge tube; applying a potential across the nanopore through the drive circuit; applying a centrifugal force across the nanopore through the operation of the centrifuge tube; driving a molecule to be measured through the nanopore by inertial - kinetic migration; and capturing a digital representation of the sensing signal passing through the nanopore over time through the sensing circuit.

13. The method according to claim 12, comprising: transmitting the digital representation of the sensing signal through the communication device; and receiving the digital representation of the sensing signal at a location outside the centrifuge tube.

14. The method according to claim 12, comprising: extracting features from the digital representation of the sensing signal.

15. The method according to claim 14, wherein The feature is selected from the group consisting of: the amplitude of the signal pulse amplitude; the dwell time of the signal pulse; local peaks in one of the above; the mean, median, or standard deviation of any of the above series; any of the above decay times; the variation in one of the above; the difference between any two of the above; and any of the above counts.

16. The method according to claim 14, wherein, the feature includes the dwell time or the pulse amplitude.

17. The method according to claim 14, wherein, the feature includes the ratio of the second peak amplitude to the first peak amplitude.

18. The method according to claim 14, wherein, the feature includes the count of the number of peaks for each molecular translocation.

19. A nanopore sensing device, comprising: a centrifuge rotor (1); a centrifuge tube (2); a nanopore module including a single or multiple nanopores (4) located within the centrifuge tube (2); single or multiple flow cell modules (3) and (5) separated by the nanopore module (4); a signal detection module (6) spanning the nanopore (4); a signal amplifier module (7); a control module (8); and a wireless communication module (9); wherein the signal amplifier module (7), the control module (8), and the wireless communication module (9) are respectively mounted within the centrifuge tube (2) and are configured and adapted to rotate with the centrifuge tube (2); wherein the centrifuge rotor (1) is configured and adapted to provide a rotational speed between 1,000 and 4,000 revolutions per minute (rpm) to the centrifuge tube (2); wherein the nanopore structure has a wide range of applications; wherein the nanopore has an adjustable pore size; and wherein the adjustable pore size can be adjusted within the nanometer range.

20. The nanopore sensing device according to claim 19, wherein, the signal detection module (6) spanning the nanopore module (4) is configured and adapted to provide a voltage potential between 0.3 volts (V) and 0.6 V; wherein the preamplifier circuit board (7) is configured and adapted to detect the sensing signal flowing over time through the signal detection module (6) spanning the nanopore module (4); and wherein the microcontroller with the control module (8) is configured and adapted to form a digital representation of the sensing signal detected by the signal amplifier module (7).

Images