Single-molecule, real-time, label-free dynamic biosensing using nanoscale magnetic field sensors

Abstract

Disclosed herein are devices, systems, and methods for monitoring single-molecule biological processes using magnetic sensors and magnetic particles (MNPs). The MNPs are conjugated to biopolymers (e.g., nucleic acids, proteins, etc.), and the movement of the MNPs is detected and / or monitored using magnetic sensors. Because the MNPs are small (e.g., comparable in size to the molecule being monitored) and tethered to the biopolymer, changes in the volume of the MNPs' Brownian motion in solution can be monitored to monitor the movement of the MNPs, and by inference, the movement of the tethered biopolymer. The magnetic sensors are small (e.g., nanoscale or on the order of the size of the MNPs and biopolymers), and can be used to detect even small changes in the position of the MNPs within the sensing region of the magnetic sensors.

Description

[Background technology]

[0001] background The ability to quantify interactions between biomolecules is of interest for a variety of applications, including diagnosis, screening, disease staging, forensic analysis, pregnancy testing, drug development and testing, and scientific and medical research. Examples of measurable features of biomolecular interactions include interaction affinity (e.g., how strongly molecules bind / interact) and reaction rate (e.g., the rate at which molecular association and dissociation occur).

[0002] Conventional enzyme-linked immunosorbent assay (ELISA) systems are analog systems that require large volumes of reaction product to be ultimately diluted, and require millions of enzyme labels to produce a detectable signal using conventional plate readers. Consequently, the sensitivity of conventional ELISA is limited to the picomolar (pg / mL) range and above.

[0003] In contrast to ELISA systems, single-molecule systems are inherently digital because they provide a separate signal for each molecule that can be detected and counted. Single-molecule systems have the advantage that it is easier to determine the presence or absence of a signal than to detect its absolute amount or amplitude. In other words, counting is easier than integrating them.

[0004] In recent years, there has been growing interest in the detection of single molecules. For example, the COVID-19 pandemic has exposed cancer patients to a higher-than-usual risk due to their increased susceptibility to viral infections after chemotherapy, stem cell transplantation, or surgery. Another example is the need for highly sensitive detection of viruses and pathogens, such as detecting COVID-19 antibodies or human SARS-CoV-2 antibodies. Another example of an application where single-molecule detection can be beneficial is single-molecule immunoassays to provide simple and highly sensitive detection of protein biomarkers.

[0005] In some applications, the detection of single molecules has become possible. For example, the use of tethered particle motion (TPM) technology has made it possible to detect the binding of a single biomolecule to a receptor immobilized on the surface of a sensing device. In TPM, one end of a biopolymer (e.g., DNA, RNA, etc.) is immobilized on a solid support, thereby creating a "tethered biopolymer," to which small particles (e.g., micrometer or nanometer size) are bound. In solution, the tethered biopolymer and bound particles move due to constrained Brownian motion (random motion of particles suspended in a medium). The volume occupied by the tethered biopolymer (and bound particles) is limited and depends on the size and shape of the tethered biopolymer. Enzymes that directly interact with the biopolymer can alter the structure of the biopolymer at any given time. For example, in the case of DNA and RNA, the volume occupied by the bound particles changes in response to DNA deformation (e.g., DNA loop formation or DNA elongation). By observing and interpreting changes in particle position as a function of time, it is possible to describe, for example, the dynamics and biochemical dynamics of interactions between biopolymers and enzymes in solution.

[0006] Tethered biopolymers can be nucleotide sequences, such as DNA fragments. The binding event typically alters the molecular dynamics of the receptor. Before the complementary nucleotide is incorporated, the DNA fragment may adopt a coiled or U-shaped (loop-shaped) conformation (e.g., due to the presence of (partial) palindromes in the nucleotide sequence), and then, upon integration of the complementary nucleotide, it may adopt a more linear or elongated conformation. This conformational change affects the volume of Brownian motion in which the tethered biopolymer resides. In TPM, volume changes can be detected by binding particles (sometimes called labels) to the receptor and observing the particle's movement using optical techniques.

[0007] Data acquisition in TPM systems typically involves tracking and recording nanoscale variations in particle mean velocity and range of motion caused by localized changes in the microenvironment using high-resolution, high-speed video microscopy. This single-molecule analysis technique is employed, for example, for dynamic in vitro monitoring of DNA-protein interactions and for detecting biochemically induced conformational changes in proteins, DNA, and RNA.

[0008] Because TPM relies on its ability to resolve small variations in stochastic motion patterns, image contrast must be sufficient to enable particle tracking and subsequent analysis, and the frame acquisition rate must be sufficiently high. State-of-the-art TPM systems can optically track nanoscale particles coupled to short (e.g., about 50 nm) tethers with localization accuracy of 1-2 nm. While the high resolution is impressive, the number of particles that can be tracked and analyzed simultaneously within a small field of view is limited to a few hundred. Thus, the throughput of such systems is limited. Increasing the field of view to enable monitoring of 10,000 nanoparticles reduces localization accuracy to over 100 nm. This limitation, coupled with the technical complexity of high-throughput real-time motion tracking at the nanoscale, has so far limited the use of TPM to the realm of academic scientific interest, hindering its widespread use in commercial applications such as diagnostics and drug discovery.

[0009] Particle size plays a crucial role in TPM measurements. Larger particles are easier to observe and track than smaller particles, but their stochastic motion is only slightly affected by single-molecule processes due to the large size difference between the particle and the receptor. Furthermore, if large tethered particles are close to a solid surface (e.g., where the receptor is bound), tensile forces are generated in the biopolymer, altering its biophysical properties and potentially causing significant fluctuations in the binding equilibrium if the molecule is involved in the biomarker binding reaction. Therefore, to accurately reproduce the in vivo process, it is desirable to make the tethered particles as small as possible. The stochastic motion patterns of smaller particles are also more sensitive to perturbations caused by the binding of individual biomolecules. However, the problem with small particles is that they are more difficult to observe using optics. Strongly scattering 10 nm gold nanoparticles confined within a two-dimensional biomembrane have been observed and optically tracked. Larger sizes (typically larger than 40 nm in diameter) are preferred for reliable tracking when particles are tethered to a surface in a biopolymer and can move in and out of the focal plane. However, these dimensions make the particles considerably larger than the molecules involved in many biomedically relevant processes. Since the amount of light scattering at these length scales is proportional to the sixth power of the diameter, further reducing the particle size to match molecular dimensions would make it untrackable even with the most advanced optics available today.

[0010] Therefore, improved single-molecule devices, systems, and methods are needed to monitor and / or quantify interactions between biomolecules. [Overview of the project]

[0011] overview This summary represents non-limiting embodiments of the present disclosure.

[0012] This specification discloses apparatus, systems, and methods for monitoring single-molecule processes using magnetic sensors. In some embodiments, magnetic particles (e.g., magnetic nanoparticles), referred herein as MNPs, are attached to a biopolymer (e.g., nucleic acids, proteins, etc.), also called a tether, to detect the movement of the MNPs. For example, the binding of individual molecules, antibody / antigen reactions, and / or conformational changes of proteins or nucleic acids can be detected by observing, tracking, or tracing the position and / or movement of the MNPs using a magnetic sensor. The MNPs are small (e.g., their size is comparable to the size of the molecule being monitored) and tethered to the biopolymer. The volume of Brownian motion of the MNPs in solution changes due to collisions between the MNPs and molecules in the solution, thereby changing the position of the MNPs, and the movement of the MNPs, and inference, allows for observation and / or monitoring of the tethered biopolymer. Changes in the position and / or movement of the MNPs can be inferred from changes in the signal obtained from the magnetic sensor. For example, analysis of the autocorrelation function or power spectral density of the signal obtained from the magnetic sensor can reveal the presence, position, and / or movement of the MNPs.

[0013] Magnetic sensors (e.g., nanoscale, or having a size comparable to the size of MNPs and / or biopolymers) can be used to detect even small changes in the position of MNPs within the detection area of ​​the magnetic sensor. The baseline response (e.g., signal) of the magnetic sensor can be determined in the absence of MNPs, and then, after MNPs bind to the biopolymer within the detection area of ​​the magnetic sensor, the signal provided by the magnetic sensor is a superposition of the Brownian motion of the MNPs and the baseline sensor response. Thus, the effect of MNPs moving according to a random process is to add noise to the baseline sensor response. By detecting and / or analyzing the noise contribution from MNPs in the sensor signal in either or both the time domain and / or frequency domain (e.g., by detecting fluctuations around the mean, examining / processing / analyzing the autocorrelation function or power spectral density, etc.), conclusions can be drawn about the presence, position, and / or movement of MNPs. In this way, MNPs can serve as reporters for biopolymer activity (e.g., conformational changes).

[0014] Because the disclosed apparatus, systems, and methods are imaging-independent, the MNP can be substantially smaller than that used in TPM systems, thereby providing higher resolution and enabling higher throughput from an apparatus of a selected size. Furthermore, magnetic sensors and MNPs can be used to reliably detect nanoscope motion with high precision (e.g., motion on the order of a few nanometers). The disclosed apparatus, systems, and methods can be used for a variety of single-molecule applications, including but not limited to diagnostics, screening, disease staging, forensic analysis, pregnancy testing, drug development and testing, immunoassays, nucleic acid sequencing, and scientific and medical research. They offer potentially higher throughput and higher sensitivity and accuracy than conventional TPM or conventional ELISA methods that rely on optics. [Brief explanation of the drawing]

[0015] The objectives, features, and advantages of the present disclosure will become readily apparent from the following description of specific embodiments in conjunction with the accompanying drawings.

[0016] [Figure 1A] Schematic diagram of the nanoscale monitoring of the movement of MNPs bound to biopolymers according to some embodiments. [Figure 1B] An example of a recorded sensor signal according to some embodiments is shown. [Figure 2A] Examples of four reversible biomolecular single-molecule processes that affect the MNP velocity and motility pattern according to some embodiments are shown. [Figure 2B] Examples of four reversible biomolecular single-molecule processes that affect the MNP velocity and motility pattern according to some embodiments are shown. [Figure 2C] Examples of four reversible biomolecular single-molecule processes that affect the MNP velocity and motility pattern according to some embodiments are shown. [Figure 2D] Examples of four reversible biomolecular single-molecule processes that affect the MNP velocity and motility pattern according to some embodiments are shown. [Figure 3] A part of a magnetic sensor according to some embodiments is shown. [Figure 4A] The resistance of a magnetoresistive (MR) sensor that can be used according to some embodiments is shown. [Figure 4B] The resistance of a magnetoresistive (MR) sensor that can be used according to some embodiments is shown. [Figure 5A] A spin torque oscillator (STO) sensor that can be used according to some embodiments is shown. [Figure 5B] The experimental response of an STO under exemplary conditions is shown. [Figure 5C] A short nanosecond field pulse of an STO that can be used according to some embodiments is shown. [Figure 5D]This shows short nanosecond field pulses of STO that can be used according to several embodiments. [Figure 6] This is a partial diagram of an exemplary readout head, including a magnetic sensor used for perpendicular magnetic recording (PMR) applications. [Figure 7] Figure 7A shows a magnetic sensor without an MNP in its vicinity, according to several embodiments. Figure 7B shows a magnetic sensor with an MNP located directly above it, according to several embodiments. Figure 7C shows a magnetic sensor with an MNP offset laterally, according to several embodiments. [Figure 8] The results of nanomagnetic simulations of exemplary magnetic sensors, according to several embodiments, where MNPs are present at various positions relative to the magnetic sensor, are shown. [Figure 9A] This is a plan view scanning electron microscope (SEM) image of an exemplary magnetic sensor in which the MNP is located within its detection area, according to several embodiments. [Figure 9B] Figure 9A shows the behavior of an exemplary magnetic sensor according to several embodiments. [Figure 9C] Figure 9A shows the behavior of an exemplary magnetic sensor according to several embodiments. [Figure 10A] This document presents exemplary models for analyzing the behavior of MNPs according to several embodiments. [Figure 10B] This is a graphical representation of a single particle diffusing under a harmonic potential applied by a DNA strand. [Figure 11A] This presents a thought experiment. [Figure 11B] This presents a thought experiment. [Figure 12A] This shows an exemplary magnetic sensor according to several embodiments. [Figure 12B] The expected noise power spectral density (PSD) of an exemplary magnetic sensor is plotted against the Lorentz function characterizing the PSD of the restricted Brownian motion of the MNP. [Figure 13] This is a diagram illustrating an experiment conducted by the inventors of this invention. [Figure 14] The measured PSD of the three magnetic sensors tested is shown. [Figure 15A] This shows the test results investigating the effect of magnetic sensor bias voltage. [Figure 15B] This shows the test results investigating the effect of magnetic sensor bias voltage. [Figure 15C] This shows the test results investigating the effect of magnetic sensor bias voltage. [Figure 15D] This shows the test results investigating the effect of magnetic sensor bias voltage. [Figure 15E] This shows the test results investigating the effect of magnetic sensor bias voltage. [Figure 16] This shows a one-dimensional model that includes the force component from a magnetic sensor. [Figure 17] Figure 17A shows three states of a system according to several embodiments. Figure 17B shows three states of a system according to several embodiments. Figure 17C shows three states of a system according to several embodiments. [Figure 18A] The following examples illustrate recorded current fluctuations of two exemplary magnetic sensors and their corresponding autocorrelation functions, according to several embodiments. [Figure 18B] The following examples illustrate recorded current fluctuations of two exemplary magnetic sensors and their corresponding autocorrelation functions, according to several embodiments. [Figure 18C] The following examples illustrate recorded current fluctuations of two exemplary magnetic sensors and their corresponding autocorrelation functions, according to several embodiments. [Figure 19A] This block diagram shows the components of an exemplary monitoring system according to several embodiments. [Figure 19B] This shows some exemplary monitoring systems according to several embodiments. [Figure 19C] This shows some exemplary monitoring systems according to several embodiments. [Figure 19D]This shows some exemplary monitoring systems according to several embodiments. [Figure 19E] The magnetic sensor patterns of sensor arrays according to several embodiments are shown. [Figure 20] This is a flowchart illustrating an exemplary method for detecting the movement of a tethered MNP (Mobile Number Portability) device, according to several embodiments. [Figure 21] The following are some of the components involved in multiplex magnetic digital homogeneous non-enzymatic (HoNon) ELISA according to several embodiments. [Figure 22A] This document shows some exemplary procedures for multiplexed magnetic digital HoNon ELISA according to several embodiments. [Figure 22B] This document shows some exemplary procedures for multiplexed magnetic digital HoNon ELISA according to several embodiments. [Figure 23-1] Further steps of an exemplary procedure for a multiplexed magnetic digital HoNon ELISA according to several embodiments are shown. [Figure 23-2] Further steps of an exemplary procedure for a multiplexed magnetic digital HoNon ELISA according to several embodiments are shown. [Figure 24A] The addition of a complex biological solution containing multiple biomarkers according to several embodiments is shown. [Figure 24B] This is a description of how the sensor array appears after the addition of a complex biological solution containing multiple biomarkers, according to several embodiments. [Figure 25] This describes how the binding of biomarkers can be detected from the noise PSD detected by a particular magnetic sensor, according to several embodiments. [Figure 26] This is a flowchart illustrating a method of using a magnetic sensor array according to several embodiments.

[0017] For ease of understanding, the same reference numerals are used to indicate identical elements common to the figures, where possible. It is intended that elements disclosed in one embodiment can be usefully used in other embodiments without specific description. Furthermore, the description of an element in the context of one drawing is applicable to other drawings showing that element. [Modes for carrying out the invention]

[0018] Detailed explanation The stochastic motion of freely diffused or tethered particles embedded in biological systems reveals a wealth of information. Statistical analysis of particle motion can facilitate the understanding of important in vivo processes through their in vitro results. While tracking small, strongly scattering particles of about 10 nm in size is a powerful tool for studying biological membranes, tracking tethered particles reveals a much wider range of single-molecule behavior. TPM experiments use biopolymers (e.g., DNA, RNA, proteins) with one end fixed to a solid surface and the other end bound to a particle to monitor various biophysical and biochemical processes; however, the throughput and accuracy of conventional TPM systems are limited because they rely on optical techniques for tracking particles.

[0019] This specification discloses apparatus, systems, and methods for dynamically detecting biochemically induced changes in the motion patterns of tethered nanoparticles, without imaging. Instead, embodiments disclosed herein use magnetic sensors and monitor the response of those magnetic sensors to detect limited diffusion of magnetic particles as the tethered magnetic particles move probabilistically within or outside the detection area of ​​each magnetic sensor. The magnetic sensor may be, for example, a nanoscale magnetic field sensor (MFS). The detected response or characteristic of the magnetic sensor may be, for example, a detected tunnel current, voltage, or resistance in the time or frequency domain, or any other characteristic of the detectable magnetic sensor. The detection area of ​​the magnetic sensor may be, for example, about 10 5 nm 3 From 5x105 nm 3 It can have a volume of .

[0020] Magnetic particles can be, or may include, magnetic nanoparticles (MNPs), such as molecules, superparamagnetic nanoparticles, or ferromagnetic particles. As will be understood by those skilled in the art, magnetic nanoparticles are generally considered to be particles of a material with a diameter between 1 and 100 nanometers (nm). Magnetic particles may also be nanoparticles with high magnetic anisotropy. Examples of magnetic particles with high magnetic anisotropy include, but are not limited to, Fe3O4, FePt, FePd, and CoPt. In some applications involving nucleotides, magnetic particles can be synthesized and coated with, for example, SiO2. See, for example, M. Aslam, L. Fu, S. Li, and VPDravid, "Silica encapsulation and magnetic properties of FePt nanoparticles," Journal of Colloid and Interface Science, Volume 290, Issue 2, October 15, 2005, pp. 444-449.

[0021] Magnetic particles can be, for example, organometallic compounds, or may contain organometallic compounds. As understood, organometallic compounds are any member of the class of substances that contain at least one metal-carbon bond in which carbon is part of an organic group. Examples of organometallic compounds include Gilman reagents (containing lithium and copper), Grinal reagents (containing magnesium), tetracarbonyl nickel, ferrocene (containing transition metals), organolithium compounds (e.g., n-butyllithium (n-BuLi)), organozinc compounds (e.g., diethylzinc (Et2Zn)), organotin compounds (e.g., tributyltin hydride (Bu3SnH)), organoborane compounds (e.g., triethylborane (Et3B)), organoaluminum compounds (e.g., trimethylaluminum (Me3Al)), and the like.

[0022] The magnetic particles may be, for example, charged molecules or any other functional molecular groups that can be detected by a nanoscale magnetic sensor, or may include such groups. In other words, if a magnetic sensor can detect the presence of candidate magnetic particles and the candidate magnetic particles can be bound to a biopolymer of interest, then the candidate magnetic particles are suitable for use in the apparatus, systems, and methods described herein.

[0023] While magnetic particles used in many applications are likely to be nanoparticles, being comparable in size to the observed biopolymers, the systems, apparatus, and methods described herein generally apply to magnetic particles. Therefore, for convenience, the abbreviation “MNP” is used herein, and it should be understood that “MNP” can generally refer to magnetic particles. Accordingly, unless otherwise indicated by context, any disclosure herein referring to or illustrating MNPs is not necessarily limited to nanoparticles. Similarly, while MNPs are expected to be superparamagnetic, this disclosure is not limited to use with superparamagnetic MNPs.

[0024] Figures 1A and 1B illustrate the principle of nanoscale monitoring of MNP movement using a magnetic sensor, according to several embodiments. As shown in Figure 1A, the MNP 102 is tethered to the solid surface 117 of the monitoring device by a biopolymer 101 (e.g., ssDNA, dsDNA, RNA, protein, etc.). The biopolymer 101 is sometimes referred to as the "tether". Due to interactions with molecules in the surrounding fluid, the MNP 102 moves at a certain average distance from the magnetic sensor 105. <r>Within the constrained motion region 203, which is the surrounding volume, it undergoes stochastic (random) motion represented by arrow 103 in Figure 1A. MNP 102 moves within or outside the detection region 206 of the magnetic sensor 105. In some biosensing applications, the detection region 206 is, for example, about 10 5 nm 3 From approximately 5 x 10 5 nm 3 The volume of the sensing region 206 can be selected to suit a particular application and may be larger or smaller than these values. Depending on the design of the magnetic sensor 105 (e.g., its sensitivity), the bias voltage applied to the magnetic sensor 105, the characteristics of the MNP 102 (e.g., its size), the characteristics of the biopolymer 101 (e.g., its length), and the position where the biopolymer 101 is tethered to the surface 117 relative to the magnetic sensor 105, the constrained motion region 203 and the sensing region 206 may substantially overlap or be offset, as shown in the example in Figure 1A. Similarly, the volumes of the constrained motion region 203 and the sensing region 206 may be the same or different. In the example shown in Figure 1A, the constrained motion region 203 is larger than the sensing region 206 and is offset laterally by ρ.

[0025] Figure 1B shows an example of a recorded sensor signal 207 according to several embodiments. In this example, the sensor signal 207 is recorded as a statistically steady variation of several detectable characteristics of the magnetic sensor 105, which may be, for example, a measured current, voltage, resistance, oscillation frequency, phase noise, frequency noise, or any other characteristic of the magnetic sensor 105 indicating a detected change in the magnetic environment of the magnetic sensor 105 (e.g., within the sensing region 206, due to the presence, absence, and / or movement of MNP 102), as will be further described below. One advantage of using the magnetic sensor 105 is that the MNP 102 can be considerably smaller than the particles used in TPM systems that rely on optical tracking. In some embodiments, for example, the MNP 102 has biomolecular dimensions (e.g., its size can be about 5 nm or less).

[0026] To enable the detection of MNP102, the response of the magnetic sensor 105, represented by the sensor signal 207, should change due to the mobility of MNP102, which is affected by interactions with individual single molecules (e.g., the surrounding solution). Therefore, it is desirable that the mobility of MNP102 is small enough that it is not affected by other molecules. The sensor signal 207 (e.g., the noise component of the sensor signal 207 due to the movement of MNP102) should change when a biomolecule of comparable size binds to the molecule bound to MNP102, or when the bound molecule (biopolymer 101) changes its conformation, for example, as described later in the explanations of Figures 18A, 18B, and 18C. In both cases, the effective hydrodynamic radius of the tethered MNP102 changes, and its statistical velocity and range of motion also change. Therefore, both the amplitude and noise of the sensor signal 207 should change both when the tethered MNP 102 is immobilized on or near the surface of the magnetic sensor 105 by specific target binding, and when the conformational state of the tether / biopolymer 101 (e.g., dsDNA, ssDNA, RNA, protein) changes.

[0027] The systems, apparatus, and methods disclosed herein can be used to detect and / or monitor various changes in biomolecular processes, such as, for example, loop formation (connection and cleavage), protein folding and unfolding, antibody / antigen interactions, and their conformational dynamics. Figures 2A, 2B, 2C, and 2D show examples of four reversible single-molecule biomolecular processes affecting the velocity and migration range pattern of MNP 102, according to several embodiments. Each of Figures 2A, 2B, 2C, and 2D shows a magnetic sensor 105 together with a biopolymer 101, with one end of the biopolymer bound to the surface 117 of a monitoring device near the magnetic sensor 105 (e.g., at a binding site 116 discussed below), and the other end of the biopolymer bound to MNP 102. Figures 2A and 2C show exemplary antibody-antigen reactions, and Figures 2B and 2D show exemplary conformational changes. Figure 2A shows that binding of large biomolecules such as proteins, DNA, or RNA to MNP102 increases the mass of MNP102 and its effective hydrodynamic radius, causing a change in detectable limited diffusion. (As will be described in more detail below, binding of molecules of comparable size to MNP102 can be detected by detecting changes in the corner frequencies of the Lorentz function that characterize the noise PSD of the limited Brownian motion of MNP102.) Figure 2B shows that significant conformational changes, such as the folding and unfolding of proteins or nucleic acids, also change the effective hydrodynamic radius of MNP102, and these can also be detected. Figure 2C shows, similar to Figure 2A, that MNP102 can bind to molecules immobilized on the surface 117 of the monitoring device (shown as antigens in the example in Figure 2C). The strength of the interaction can be studied according to several embodiments. Figure 2D shows that conformational changes of the tether (biopolymer 101), such as DNA or RNA hairpin formation, also limit the motion of MNP102. For example, how nucleic acids behave as a function of temperature (e.g., wrap-and-unwrap) can be important.The apparatus, systems, and methods disclosed herein can be used to detect and / or monitor changes, including but not limited to those shown in Figures 2A, 2B, 2C, and 2D.

[0028] Magnetic sensor Embodiments disclosed herein utilize at least one magnetic sensor 105 (e.g., a magnetoresistive nanoscale sensor or any other type of magnetic sensor) to detect the presence of one or more MNPs 102 (e.g., magnetic nanoparticles, organometallic complexes, charged molecules, etc.) bound to a biopolymer 101. Figure 3 shows some exemplary magnetic sensors 105 according to several embodiments. The exemplary magnetic sensor 105 in Figure 3 has a bottom surface 108 and a top surface 109 and comprises three layers: a first ferromagnetic layer 106A, a second ferromagnetic layer 106B, and a non-magnetic spacer layer 107 between the first ferromagnetic layer 106A and the second ferromagnetic layer 106B. Suitable materials for use in the first ferromagnetic layer 106A and the second ferromagnetic layer 106B include, for example, alloys of Co, Ni, and Fe (which may also be mixed with other elements). In some embodiments, the magnetic sensor 105 is implemented using thin-film technology, and the first ferromagnetic layer 106A and the second ferromagnetic layer 106B are operated so that their magnetic moments are oriented in the plane of the film or perpendicular to the plane of the film. The non-magnetic spacer layer 107 may be a metallic material such as copper or silver, in which case the structure is called a spin valve (SV), or it may be an insulator such as alumina or magnesium oxide, in which case the structure is called a magnetic tunnel junction (MTJ).

[0029] Additional materials can be deposited both below and above the first ferromagnetic layer 106A, the second ferromagnetic layer 106B, and the non-magnetic spacer layer 107 shown in Figure 3, to serve purposes such as interface smoothing, texture processing, and / or protection from processes used to pattern the apparatus into which the magnetic sensor 105 is incorporated. Furthermore, as will be further described below, the magnetic sensor 105 may be wrapped or covered with material to protect it from fluids used in single-molecule analysis. Nevertheless, the active area of ​​the magnetic sensor 105 is located in the three-layer structure shown in Figure 3. Thus, components that come into contact with the magnetic sensor 105 (e.g., a readout circuit) may be in contact with any of the first ferromagnetic layer 106A, the second ferromagnetic layer 106B, or the non-magnetic spacer layer 107, or with other parts of the magnetic sensor 105.

[0030] As shown in Figures 4A and 4B, the resistance of a magnetoresistive sensor (e.g., one possible type of magnetic sensor 105) is proportional to 1-cos(θ), where θ is the angle between the moments of the first ferromagnetic layer 106A and the second ferromagnetic layer 106B shown in Figure 3. To maximize the signal generated by the magnetic field and provide a linear response of the magnetic sensor 105 to the applied magnetic field, the magnetic sensor 105 may be designed such that the moments of the first ferromagnetic layer 106A and the second ferromagnetic layer 106B are oriented relative to each other at π / 2 radians or 90 degrees in the absence of a magnetic field. This orientation can be achieved by any number of methods known in the art. For example, one solution involves using an antiferromagnetic material to "pin" the magnetization direction of one of the ferromagnetic layers (either the first ferromagnetic layer 106A or the second ferromagnetic layer 106B, called "FM1") by an effect called exchange bias, and then coating the magnetic sensor 105 with a double layer having an insulating layer and a permanent magnet. The insulating layer prevents electrical short circuits in the magnetic sensor 105, and the permanent magnet supplies a "hard bias" magnetic field perpendicular to the pinning direction of FM1, which then rotates the second ferromagnetic material (either the second ferromagnetic layer 106B or the first ferromagnetic layer 106A, called "FM2") to produce the desired configuration. A magnetic field parallel to FM1 then rotates FM2 around this 90-degree configuration, and the change in the resistance of the magnetic sensor 105 yields a voltage (or current) signal (e.g., sensor signal 207) that can be calibrated to measure the magnetic field acting on the magnetic sensor 105. In this way, the magnetic sensor 105 functions as a magnetic field voltage transducer.

[0031] For biosensing applications, the magnetic sensor 105 should be designed so that FM1 and FM2 are weakly coupled, allowing the sensor signal 207 to detect perturbations to the position of FM2 caused by the presence of MNP 102. If the coupling between FM1 and FM2 is too strong, the presence of MNP 102 will not produce sufficient perturbation in the sensor signal 207 to be detected. On the other hand, if the coupling between FM1 and FM2 is too weak, the magnetic sensor 105 will be thermally unstable, thermal fluctuations will become dominant, and the signal-to-noise ratio (SNR) may decrease. As will be further described below, certain magnetic sensors 105 designed for use in magnetic recording have characteristics that enable their use in certain biosensing applications.

[0032] The example discussed above illustrates the use of ferromagnets in which their moments are oriented in the plane of the film at 90 degrees to each other. However, it should be noted that, alternatively, a perpendicular configuration can be achieved by oriented the moments of one of the ferromagnetic layers (the first ferromagnetic layer 106A or the second ferromagnetic layer 106B) out of the plane of the film, which can be achieved using something called perpendicular magnetic anisotropy (PMA).

[0033] In some embodiments, the magnetic sensor 105 utilizes a quantum mechanical effect known as spin transfer torque. In such a magnetic sensor 105, a current passing through the first ferromagnetic layer 106A (or the second ferromagnetic layer 106B) in the SV or MTJ allows electrons with spins parallel to the layer's moment to pass through preferentially, while electrons with antiparallel spins are more easily reflected. In this way, the current is spin-polarized such that there are more electrons of one spin type than the other. This spin-polarized current then interacts with the second ferromagnetic layer 106B (or the first ferromagnetic layer 106A), exerting a torque on its moment. This torque can, under different circumstances, cause the moment of the second ferromagnetic layer 106B (or the first ferromagnetic layer 106A) to precess around the effective magnetic field acting on the ferromagnet, or it can reversibly switch the moment between two orientations defined by uniaxial anisotropy induced in the system. The resulting spin-torque oscillators (STOs) are frequency-tunable by changing the magnetic field acting on them. Therefore, they have the ability to act as magnetic field-to-frequency (or phase) converters (thus generating AC signals with frequency), as shown in Figure 5A, which illustrates the concept of using STO sensors for magnetic recording. Figure 5B shows the experimental response of an STO through a delay detection circuit when an AC magnetic field with a frequency of 1 GHz and a peak-to-peak amplitude of 5 mT is applied across the STO. This result, as well as the results shown in Figures 5C and 5D for short nanosecond magnetic field pulses, demonstrates how these oscillators can be used as nanoscale magnetic field detectors.Further details can be found in T. Nagasawa, H. Suto, K. Kudo, T. Yang, K. Mizushima, and R. Sato, "Delay detection of frequency modulation signal from a spin-torque oscillator under a nanosecond-pulsed magnetic field," Journal of Applied Physics, Vol. 111, 07C908 (2012), which is incorporated herein by reference in its entirety for all purposes.

[0034] In some embodiments, the magnetic sensor 105 includes a STO for detecting a magnetic field caused by an MNP 102 coupled to a biopolymer 101. The magnetic sensor 105 is configured to detect the magnetic field of the MNP 102 by detecting a change or presence of a precessional oscillation frequency of the magnetization of the magnetic layers of the magnetic sensor 105. The magnetic sensor 105 may include a magnetic free layer (e.g., a first ferromagnetic layer 106A or a second ferromagnetic layer 106B), a magnetic pinning layer (e.g., a second ferromagnetic layer 106B or a first ferromagnetic layer 106A), and a non-magnetic layer between the free layer and the pinning layer (e.g., a non-magnetic spacer layer 107), as described above in the description of Figure 3. In some embodiments, during operation, a detection circuit coupled to the magnetic sensor 105 induces an electric (DC) current through the layers of the magnetic sensor 105. The spin polarization of electrons moving through the magnetic sensor 105 causes a spin-torque-induced precession of one or more magnetizations in the layers. The frequency of this oscillation changes in response to the magnetic field generated by the MNP 102 near the magnetic sensor 105. In some embodiments, the change in the frequency of the sensor's oscillation or noise in the oscillation frequency (referred to as phase noise or frequency noise) can be used to detect the presence, absence, or change of the magnetic field, and therefore the MNP 102.

[0035] In some embodiments, the magnetic sensor 105 includes an MTJ, and changes in the resistance, current, or voltage of the magnetic sensor 105 are used to detect the presence, absence, or movement of MNPs 102 within the detection region 206 of the magnetic sensor 105. For example, an MTJ similar to those used in hard disk drives is an example of a magnetic sensor 105 suitable for use in the apparatus, systems, and methods described herein. Such a magnetic sensor 105 can be used to monitor nanoscale changes in the motion patterns of any suitable MNPs 102, such as 20 nm superparamagnetic iron oxide nanoparticles, as will be further described below. Other MNPs 102, such as Fe3O4 and FePt, can also be used, but it should be understood that the experimental results below relate to iron oxide nanoparticles, as other particles (e.g., Fe3O4 and FePt) are more difficult to functionalize for tethering and may be difficult or impossible to image using a scanning electron microscope to confirm the presence of MNPs 102 within the detection region 206. Similarly, MNPs 102 larger or smaller than 20 nm can be used.

[0036] To illustrate specific concepts applicable to the magnetic sensor 105 used in the apparatus, systems, and methods described herein, Figure 6 shows the operation of a magnetic sensor capable of reading data previously recorded on a magnetic recording medium. Specifically, Figure 6 is a partial diagram of an exemplary read head 240 containing a magnetic sensor used in perpendicular magnetic recording (PMR) applications. The surface of the recording medium 250 lies in the xz plane, as does the air support surface (ABS) of the exemplary read head 240 from which information stored in the recording medium 250 is read. The recording medium 250 may have a plurality of concentric tracks on which information can be recorded, including track 251, which is the track being read in Figure 6. The exemplary read head 240 includes a plurality of layers within the wafer surface, which is the xy plane using the coordinates shown in Figure 6. The plurality of layers include a free layer 260, a reference layer 262, and a pinning layer 264. The free layer 260, the reference layer 262, and the pinned layer 264 can correspond to the first ferromagnetic layer 106A, the non-magnetic spacer layer 107, and the second ferromagnetic layer 106B (or, equivalently, the second ferromagnetic layer 106B, the non-magnetic spacer layer 107, and the first ferromagnetic layer 106A), respectively. The magnetic moment 263 of the reference layer 262 is in a specific direction, shown as being in the positive y direction in Figure 6. The magnetic moment 265 of the pinned layer 264 may be pinned (fixed in a specific direction) by the antiferromagnetic material 266, as described above. In Figure 6, the magnetic moment 265 of the pinned layer 264 is pinned in the negative-y direction. The magnetic moment 261 of the free layer 260 rotates freely in response to the applied or induced magnetic field. The hard bias regions 268A and 268B can be located laterally (in a direction called the sidetrack direction) from the free layer 260, the reference layer 262, and / or the pinning layer 264 to supply a magnetic field perpendicular to the direction of the magnetic moment 265 of the pinning layer 264. In Figure 6, the moments 269A and 269B of the hard bias regions 268A and 268B are oriented to the right of the page in the positive x-direction. A circuit 270 coupled to the layer provides a bias voltage (or, equivalently, a bias current) to read the information stored in the recording medium 250.

[0037] As shown in Figure 6, the magnetic moment 261 of the free layer 260 is oriented in some default or equilibrium direction to the right of the page, perpendicular to the magnetic moment 263 of the reference layer 262 and perpendicular to the magnetic moment 265 of the pinning layer 264, along the x-axis in Figure 6. As shown in Figure 6, when a “bit” on the recording medium 250 generates an upward magnetic field toward the exemplary read head 240, the magnetic moment 261 of the free layer 260 rotates upward, constructively adding a component to the magnetic field generated by the bias applied to the exemplary read head 240 by the circuit 270. As a result, the resistance of the exemplary read head 240 decreases. Conversely, when a “bit” on the recording medium 250 generates a downward magnetic field away from the exemplary read head 240, the magnetic moment 261 of the free layer 260 rotates downward in the opposite direction, thereby adding a destructive component to the magnetic field generated by the bias applied by the circuit 270. As a result, the resistance of the exemplary read head 240 increases. Therefore, the change in resistance indicates which of the two possible "bits" (upper or lower, which can be interpreted as 0 or 1 (or vice versa)) on the recording medium 250 has been detected.

[0038] Figures 7A, 7B, and 7C illustrate how these same principles can be applied to single-molecule detection devices, systems, and methods according to several embodiments disclosed herein. Figure 7A shows a portion of the magnetic sensor 105 where MNP 102 is not in the vicinity. In the presence of an applied magnetic field H oriented in the positive z direction (e.g., caused by a bias voltage), the magnetic moment 261 of the free layer 260 is at an angle from the x-axis.

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[0039] Figure 7B shows a magnetic sensor 105 with an MNP 102 located directly above (in the z direction) the free layer 260 of the magnetic sensor 105. As shown in the panel above, the applied magnetic field H in the positive z direction aligns the magnetic moment of the MNP 102 in substantially the same direction as the applied magnetic field H. As a result, at the location of the free layer 260, the magnetic field caused by the MNP 102 constructively adds to the applied magnetic field H, and the magnetic moment 261 of the free layer 260 is, here at an angle from the x-axis.

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[0040] Figure 7C shows a magnetic sensor 105 having an MNP 102 that is laterally offset from the free layer 260 of the magnetic sensor 105 (specifically, offset in the x-direction). As shown in the upper panel of Figure 7C, an applied magnetic field H in the positive z-direction orients the magnetic moment of the MNP 102 substantially in the same direction as the applied magnetic field H. However, here, since the MNP 102 is laterally offset from the free layer 260, the magnetic field caused by the MNP 102 is in the opposite direction to the applied magnetic field H at the location of the free layer 260. Therefore, the magnetic field caused by the MNP 102 reduces the effect of the applied magnetic field H on the free layer 260, and the magnetic moment 261 of the free layer 260 rotates from that direction in Figure 7B. Here, the magnetic moment 261 of the free layer 260 is at an angle from the x-axis.

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[0041] Therefore, by monitoring the current flowing through the magnetic sensor 105 (or any substitute for current such as resistance or voltage, or, in the case of different types of magnetic sensors 105, any other property representing the magnetic environment sensed by the magnetic sensor 105), the presence and position of the MNP 102 relative to the free layer 260 (and thus the magnetic sensor 105) can be detected and monitored, as will be further described below. Figure 8 shows the results of a nanomagnetic simulation of an exemplary magnetic sensor 105 in the presence of the MNP 102 at various positions relative to the magnetic sensor 105, according to several embodiments. The contour plot 402 shows the magnetic field acting on the magnetic sensor 105 for various lateral positions of the MNP 102 in the xy plane in Figures 7A, 7B, and 7C, when the MNP 102 is 10 nm above the xy plane (z value 10 nm). As shown by the cross section 406, the magnetic sensor 105 is centered at coordinate (0,0) in the xy plane, indicated as position 404. Cross section 406 shows the magnitude of the magnetic field as a function of the lateral position of MNP 102 along the x-axis at the position y=0 (shown by the dashed line 416 in contour plot 402) and at various positions along the z-axis in the range of 10 nm to 60 nm from the surface of the magnetic sensor 105. Plot 408 shows the magnitude of the magnetic field along the dashed line 420 in cross section 406. As shown, when MNP 102 is 10 nm directly above the magnetic sensor 105, the magnetic field amplitude is approximately 100 oorsted, and when MNP 102 is 60 nm directly above the magnetic sensor 105, the magnetic field amplitude is near 0.

[0042] Cross-section 412 shows the magnitude of the magnetic field as a function of the lateral position of MNP 102 along the y-axis at the position of x = 0 (indicated by dashed line 418 of contour plot 402) and at various positions along the z-axis ranging from 10 nm to 60 nm from the surface of magnetic sensor 105. Plot 414 shows the magnitude of the magnetic field along dashed line 422 of cross-section 412 at position 410 shown in contour plot 402 that is at a lateral offset of 39 nm along the y-axis. As shown, when MNP 102 is 10 nm above the surface of magnetic sensor 105 and offset laterally by 39 nm, the magnetic field amplitude is approximately -4 Oersted, and when MNP 102 is 60 nm above magnetic sensor 105 and offset laterally by 39 nm, the magnetic field amplitude is near 0. Thus, FIG. 8 shows that as MNP 102 changes its position within the three-dimensional space, the magnitude of the magnetic field changes substantially. Even a slight change in position causes a significant change in the detected magnetic field. Changes in both its amplitude and direction, and these changes can be detected by free layer 260 of magnetic sensor 105. Thus, the position of MNP 102 can be inferred by interpreting the signal from magnetic sensor 105 rather than directly observing it using an imaging system.

[0043] FIG. 9A is a plan view scanning electron microscope (SEM) image of an exemplary magnetic sensor 105 that is a MTJ having a surface area of about 30×40 nm in the x-y plane with MNP 102 coupled within sensing region 206 (the dashed line indicates the estimated or approximate boundary of sensing region 206 in the x-y plane). In the illustrated exemplary embodiment, the junction region is parallel to the x-z plane (out of the plane of the paper), and the tunnel current flows in the y-axis direction. FIG. 9A shows a single 20 nm MNP 102 within sensing region 206. The effective sensing region 206 of an exemplary magnetic sensor 105 initially developed for magnetic recording applications is very small (e.g., about 10 2 nm 5 nm 3 from about 5×10 5 nm 3 It is designed to be such that, therefore, it is well suited for detecting the stochastic motion of MNP102 as described herein. The volume of the detection region 206 can be any appropriate value, and it should be understood that the above range is merely an example.

[0044] Figures 9B and 9C show cross-sectional views of the magnetic sensor 105 showing an external magnetic field H applied perpendicularly to the surface of the magnetic sensor 105. In Figure 9B, MNP 102 (shown as a circle, but unlabeled to avoid obscuring the drawing) is fixed above the magnetic sensor 105 (also unlabeled, but shown as a filled-in diagonal line), and near the magnetic sensor 105, the magnetic field line is aligned with the external magnetic field, shown as a thick arrow within the sensor region. As mentioned above, the presence of MNP 102 constructively increases the effective magnetic field measured by the magnetic sensor 105 because the magnetic field is added constructively.

[0045] In Figure 9C, MNP102 (also unlabeled but shown as a diagonal fill) is positioned laterally away from the magnetic sensor 105, and the magnetic field lines affecting the free layer 260 point in the opposite direction to the external magnetic field. In this case, as described above, the effective magnetic field measured by the magnetic sensor 105 decreases. Therefore, the perturbation to the sensor signal 207 due to the presence of MNP102 changes rapidly from positive to negative as MNP102 moves laterally away from the magnetic sensor 105. As shown in Figures 9B and 9C, the magnetic field perturbation is extremely sensitive to the position of MNP102 relative to the magnetic sensor 105. The magnetic field lines of MNP102 align with the external magnetic field when MNP102 is above the magnetic sensor 105, as shown in Figure 9B, but point in the opposite direction when MNP102 is displaced laterally, as shown in Figure 9C.

[0046] The effect of the movement of MNP102 on the sensor signal 207 is schematically shown by curve 209 in Figures 9B and 9C. An MNP102 tethered near a magnetic sensor 105 induces a dynamic stochastic perturbation in the sensor signal 207 when the MNP102 moves around while an external magnetic field is applied to fix the magnetic moment of the MNP102 in a specific direction. The response of the magnetic sensor 105 is affected by both the in-plane (in the xy-plane) and out-of-plane (along the z-axis) motion of the MNP102. Even when no external magnetic field is applied, the presence of an MNP102 with a sufficiently high magnetic moment can be detected by the magnetic sensor 105. In other words, the disclosed embodiments can be used, for example, with superparamagnetic and ferromagnetic MNPs.

[0047] In conventional TPM systems, the results of time averaging (exposure time) and observation frequency (frame rate) are well understood in imaging systems. While exposure time and frame rate do not limit the tracking of freely diffusing Brownian particles, they have serious implications for observing particles that undergo anomalous (or restricted) diffusion, such as tethered nanoparticles in biological systems. Time averaging in imaging of such particles can have significant consequences for the apparent characteristics of the reported motion, as the observed velocity depends on the duration of the observation. In extreme cases where the exposure time is too long, the particles appear blurred and stationary at some equilibrium position. These drawbacks can be mitigated or overcome by systems, apparatus, and methods using the magnetic sensor 105 described herein.

[0048] The ability of the magnetic sensor 105 to detect changes in the sensor signal 207 depends on the responsiveness of the detection circuit (e.g., a detection amplifier circuit, other detection electronic circuits, as described below). For example, if the response of the magnetic sensor 105 is too slow (e.g., due to limitations of the detection circuit such as the sampling rate), the monitoring device or system may be able to detect when MNP 102 moves to a different equilibrium position during the processes shown in Figures 2C and 2D, but may not be able to detect processes that change the statistical rate of MNP 102 without affecting the equilibrium position, such as changes in molecular bonding and conformation shown in Figures 2A and 2B.

[0049] Unlike image imaging systems that generate a series of particle images to track the position of particles in both space and time, the magnetic sensor 105 generates a time response to a random series of similar (but not identical) impacts or pulses caused by solution molecules colliding with the MNP 102. Free-diffusing MNP 102 can be considered to estimate the response time and sampling rate of the magnetic sensor 105 that can detect the motion of the MNP 102. Free-diffusing MNP 102 is a good first approximation for the case of MNP 102 tethered to the surface of the magnetic sensor 105 by a long flexible polymer (e.g., biopolymer 101). The length of the polymer is assumed to be considerably longer than the dimensions of the sensing area 206. This constraint increases the probability of detection by preventing the MNP 102 from diffusing too far from the magnetic sensor 105 (e.g., outside the sensing area 206 for a long period of time), but does not constrain its motion, which can still be considered simple Brownian motion.

[0050] The random motion of particles in a fluid due to collisions with fluid molecules can be mathematically described by solving the Langevin equation. This is an equation of motion with a velocity damping term that accounts for viscosity or friction. The mean squared displacement (MSD) of a particle on a short-time scale is given by:

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[0051] The response of the magnetic sensor 105 to the motion of tightly confined nanoparticles (e.g., tether length ≈ size of the sensing region 206 of the magnetic sensor 105 ≈ size of MNP 102) is quite difficult to interpret. The MNP 102 diffuses only locally within the sensing region 206, and its apparent diffusion coefficient (free diffusion equivalent) is significantly affected by time averaging. The signal pulses (e.g., of the sensor signal 207) resulting from the motion of the MNP 102 are neither discrete nor clearly defined. The motion of the MNP 102 is added to the inherent noise of the magnetic sensor 105, generating another source of random noise that alters the noise characteristics of the detected sensor signal 207. To detect changes in the motion of the MNP 102, the difference between the signal spectrum and the noise spectrum across the sensing bandwidth can be utilized, as further described below. Various advanced sensing schemes, such as energy detection or autocorrelation, have been developed and implemented, as described below, to improve detection under low signal-to-noise ratio (SNR) conditions.

[0052] To help understand how the presence and location of MNP102 affect the magnetic sensor 105, a physical problem can be defined. Figure 10A presents an exemplary model. MNP102 is bonded to the surface of the magnetic sensor 105 by a tether. (It should be understood that the surface of the magnetic sensor 105 itself may actually be physically separated from any fluid acting on MNP102 by some kind of protective barrier such as a tether (e.g., biopolymer 101), MNP102, and an insulator, which will be further explained elsewhere in this specification. When this specification refers to the “surface of the magnetic sensor 105”, it is for simplicity, and it should be understood that the surface of the magnetic sensor 105 may not be exposed, but is physically close.) For example, the tether (biopolymer 101) may contain peg / biotin / streptavidin as shown in Figure 10A. When molecules from the surrounding solution collide with MNP102, MNP102 moves via a stochastic Brownian perturbation. The motion can be approximated as a one-dimensional harmonic potential. Specifically, as shown in Figure 10A, MNP102 can be considered as a mass on a spring (e.g., biopolymer 101). Ignoring gravity, the driving force is Brownian and stochastic, caused by collisions between molecules of the surrounding solution and MNP102. The Brownian driving force is a function of the diameter and temperature (in Kelvin) of MNP102.

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[0053] The one-dimensional time-open method for the distribution probability P of a diffuse spherical particle at position x and time t, given an initial position x0 at time t0 in a harmonic potential field, is given by the equation of motion:

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[0054] Figure 10B is a reproduction of Figure 1 from the paper by M. Lindner et al. entitled "Dynamic analysis of a diffusing particle in a trapping potential." (See M. Lindner et al., "Dynamic analysis of a diffusing particle in a trapping potential," Physical Review E 87, 022716 (2013).) Figure 10B is a graphical representation of a single particle diffusing at a harmonic potential applied by a DNA strand. The upper panel shows two conformations, and the lower panel shows the Boltzmann steady-state distribution, as well as the 0.01τ, 0.1τ, and 10τ values ​​with x0 = -650 nm.

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[0055] To illustrate how the presence and movement of MNP102 affect the sensor signal 207 provided by the magnetic sensor 105, we first consider a thought experiment using an optical method, as shown in Figure 11A. Assume that MNP102 has a diameter of 20 nm and is coupled to the surface of the device by a tether (e.g., PEG / biotin / streptavidin). Furthermore, assume that there is a light source capable of producing light with a wavelength comparable to the diameter of MNP102, and that photodiode 502 detects photons reflected in a specific direction by MNP102 coupled to the surface of the device. If MNP102 is stationary and illuminated by the light source, the intensity of the reflected light remains constant over time. Therefore, the PSD of the signal 505 from photodiode 502 provides an indication of the noise introduced by photodiode 502. In other words, as long as MNP102 is not moving, the noise in the photodiode 502 signal is entirely due to the characteristics of photodiode 502. If the noise floor of photodiode 502 is white (e.g., thermal noise or Johnson-Nyquist noise), the noise spectrum becomes nearly flat at a relatively low level, as shown by the dashed line in Figure 11B. Once MNP 102 is able to move, the stochastic perturbation causes the MNP to move in restricted Brownian motion (because the tether prevents MNP 102 from floating). The PSD of the restricted Brownian motion is a Lorentz function, which has the following form of PSD:

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[0056] Once it is known that the PSD (which can be thought of as a signature) of restricted Brownian motion is a Lorentz function, the expected PSD of the sensor signal 207 from magnetic sensor 105 in the absence of a moving MNP 102 and in the presence of a moving MNP 102 can be similarly determined by first considering the noise PSD of magnetic sensor 105 without an MNP 102 in its vicinity, and then evaluating what the effect of MNP 102 should be on that noise PSD. Figure 12A shows an exemplary magnetic sensor 105 having a configuration similar to that described in the description of Figure 6. The description of the components of Figure 6, also shown in Figure 12A, applies to Figure 12A and is not repeated.

[0057] The noise PSD of a complete MTJ exhibits 1 / f behavior (it decreases by only 10 dB / decimal). Figure 12B plots the expected noise PSD of an exemplary magnetic sensor 105, which is a complete MTJ driven by a selected bias voltage (further described below), against the Lorentz function characterizing the PSD of the restricted Brownian motion of MNP 102. In the example in Figure 12B, the Lorentz function exceeds the noise PSD of magnetic sensor 105 in the frequency range of approximately 2 kHz to approximately 70 kHz. As a result, on a logarithmic / logarithmic scale, the overall PSD has an identifiable "bump" labeled 140 in this frequency range. Thus, if magnetic sensor 105 is sensitive to the presence of MNP 102, its sensitivity manifests as an identifiable bump 140 in the PSD of the sensor signal 207. As will be explained in more detail below, whether the Lorentz function exceeds the noise PSD of the magnetic sensor 105, and over what frequency range, depends on various factors, including the design of the magnetic sensor 105 and the bias voltage (or current) used to drive the magnetic sensor 105, as well as the aforementioned factors that determine the corner frequency of the Lorentz function (e.g., the spring constant of the molecular tether, the diameter of MNP 102, and the kinematic viscosity of the liquid surrounding MNP 102).

[0058] To verify the theoretical analysis above, the inventors conducted experiments using a magnetic sensor 105 in the form of an MTJ to determine whether the PSD of the collected sensor signal 207 actually exhibited the behavior derived above. Figure 13 illustrates the experiment. First, an external magnetic field was applied, as shown by the leftmost panel, and the sensor signal 207 was captured to determine the noise PSD of the magnetic sensor 105 in the absence of MNP 102 (ideally having a 1 / f profile, as described above). Next, the external magnetic field was turned off, and MNP 102 (20 nm in diameter) was tethered to surface 117 using peg / biotin / streptavidin as described above. A bias voltage was applied to the magnetic sensor 105, generating a magnetic field in its vicinity. In response to this magnetic field, the magnetization of MNP 102 orients itself in alignment with the magnetic field and then moves in constrained Brownian motion as described above. As shown graphically in the center and rightmost panels of Figure 13, the sensor signal 207 was captured to capture the dipole interaction between the magnetic moment of the magnetic sensor 105 and the magnetic moment 261 of the free layer 260 of the magnetic sensor 105 as the MNP 102 moved around.

[0059] Figure 14 shows the measured PSDs of the three tested magnetic sensors 105. Each dashed line with a circle labeled 161 is the noise PSD of one of the tested magnetic sensors 105 (without any MNP 102), and each solid line with a diamond labeled 162 is the combined PSD of MNP 102 and magnetic sensor 105. As shown in the plot in Figure 14, each of the combined PSDs has a characteristic bump 140 that is expected when MNP 102 is detected. Thus, the experiment confirmed that, for a bias voltage of about 10 mV, the tethered MNP 102 behaves like a particle confined in a harmonic potential. Furthermore, its PSD can be expressed as a Lorentz function in the range of about 488 Hz to 120 kHz, as shown in Figure 14. As shown in Figure 14, the corner frequencies of each Lorentz function differ slightly for each magnetic sensor 105, but are all around 45 kHz. Figure 14 shows data from only three exemplary magnetic sensors 105, but other tested magnetic sensors 105 behaved similarly. In all experiments, the corner frequency of the Lorentz function due to the limited Brownian motion of MNP102 was found to be approximately 45 kHz.

[0060] As explained above, the corner frequency depends on the selected tether (e.g., biopolymer 101), specifically its spring constant. A polymer tether can be thought of as an "entropy" spring, as described in "Scaling Concepts in Polymer Physics" by P.G. de Gennes (Cornell University Press, Ithaca, 1979). Stretching or compressing a coil from its equilibrium size reduces the number of possible conformations and therefore decreases the entropy. As a result, the free energy increases. The free energy is quadratic with respect to the change in chain size, and the spring constant is given by:

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[0061] As described above, the bias voltage applied to the magnetic sensor 105 affects whether and to what extent the overall characteristic bump 140 of the PSD is evident in the sensor signal 207 measured when MNP 102 is present. To detect the presence and movement of MNP 102, it is desirable to find a Lorentz function that can yield the detected overall PSD in addition to the noise PSD of the magnetic sensor 105. Figures 15A, 15B, 15C, 15D, and 15E show the results of experiments conducted to investigate the effect of bias voltage on this procedure. Figure 15A shows the results when the bias voltage is 11mV. Figure 15B shows the results when the bias voltage is 25mV. Figure 15C shows the results when the bias voltage is 50mV. Figure 15D shows the results when the bias voltage is 75mV. Figure 15E shows the results when the bias voltage is 100mV.

[0062] As the comparison between Figures 15A, 15B, 15C, 15D, and 15E shows, at higher bias voltages, it becomes increasingly difficult to fit the Lorentz function representing the limited Brownian motion of MNP102 to the measured data. The use of higher bias voltages may trigger the onset of hyperspreading, in which case the motion of MNP102 is no longer Brownian motion but driven motion (e.g., MNP102 is affected by an additional force and moves faster than it would in limited Brownian motion). Hyperspreading can occur if the magnetic sensor 105 influences (drives) the motion of MNP102 rather than simply observing it. As a result of higher bias voltages, the slope of the high-frequency tail of the entire PSD is greater than 2, which is characteristic of hyperspreading. In our experiments, we found that for higher bias voltages, the PSD of MNP102 cannot be represented by a function rather than by a Lorentz function.

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[0063] To adjust the mathematical model to account for hyperdiffusion, the one-dimensional harmonic potential approximation derived above can be modified to include a component representing the magnetic force caused by the bias voltage of the magnetic sensor 105. Figure 16 shows how the model can be modified to include a component resulting from the magnetic sensor 105 influencing the motion of MNP 102. Here again, MNP 102 is considered to be the mass on the spring, which is a tether (e.g., biopolymer 101). The Brownian driving force, liquid damping force, and spring restoring force are the same, shown in Figure 10A and explained in the description of the figure above. In addition to these forces, the model in Figure 16 adds the magnetic force caused by the magnetic sensor 105, which is expressed as follows:

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[0064] To avoid the onset of hyperdiffusion and allow the MNP 102 to move in restricted Brownian motion without substantially affecting the movement of the magnetic sensor 105, the bias voltage of the magnetic sensor 105 should be kept low enough that the characteristic bump 140 caused by the presence of the MNP 102 is present throughout the PSD and can be fitted to a Lorentz function representing the restricted Brownian motion of the MNP 102 as described above. In other words, if it is not possible to fit the measured PSD data to a Lorentz function, the bias voltage used to drive the magnetic sensor 105 may be too high and may need to be reduced.

[0065] While the above description primarily focuses on MTJ sensors, some explanation regarding SV sensors is provided. It should be understood that magnetic sensor 105 can be any type of magnetic sensor. The use of MTJ in experiments and examples is not intended to be limiting. Suitable magnetic sensors 105 include, but are not limited to, giant magnetoresistance (GMR) sensors, Hall effect devices, spin valves, and spin storage sensors. In general, magnetic sensor 105 can be any magnetic sensor that can enable the detection of the presence and / or movement of MNP 102 from the sensor signal 207.

[0066] Further Examples To demonstrate the feasibility and implementation of the dynamic spectral biosensing technique described herein, conformational changes of exemplary biopolymer 101 and ssDNA induced by altering the ionic strength of a buffer were monitored using a magnetic sensor 105 located within a flow cell.

[0067] The three-stage experiment performed is schematically shown in Figures 17A, 17B, and 17C. First, as shown in Figure 17A, the 5' end of a 150-nucleotide (nt) ssDNA was initially bound to the surface 117 of the device within the detection region 206 of the magnetic sensor 105 using copper-catalyzed azide-alkyne click chemistry. Next, a 3'-terminal biotinylated 20-mer was hybridized to the 3' end of the ssDNA. Thus, Figure 17A shows an exemplary 150-nt ssDNA bound to the surface 117 near the magnetic sensor 105 before the MNP 102 is bound. The ssDNA is bound to the surface 117 near the magnetic sensor 105 so that the magnetic sensor 105 can detect the MNP 102 bound to the other end of the ssDNA. In the experiment, a uniform 15 Oersted external magnetic field was then applied perpendicular to the exposed surface of the magnetic sensor 105 (both positive and negative directions along the z-axis in Figure 17A), and the sensor signal 207 was recorded in the absence of MNP 102.

[0068] Next, a streptavidin-coated 20 nm MNP102 was bound to the end of an ssDNA tether (biopolymer 101). Figure 17B shows the ssDNA tether to which the streptavidin-coated 20 nm MNP102 was bound. As shown in Figure 17B, the tethered 20 nm MNP102 is located near the magnetic sensor 105 (e.g., generally within its detection region 206). The streptavidin coating of MNP102 allows it to bind strongly to the ssDNA tether. The arrows overlapping MNP102 represent the degree of stochastic motion of MNP102. The sensor signal 207 was recorded in 10 mM Tris buffer.

[0069] For example, Mg 2+ The addition of ions causes compression of ssDNA. Therefore, the limited stochastic motion of MNP102 bound to ssDNA is Mg 2+ Attenuation should occur upon ion addition. (Similar behavior was observed in polyuridine (U) messenger (m)RNA by TPM.) Therefore, in the experiment, magnesium ions were added to the solution. Figure 17C shows an exemplary state after the addition of magnesium ions and subsequent compression of the ssDNA tether. Compared to Figure 17B, the stochastic motion of MNP102 is attenuated, as represented by the short arrow covering MNP102. The sensor signal 207 was recorded in 15 mM Tris-MgCl2 buffer.

[0070] Although the above-described explanations in Figures 17A, 17B, and 17C describe only one MNP102 and only one magnetic sensor 105, the tests used a series of magnetic sensors 105, multiple MNP102s, and multiple ssDNA fragments (biopolymer 101). In the tests, the density of ssDNA immobilized on the surface of the flow cell was not controlled, and it is possible that a particular observed MNP102 was bound to the surface by multiple DNA strands. (Single-molecule systems to mitigate or eliminate this possibility are described below, for example, in the context of Figures 19A, 19B, 19C, 19D, and 19E.) Therefore, the density of bound MNP102s was adjusted to ensure that only one or a few MNP102s were tethered to the magnetic sensor 105, so as to ensure that only one MNP102 was present within the sensing region 206. Several such magnetic sensors 105 were identified, and the recorded sensor signals 207 of those magnetic sensors 105 were sampled at a moderate sampling rate of 6 kHz. The recorded sensor signals 207 and the corresponding autocorrelation functions of two such representative magnetic sensors 105 are shown in Figures 18A, 18B, and 18C.

[0071] Figure 18A shows exemplary recorded current fluctuations (e.g., sensor signals 207) for two different exemplary magnetic sensors 105, labeled "Sensor 1" and "Sensor 2," over a 2-second period in the presence of an applied external magnetic field H, after 150 nt of ssDNA (e.g., each biopolymer 101) was bound (immobilized) to the surface 117 of each of the two magnetic sensors 105, but before binding of any MNP 102. That state is shown in the top row (unplotted) of Figure 18A. In other words, the recorded current fluctuations for each of the two magnetic sensors 105, indicated by the intensity vs. time plot, are the background or baseline sensor signals 207 for the two magnetic sensors 105, Sensor 1 and Sensor 2, for the stage shown in Figure 17A. The positive and negative autocorrelation functions of the measured sensor signals 207 are also shown in Figure 18A for Sensor 1 and Sensor 2, respectively. The smooth dashed-dotted curves in each autocorrelation plot are the mean autocorrelation of the respective baseline measured sensor signals 207.

[0072] Figure 18B shows the measured sensor signals 207 (intensity vs. time) for sensor 1 and sensor 2, as well as their autocorrelation functions after binding of MNP102, which were 20 nm Fe3O4 particles attached to each end of the DNA strand in the test, and after the addition of Tris buffer. Figure 18B provides results when ssDNA is in its extended conformation, corresponding to the stage shown in Figure 17B. The stage is shown in the top row (unplotted) of Figure 18B. The introduction of MNP102 alters both the recorded current fluctuations and autocorrelation functions in each sensor signal 207 compared to Figure 18A. For example, as the comparison between Figure 18A and Figure 18B shows, the positive and negative autocorrelation functions of sensor 1 shift upward relative to the baseline sensor signal 207 for a delay time of approximately 1 ms to 200-300 ms, while the autocorrelation function of sensor 2 generally shifts downward relative to the baseline sensor signal 207 for a delay time of approximately 1 ms to 50 ms. Therefore, the presence of MNP102 within detection region 206 can be inferred from the shift of the autocorrelation function relative to the baseline in Figure 18A (if MNP102 is not present).

[0073] Figure 18C shows the measured sensor signals 207 (intensity vs. time) of sensor 1 and sensor 2, as well as the DNA tether (e.g., biopolymer 101) with Mg 2+ The autocorrelation functions of the samples when compressed by the introduction of ions are shown. That is, Figure 18C corresponds to the stages shown in Figure 17C. The stages are indicated by the top (unplotted) portion of Figure 18C. Comparing the autocorrelation function of Figure 18B with that of Figure 18C and / or Figure 18A reveals that conformational changes are detectable in the autocorrelation function. For example, compared to the autocorrelation function shown in Figure 18B for sensor 1, the positive and negative autocorrelation functions are Mg 2+ After ion addition, there is a slight downward shift during the delay period from 1 ms to approximately 60-70 ms, and it becomes closer to the average autocorrelation function for delay periods exceeding approximately 300 ms. Similarly, for sensor 2, compared with the autocorrelation function shown in Figure 18B, Mg 2+ The conformational changes of ssDNA caused by the addition of ions manifest as downward shifts in the positive and negative autocorrelation functions for delay times of approximately 1 ms to 50 ms, and upward shifts for delay times of approximately 200–300 ms. Thus, as shown in Figures 18A, 18B, and 18C, significant changes in the noise autocorrelation functions are observed between the three states, thereby enabling the detection and / or monitoring of both the presence and movement of MNP102 within the detection regions 206 of sensors 1 and 2.

[0074] The results described and shown in Figures 18A, 18B, and 18C confirm that the magnetic sensor 105 can not only detect changes in the mean equilibrium position of MNP 102, but can also monitor small, reversible fluctuations in noise variations induced by single-molecule processes. Billions of such magnetic sensors 105 with single-molecule sensitivity can potentially be integrated on CMOS platforms (e.g., Toshiba's 4Gbit-density STT-MRAM chips) to create next-generation high-throughput systems for diagnostics and drug discovery, while leveraging existing mature technologies and mass production capabilities developed by the semiconductor and data storage industries.

[0075] As demonstrated by the experiments described herein, the coupling between the fixed and free layers of certain tested magnetic sensors 105 is suitable for biosensing. These magnetic sensors 105 are examples of suitable magnetic sensors 105. Other magnetic sensors 105 with coupling between FM1 and FM2 optimized for biosensing applications or specific types of MNP 102 can also be used and may perform better than the exemplary magnetic recording sensors used in the experiments.

[0076] Monitoring devices and systems As will be further described below, in some embodiments, a system 100 for monitoring the movement of MNPs 102 bound to a biopolymer 101 may comprise a fluid chamber 115, at least one processor 130, and a magnetic sensor 105. The fluid chamber includes a binding site 116 configured to fix the end of the biopolymer 101 to the surface of the fluid chamber 115 and to allow the MNPs 102 to move (for example, when molecules of the surrounding fluid collide with it). The binding site 116 may include a structure (e.g., a cavity or a protrusion) configured to fix the biopolymer 101 to the binding site 116.

[0077] The magnetic sensor 105 may include, for example, an MTJ or STO. The magnetic sensor 105 has a detection region 206 that can detect MNP 102 in the fluid chamber 115. The detection region 206 is, for example, about 10 5 nm 3 From approximately 5 x 10 5 nm 3 The sensing region 206 may have a volume between the two. The sensing region 206 includes the coupling portion 116. The magnetic sensor 105 is configured to generate a sensor signal 207 that characterizes the magnetic environment within the sensing region 206 (e.g., the presence and / or location of MNP 102) and to provide the sensor signal 207 to at least one processor 130. The sensor signal 207 may transmit (e.g., report) one or more of the following: current, voltage, resistance, noise (e.g., frequency noise or phase noise), frequency or frequency change (e.g., oscillation frequency or Lorentz corner frequency).

[0078] In some embodiments, at least one processor 130 is configured to (a) acquire a first portion of the sensor signal 207 representing the magnetic environment in the detection area 206 during a first detection period, (b) acquire a second portion of the sensor signal 207 representing the magnetic environment in the detection area 206 during a second detection period, which is after the first detection period, and (c) analyze the first and second portions of the sensor signal 207 to execute machine-executable instructions that enable the detection of the movement of the tethered MNP 102. For example, as will be further described below, at least one processor 130 can determine a first autocorrelation function of the first portion of the signal, determine a second autocorrelation function of the second portion of the signal, and analyze the first and second autocorrelation functions (e.g., comparing the first and second autocorrelation functions) to detect the movement of the tethered MNP 102. At least one processor 130 can process the sensor signal 207 or any portion thereof in the time domain, the frequency domain, or both. In some embodiments, at least one processor 130 is configured to determine a Lorentz function that characterizes the limited Brownian motion of MNP 102.

[0079] The system 100 may further include a detection circuit 120 coupled to a magnetic sensor 105 and at least one processor 130. The circuit 120 may include, for example, one or more lines that allow at least one processor 130 to read or query the magnetic sensor 105. The circuit 120 may include components such as analog-to-digital converters and / or amplifiers.

[0080] In some embodiments, the monitoring system 100 comprises a plurality of magnetic sensors 105, each functionalized by an individual single biomolecule during use, so that the monitoring system 100 can detect a single-molecule process at each magnetic sensor 105. Figure 19A is a block diagram showing the components of an exemplary monitoring system 100 according to some embodiments. As shown, the exemplary monitoring system 100 includes a sensor array 110 coupled to a circuit 120 coupled to at least one processor 130. The sensor array 110 comprises a plurality of magnetic sensors 105, which can be arranged in any suitable manner, as will be further described below. (It should be understood that the sensor array 110 includes at least one magnetic sensor 105.)

[0081] The circuit 120 may include one or more lines that enable magnetic sensors 105 in the sensor array 110 to be investigated by at least one processor 130 (with the help of other components known in the art, such as current or voltage sources, amplifiers, analog-to-digital converters, etc.). For example, during operation, the processor 130 may cause the circuit 120 to apply a bias voltage or current to such lines to detect a sensor signal 207 that reports the magnetic environment of at least one magnetic sensor 105 in the sensor array 110. The sensor signal 207 indicates the presence, position, and / or movement of MNP 102 within the detection area 206. In other words, the sensor signal 207 indicates some characteristic of the magnetic sensor 105 (e.g., magnetic field, resistance, voltage, current, oscillation frequency, signal level, noise level, frequency noise, phase noise, etc.). The sensor signal 207 can be inspected and / or processed to determine whether the magnetic sensor 105 has detected MNP 102 or movement of MNP 102 (e.g., change of position) over time. For example, at least one processor 130 can monitor one or more time-domain, frequency-domain, deterministic, and / or statistical characteristics of the sensor signal 207 (e.g., peak or mean amplitude, fluctuation, deviation from mean or expected peak, autocorrelation, power spectral density, etc.) and determine whether MNP 102 or the movement of MNP 102 has been detected (or not detected). Specifically, at least one processor 130 can compare the form of the sensor signal 207 of the magnetic sensor 105 (e.g., autocorrelation, PSD, etc.) at an earlier time or over a selected period of time with the form of the sensor signal 207 at an earlier time or over an earlier time or over a different period of time (e.g., baseline autocorrelation as described above in the descriptions of Figures 17A, 17B, and 17C, or baseline noise PSD as described below in the descriptions of Figures 21 to 26, etc.) and determine whether MNP 102 has been detected or not, or whether it has moved or not, based on the changes in the sensor signal 207.For example, at least one processor 130 can determine a first overall noise PSD of the sensor signal 207 during a first detection period and a second overall noise PSD of the sensor signal 207 during a second detection period, and analyze whether MNP 102 is present and / or has moved. In some embodiments, at least one processor 130 determines a Lorentz function that, when added to the baseline noise PSD of the magnetic sensor 105, results in the overall noise PSD of the sensor signal 207 during one or both of the first and second detection periods.

[0082] The sensor signal 207 and the information it transmits to characterize the magnetic environment of the magnetic sensor 105 may depend on the type of magnetic sensor 105 used in the monitoring system 100. In some embodiments, the magnetic sensor 105 is a magnetoresistive (MR) sensor (e.g., MTJ, SV, etc.) that can detect, for example, a magnetic field or resistance, a change in the magnetic field or a change in resistance, or a noise level. In some embodiments, each of the magnetic sensors 105 in the sensor array 110 is a thin-film device that can use the MR effect to detect an MNP 102 bound to a biopolymer 101 bound to a respective binding site 116 associated with the magnetic sensor 105. The magnetic sensor 105 can operate as a potentiometer having a resistance that changes as the strength and / or direction of the detected magnetic field changes. In some embodiments, the magnetic sensor 105 comprises a magnetic oscillator (e.g., STO), and the sensor signal 207 reports the frequency, or a change in frequency, frequency noise, or phase noise generated by the magnetic oscillator.

[0083] In some embodiments, at least one processor 130, with the help of circuit 120, detects deviations or fluctuations in the magnetic environment of some or all of the magnetic sensors 105 in the sensor array 110. For example, an MR-type magnetic sensor 105 in the absence of MNP 102 should have relatively small noise above a certain frequency compared to the magnetic sensor 105 in the presence of MNP 102, since magnetic field fluctuations from MNP 102 cause fluctuations in the moment of the detected ferromagnet. These fluctuations can be measured, for example, using heterodyne detection (e.g., by measuring noise power density) or by directly measuring the current or voltage of the magnetic sensor 105 and can be evaluated using a comparator circuit to compare with another sensor element that does not detect the coupling site 116. In some embodiments, the magnetic sensor 105 includes an STO element, and the fluctuating magnetic field from MNP 102 causes a phase jump in the magnetic sensor 105 due to instantaneous changes in frequency that can be detected using a phase detection circuit.

[0084] It should be understood that the examples of MNP102 and magnetic sensors 105 provided herein are for illustrative purposes only. In general, any type of MNP102 that can be bound to the biopolymer 101 can be used with an array 110 of any type of magnetic sensors 105 that can detect that type of MNP102.

[0085] It should also be understood that the components of the monitoring system 100 may be distributed or contained within a single physical device. For example, if at least one processor 130 includes multiple processors, the first processor may be part of a device (e.g., a chip) containing a sensor array 110 of at least one magnetic sensor 105, and the second processor may be in a different physical location (e.g., off-chip in an attached computer). Specifically, the first processor in the monitoring system 100 may be configured to extract a sensor signal 207 from the magnetic sensor 105, and the second processor in the monitoring system 100, which is not necessarily part of the same physical device as the first processor, may process the sensor signal 207 (e.g., calculate an autocorrelation function, PSD, Lorentz function, etc., and / or perform signal processing and / or analysis, etc.) to detect the presence and / or movement of the MNP 102. Thus, the components shown in Figure 19A can be located in the same place or distributed. In other words, the system may have the components shown in Figure 19A in a single physical device, or the components of Figure 19A may be distributed. Similarly, the monitoring system 100 may include other components, such as memory for storing sensor signals 207 or sampled or processed versions of sensor signals 207, or instructions for execution by at least one processor 130.

[0086] Figures 19B, 19C, and 19D show exemplary monitoring systems 100 for the detection and monitoring of single-molecule processes according to several embodiments. Figure 19B is a plan view of a portion of the monitoring system 100. Figure 19C is a cross-sectional view at the location of the dashed line labeled "19C" in Figure 19B, and Figure 19D is a cross-sectional view at the location of the dashed line labeled "19D" in Figure 19B.

[0087] The exemplary portion of the monitoring system 100 shown in Figures 19B, 19C, and 19D includes a sensor array 110 for detecting MNP 102 in the fluid chamber 115 of the monitoring system 100. The sensor array 110 includes a plurality of magnetic sensors 105, with 16 magnetic sensors 105 shown in the array 110 of Figure 19B. It should be understood that the implementation of the monitoring system 100 may include any number of magnetic sensors 105 (e.g., just one, or hundreds, thousands, millions, or billions of magnetic sensors 105). To avoid obscuring the drawing, only seven of the magnetic sensors 105, namely magnetic sensors 105A, 105B, 105C, 105D, 105E, 105F, and 105G, are labeled in Figure 19B. (For simplicity, in this document, magnetic sensors 105 are generally referred to by reference numeral 105. Individual magnetic sensors 105 are denoted by letters following the reference numeral 105.) As described above, magnetic sensors 105 can detect the presence or absence of MNP 102 and the movement of MNP 102 within their respective detection areas 206. In other words, each magnetic sensor 105 can detect whether MNP 102 is in its vicinity (for example, in the detection area 206), and the sensor signal 207 provided by the magnetic sensor 105 also provides indication of whether and how MNP 102 is moving.

[0088] Referring here to Figures 19C and 19D in relation to Figure 19B, each magnetic sensor 105 is shown as having a cylindrical shape in an exemplary embodiment of the monitoring system 100. However, it should be understood that in general, the magnetic sensor 105 can have any suitable shape. For example, the magnetic sensor 105 may be a three-dimensional rectangular parallelepiped. Furthermore, different magnetic sensors 105 may have different shapes (for example, some may be rectangular parallelepipeds and others cylindrical, etc.). It should be understood that the drawings are for illustrative purposes only.

[0089] As shown in Figures 19C and 19D, the monitoring system 100 includes a fluid chamber 115. The fluid chamber 115 includes a plurality of binding sites 116 on its surface 117. The fluid chamber 115 holds a fluid (e.g., buffer, nucleotide precursor, other fluid or solution). In the illustrated embodiment, each magnetic sensor 105 is associated with its respective binding site 116. (For simplicity, in this document, binding sites are generally denoted by reference number 116. Individual binding sites are denoted by a letter following reference number 116.) In other words, there is a one-to-one relationship between the magnetic sensor 105 and the binding site 116. As shown in Figure 19B, magnetic sensor 105A is associated with coupling portion 116A, magnetic sensor 105B is associated with coupling portion 116B, magnetic sensor 105C is associated with coupling portion 116C, magnetic sensor 105D is associated with coupling portion 116D, magnetic sensor 105E is associated with coupling portion 116E, magnetic sensor 105F is associated with coupling portion 116F, and magnetic sensor 105G is associated with coupling portion 116G. Each of the other unlabeled magnetic sensors 105 shown in Figure 19B is also associated with their respective coupling portion 116. In the exemplary embodiments of Figures 19B, 19C, and 19D, each magnetic sensor 105 is shown positioned below its respective coupling portion 116, but it should be understood that the coupling portion 116 may be in other positions relative to each of those magnetic sensors 105. For example, the coupling portion 116 may be on the side of each magnetic sensor 105.

[0090] Each binding site 116 is configured to bind one or fewer biopolymers 101 (e.g., ssDNA, RNA, protein, etc.) to a surface 117 within the fluid chamber 115. In other words, each binding site 116 has properties and / or features intended to allow the binding of one and only biopolymers 101 for detection and monitoring by each magnetic sensor 105 (or multiple magnetic sensors 105 as described later), thereby enabling the system 100 to be a single-molecule system. Each magnetic sensor 105 can then detect and monitor the movement of MNPs 102 bound to the biopolymers 101 bound to the binding site 116. In some embodiments, the binding site 116 has a structure (or multiple structures) configured to fix the biopolymers 101 to the binding site 116. For example, the structure (or multiple structures) may include cavities or protrusions. Although Figures 19C and 19D show the coupling portion 116 extending from the surface 117 of the fluid chamber 115, it should be understood that the coupling portion 116 may be coplanar with the surface 117 of the fluid chamber 115, or may be etched.

[0091] The binding sites 116 can have any suitable size and shape that facilitates the binding of a single biopolymer 101 to each binding site 116. For example, the shape of the binding sites 116 can be similar to or identical to the shape of the magnetic sensor 105 (for example, if the magnetic sensor 105 is three-dimensional and cylindrical, the binding sites 116 may be cylindrical and protrude from the surface 117 of the fluid chamber 115 or form a fluid container within the surface 117 of the fluid chamber 115, and have a radius that can be larger than, smaller than, or the same size as the radius of each magnetic sensor 105. If the magnetic sensor 105 is three-dimensional and rectangular, the binding sites 116 may be rectangular and may be larger than, smaller than, or the same size as the nearest part of the magnetic sensor 105, etc.). Generally, the binding sites 116 and the surface 117 of the fluid chamber 115 can have any shape and properties that facilitate the binding of a single biopolymer 101 to each binding site 116, and enable the magnetic sensor 105 to detect the presence and movement of MNPs 102 bound to the biopolymer 101 bound to each binding site 116.

[0092] Figures 19C and 19D show an enclosed fluid chamber 115 having a apex extending in the xy plane, although the fluid chamber 115 does not necessarily need to be enclosed. In some embodiments, the surface 117 of the fluid chamber 115 has properties and characteristics that protect the sensor 105 from any fluid within the fluid chamber 115, while allowing the biopolymer 101 to bond to the binding site 116 and enabling the magnetic sensor 105 to detect the MNP 102 bonded to the biopolymer 101 bonded to the binding site 116. The material of the fluid chamber 115 (and optionally the material of the binding site 116) can be an insulator or may include an insulator. In some embodiments, the surface 117 of the fluid chamber 115 includes an organic polymer, a metal, or a silicate. The surface 117 of the fluid chamber 115 may include, for example, a metal oxide, silicon dioxide, polypropylene, gold, glass, or silicon. The thickness of the surface 117 of the fluid chamber 115 can be selected so that the magnetic sensor 105 can detect MNPs 102 bound to the biopolymer 101 bound to the binding site 116 within the fluid chamber 115. In some embodiments, the surface 117 is about 3 nm to 20 nm thick so that each magnetic sensor 105 is between about 5 nm and about 50 nm from any MNPs 102 bound to the biopolymer 101 bound to its respective binding site 116. It should be understood that these values ​​are for illustrative purposes only. Implementations may have fluid chambers 115 with thicker or thinner surfaces 117, and it should be understood that the sensing area 206 can be of any appropriate size, as described above.

[0093] The circuitry 120 of the monitoring system 100 may include one or more lines 125, or thereby be attached to the sensor array 110. In some embodiments, each magnetic sensor 105 is coupled to at least one line 125. In the examples shown in Figures 19B, 19C, and 19D, the monitoring system 100 includes eight lines 125A, 125B, 125C, 125D, 125E, 125F, 125G, and 125H. (For simplicity, this document generally refers to lines by reference numeral 125. Each line is given reference numeral 125 followed by a letter.) In the exemplary embodiments shown in Figures 19B, 19C, and 19D, a pair of lines 125 are used to access (e.g., read from or query) the individual magnetic sensors 105. In the exemplary embodiments shown in Figures 19B, 19C, and 19D, each magnetic sensor 105 of the sensor array 110 is coupled to two lines 125. For example, magnetic sensor 105A is coupled to lines 125A and 125H. Magnetic sensor 105B is coupled to lines 125B and 125H. Magnetic sensor 105C is coupled to lines 125C and 125H. Magnetic sensor 105D is coupled to lines 125D and 125H. Magnetic sensor 105E is coupled to lines 125D and 125E. Magnetic sensor 105F is coupled to lines 125D and 125F. Magnetic sensor 105G is coupled to lines 125D and 125G. In the exemplary embodiments of Figures 19B, 19C, and 19D, lines 125A, 125B, 125C, and 125D are shown to be located below magnetic sensor 105, and lines 125E, 125F, 125G, and 125H are shown to be located above magnetic sensor 105. Figure 19C shows magnetic sensors 105E associated with lines 125D and 125E, magnetic sensors 105F associated with lines 125D and 125F, magnetic sensors 105G associated with lines 125D and 125G, and magnetic sensors 105D associated with lines 125D and 125H.Figure 19D shows magnetic sensors 105D associated with lines 125D and 125H, magnetic sensors 105C associated with lines 125C and 125H, magnetic sensors 105B associated with lines 125B and 125H, and magnetic sensors 105A associated with lines 125A and 125H.

[0094] The magnetic sensors 105 of the exemplary monitoring system 100 shown in Figures 19B, 19C, and 19D are arranged in a sensor array 110 having a rectangular pattern. (It should be understood that a square pattern is a special case of a rectangular pattern.) Each of the lines 125 identifies a row or column of the sensor array 110. For example, each of lines 125A, 125B, 125C, and 125D identifies a different row of the sensor array 110, and each of lines 125E, 125F, 125G, and 125H identifies a different column of the sensor array 110. As shown in Figure 19C, each of lines 125E, 125F, 125G, and 125H is in contact with one of the magnetic sensors 105 along its cross-section (i.e., line 125E is in contact with the top of magnetic sensor 105E, line 125F is in contact with the top of magnetic sensor 105F, line 125G is in contact with the top of magnetic sensor 105G, and line 125H is in contact with the top of magnetic sensor 105D), and line 125D is in contact with the bottom of sensors 105E, 105F, 105G, and 105D, respectively. Similarly, as shown in Figure 19D, each of lines 125A, 125B, 125C, and 125D is in contact with the lower part of one of the sensors 105 along the cross section (i.e., line 125A is in contact with the lower part of magnetic sensor 105A, line 125B is in contact with the lower part of magnetic sensor 105B, line 125C is in contact with the lower part of magnetic sensor 105C, and line 125D is in contact with the lower part of magnetic sensor 105D), and line 125H is in contact with the upper parts of magnetic sensors 105D, 105C, 105B, and 105A, respectively.

[0095] Parts of the lines 125 connecting to the magnetic sensor 105 and the sensor array 110 are shown in Figure 19B using dashed lines to indicate that they may be embedded within the monitoring system 100. As mentioned above, the magnetic sensor 105 can be protected (e.g., by an insulator) from the contents of the fluid chamber 115 in which it may be surrounded. It should be understood that various illustrated components (e.g., lines 125, magnetic sensor 105, coupling portion 116, etc.) are not necessarily visible in the physical instance of the monitoring system 100 (e.g., they may be embedded in or covered by protective materials such as an insulator).

[0096] In some embodiments, part or all of the bonding site 116 is located within a nanocell or trench of line 125 passing through the magnetic sensor 105. For example, as shown in the example in Figure 19D, line 125H may be thinner on the magnetic sensor 105 than between the magnetic sensors 105. For example, line 125H has a first thickness above magnetic sensor 105D, a second greater thickness between magnetic sensors 105D and 105C, and a first thickness above magnetic sensor 105C. Such configurations can be advantageously fabricated using conventional thin-film fabrication methods (e.g., by depositing a material, applying a mask to the deposited material, and removing a portion of the deposited material according to the mask (e.g., by etching)). Both the bonding site 116 and the nanowells, if present, can be fabricated using conventional techniques.

[0097] For simplicity of explanation, Figures 19B, 19C, and 19D show an exemplary monitoring system 100 having only 16 magnetic sensors 105, only 16 corresponding binding sites 116, and 8 lines 125 within a sensor array 110. It should be understood that the monitoring system 100 may have fewer or more magnetic sensors 105 within the sensor array 110, and therefore may have more or fewer binding sites 116. Similarly, embodiments having lines 125 may have more or fewer lines 125. In general, any configuration of the circuitry 120 (e.g., including lines 125) that enables the magnetic sensors 105 to detect the magnetic sensors 105, the binding sites 116, and the MNP 102 bound to the biopolymer 101 bound to the binding sites 116 can be used. Similarly, any configuration of one or more lines 125 or any other mechanism that enables the sensor signal 207 to be extracted from the magnetic sensors 105 can be used. The examples presented herein are not intended to be limiting.

[0098] The magnetic sensor 105 shown in Figures 19B, 19C, and 19D is in close proximity to the binding site 116, and therefore also in close proximity to the biopolymers 101 and MNP 102 bound to the binding site 116.

[0099] Figures 19B, 19C, and 19D show a one-to-one relationship between the magnetic sensors 105 and the coupling points 116, but it should be understood that each coupling point 116 can be detected by multiple magnetic sensors 105. For example, if the monitoring system 100 has more magnetic sensors 105 than coupling points 116, it may be possible for at least some MNPs 102 to be detected by multiple magnetic sensors 105 (e.g., to improve the detection accuracy of MNPs 102 and their motion). Such a technique can improve the SNR by providing diversity of observation.

[0100] The exemplary sensor array 110 illustrated and described in the context of Figures 19B, 19C, and 19D is a rectangular array, and the magnetic sensors 105 are arranged in rows and columns. In other words, the multiple magnetic sensors 105 of the sensor array 110 are arranged in a rectangular grid pattern. In some embodiments, adjacent rows and columns of the rectangular grid pattern are equidistant from each other, and as a result, the magnetic sensors 105 are arranged in a square grid (or lattice) pattern, as shown in Figure 19E. In embodiments where the magnetic sensors 105 are arranged in a square grid pattern, each magnetic sensor 105 has up to four nearest neighbors. For example, as shown in Figure 19E, magnetic sensor 105A has four nearest neighbors labeled 105B, 105C, 105D, and 105E. The closest sensor 105 is located a nearest neighbor distance of 112, as shown in Figure 19E. Therefore, each of the sensors 105B, 105C, 105D, and 105E is located at a nearest distance of 112 from the magnetic sensor 105A.

[0101] According to some embodiments, the exemplary monitoring system 100 can utilize high-precision nanoscale fabrication of high-density packed nanoscale magnetic sensors 105 capable of detecting individual MNPs 102, as described above in the descriptions of Figures 18A, 18B, and 18C. The size of the functionalization binding site 116 can be similar to the size of the biopolymer 101 to which the MNPs 102 are bound, for example, so that multiple biopolymers 101 cannot bind to the same binding site 116 or be detected by the same magnetic sensor 105 (e.g., so that each magnetic sensor 105 detects / senss only one MNP 102). A suitable value for the nearest neighbor distance 112, which can then be used to determine the size of the sensor array 110 and / or the maximum number of magnetic sensors 105 that can be fitted into a sensor array 110 of a selected size, can be determined based on the characteristics of the magnetic sensors 105 (e.g., sensitivity, size, etc.), the characteristics of the biopolymer 101 that the monitoring system 100 is intended to monitor (e.g., length, softness, etc.), and the characteristics of the MNPs 102 used (e.g., size, type, etc.). For example, the total length of the biopolymer 101 used and the size of the MNP 102 can provide physical constraints on how close two magnetic sensors 105 can be positioned within the sensor array 110. In some embodiments, the size of the magnetic sensors 105 can be limited by the nanoscale patterning capability of the process used to manufacture the sensor array 110. For example, using techniques available at the time of writing, the size of each magnetic sensor 105 (e.g., the diameter of the sensor 105 in the xy plane, assuming a cylindrical sensor 105) may be around 20 nm. Assuming that the type of biopolymer 101 to be monitored is ssDNA and it is desirable to monitor fragments up to 150 nt in length, the maximum length of the biopolymer 101 to be sequenced is about 50 nm in the extended state, although the ssDNA conformation can vary between the extended and coiled states depending on the ionic strength of the buffer. Since the MNP 102 is involved in single-molecule reactions, the MNP 102 should have molecular dimensions.As described above, MNP102 can be, for example, superparamagnetic nanoparticles, organometallic compounds, or any other functional molecular group that can be detected by the nanoscale magnetic sensor 105.

[0102] As described above, the exemplary monitoring system 100 can be implemented using magnetic sensors 105 of various configurations. For example, in some embodiments of the monitoring system 100, the magnetic sensors 105 (e.g., MTJs) are arranged in a square grid compatible with existing crosspoint MRAM sensor shapes. As a specific example, a sensor array 110 having a configuration similar to that of Toshiba's 4Gbit-density STT-MRAM chip, which was first introduced at the International Electronic Devices Conference (IEDM) in 2016, can be used. In this case, the region of each nanoscale magnetic sensor 105 or its immediate vicinity can be functionalized to function as its respective bonding site 116. The minimum nearest neighbor distance 112 between magnetic sensors 105 on the Toshiba platform is 90 nm, which is sufficient spacing assuming that MNP 102 are superparamagnetic nanoparticles (e.g., iron oxide, platinum iron, etc.), the biopolymer 101 is 150 nt in length, and the sensor array 110 is a rectangular (e.g., square) array of magnetic tunnel junctions (MTJs) similar to those used in non-volatile data storage applications.

[0103] It should be understood that the arrangement of the magnetic sensors 105 in a grid pattern (e.g., a square grid as shown in Figure 19B) is one of many possible arrangements. Other arrangements of the magnetic sensors 105 are also possible and will be understood by those skilled in the art as being within the scope of the disclosure herein. For example, the magnetic sensors 105 may be arranged in a hexagonal pattern, in which case each magnetic sensor 105 has up to six nearest neighbors, all within a nearest neighbor distance 112. As will be understood by those skilled in the art, the sensor filling limit (e.g., the minimum nearest neighbor distance 112) of the monitoring system 100 having a hexagonal arrangement of the bonding site 116 and magnetic sensors 105 can be derived from knowledge of the size, shape, and properties of the magnetic sensors 105, the expected length of the biopolymer 101, and the size and type of MNP 102 used.

[0104] Exemplary monitoring method As described above (for example, in the descriptions of Figures 17A, 17B, 17C, 18A, 18B, and 18C), the magnetic sensor 105 described herein can be used in a method for monitoring a single-molecule process. Figure 20 is a flowchart of an exemplary method 300 for detecting the movement of a tethered MNP 102, according to several embodiments. In 302, optionally, the noise PSD of the magnetic sensor 105 is determined when there are no MNP 102s in its vicinity. As described above, this step establishes a baseline sensor PSD that can be compared to other PSDs to determine whether MNP 102s are present, if performed.

[0105] In 304, MNP102 is bonded to the first end of the biopolymer 101 (e.g., nucleic acid, protein, etc.). As described above, MNP102 can be any suitable particle, including, for example, superparamagnetic particles and / or particles having a diameter of a few nanometers (e.g., less than about 5 nm). MNP102 may be of different sizes (e.g., 20 nm). MNP102 can or may contain any suitable material that can be detected by the magnetic sensor 105. For example, MNP102 can be or may contain iron oxide (FeO), Fe3O4, or FePt.

[0106] In 306, the second end (other end) of the biopolymer 101 is bound to a binding site 116 detected by the magnetic sensor 105. As described above, the binding site 116 may be located within the fluid chamber 115 of the monitoring system 100. Also as described above, the magnetic sensor 105 may be any suitable sensor. For example, the magnetic sensor 105 may include an MTJ or STO.

[0107] In 308, a sensor signal 207 is acquired from the magnetic sensor 105 during the first detection period and the second detection period. As described above, the sensor signal 207 can be, or can represent, current, voltage, resistance, noise (e.g., frequency noise or phase noise), frequency (e.g., oscillation frequency of STO), magnetic field, etc. The first detection period and the second detection period may be partially overlapping or not overlapping, in which case (for example, as described above in the descriptions of Figures 17B and 17C and Figures 18B and 18C) a solution (e.g., Mg) may be obtained between the first and second periods. 2+ A (containing ions) may be added (for example, to the detection device fluid chamber 115).

[0108] In 310, the movement of MNP 102 is detected based on an analysis of the change in the sensor signal 207 between a first detection period and a second detection period. The change in the sensor signal 207 between the first detection period and the second detection period may be detected, for example, by obtaining a first autocorrelation of a portion of the signal corresponding to the first detection period, obtaining a second autocorrelation of a portion of the signal corresponding to the second detection period, and identifying at least one difference between the first autocorrelation and the second autocorrelation (for example, by comparing the autocorrelation functions described above in the descriptions of Figures 18A, 18B, and 18C). As another example, the change in the sensor signal 207 between the first detection period and the second detection period may be detected in part by determining at least one Lorentz function that, when added to the noise PSD of the magnetic sensor 105, results in the PSD of the sensor signal 207 during the first and / or second detection period. The motion of MNP102 can be determined based on a comparison of a Lorentz function fitted to the sensor signal 207 captured during a first detection period and a Lorentz function fitted to the sensor signal 207 captured during a second detection period. Processing and / or analysis of the sensor signal 207 can be performed in the time domain, frequency domain, or a combination thereof. For example, as described above, the autocorrelation function of portions of the sensor signal 207 taken at different times can reveal the motion of MNP102 detected by the magnetic sensor 105. In some situations, time-domain processing may be preferred for this analysis. As another example, as described above, the PSD of the sensor signal 207 may be processed and / or fitted by a Lorentz function, and / or different Lorentz functions may be compared. In some situations, this processing may be more convenient in the frequency domain. As yet another example, if the sensor signal 207 transmits a frequency (e.g., the oscillation frequency of the STO of the magnetic sensor 105), frequency-domain processing (e.g., Fourier transform of the time-domain data below) may be preferred. As yet another example, the autocorrelation function can be calculated or determined and then converted to the frequency domain for further analysis.

[0109] While the steps of Method 300 are shown in an exemplary order, it will be understood that at least some of the steps can be performed in a different order. For example, step 306 can be performed before step 304 (as described above, e.g., in the descriptions of Figures 17A, 17B, and 17C). It will also be understood that some of the steps shown in Figure 20 can be performed in real time (or near real time) or afterward. For example, step 302, if performed, can be performed much earlier than any of the other steps, or even after all the other steps have been completed (e.g., after MNP 102 has been rinsed off). As another example, one or more signals collected during step 308 can be recorded, and step 310 can be performed on the recorded data. Specifically, the magnetic sensor 105 can be read / queried during the test or experiment, and the collected sensor signal 207 can be recorded in either its native form or another form (e.g., sampled, amplified, normalized, etc.) (e.g., stored in memory). At some point later, one or more processors (e.g., at least one processor 130) can acquire and process the recorded sensor signals 207 to determine whether, and / or when, and / or how, the MNP 102, which is being monitored by the magnetic sensor 105, has moved during the test or experiment.

[0110] Multiplex magnetic digital homogeneous, non-enzymatic (HoNon) ELISA As explained above, conventional ELISA (analog) readout systems require a large volume to ultimately dilute the reaction product and millions of enzyme labels to generate a detectable signal using conventional plate readers. Conventional ELISA sensitivity is limited to the range of picomolar concentrations (e.g., pg / mL) and above.

[0111] In contrast, single-molecule measurements are inherently digital. Each molecule generates a signal that can be detected and counted. It is easier to measure the presence or absence of a signal (1s and 0s) than to detect the absolute amount of the signal. Digital ELISA sensitivity ranges from attomolecule (aM) to subfemtomolecule (fM).

[0112] An example of single-molecule digital ELISA technology is the assay based on Quanterix's Simoa beads. (https: / / www.quanterix.com / simoa-technology / , last accessed June 30, 2021.) In Simoa, paramagnetic particles are bound to antibodies designed to bind to specific targets. These particles are added to the sample. A detection antibody capable of generating fluorescence is then added, with the aim of forming an immune complex consisting of the beads, binding proteins, and detection antibody. At sufficiently low concentrations, each bead contains either one or zero binding proteins. The sample is then loaded into an array with a large number of microwells, each large enough to hold one bead. After enzyme signal amplification and fluorescence imaging using a fluorescent substrate, the data can be analyzed.

[0113] Both conventional and digital ELISAs are heterogeneous assays that involve enzyme signal amplification and multiple time-consuming incubation, reaction, and washing steps, typically lasting several hours. Homogeneous assays are assay formats that allow for assay measurements using a simple mix-and-read procedure without the need to process the sample with separation or washing steps, significantly reducing analysis time. However, short detection times typically correlate with reduced sensitivity and dynamic range.

[0114] The simplicity of homogeneous assays makes it possible to obtain highly sensitive detection comparable to digital ELISA. For example, homogeneous entropy-driven biomolecular assays (HEBA) achieve one-pot catalytic amplification signal generation without the use of enzymes or precise temperature cycling. (See, for example, Donghyuk Kim et al., "Homogeneous Entropy-Driven Amplified Detection of Biomolecular Interactions," ACS Nano, July 2016, 10(8), 7467-75.)

[0115] Digital homogeneous non-enzymatic (HoNon) immunoadsorption assays (ELISAs) without signal amplification have been demonstrated. (See, for example, Kenji Akama et al., "Wash-and Amplification-Free Digital Immunoassay Based on Single-Particle Motion Analysis," ACS Nano, November 2019, 13(11), 13116-26; Kenji Akama and Hiroyuki Noji, "Multiplexed homogeneous digital immunoassay based on single-particle motion analysis," Lab on a Chip, December 2020; and Kenji Akama and Hiroyuki Noji, "Multiparameter single-particle motion analysis for homogeneous digital immunoassay," Lab on a Chip, December 2020.)

[0116] Compared to optical, plasmon, and electrochemical biosensors, magnetic biosensors (e.g., magnetic sensor 105 described herein) exhibit low background noise because the majority of the biological environment is non-magnetic. The sensor signal 207 is also less affected by the type of sample matrix, thereby enabling an accurate and reliable detection process. Thus, embodiments of the systems (e.g., system 100), apparatus, and methods described herein can be used to provide what can be called a “multiplexed magnetic digital HoNon ELISA”.

[0117] Figure 21 shows some components involved in a multiplexed magnetic digital HoNon ELISA according to several embodiments. For the purposes of the example, let's assume there are three biomarkers A, B, and C to be tested, as shown in Figure 21. Three anti-biomarker beads A, B, and C are also shown to test these three biomarkers. Each bead contains MNP102 and a tethering group (shown as a small circle) that allows it to bind to a flexible molecular tether. The same type of MNP102 can be used for each bead, or different beads can contain different types of MNP102. For example, the MNP102 contained in anti-biomarker beads A, B, and C can be of the same type (e.g., they can have the same chemical composition (e.g., FeO, Fe3O4, FePt, etc.)) and a single type of MNP102 can be used for all of anti-biomarker beads A, B, and C. Alternatively, two or more types of MNP102 can be used for different anti-biomarker beads (e.g., FeO can be used for anti-biomarker bead A, FePt can be used for anti-biomarker bead B, etc.). In Figure 21, anti-biomarker A beads contain a first type of MNP102A, anti-biomarker B beads contain a second type of MNP102B which may be the same as or different from the first type, and anti-biomarker C beads contain a third type of MNP102C which may be the same as or different from the first and / or second types. Different types of anti-biomarkers are shaded differently in the drawing to allow them to be distinguished from one another, but it should be understood that shading in the drawing does not necessarily mean that the chemical composition of MNP102 in use is different.

[0118] As described above, the monitoring system 100 may include a sensor array 110. Figure 21 shows a portion 118 of such a sensor array 110 according to several embodiments. The portion 118 includes three magnetic sensors 105, namely magnetic sensor 105A, magnetic sensor 105B, and magnetic sensor 105C. Each magnetic sensor 105 has a corresponding binding site 116 on the surface 117 of the sensor array 110, which may be located within a fluid chamber 115 (i.e., magnetic sensor 105A has a binding site 116A, magnetic sensor 105B has a binding site 116B, and magnetic sensor 105C has a binding site 116C). A corresponding flexible molecular tether (e.g., biopolymer 101) is bound to the surface 117 at each binding site 116. For example, junction site 116A is tether 101A, junction site 116B is tether 101B, and junction site 116C is tether 101C.

[0119] Figures 22A and 22B show some exemplary procedures for a multiplexed magnetic digital HoNon ELISA according to several embodiments. Figure 22A shows the introduction of multiple anti-biomarker A beads containing MNP 102A into a sensor array 110 (for example, by adding a solution to a fluid chamber 115 of a monitoring system 100). As shown on the right side of Figure 22A, the anti-biomarker A beads containing MNP 102A bind to the tether 101A at a binding site 116A detected by a magnetic sensor 105A. Figure 22B shows how the binding of MNP 102A to the tether 101A affects the sensor signal 207 detected by the magnetic sensor 105 (assumed to be an MTJ for the example). As shown by sensor signal 207 and plotted on the left side of Figure 22B, before the anti-biomarker A bead containing MNP102A is coupled to tether 101A, the noise PSD of sensor signal 207 exhibits the 1 / f characteristics expected for the MTJ sensor in the absence of MNP102. The right side of Figure 22B shows that after MNP102A is coupled to tether 101A, the noise PSD of sensor signal 207 exhibits the expected characteristic bump 140 due to the presence of a Lorentz function throughout the noise. The presence of bump 140 in the overall noise PSD indicates that MNP102 is coupled to tether 101A in magnetic sensor 105A. At this point, since only the anti-biomarker A beads have been added, all magnetic sensors 105 in the sensor array 110 are queryed to identify which of their entire PSDs have the bump 140, thereby determining the location of the anti-biomarker A beads (for example, to determine which of all the tethers 101 incorporate the type A anti-biomarker beads).

[0120] Figure 23 shows additional possible steps in the exemplary procedure shown in Figures 22A and 22B. The portions of Figure 23 labeled "(a)" and "(b)" are described above in the descriptions of Figures 22A and 22B. That description applies to Figure 23 and is not repeated. After recording the positions of the anti-biomarker A beads in the sensor array 110, several other anti-biomarker beads may be optionally added. For example, Figure 23 shows the addition of several anti-biomarker B beads, one of which then contains MNP102B. As shown in the portion labeled "(c)" in Figure 23, the anti-biomarker B beads containing MNP102B are coupled to tether 101 C in the magnetic sensor 105C. As described above, the presence of MNP102B can be detected in the sensor signal 207 of the magnetic sensor 105C, and the overall noise PSD has a bump 140 due to the Lorentz component brought about by MNP102B. Therefore, the location of the anti-biomarker B beads in the sensor array 110 can be determined by querying the magnetic sensor 105 of the sensor array 110 that did not previously detect anti-biomarker A beads. After the identification information of the magnetic sensor 105 that detects anti-biomarker B beads is determined, the identification information / location of the magnetic sensor 105 that detects anti-biomarker A beads and the identification information / location of the magnetic sensor 105 that detects anti-biomarker B beads in the sensor array 110 are known.

[0121] Next, optionally, multiple other anti-biomarker beads can be added. For example, Figure 23 shows the addition of multiple anti-biomarker C beads, one of which contains MNP102C. As shown in the portion labeled "(d)" in Figure 23, the anti-biomarker C beads containing MNP102C are coupled to the tether 101B at the magnetic sensor 105B. As described above, the presence of MNP102C can be detected in the sensor signal 207 of the magnetic sensor 105B, and the overall noise PSD has a bump 140 due to the Lorentz component introduced by MNP102C. Thus, the location of the anti-biomarker C beads can be determined by examining the magnetic sensor 105 of the sensor array 110, which has not previously detected anti-biomarker A beads or anti-biomarker B beads. After the identification information of the magnetic sensor 105 that detects anti-biomarker C beads is determined, the identification information / location of the magnetic sensor 105 that detects anti-biomarker A beads, the identification information / location of the magnetic sensor 105 that detects anti-biomarker B beads, the identification information / location of the magnetic sensor 105 that detects anti-biomarker C beads, and the location / identification information of the magnetic sensor 105 that did not detect MNP 102 in the sensor array 110 are all known.

[0122] Optionally, additional types of anti-biomarker beads can be added (for example, more or fewer than three biomarkers can be tested), and the positions of these additional anti-biomarker beads can be determined as described above.

[0123] Next, as shown in Figure 24A, biomarkers corresponding to the previously added anti-biomarker beads can be added (for example, to the fluid chamber 115 of the monitoring system 100). Figure 24A shows the addition of a complex biological solution containing all of biomarkers A, B, and C. The positions of the anti-biomarker A beads, anti-biomarker B beads, and anti-biomarker C beads are known, and since each type of biomarker binds only to beads of the same type of anti-biomarker, all biomarkers to be tested can be added simultaneously without interference. As shown in the example in Figure 24A, a type A biomarker binds to an anti-biomarker A bead containing MNP102A bound to tether 101A. Similarly, a type B biomarker binds to an anti-biomarker B bead containing MNP102B bound to tether 101C, and a type C biomarker binds to an anti-biomarker C bead containing MNP102C bound to tether 101B. Figure 24B shows an example of what the entire sensor array 110 looks like after the addition of all three biomarkers A, B, and C (as mentioned above, it should be understood that implementations of the sensor array 110 can have more magnetic sensors 105 than shown in the figures herein (e.g., thousands, millions, etc.)).

[0124] Figure 25 illustrates how biomarker binding can be detected from the detected noise PSD of the sensor signal 207 of a specific magnetic sensor 105. The left side of Figure 25 shows an exemplary noise PSD of magnetic sensor 105A after MNP 102A is coupled to tether 101A (corresponding, for example, to the state of sensor array 110 shown on the right side of Figure 22A). The size on the left side of Figure 25 shows the component sensor noise PSD (caused by magnetic sensor 105A) and the Lorentz function (caused by MNP 102A), which, when added to the sensor noise PSD, generate the overall noise PSD in the sensor signal 207. In the illustrated example, the corner frequency of the Lorentz function is approximately 10 kHz, which is a function of the diameter of MNP 102A, as described above:

number

number

[0125] The right side of Figure 25 shows an example of the noise PSD of magnetic sensor 105A after the addition of the complex biological solution and after the type A biomarker was bound to anti-biomarker A beads containing MNP102A bound to tether 101A in magnetic sensor 105A. Also shown are the component sensor noise PSD and Lorentz function that, when added to the sensor noise PSD, generate the overall noise PSD in the sensor signal 207. The sensor noise PSD is the same as on the left side of Figure 25, but the Lorentz function has changed due to the incorporation of the type A biomarker. Assuming that the diameter of the type A biomarker is approximately the same as the diameter of MNP102A, the corner frequencies of the Lorentz function shift to lower frequencies given by:

number

[0126] Figure 26 is a flow chart of a process 600 for detecting biomarker binding, according to several embodiments. For example, process 600 can be used to detect biological events such as those described in the context of Figure 2A. In 602, the noise PSD of the magnetic sensor 105 of the sensor array 110 is determined when MNP 102 is absent (e.g., there is no MNP 102 at all in the detection area 206). In 604, the biopolymer 101 (tether) is bound to each binding site 116 that is detected by each magnetic sensor 105. In 606, multiple anti-biomarker beads are prepared. As described above in the discussion of Figure 21, the anti-biomarker beads contain MNP 102. In 608, a first set of anti-biomarker beads (e.g., a first type to be tested) is added to the fluid chamber 115 of the monitoring system 100. In 610, the identity (or location) of the magnetic sensor 105 that detects the anti-biomarker beads is determined. As described above (for example, in the description of Figures 22A and 22B), the presence of anti-biomiger beads in a particular magnetic sensor 105 can be detected by determining whether the overall noise PSD of the sensor signal 207 after the addition of the anti-biomiger beads (and thus MNP 102) has a bump 140 due to the addition of a Lorentz function that characterizes the PSD of the noise caused by MNP 102.

[0127] In step 612, it is determined whether there are any more antibiomarker beads to be tested (for example, whether there are antibiomarker B beads or antibiomarker C beads, as shown in Figure 23). If so, process 600 repeats steps 608 and 610. Once there are no more antibiomarker beads to add, the monitoring system 100 has a map of which magnetic sensors 105 of the sensor array 110 are detecting the tether 101 incorporating the antibiomarker beads, and, in the case of multiple types of antibiomarker beads, which magnetic sensors 105 are detecting which type of antibiomarker bead.

[0128] In 614, a solution containing a biomarker corresponding to the anti-biomarker beads in the fluid chamber 115 is added to the fluid chamber 115. As described above, one advantage of some embodiments is that multiple biomarkers can be tested at once. Therefore, if the fluid chamber 115 contains multiple types of anti-biomarker beads, the added solution can contain multiple types of biomarkers, and all of them can be added to the fluid chamber 115 simultaneously. (Of course, it should be understood that if there are multiple biomarkers to be tested, they can be added separately.)

[0129] In step 616, the sensor signals 207 are obtained from at least their magnetic sensors 105 that detect each MNP 102. In step 618, biomarker binding is detected based on a comparison between the sensor signals 207 collected in step 610 and the sensor signals collected in step 616. For example, as described above in the explanation of Figure 25, the corner frequencies of the Lorentz function fitted to the overall noise PSD of the sensor signal 207 from step 610 can be compared with the corner frequencies of the Lorentz function fitted to the overall noise PSD of the sensor signal 207 from step 616 to determine whether the corner frequencies have changed. Specifically, as described above, biomarker integration can be detected from a decrease in corner frequencies due to an increase in the effective diameter of the MNP 102 (for example, the effective mass of the biopolymer 101 increases, and the motion frequency of the MNP 102 decreases).

[0130] While the steps of process 600 are shown in an exemplary order, it should be understood that some steps can be performed in a different order. For example, the order of steps 602, 604, and 606 may differ (for instance, step 604 can be performed before step 602 or after step 606; step 606 can be performed before step 602 and / or before step 604, etc.).

[0131] The preceding description and accompanying drawings use certain terms to provide a complete understanding of the disclosed embodiments. In some cases, terms or drawings may refer to certain details that are not necessary to carry out the invention.

[0132] To avoid unnecessarily obscuring this disclosure, well-known components are shown in block diagram form and / or are not described in detail, or in some cases not described at all.

[0133] Unless otherwise explicitly defined herein, all terms should be given the broadest possible interpretation, including the meaning implied by this specification and the drawings, as well as the meaning understood by those skilled in the art, and / or the meaning defined in dictionaries, papers, etc. Some terms may not correspond to their usual or conventional meanings, as explicitly stated herein.

[0134] Where used herein, the singular forms “a,” “an,” and “the” do not exclude multiple referents unless otherwise specified. The word “or” should be interpreted as inclusive unless otherwise specified. Thus, the phrase “A or B” should be interpreted as meaning all of the following: “both A and B,” “A but not B,” and “B but not A.” No use of “and / or” herein implies that the word “or” alone implies exclusivity.

[0135] As used herein, the phrases “at least one of A, B, and C,” “at least one of A, B, or C,” “one or more of A, B, or C,” and “one or more of A, B, and C” are interchangeable and each encompasses all of the following meanings: “A only,” “B only,” “C only,” “A and B but not C,” “A and C but not B,” “B and C but not A,” and “all of A, B, and C.”

[0136] To the extent that the terms “include,” “having,” “has,” and “with,” and their variations thereof are used herein, such terms are intended to be inclusive in a similar manner to the term “comprising,” that is, to mean “including but not limited to.” The terms “exemplary” and “embodiment” are used to represent examples and not to represent preferences or requirements. The term “coupled” is used herein to represent direct connection / attachment and connection / attachment via one or more intervening elements or structures. The terms “over,” “under,” “between,” and “on” refer herein to the relative position of one feature to another feature. For example, one feature positioned “over” or “under” another feature may be in direct contact with the other feature or may have intervening material. Furthermore, a feature positioned "between" two features may be in direct contact with the two features, or it may have one or more intervening features or materials. In contrast, a first feature "on" a second feature is in contact with that second feature.

[0137] The term "substantially" is used to describe structures, configurations, dimensions, etc., that are largely or nearly as described, but due to manufacturing tolerances, etc., there may be situations where the structure, configuration, dimensions, etc., are not always or necessarily as described. For example, describing two lengths as "substantially equal" means that the two lengths are the same for all practical purposes, but they do not have to be (and do not need to be) exactly equal on a sufficiently small scale. As another example, a structure that is "substantially perpendicular" is considered perpendicular for all practical purposes even if it is not exactly 90 degrees to the horizontal.

[0138] The drawings are not necessarily to scale, and the dimensions, shapes, and sizes of features may differ substantially from those shown in the drawings.

[0139] While specific embodiments are disclosed, it will be apparent that various modifications and variations can be made without departing from the broader spirit and scope of this disclosure. For example, any feature or aspect of an embodiment can be applied in combination with or instead of any other embodiment, at least where feasible. Accordingly, this specification and the drawings should be considered illustrative rather than restrictive.< / r>

Claims

1. A method (300) for monitoring a single molecular biological process using a magnetic sensor (105) having a detection area, The biopolymer is attached to the binding site detected by the magnetic sensor (304), Bonding magnetic particles to the biopolymer (306), Acquiring signals from the magnetic sensor during the first detection period and the second detection period (308), Using the signal acquired from the magnetic sensor during the first detection period, the first power spectral density (PSD) of the signal acquired during the first detection period is determined. The first power spectral density (PSD) is fitted with a first Lorentz function characterized by a first corner frequency, The second power spectral density (PSD) of the signal acquired during the second detection period is determined using the signal acquired from the magnetic sensor during the second detection period. The second power spectral density (PSD) is fitted with a second Lorentz function characterized by a second corner frequency, Detecting the movement of the magnetic particles based on the change in the signal between the first detection period and the second detection period (310), which includes identifying the difference between the first corner frequency and the second corner frequency. Methods that include...

2. The method according to claim 1, wherein the magnetic particles are magnetic nanoparticles.

3. The method according to claim 1, wherein the magnetic particles are superparamagnetic.

4. The method according to claim 1, wherein the size of the magnetic particles is less than about 5 nm.

5. The magnetic particles are iron oxide (FeO), Fe ３ O ４ The method according to claim 1, or comprising FePt.

6. The method according to claim 1, wherein the biopolymer is a nucleic acid or a protein.

7. The method according to claim 1, wherein the signal represents current, voltage, or resistance.

8. The method according to claim 1, wherein the signal represents a detected magnetic field.

9. Based on the change in the signal between the first detection period and the second detection period, the movement of the magnetic particles is detected. Obtaining a first autocorrelation of a portion of the signal corresponding to the first detection period, To obtain a second autocorrelation of a portion of the signal corresponding to the second detection period, Identifying at least one difference between the first autocorrelation and the second autocorrelation. The method according to claim 1, including the method described in claim 1.

10. The method according to claim 9, further comprising sampling the signal.

11. The method according to claim 1, wherein the first detection period and the second detection period do not overlap.

12. The method according to claim 1, wherein the signal represents noise.

13. The method according to claim 12, wherein the noise is frequency noise or phase noise.

14. The method according to claim 1, wherein the signal represents the oscillation frequency of the magnetic sensor.

15. The method according to claim 1, further comprising sampling the signal.

16. The method according to claim 1, wherein the magnetic sensor comprises a magnetic tunnel junction (MTJ).

17. The method according to claim 1, wherein the magnetic sensor comprises a spin torque oscillator (STO).

18. The method according to claim 1, wherein the magnetic sensor comprises a spin valve.

19. The volume of the detection area of ​​the magnetic sensor is approximately 10 ５ nm ３ From approximately 5 x 10 ５ nm ３ The method according to claim 1, wherein the method is between the two.

20. The method according to claim 1, further comprising the coupling portion being located within a fluid chamber of a detection system, and adding a solution to the fluid chamber.

21. The method according to claim 20, wherein the addition of the solution to the fluid chamber is performed between the first detection period and the second detection period.

22. The aforementioned solution contains Mg ２＋ The method according to claim 20, comprising ions.

23. The method according to claim 20, wherein the solution comprises at least one biomarker.

24. The method according to claim 1, further comprising applying a magnetic field to the magnetic particles.

25. The method according to claim 1, further comprising acquiring the signal from the magnetic sensor during a third detection period, wherein the third detection period is performed while the magnetic particles are outside the detection area.

26. The method according to claim 25, further comprising determining the noise power spectral density (PSD) of the magnetic sensor using the signal detected during the third detection period.

27. The sum of the first Lorentz function and the noise power spectral density (PSD) of the magnetic sensor is approximately equal to the first power spectral density (PSD). The sum of the second Lorentz function and the noise power spectral density (PSD) of the magnetic sensor is approximately equal to the second power spectral density (PSD). The method according to claim 26, further comprising concluding that a biological process has occurred based on the fact that the first corner frequency is different from the second corner frequency.

28. The method according to claim 27, wherein the biological process includes binding a biomarker to the biopolymer, and the second detection period follows the addition of a complex biological solution containing a plurality of biomarkers, wherein the first corner frequency is greater than the second corner frequency.

29. The first Lorentz function represents the first noise PSD resulting from the movement of the magnetic particles during the first detection period. The second Lorentz function represents the second noise PSD resulting from the movement of the magnetic particles during the second detection period. The method according to claim 1.

30. The method according to claim 29, wherein the second detection period is followed by the addition of a complex biological solution containing multiple biomarkers, and the first corner frequency is greater than the second corner frequency.

31. A system (100) for monitoring the movement of magnetic particles (102) bound to a biopolymer (101), A fluid chamber (115) comprising binding sites (116) for holding one or fewer biopolymers at a time, wherein the binding sites are configured to bind the ends of the biopolymers to the surface (117) of the fluid chamber and to allow the magnetic particles to move, At least one processor (130), A magnetic sensor (105) configured to detect a detection region (206) within the fluid chamber, wherein the detection region includes the coupling portion, the magnetic sensor is configured to generate a signal (207) characterizing the magnetic environment within the detection region, and to provide the signal to the at least one processor, and the magnetic sensor is one of a plurality of magnetic sensors arranged in a sensor array (110), At least one line connecting the sensor array to the at least one processor, wherein the connection portion is located within the trench of the first line of the at least one line and Equipped with, The aforementioned at least one processor, To acquire a first portion of the signal, wherein the first portion of the signal represents the magnetic environment within the detection area during a first detection period. To obtain a second portion of the signal, wherein the second portion of the signal represents the magnetic environment within the detection area during a second detection period, and the second detection period is after the first detection period. The first portion and the second portion of the signal are analyzed to detect the movement of the magnetic particles. It is configured to do the following: Analyzing the first portion and the second portion of the signal is: Determining the first power spectral density (PSD) of the first portion of the signal, Fitting the first power spectral density (PSD) to the first Lorentz function, Determining the second power spectral density (PSD) of the second portion of the signal, Fitting the second Lorentz function to the second power spectral density (PSD), A system that includes this.

32. The system according to claim 31, wherein the signal represents current, voltage, or resistance.

33. The system according to claim 31, wherein the signal represents noise.

34. The system according to claim 33, wherein the noise is frequency noise or phase noise.

35. The system according to claim 31, wherein the signal represents the oscillation frequency of the magnetic sensor.

36. The system according to claim 31, wherein the magnetic sensor comprises a magnetic tunnel junction (MTJ).

37. The system according to claim 31, wherein the magnetic sensor comprises a spin torque oscillator (STO).

38. The system according to claim 31, wherein the magnetic sensor comprises a spin valve.

39. The volume of the detection region is about 10 ５ nm ３ to about 5×10 ５ nm ３ between, the system according to claim 31.

40. The aforementioned at least one processor, Determine the first autocorrelation function of the first portion of the signal, and The second autocorrelation function of the second portion of the signal is determined. It is further configured in this way, The system according to claim 31, wherein detecting the movement of the magnetic particles by analyzing the first and second portions of the signal includes comparing the first autocorrelation function with the second autocorrelation function.

41. The system according to claim 31, further comprising the magnetic sensor and a detection circuit coupled to the at least one processor.

42. The system according to claim 41, wherein the detection circuit comprises at least one line.

43. The system according to claim 41, wherein the detection circuit comprises at least one of an amplifier or an analog-to-digital converter.

44. The system according to claim 31, wherein the binding site includes a structure configured to fix the biopolymer to the binding site.

45. The system according to claim 44, wherein the structure comprises a cavity or a protruding portion.

46. The magnetic particles are first magnetic particles, the biopolymer is a first biopolymer, the magnetic sensor is a first magnetic sensor, the detection area is a first detection area, the signal is a first signal, the fluid chamber further includes a second binding site for holding one or fewer biopolymers at a time, the second binding site is configured to bind the end of the second biopolymer to the surface of the fluid chamber, and to allow the second magnetic particles bound to the second biopolymer to move. A second magnetic sensor having a second detection region within the fluid chamber, wherein the second detection region includes a second coupling portion but does not include other coupling portions, and the second magnetic sensor is configured to generate a second signal characterizing the magnetic environment within the second detection region and to provide the second signal to the at least one processor, further comprising the second magnetic sensor. The aforementioned at least one processor, To obtain a first portion of the second signal, wherein the first portion of the second signal represents the magnetic environment within the second detection region during the third detection period. To obtain a second portion of the second signal, wherein the second portion of the second signal represents the magnetic environment within the second detection region during the fourth detection period, The first portion of the second signal and the second portion of the second signal are analyzed to detect the movement of the second magnetic particle. The system according to claim 31, further configured to perform the following:

47. The system according to claim 46, wherein the first detection period and the third detection period are the same, and the second detection period and the fourth detection period are the same.

48. The system according to claim 31, wherein the plurality of magnetic sensors are arranged in a rectangular grid pattern.

49. The system according to claim 31, wherein the at least one processor comprises at least two processors, the first of the at least two processors configured to acquire the first and second portions of the signal, and the second of the at least two processors configured to analyze the first and second portions of the signal to detect the movement of the magnetic particles.

50. The system according to claim 49, wherein the first processor is located within the device equipped with the magnetic sensor, and the second processor is located outside the device.

51. The system according to claim 31, wherein the at least one processor is further configured to determine the noise power spectral density of the magnetic sensor.

52. The system according to claim 31, wherein detecting the motion of the magnetic particle by analyzing the first and second portions of the signal further includes comparing the first corner frequency of the first Lorentz function with the second corner frequency of the second Lorentz function.

53. The system according to claim 31, wherein the at least one processor is further configured to determine that a particular biomarker is bound to the biopolymer based on a comparison between a first corner frequency of the first Lorentz function and a second corner frequency of the second Lorentz function.

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