Device, method and system for biomarker detection

Abstract

A point-of-care device and method for simultaneously and quantitatively assessing a magnetically-labelled analyte immobilised on a lateral flow test strip are provided. The method comprises the steps of sensing a magnetic signal from the magnetically-labelled analyte on the test strip and converting the magnetic signal into an electric signal; processing the electric signal to obtain a quantitative value of the amount of analyte; and processing the values to provide a differential assessment between the magnetically-labelled analyte. The device includes a magnetic reader with at least one magnetic sensor, such as a giant magnetoresistive sensor. The magnetic sensor can be directly or indirectly coupled to a lock-in amplifier configured to perform phase-sensitive detection and enhance the signal-to-noise ratio of weak magnetic signals. A system, microfluidics device, lateral flow immunoassay test strip and kit are also provided.

Description

[0001] DEVICE, METHOD AND SYSTEM FOR BIOMARKER DETECTION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority from UK patent application number 2414207.7 filed on 27 September 2024, which is incorporated by reference herein.

[0004] FIELD

[0005] A device, method and system are described for rapid, quantitative and cost-effective detection of biological markers. The device may be used to assess dynamic changes in biomarkers over time, measure autophagy flux, support wellness tracking, personalize medical treatments or lifestyle interventions, enable early detection of disease or aging- related changes, assess drug efficacy, and the like.

[0006] BACKGROUND TO THE INVENTION

[0007] The 21 st century has been characterised by increasing life expectancy, yet a variety of health challenges, including cancer, neurodegeneration, metabolic disorders, infections, inflammatory conditions, and cardiovascular disease, continue to impact global health. There is growing interest in monitoring biological markers to support wellness, personalise medical interventions, and enable early detection of disease or age-related decline.

[0008] Dynamic biological processes, such as protein turnover, enzymatic activity, receptor trafficking, and cellular maintenance pathways, play a critical role in maintaining health. Autophagy, for example, is a cellular recycling mechanism that removes damaged organelles and protein aggregates, supporting proper organ function and cellular homeostasis. Dysregulation of autophagy has been linked to a range of diseases, including neurodegenerative disorders, cancer, and age-related conditions. Modulation of autophagy, whether through lifestyle factors or pharmacological interventions, has attracted considerable attention in biomedical research. While autophagy is highlighted herein as an illustrative example, similar considerations apply broadly to other dynamic cellular and molecular processes.

[0009] Existing approaches to assess these biological processes are generally complex, labour- intensive, costly, and require specialised laboratory facilities and skilled personnel. Techniques such as Western blot analysis, electron microscopy, and fluorescence-based assays may provide detailed information on cellular processes, but are time-consuming, require substantial sample preparation, and are unsuitable for routine or frequent measurement in humans. Western blot and electron microscopy provide static snapshots of cellular components, while even advanced livecell imaging approaches are limited by specialised equipment requirements, labour intensity, and cost.

[0010] Moreover, current systems are typically not portable, preventing point-of-care monitoring or routine assessments outside specialised laboratory settings. These limitations present barriers to wider adoption for personalised healthcare, wellness tracking, and rapid evaluation of biological interventions.

[0011] Consequently, there remains significant room for improvement in methods and systems that enable accessible, rapid, quantitative, and reliable monitoring of biological markers and dynamic cellular processes in humans. Development in this area has the potential to facilitate research into disease mechanisms, screen candidate drugs, monitor the effects of interventions, and support more proactive approaches to health and wellbeing.

[0012] The preceding discussion of the background to the invention is intended only to facilitate an understanding of the present invention. It should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was part of the common general knowledge in the art as at the priority date of the application.

[0013] SUMMARY

[0014] In accordance with a first aspect there is provided a device for quantitatively assessing one or more magnetically-labelled analytes captured on at least two lateral flow immunoassay test strip test line regions, the device including a magnetic reader comprising: a) means for receiving at least one lateral flow test strip having a test line; b) at least one magnetic sensor; c) one or more magnets which bias the magnetic sensor; and d) one or more magnets which magnetise magnetic particles on the test line of each lateral flow test strip.

[0015] The device may include a housing. The magnetic reader may include a lock-in amplifier electrically connected to the magnetic sensor for enhancing a signal-to-noise ratio of magnetic readings from the test line.

[0016] In some embodiments the lock-in amplifier may be configured to selectively detect magnetic readings from the magnetic sensor corresponding to the magnetisation of magnetic particles on the test line, so as to discriminate the particle signal from background noise.

[0017] The lock-in amplifier may further be configured to perform phase-sensitive detection of the magnetic readings obtained from the magnetic sensor, such that only readings having a defined phase relationship with the applied magnetic field used to magnetise the magnetic particles are detected.

[0018] In some embodiments the lock-in amplifier may be configured to enable detection of low concentrations of magnetically-labelled biorecognition molecules on the lateral flow test line by amplifying magnetic readings from the magnetic sensor corresponding to the magnetised magnetic particles and rejecting broadband electronic noise.

[0019] In a preferred embodiment the lock-in amplifier is configured to reduce the effect of environmental magnetic interference on the magnetic readings obtained from the magnetic sensor by selectively filtering readings at the frequency of the applied magnetisation.

[0020] The lock-in amplifier may even further be configured to detect magnetic readings corresponding to magnetic particles located on multiple test lines of the same lateral flow test strip or on multiple lateral flow test strips.

[0021] The magnetic reader may be configured so that the test line of the at least one lateral flow test strip may be positioned in line with the magnetic sensor when the lateral flow test strip is received by the magnetic reader.

[0022] The magnetic sensor may be a giant magnetoresistive (GMR) sensor.

[0023] The electromagnet may be a Helmholtz coil.

[0024] The device may be an autophagy flux measurement device which includes a processing means for quantitatively assessing or comparing biomarker levels from magnetic readings obtained by the magnetic reader and an output means for communicating the assessment or comparison to a user. The device may include a housing configured to at least house the processing means and the magnetic reader. In accordance with a second aspect there is provided a device for simultaneously and quantitatively assessing one or more magnetically-labelled analytes captured on at least two lateral flow immunoassay test strip test line regions, the device comprising: a housing; a magnetic reader including: a) means for receiving at least one lateral flow test strip having one or more test lines; b) at least one magnetic sensor; c) one or more magnets which bias the magnetic sensor; d) one or more magnets which magnetise magnetic particles on the test line(s) of each lateral flow test strip; and e) a lock-in amplifier electrically connected to the magnetic sensor for enhancing a signal- to-noise ratio of magnetic readings from each test line; a processing means for quantitatively assessing or comparing analyte levels from magnetic readings obtained by the magnetic reader; and an output means for communicating the assessment or comparison to a user.

[0025] In accordance with a third aspect there is provided a system comprising: a) a magnetic reader substantially as described above; b) a processing means for processing one or more outputs received from the magnetic reader; and c) an output means in data communication with the processing means for outputting the processed outputs.

[0026] The output means may be a digital display.

[0027] The lock-in amplifier may be implemented as an analog or digital lock-in amplifier within the magnetic reader or the processing means.

[0028] In some embodiments processing may include processing the one or more outputs into readable results and outputting the processed outputs may include outputting the readable results.

[0029] In accordance with a further aspect there is provided a method of simultaneously and quantitatively assessing one or more magnetically-labelled analytes captured on at least two lateral flow immunoassay test strip test line regions, the method comprising the steps of: a) sensing a magnetic signal from the magnetically-labelled analyte on each test line region and converting the magnetic signal for each test line region into an electric signal; b) processing the electric signal to obtain a quantitative value of the amount of magnetically-labelled analyte on each test line region; c) processing the values obtained in step (b) to provide a differential assessment between the magnetically-labelled analyte on each test line, wherein the differential assessment is obtained by subtracting one value from the other or by comparing one value to the other.

[0030] The electric signal in step (b) may be processed by:

[0031] - amplifying the electric signal;

[0032] - filtering the amplified signal with a band-pass filter;

[0033] - splitting the filtered signal into two paths and:

[0034] - multiplying the signal of one path with a sine reference;

[0035] - multiplying the signal of the other path with a cosine reference;

[0036] - low-pass filtering each multiplied signal to attenuate frequency components and noise above a baseband and obtain demodulated baseband signals;

[0037] - amplifying each demodulated baseband signal to obtain output signals; and

[0038] - digitising the output signals for processing.

[0039] Each test line region may be on a different lateral flow immunoassay test strip. Alternatively, the test line regions may be on the same lateral flow immunoassay test strip.

[0040] The magnetically-labelled analytes captured on each test line region may be different or may be the same analyte but treated differently.

[0041] The analyte may be a biomarker, pathogen, antigen, toxin, allergen or other compound such as a drug.

[0042] The magnetic labels may be superparamagnetic nanoparticles (SMNPs).

[0043] The method may be performed using a magnetic reader or system substantially as described above.

[0044] In accordance with a further aspect there is provided a method of quantitatively assessing an analyte in a sample, the method comprising the steps of: a) dividing a sample from a subject into at least first and second portions; b) treating at least one of the portions with a treatment, wherein if both the portions are treated, the treatments of the first and second portions are different; c) applying the first and second samples to separate lateral flow test strips, each lateral flow test strip having a conjugate pad comprising magnetically-labelled biorecognition molecules which can specifically bind to the analyte and a test line region comprising biorecognition molecules which can specifically bind to the analyte, wherein the biorecognition molecules are immobilized on the test line region; d) magnetically sensing the magnetically-labelled biorecognition molecules on the test line for each portion and obtaining a quantitative value for each portion; e) processing the values obtained in step (d) to provide a differential and quantitative assessment of the quantity of analyte in the portions.

[0045] The analyte may be a biomarker, pathogen, antigen, toxin, allergen or other compound such as a drug.

[0046] The biorecognition molecule may be an antibody.

[0047] The magnetic labels may be superparamagnetic nanoparticles (SMNPs).

[0048] The method may be performed using a magnetic reader or system substantially as described above.

[0049] In accordance with an even further aspect there is provided a method of measuring autophagy flux, the method comprising the steps of: a) dividing a sample from a subject into first and second portions; b) contacting only the first portion with a sufficient amount of an autophagosome-lysosome fusion inhibitor to inhibit fusion of autophagosomes and lysosomes in the first portion for a period of time; c) determining the level of LC3 (i.e. LC3-I and LC3-II) or LC3-II in each portion; and d) calculating the autophagy flux by comparing the level of LC3 or LC3-II in the first portion with the level of LC3-II in the second portion with respect to time.

[0050] The autophagosome-lysosome fusion inhibitor may be bafilomycin or chloroquine.

[0051] The period of time may be from about 5-30 minutes, such as from about 10-20 minutes, or more particularly about 15 minutes.

[0052] The method may further comprise the step of isolating the white blood cells in each sample after step a). The method may further comprise a step of contacting the isolated white blood cells with a lysing agent.

[0053] In step c), the level of LC3 in each portion may be determined by contacting each portion with magnetically labelled antibodies which bind specifically to LC3 proteins, so as to form labelled- antibody-LC3 complexes, and magnetically sensing the amount of labelled-antibody-LC3 complex in each portion.

[0054] The magnetic labels may be superparamagnetic nanoparticles (SMNPs).

[0055] A lateral flow immunoassay (LFIA) and a magnetic reader as described above may be used to determine the level, number, concentration or relative magnetic intensity of LC3 or LC3-II in each portion.

[0056] In accordance with a further aspect there is provided a lateral flow immunoassay (LFIA) test strip for use in measuring autophagy flux, comprising: a) a sample pad for receiving a fluid sample; b) a conjugate pad comprising magnetically-labelled biorecognition molecules which can specifically bind to LC3-I and LC3-II proteins; c) a test line region comprising biorecognition molecules which can specifically bind to LC3-I and LC3-II proteins, wherein the biorecognition molecules are immobilized on the test line region.

[0057] The biorecognition molecules may be antibodies.

[0058] In accordance with a further aspect there is provided a microfluidic device for separating a fluid sample into at least two portions and differentially treating the portions, the device comprising: a) a zone for receiving a fluid sample; b) two passages; and c) means for dividing the fluid sample into two portions and directing one portion into one passage and the other portion into the other passage; wherein at least one of the passages has a reagent which will come into contact with the sample in that passage, and wherein the other passage does not have the same reagent.

[0059] Each passage may be configured to be in fluid communication with a sample pad of a lateral flow test strip. In accordance with a further aspect there is provided a microfluidic device for use in a method of measuring autophagy flux, the device comprising: a) a zone for receiving a fluid sample; b) a test passage; c) a control passage; and d) means for dividing the fluid sample into two substantially equal portions and directing one portion into the test passage and the other portion into the control passage; wherein the test passage comprises:

[0060] (i) a zone containing an autophagosome-lysosome fusion inhibitor; and

[0061] (ii) a zone containing a lysing agent for lysing the white blood cells; and wherein the control passage comprises:

[0062] (iii) a zone which does not contain an autophagosome-lysosome fusion inhibitor; and

[0063] (iv) a zone containing a lysing agent for lysing the white blood cells.

[0064] The device may further comprise one or more zones in which red blood cells are removed from the sample. One such zone may be located between the sample receiving zone and the means for dividing the sample into two portions. Alternatively, a zone for removing red blood cells may be located in each of the test and control passages, e.g. before the lysing zone.

[0065] The test and control passages may further comprise yet a further zone for depositing the respective portion of the sample onto a sample pad of a lateral flow immunoassay test strip.

[0066] The device may further comprise a lateral flow immunoassay test strip leading from each of the test and control passages, each lateral flow test strip comprising: a) a sample pad for receiving the sample from the test or control passage; b) a conjugate pad comprising magnetically-labelled biorecognition molecules which can specifically bind to LC3-I and LC3-II proteins; c) a test line region comprising biorecognition molecules which can specifically bind to LC3-I and LC3-II proteins, wherein the biorecognition molecules are immobilized on the test line region.

[0067] In accordance with a further aspect there is provided a kit comprising: one or more lateral flow immunoassay test strips substantially as described above; one or more microfluidic devices substantially as described above; a magnetic reader device substantially as described above; one or more cartridges for housing a lateral flow test strip and configured to engage with the magnetic reader device; a fusion inhibitor which inhibits fusion of autophagosomes and lysosomes; a white blood cell lysing composition; one or means for collecting a sample from a subject; and / or instructions for performing the method substantially as described above.

[0068] In accordance with a further aspect there is provided a use of a magnetic reader, system, device or lateral flow immunoassay test strip as described above for quantitatively and / or differentially analysing one or more analytes in a sample or for presenting information related thereto.

[0069] In accordance with a further aspect there is provided a use of magnetically-labelled biorecognition markers in the manufacture of a lateral flow immunoassay test strip, device or kit for quantitatively and / or differentially analysing one or more analytes in a sample.

[0070] ABBREVIATIONS

[0071] AF Antiferromagnetic

[0072] BAF Bafilomycin A1

[0073] BJT Bipolar junction transistor

[0074] CCD Charge-coupled devices

[0075] CMA Chaperone-mediated autophagy

[0076] CMOS Complementary metal-oxide semiconductor

[0077] DNA Deoxyribonucleic acid

[0078] EDC 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0079] Fab Antigen-binding fragment

[0080] Fc Crystallisable fragment

[0081] GMR Giant Magnetoresistance

[0082] HCL Hydrochloric acid

[0083] HTM High temperature multilayer

[0084] Ig Immunoglobulin

[0085] IA Instrumentation amplifier

[0086] LAMP1 Lysosomal-associated membrane protein 1

[0087] LC3-I Microtubule-associated protein 1 light chain 3

[0088] LC3-II LC3-phosphatidylethanolamine

[0089] LC3 Both LC3-I and LC3-II

[0090] LFIA Lateral flow immunoassay, also commonly referred to as a lateral flow assay (LFA), lateral flow test (LFT) and lateral flow device (LFD), all of which can be used interchangeably

[0091] LFTS Lateral flow test strip LHHTM Low hysteresis high temperature multilayer mAb Monoclonal antibody

[0092] MES 2-(N-morpholino)ethanesulfonic acid

[0093] MNP Magnetic nanoparticles

[0094] NC Nitrocellulose

[0095] NHS N-Hydroxysuccinimide

[0096] NIR Near infrared pAb Polyclonal antibody

[0097] PBS Phosphate-buffered saline

[0098] PCB Printed circuit board

[0099] PCT Patent Cooperation Treaty

[0100] PEG Polyethylene glycol

[0101] POC Point-of-care

[0102] QD Quantum dots

[0103] RIPA Radioimmunoprecipitation assay buffer

[0104] RNA Ribonucleic acid

[0105] SML Standard multilayer

[0106] SMNP Superparamagnetic nanoparticles

[0107] SQSTM1 Sequestosome 1

[0108] Sulfo-NHS N-Hydroxysulphosuccinimide

[0109] SV Spin valve

[0110] TEM Transmission Electron Microscopy

[0111] TMR Tunnel Magnetoresistance

[0112] UCP Up-converting phosphor

[0113] BRIEF DESCRIPTION OF THE FIGURES

[0114] Figure 1 : shows a side view of components of a magnetic reader in one embodiment of the invention.

[0115] Figure 2: shows a signal-flow diagram of an example lock-in amplifier (LIA) dual-phase demodulation chain for reading a GMR sensor.

[0116] Figure 3: is a second-order multiple-feedback active bandpass filter.

[0117] Figure 4: shows an example embodiment of ADG1208 multiplexer devices in a dual-phase lock-in amplifier. Figure 5: is a circuit diagram showing two low-pass Butterworth filters in a Sallen-Key topology.

[0118] Figure 6: shows a schematic representation of the components of a device for reading test and control LFIAs of the invention, calculating autophagy flux and communicating the result to a user.

[0119] Figure 7: shows a magnetic sensor circuit diagram, where VCC = 5 V.

[0120] Figure 8: shows a unity gain Sallen-key second order low pass Butterworth filter circuit configuration.

[0121] Figure 9: shows Helmholtz coil design parameters.

[0122] Figure 10: is a circuit diagram showing the voltage controlled current source for the Helmholtz coil, where VCC = 5 V.

[0123] Figure 11 : shows the autophagic multistep pathway involving the sequestration of cytoplasm within a cell by a phagophore to form an autophagosome, autolysosome formation by fusion of an autophagosome with a lysosome, and the breakdown of cytoplasm into amino acids. A cross shows the step in the pathway that is blocked in the method of this invention.

[0124] Figure 12: shows a schematic representation of a microfluidics system for handling and preparing a sample before it is applied to a lateral flow assay test strip.

[0125] Figure 13: shows an overview of components of a system of the invention for indirectly determining the level of autophagosome biomarker in test and control samples.

[0126] Figure 14: shows an overview of an autophagy flux measurement system, in which a sample is prepared by splitting and being exposed to a buffer (RIPA) in designated wells (A); the prepared sample is used in LFIAs for autophagosome biomarker detection (B); and the autophagosome biomarkers are quantified using a magnetic transducer mechanism (C).

[0127] Figure 15: shows fluorescence microscopy images for the conjugation of anti-LC3B antibodies to magneticparticles and the detection of mCherry LC3 or LC3-II protein. The TPMT, mCherry-LC3, Alexa Fluor 488 and merged channels are shown along with a region of interest (ROI). Figure 16: shows grayscale colocalisation plots of fluorescence images, where white regions indicate high signal overlap, grey regions partial overlap, and black regions no overlap, demonstrating specific signal amplification in antigen-containing groups with aggregated 30 nm magnetic nanoparticles for visualization.

[0128] DETAILED DESCRIPTION

[0129] A device and method for simultaneously and quantitatively assessing one or more magnetically- labelled analytes immobilised on at least two regions of at least one lateral flow immunoassay test strip are described herein. The method comprises the steps of: a) sensing a magnetic signal from the magnetically-labelled analyte on each region and converting the magnetic signal for each region into an electric signal; b) processing the electric signal to obtain a quantitative value of the amount of magnetically-labelled analyte on each region; and c) processing the values obtained in step (b) to provide a differential assessment between the magnetically-labelled analyte on each region. The differential assessment may be obtained by, for example, subtracting one value from the other or by comparing one value to the other.

[0130] The electric signal in step (b) can be processed by amplifying the electric signal, filtering the amplified signal with a band-pass filter, splitting the filtered signal into two paths, multiplying the signal of one path with a sine reference, multiplying the signal of the other path with a cosine reference, low-pass filtering each multiplied signal to attenuate frequency components and noise above a baseband and obtain demodulated baseband signals, amplifying each demodulated baseband signal to obtain output signals, and digitising the output signals for processing.

[0131] A system, microfluidics device, lateral flow immunoassay test strip and kit are also described herein.

[0132] The device comprises a magnetic reader includes at least one magnetic sensor, such as a giant magnetoresistive (GMR) sensor, tunneling magnetoresistance (TMR) sensor, anisotropic magnetoresistance (AMR) sensor or Hall-effect sensor. The magnetic sensor can be directly or indirectly coupled to an instrument amplifier (IA), in particular a lock-in amplifier (LIA) configured to perform phase-sensitive detection and enhance the signal-to-noise ratio of weak magnetic signals in a portable, point-of-care (POC) format. Figure 1 shows a side view of the components of a magnetic reader (100) according to one embodiment. The magnetic reader (100) comprises one or more magnets (1 10), which may be permanent magnets, electromagnets, or combinations thereof. The magnets (1 10) provide a biasing field for a magnetic sensor (120) of the magnetic reader and magnetize magnetic nanoparticles (MNPs) present on a test or control line (130) of a lateral flow immunoassay (LFIA) test strip (140). If the magnets are electromagnets, it should be appreciated that the electromagnets may be connected to DC or AC current sources. The magnetic reader (100) includes at least one slot (not shown) configured to receive the lateral flow test strip (140), positioning the test or control line (130) beneath the magnetic sensor (120). In some embodiments a second slot may be provided to accommodate an additional lateral flow test strip for a differential measurement.

[0133] The magnetic fields (150) applied to lateral flow test strip test or control lines (130) may be oriented vertically (150a), horizontally (150b), or in any combination, generated by permanent magnets, electromagnets, or both.

[0134] The magnetic reader (100) has a Helmholtz coil (160) which produces the horizontal magnetic field (150b) to bias the sensor in its sensitive direction. The vertical magnetic field (150a) is produced using one or more permanent magnets (110) to magnetise the magnetic nanoparticles. The permanent magnets (110) are positioned to create a field orthogonal to the sensitive axes of the sensor (120) such that minimal interference on the output of the sensor is incurred. The sensitive axes of the sensor are in the horizontal plane. Thus, the permanent magnets (1 10) are positioned vertically in relation to the sensor to produce a vertical magnetic field (150a). The lateral flow test strip (140) is positioned parallel to the field produced by the Helmholtz coil (160). The magnetic sensor (120), which in this embodiment is a GMR sensor, is mounted on a printed circuit board (PCB) (170) directly above or below the test or control line (130) on the lateral flow test strip (140) when it is inserted into the magnetic reader (100), such that the GMR sensor (120) can read the magnetic signal produced by the magnetic nanoparticles captured in the test line region.

[0135] During operation, the magnetic sensor (120) detects the magnetic response of magnetic nanoparticles on the lateral flow test strip test or control line (130) in the presence of the biasing field, producing a voltage output proportional to the number of magnetic nanoparticles. To avoid weak signals from the magnetic sensor from being obscured by noise arising from environmental sources, thermal effects, or the inherent limitations of low-frequency detection, a lock-in amplifier can be integrated as a signal processing component of the magnetic reader. The lock-in amplifier may be configured to perform phase-sensitive detection by multiplying the sensor output with a reference waveform of known frequency. This may enable the selective recovery of the signal at the reference frequency, while noise components at other frequencies are attenuated. The recovered signal may provide both amplitude and phase information, which can be correlated with the presence or quantity of an analyte under investigation.

[0136] A signal-flow diagram of an example lock-in amplifier dual-phase demodulation chain (200) for reading the magnetic sensor (120) is shown in Figure 2. The output of a magnetic sensor (120), for example a GMR sensor, is amplified using an instrumentation amplifier (IA) (210) and bandlimited by a band-pass filter (BPF) (230). The filtered signal is then split into two parallel paths, an upper path and a lower path. The upper path is multiplied with a sine reference mixer (240) and the lower path is multiplied with a cosine reference mixer (250). Each mixer output is passed through a low-pass filter (LPF) (260a, 260b) to remove high-frequency components and noise from the signals, thereby producing demodulated baseband signals labelled Vx (upper) and Vy (lower). Those baseband signals are passed through a final amplification stage using another instrumentation amplifier (IA) (270a, 270b) in each signal path to produce the output signals labelled Vxx (upper) and Vyy (lower). This configuration is discussed in more detail below.

[0137] The lock-in amplifier (200) is configured to receive a magnetic sensor output signal and a reference signal corresponding to a magnetic excitation applied to a device under test. The LIA (200) isolates the frequency component of the sensor signal that matches the reference signal while suppressing other frequency components. The lock-in amplifier (200) produces outputs corresponding to the in-phase (X) and quadrature (Y) components of the demodulated signal, from which amplitude R and phase 0 are calculated according to: where 0 can be defined by a piecewise function as follows:

[0138] At the core of lock-in detection is phase-sensitive demodulation. The input signal can be expressed as

[0139] Fs(t) = Rcos and is multiplied by a reference of known frequency,

[0140] Mixing, or multiplying, the signals yields

[0141] Low-pass filtering removes the high-frequency term at ws+ wr, leaving only the coherent contribution at the frequency difference:

[0142] In the matched-frequency case ws= wr, the demodulated signal simplifies to a stationary phasor: Z(t) = Reie. (6) with Cartesian components

[0143] X = Rcose, Y = Rsine. (7)

[0144] Thus, dual-phase demodulation directly yields both amplitude and phase relative to the reference.

[0145] In the frequency domain, this process is equivalent to applying a highly selective bandpass filter centred at the reference frequency:

[0146] Z (w) = Vs(w - wr)Hn(w) , (8) where Hn(w) is the transfer function of an nth-order low-pass filter. Adjusting the filter order and time constant T sets the effective noise-equivalent bandwidth, balancing signal-to-noise ratio (SNR) against time resolution.

[0147] In other words, the magnetic sensor (120) output may be conditioned by a band-pass filter (BPF) (230) and / or a low-pass filter (260a, 260b) to isolate a frequency component corresponding to a magnetic excitation applied to a lateral flow immunoassay (LFIA).

[0148] In an example embodiment the BPF (230) is a second-order active multiple-feedback section centred at approximately 10 kHz (Q«0.5) and receiving the output of the initial gain / IA stage, as shown in Figure 3.

[0149] The conditioned sensor output may be supplied to a lock-in-amplifier (200) arranged for phasesensitive detection, as discussed above. The lock-in amplifier (200) receives the conditioned sensor signal and a reference waveform derived from the applied magnetic excitation and performs multiplication followed by low-pass filtering to retain a coherent (difference-frequency) component while suppressing high-frequency mixing products and out-of-band noise. In a dual-phase lock-in amplifier implementation, as shown in Figure 2, the conditioned sensor signal (i.e., the sensor output after band-pass filtering) is divided into two parallel paths. In an example embodiment the first path is multiplied by an in-phase reference waveform, and the second path is multiplied by a quadrature reference waveform phase-shifted by 90°. The multiplication may be performed by commutating analog mixers (240, 250) implemented using CMOS analog multiplexers. In an example embodiment, ADG1208 multiplexer devices are used, with the band-pass-filtered sensor signal applied to the multiplexer inputs and the selection lines driven by a reference square wave and its 90° phase-shifted counterpart, thereby achieving commutating multiplication, as shown in Figures 4A and B.

[0150] The outputs of the mixers (240, 250) are supplied to low-pass demodulation filters (260a, 260b). In one embodiment, two second-order Butterworth low-pass filters arranged in a Sallen-Key topology is employed. These filters (260a, 260b) are configured with an effective passband Fp«1 Hz and stopband Fs«10 Hz, thereby providing strong attenuation of the 2co mixing product and residual noise. The filtered outputs yield near-DC signals proportional to the in-phase (Vx) and quadrature (Vy) components of the sensor output signal, as shown in Figures 5A and B.

[0151] In some embodiments optional instrumentation amplifiers (lAs) (270a, 270b) may also be provided downstream of the filters (260a, 260b) to amplify Vx and Vy, thereby generating outputs designated Vxx (in-phase) and Vyy (quadrature), for example during an IA stage. These outputs may then be digitised for downstream processing and numerical computation of amplitude and phase.

[0152] The commutating multiplexer arrangement alternately routes the conditioned sensor signal between positive and negative paths under the control of reference selection signals. This approach provides phase-sensitive multiplication without the need for transconductance stages or bias currents. The configuration exhibits low input-referred noise and negligible offset, is compatible with standard CMOS processes, and has been demonstrated to achieve microvoltlevel resolution at the mixer stage.

[0153] The magnetic reader (100) therefore provides for full lock-in detection, including phase-sensitive demodulation and quadrature recovery, to be realised in a compact, low powering analog architecture. By employing CMOS multiplexers as mixers in place of conventional Gilbert-cell or dedicated multiplier integrated circuits (ICs), the architecture achieves both size and power reduction while retaining the essential performance advantages of a lock-in amplifier. This miniaturisation makes lock-in detection directly compatible with portable, point-of-care biosensing platforms, as disclosed herein.

[0154] Given the above, a person skilled in the art would appreciate that the inclusion of the lock-in amplifier in the device and system enables the detection of signals at an operating frequency, such as 10 kHz, which is typically selected to be higher than frequencies where noise from environmental and electronic sources is expected to be significant. The architecture is optimised to reject 1 / f noise and other interfering signals while retaining sensitivity to the magnetic nanoparticle signal. However, it should be appreciated that the frequency of operation is not limited to a specific range. The excitation frequency applied to the magnetic nanoparticles, as well as the filter characteristics of the lock-in amplifier, may be selected based on the application, enabling adaptation to different noise environments. Component values in the band-pass and low-pass filters may be altered accordingly to tune the system to the desired operating frequency.

[0155] It should further be appreciated that various lock-in amplifier configurations may be supported by this framework. For example, two independent lock-in amplifiers may be used for parallel differential channels, or a single lock-in amplifier may be time-multiplexed across channels with differential subtraction performed at the sensor output.

[0156] It should further also be appreciated that variations in the filtering stage (e.g., Butterworth, Bessel, switched-capacitor) and mixer implementation (e.g., multiplexer-based, chopper-stabilised, or hybrid analog-digital) may also be implemented.

[0157] The magnetic reader (100) can incorporate or be connectable to a processing means, such as a microcontroller or single-board computer, and / or an output or display device (170), which together may be used to assess a biological process, such as to calculate an autophagic flux measurement or value and report this to a user.

[0158] An example system including the magnetic reader (100), as discussed above, is shown in Figure 6. In a preferred embodiment, the system includes the magnetic reader (100), a processor (180) (or other processing means), and a display (190). The system may be embodied as a single device (300) integrating the above components. The device (300) may include a housing configured to at least partially enclose the components of the system.

[0159] It should be appreciated that each of the components of the system may include one or more sub-components. For example, the magnetic reader (100) may house the lock-in amplifier (120) and its associated circuitry, processing electronics, power supply, and, in some embodiments, a user interface. The processing electronics of the magnetic reader (100) may receive the in-phase and quadrature outputs from the lock-in amplifier, calculate amplitude and phase, perform differential calculations if required, and display the results via its own user interface or a display device (190) associated with the system. In some embodiments, the processing electronics of the magnetic reader (100) may be configured to perform simple processing tasks and then transmit the processing tasks to a processor (180) of the device (300) for further, more complex processing tasks.

[0160] Preferably, the device (300) is a single point-of-care or hand-held device. In some embodiments, the display (190) may be provided by an external device in data communication with the processor (180) and / or magnetic reader (100) via a suitable wired or wireless connection. In other words, it should be appreciated that the magnetic reader (100) and / or processor (180) may be configured to communicate wirelessly with an external device, such as a smartphone or computer, enabling additional data analysis, storage, or transmission to a remote server.

[0161] It should be noted that, in some embodiments, the device (300) may simply include the magnetic reader (100) and its associated components. In such an embodiment the processor (180) and display device (190) may be external components in data communication with the magnetic reader (100) via one or more communication modules, such as a Bluetooth™ module or a Wi-Fi module.

[0162] In one embodiment, a cartridge configured to house a lateral flow test strip interfaces with the reader device. The cartridge may include alignment features to position the strip correctly with respect to the magnetic sensor, as well as shielding to reduce interference.

[0163] In some embodiments, the lock-in amplifier circuitry is partially or fully integrated into an application-specific integrated circuit (ASIC) or system-on-chip (SoC). This allows the entire detection system, including the sensor interface, amplification, mixing, filtering, and processing stages, to be realised in a compact, low-cost form factor. Integration at this level further reduces power consumption and enhances portability, making the device suitable for field deployment or home use.

[0164] In still further embodiments, hybrid architectures are contemplated, wherein the mixing and filtering stages are implemented in analog circuitry, while the demodulation and amplitude / phase calculation are performed digitally by a microcontroller or digital signal processor.

[0165] The system may also incorporate calibration and referencing features. For example, one channel of the lock-in amplifier may be reserved for a calibration signal, generated by a reference coil or magnetic source. This enables real-time verification of system performance and compensation for drift. Alternatively, calibration may be achieved by running a known standard sample on an LFA strip in parallel with the test sample, and subtracting its output from that of the test. Such calibration approaches ensure reliable and reproducible results across different devices and test conditions. An embodiment of a magnetic reader (100) is described in more detail below.

[0166] A NVE GMR AA006-02 magnetometer from NVE Corporation was selected as the GMR sensor for its sensitivity and linear range. It is designed for unipolar operation, i.e. exposure to magnetic fields of one polarity, and has maximum hysteresis of 4 %.

[0167] The AA006-02 sensor was coupled to an instrumentation amplifier (IA) (Analog Devices AD620) in a preamplifier circuit configuration adapted from the NVE GMR Sensor Catalog application notes [1], shown in the circuit diagram in Figure 7. The transfer function for the circuit is given by: where Vout is the voltage output of the sensor, Vinis the input voltage to the sensor where Vin= ( Vout+ - Vout-), Vrefis the reference voltage, and RG is the value of the gain resistor. The gain of the circuit can be calculated as:

[0168] Setting Vin= 5 V, Vref= 0 V, and G = 1000, the gain resistance was found to be 49, 5 Q. To accommodate standard resistor values available, RG = 47 Q was chosen.

[0169] A low pass filter can optionally be added to attenuate higher frequency components and noise, particularly stemming from the grid power supply with an operating frequency of 50 Hz. The filter can be placed between the GMR sensor and IA shown in Figure 7. A Butterworth filter with a Sallen-Key topology was selected. The unity-gain Sallen-Key configuration provides gain accuracy unaffected by component variations. Additionally, Butterworth filters are known for maximum passband flatness, ensuring a consistent response across the desired frequency range. This maintains signal integrity in the presence of a small output signal, where susceptibility to noise is a concern. The passband flatness ensures uniform amplification of the signal, thereby enhancing sensitivity of the sensor. The transfer function for a second order low-pass Butterworth filter is given by: where f is the frequency variable and / Cis the gain factor. Its circuit diagram is shown in Figure

[0170] 8.

[0171] The component values were designed based on a simplification proposed by an application report from Texas Instruments in which the filter components are set as ratios and the gain is set equal to 1 . By letting F?1 = mR, R2 = R, C1 = C, C2 = nC, and K = 1 , the cut-off frequency can be calculated according to: and the quality factor, Q can be calculated according to:

[0172] This keeps gain = 1 in the pass band. Design should start by choosing the ratios m and n to set Q, and then selecting C and calculating R to set fc. For a second order low pass filter, Q = 0.707. The circuit is designed for a cut-off frequency fc= 40 Hz to ensure higher frequency components stemming from the grid power supply are attenuated. The component values that yield the desired response were found through an iterative process and are listed in Table 1. The desired filter response was confirmed using an online filter design tool by Texas Instruments (ti.com).

[0173] Table 1 : Component values for the second order Sallen-key low pass Butterworth filter.

[0174] Component Series Value

[0175] R1 E96 2,87 kQ

[0176] R2 E96 3,16 kQ

[0177] R3 E96 2,49 kQ

[0178] R4 E96 210 Q

[0179] C1 E96 1 pF

[0180] C2 E96 1 ,74 pF

[0181] The magnetic nanoparticles used as labels in the lateral flow test strips require an external field in order to be magnetised. For simplicity of manufacture, a back-biasing configuration was used. Experimentation showed that a magnetic field strength of at least 4000 G is required to magnetise 1 pL of 5 mg / mL SMNPs spotted on a nitrocellulose membrane of a lateral flow test strip. A 4 x 2 N38 4454 G nickel-coated neodymium disc magnet was attached to the underside of a magnetic sensor printed circuit board (PCB). It was aligned with the GMR sensor to minimally influence the sensor response.

[0182] An electromagnet was designed to bias the sensor in its sensitive direction. Helmholtz coils were selected as they generate homogeneous, uniform magnetic fields while cancelling out external magnetic fields. An example embodiment of a Helmholtz coil (160) is shown in Figure 9. The Helmholtz coil (160) arrangement consists of two current-carrying coils with equal radii and number of turns, connected in series and spaced apart at a distance equal to the mean radius of the coil bundles. The magnetic field produced by the coils is directly proportional to the current passing through them and number of turns in each coil, and inversely proportional to the distance between the coils.

[0183] For optimal performance of the coil, the width and depth of the wound coil cross section do not exceed a tenth of the average coil diameter and a constant current is supplied to the coil to ensure stability of the magnetic flux density. Non-magnetic materials are used for the frame and fittings, and annealed high conductivity copper wire is used for the winding. To ensure leads exist on opposite edges of the coil, an odd number of winding layers is used. The design parameters for a Helmholtz coil are shown in Figure 9.

[0184] The equations governing a Helmholtz coil are derived from the Biot Savart Law, describing the on-axis field due to a single wire loop: where B is the on-axis field, b is the permeability constant of free space given by b = 4TT X 10-7T / (mA), I is the coil current in amperes, R is the coil radius in meters, and a is the distance from the coil to a point in meters. Helmholtz coils produce a highly uniform field in the central region, around:

[0185] Since the coils consist of a number of wire loops, N, the current is multiplied by this number to give the total coil current of Nl. Combining Equations 14 and 15, multiplying by two to account for the field produced by both coils, and substituting / V / for the total coil current gives the magnetic flux density at the centre of the coils as: where B is measured in Gauss (G).

[0186] A Helmholtz coil was designed to produce a horizontal magnetic field of 30 G, saturating the NVE AA005-02 GMR sensor and biasing it in its sensitive plane. The coils were wound such that current flows through them in the same direction, creating a uniform magnetic field with the primary component parallel to the axes of the two coils. Since the required magnetic flux density is known, Equation 16 was rearranged to determine the number of turns in each coil using an iterative process:

[0187] To accommodate the magnetic sensor PCB and lateral flow test strip moving through the coils, an inner coil diameter Di = 40mm was chosen. The coil width and depth were set at 4,3 mm and

[0188] 4,1 mm, respectively. The generally accepted current capacity for copper wire is

[0189] It was found that a wire gauge t = 0, 17mm, N = 600 turns and current I = 125 mA provided a magnetic flux density of 31 G and satisfied the coil design parameters and current carrying capacity of the wire.

[0190] Further calculations using Equations 18-23 were done to ensure the configuration satisfied power dissipation and temperature concerns. Copper wire has a specific heat average wire resistivity at room temperature of Rcu= 1 .68(10)-8Qm. The wire resistance is calculated as: where Awireis the cross-sectional area of the wire, and L is the length of the wire used in a coil.

[0191] Power dissipation (P ) is calculated as:

[0192] P = PRwire (19) and thermal energy absorbed per minute (Q) is calculated as:

[0193] Q= P x 60s (20)

[0194] The volume of the coil ( I / ) can be determined by:

[0195] V= TT( Ri + depth)2( width) - TT( Ri)2( width) (21 ) where depth and width refer to the parameters of one coil, and Ri is the inner radius of the coil.

[0196] The mass of the coil can be calculated using the volume as follows: m = pCuV (22)

[0197] To find the change in temperature per minute, the specific heat equation can be used:

[0198] Q = ATmc (23)

[0199] These equations result in a low power dissipation of 0,977 W and a change in temperature per minute of 6,9 °C. Since the operational time of the device to take measurements is short, this increase in temperature is acceptable and will not result in damage to the copper wire used to wind the coil.

[0200] A voltage-controlled current source, shown in Figure 10, was designed to provide a constant 125 mA to the Helmholtz coil. A TLC2272 operational amplifier was used in conjunction with a 2N2222A bipolar junction transistor (BJT).

[0201] The following assumptions were made regarding the BJT: (i) the base current is approximately equal to 0 A, (ii) the collector and emitter currents are equal, and (iii) the input voltage at the base is equal to the output voltage of the operational amplifier. Thus, controlling the emitter current through the appropriate combination of resistors and input voltages to the various components determines the collector current, i.e. the current through the coil.

[0202] The total coil resistance was measured using a multimeter as 38 Q. R1 and R2 are symbolic of a variable resistor (1000 Q) used to tune the input voltage to the operational amplifier, which will be the same for both the positive and negative input terminals, and also the potential difference over F?4. The collector current can be calculated as: where lc is the collector current and Vm is the potential difference over F?4. After simulating the circuit in LTSpice, the values for R3 and F?4 were chosen as 1 kQ and 1 Q, respectively. It was determined that the variable resistor should be adjusted to provide an input voltage of 2.86 V. Decoupling capacitors were all set to 1 nF.

[0203] The method, magnetic reader and system described above can be used to perform differential lock-in detection. By exploiting either duplicate lock-in amplifier channels or multiplexed switching, the system can quantitatively compare two related signals in real time, enabling paired test and control measurements within lateral flow immunoassays. The differential approach allows assessment of dynamic biological processes and comparative analyte detection, overcoming the limitations of conventional single-channel LFAs. This principle can be extended to multiple biosensing contexts, for example:

[0204] • Between two LFAs: Running parallel LFAs for different targets and subtracting their outputs can reduce false positives, improve specificity, and enable comparative studies (e.g., treatment vs. control conditions).

[0205] • On a single multiplexed LFA with two or more test lines. Differential lock-in detection can compare the signals from two test lines directly, improving quantification in assays with gradient or multiplexed targets. • Between control and test lines: Subtracting the control line signal from the test line compensates for systematic background contributions, sample matrix effects, or batch-to- batch variations in nanoparticle labeling. This yields a “background-free” measurement.

[0206] • Inter-assay calibration: One LFA strip may serve as a calibration or reference, while another runs the experimental sample. Differential measurement cancels common-mode noise, drift, or temperature effects.

[0207] • Side-by-side measurement of a biological marker under two conditions: A sample can be divided and processed in parallel channels / test strips, each leading to a lateral flow immunoassay (LFIA) with magnetically labelled antibodies or other biorecognition molecules. E.g., for autophagy flux measurement, one channel / test strip can be treated with a fusion inhibitor (blocking autophagosome-lysosome fusion) and one channel / test strip can be untreated. The differential signal provides a direct measure of autophagy flux, overcoming the limitations of conventional LFAs that only provide static or single-point readouts. By determining the number of magnetic nanoparticles (MNPs) used in the assay, together with their limit of detection and protein-binding capacity, it becomes possible to obtain quantitative measurements - enabling the differential signal to serve as an indicator of protein turnover.

[0208] While autophagy flux is described herein as a representative example of a dynamic biological process that can be assessed according to the invention, it will be apparent o a person of skill in the art that the principle of differential lock-in detection can be extended to other time-dependent or comparative assays. These include measurements of metabolic activity (e.g., glucose uptake or lactate production), enzyme kinetics (e.g., protease or kinase activity over time), immune activation markers that change in response to stimulation, or degradation and clearance rates of biomolecules in therapeutic monitoring. In cases where a truly dynamic process is not directly observable, differential detection remains valuable because it enables comparative quantification - for example, assessing relative expression levels or signal changes between treated and untreated conditions. The approach described herein therefore broadens the utility of magnetic lateral flow assays beyond static endpoint detection, allowing measurement of temporal or conditional differences in biological systems.

[0209] The method, magnetic reader and system can, for example, be used: to generate a differential autophagy measurement with time, to diagnose autophagy-related diseases or disorders, for wellness tracking, to personalize medical treatments or lifestyle interventions, to enable early detection of disease (e.g. inflammatory disorders, neurodegenerative diseases), to detect aging-related changes, multiplexed infectious disease panels (e.g. TB, HIV, influenza, sepsis) for drug efficacy screening, to assess protein turnover, to determine receptor trafficking, to assess enzymatic activity, to monitor infection progression, or to assess other disease-relevant molecular events, in a rapid and cost-effective manner.

[0210] For example, in the case of tuberculosis disease (TB), differential lock-in detection can be applied to compare responses to multiple Mycobacterium tuberculosis antigens such as ESAT-6, CFP-10, Ag85 complex (Ag85A / B / C), and TB7.7, which are commonly used to distinguish active from latent infection. Comparative measurements between these antigens, or between antigen and host antibody or cytokine responses (e.g., IFN-y, IP-10), can enhance diagnostic specificity and enable multiplexed detection within a single assay. Beyond TB, the same principle can be extended to other infectious and disease contexts, including HIV (p24 antigen vs. antibody), malaria (HRP2 vs. LDH), influenza (HA vs. NA antigens), sepsis or inflammatory disorders (IL-6 vs. CRP), neurodegenerative diseases (total vs phosphorylated tau), and metabolic or aging-related markers.

[0211] In one embodiment described herein, a sample containing a target analyte is applied to a lateral flow immunoassay test strip (“lateral flow test strip”), where it migrates along the strip by capillary action. Magnetic nanoparticles functionalised with recognition molecules bind to the target analytes and are captured and immobilised on one or more test lines on the strip. A magnetic sensor can be arranged in proximity to the test strip to detect the presence of the magnetic nanoparticles at the test lines. The sensor signal may then be amplified, filtered, and provided to the lock-in amplifier, where it may be demodulated to recover the amplitude and phase of the signal corresponding to the bound nanoparticles. The differential signal between two regions, such as a test line and a control line, may be calculated to remove background noise and improve specificity. The resulting differential measurement provides a quantitative assessment of the analyte concentration, and in the case of autophagy flux or other dynamic processes, can provide information over time or under modulated conditions.

[0212] Lateral flow immunoassays are widely used point-of-care tests owing to their simplicity, rapidity, low cost and well supported automated production. They are traditionally qualitative, returning binary results of a positive or negative test, and typically use gold nanoparticles as labels. They comprise at least four components: a sample pad (often made of glass fibre or woven mesh), a conjugate pad (similarly made of glass fibre or woven mesh), a nitrocellulose (NC) membrane with test and control lines, and a wicking pad (also referred to as a waste reservoir or absorbant pad and usually made of glass fibre or cellulose fibers).

[0213] In one embodiment, the lateral flow immunoassay used herein is a sandwich LFIA, which involves binding an analyte between two specific recognition molecules. A suitable sandwich assay could comprise the following steps:

[0214] Sample application: The sample, optionally dissolved in a running buffer, is applied to a sample pad. From here, the sample travels to the conjugate pad.

[0215] Conjugate pad interaction: The conjugate pad contains recognition molecules, typically antibodies, which specifically recognise the target analyte and which are labelled with magnetic nanoparticles (MNPs), preferably superparamagnetic nanoparticles (SMNPs). These antibodies are referred to as “detection antibodies” and any target analyte in the sample will bind to them.

[0216] Nitrocellulose migration: The sample, along with the bound or unbound recognition molecules, continues to flow through the nitrocellulose membrane.

[0217] Test line formation: Additional recognition molecules, typically antibodies, that specifically recognise the target analyte are permanently bound to a test line on the nitrocellulose membrane. These are referred to as “capture antibodies”. When the target analyte is present, it binds to the capture antibodies and becomes immobilised on the test line, along with the magnetically labelled detection antibodies. A magnetic field will be detectable on the test line due to the presence of the magnetically labelled detection antibodies. If there is no analyte, no binding will occur at the test line, and thus, no magnetic signal will be detectable.

[0218] The sample, along with any unbound magnetically labelled primary antibodies, will continue to run beyond the test line region towards the wicking pad or reservoir, optionally passing a control line along the way. The control line serves to confirm that fluid has passed successfully from the sample application pad, past the test line.

[0219] The number of magnetic nanoparticles captured on the test line and hence the strength of the magnetic field can be correlated to the amount of analyte present in the sample. Hence, the amount of analyte present in the sample can be quantified by measuring the magnetic field intensity produced by the magnetic nanoparticles on the test line. This can be done using a magnetic sensor or system as described above.

[0220] The detection and capture antibodies can be independently selected from monoclonal or polyclonal antibodies. In one embodiment, the detection antibodies are monoclonal antibodies, such as monoclonal antibodies derived from mice or rabbits. In one embodiment, the capture antibodies are polyclonal antibodies. Alternatively, other recognition molecules could be used instead of antibodies. These include aptamers and affinity ligands. Techniques such as carbodiimide chemistry, click-chemistry or maleimide chemistry can be used to label the detection antibodies with the magnetic nanoparticles.

[0221] The sample can be a blood sample, such as whole blood.

[0222] In one embodiment, the method is performed using a single lateral flow test strip, and magnetic field intensities are measured on at least two lines of the strip, e.g. on two test lines (for different target analytes) or on a test line and a control line.

[0223] The method can also be performed in parallel using two or more lateral flow test strips (or on a single test strip with separate channels for each target analyte). For example, a separate test strip could be used for each target analyte being measured. Alternatively, separate test strips or channels could be used where only one analyte is being measured but under different conditions (for example, where one sample is treated and another is untreated). In that case, the sample is divided into portions and each portion is treated differently before being applied to the sample pad of individual lateral flow test strips (or separate channels on the same lateral flow test strip, including multiple channels or test strips on a single backing pad). The individual lateral flow test strips or separate channels are preferably identical.

[0224] Both of the above embodiments enable the simultaneous measurement of two or more magnetic signals.

[0225] In some embodiments, a microfluidic device is used to treat, handle or process the samples before they are applied to the sample pad of the lateral flow test strip. The microfluidic device may be integrally connected with a lateral flow test strip of the type described above, so as to be in fluid contact with the sample pad, or may be a discrete entity. The microfluidic device has an area onto which the sample can be loaded, leading into at least one microfluidic passage or channel through which the sample can flow. The at least one passage or channel can contain one or more wells or zones for housing reagents to treat the sample, such as buffers, inhibitors, cell lysis agents, and so forth. The at least one passage can also be configured so as to increase the residence time of the sample within the passage, before the sample exits the device or flows onto the sample pad of the lateral flow test strip. For example, the passage may be serpentine-shaped, twisted or tortuous.

[0226] In one embodiment, the microfluidic device has two or more passages through which the sample can flow, each passage being capable of differently treating the sample (e.g. with a different reagent or residence time). Preferably, the passage leading from the sample application area will split the sample into the two or more passages. The microfluidic device can be a disposable, single-use device or a multi-use device.

[0227] A kit is also provided for use in performing the method described above. The kit can include any of the following: one or more lateral flow immunoassay test strips containing magnetically labelled detection antibodies and capture antibodies specific for target analyte(s) as described above; one or more microfluidic devices as described above; a magnetic reader as described above; one or more cartridges for inserting the lateral flow immunoassay test strips into the magnetic reader; a fusion inhibitor which inhibits fusion of autophagosomes and lysosomes; a white blood cell lysing composition; one or devices for collecting a sample from a subject (e.g. a fingerpricking device or lancet); and / or instructions for performing the method as described above.

[0228] In one embodiment, a method for measuring autophagy flux is provided. The method comprises: a) dividing a blood sample from a subject into first and second portions; b) inhibiting fusion of autophagosomes and lysosomes in only the first portion for a period of time; c) determining the level of an autophagosome biomarker in each portion; and d) calculating the autophagy flux by comparing the level of autophagosome biomarker in the first portion with the level of autophagosome biomarker in the second portion; e) and optionally also assigning an autophagosome flux level or value to the subject.

[0229] The sample can be a blood sample, such as whole blood.

[0230] Autophagy flux is the rate of flow at steady state along the autophagic pathway. Since autophagic flux is a highly dynamic process, steady state measurements of a “substrate” associated with a specific step in the process are inconclusive.

[0231] In the autophagic pathway, shown in Figure 1 1 , a phagophore expands and curves to sequester cargo within a cell, forming an autophagosome when the opposing ends of the phagophore fuse. After a brief maturation period, the autophagosome will fuse with a lysosome. The acidic lumen contained within the lysosome breaks down the sequestered cargo into its building blocks (amino acids). The method for measuring or determining autophagy flux described herein comprises splitting a sample into two (preferably equal) portions. In one portion (the test sample), the step in the autophagic pathway in which the autophagosomes and lysosomes fuse is blocked for a period of time, leading to an increase in the number of autophagosomes in the test sample. The blocked step is shown with a cross in Figure 11. The other portion (the control sample) is not treated with a fusion inhibitor. The differential measurement of the autophagosome levels of the two samples with time can be used to calculate the autophagy flux.

[0232] Instead of directly detecting the number of autophagosomes in the test and control samples, levels of a biomarker associated with autophagosomes, in particular LC3-II protein, are detected in the method described herein. When the autophagic pathway is activated, the cytosolic, proteolytically processed form of microtubule-associated protein 1 light chain 3 (LC3-I) is lipidated to form LC3-II which is bound to the phagophore membrane. LC3-II on the outer surface is removed through deconjugation during the autophagosome maturation stage, but LC3-II present in the inner vesicle remains associated with the autophagosome. Thus, the LC3-II levels correlate with the number of autophagosomes and can serve as a biomarker for the process. If fusion of autophagosomes and lysosomes is blocked, the LC3-II levels will increase. In the absence of fusion, LC3-II levels remain steady.

[0233] Autophagosome flux ( / ) can be measured with time according to the following formula: where ALC3 / / is the respective difference in LC3-II levels between a sample treated with a fusion inhibitor and a control sample with respect to time, At.

[0234] While the LC3-II levels will increase in the test sample after blocking of the autophagic pathway, the levels of LC3-I in the test and control sample will not differ significantly. Thus, for purposes of determining autophagy flux according to the method described herein, when detecting the levels of LC3-II in the test and control samples, it is possible to detect either LC3-II protein only or to detect both LC3-II and LC3-I (i.e. all LC3) proteins. Either way, the flux will be attributed to an increase in LC3-II levels specifically between the two sample lanes (test and control).

[0235] Fusion of the autophagosomes and lysosomes can be inhibited by treating the test sample with an autophagosome-lysosome fusion inhibitor, such as chloroquine or bafilomycin. The period of time in step (b) can range from about 5-30 minutes, but is preferably less than about 20 minutes, less than about 15 minutes or even less than about 10 minutes. In one embodiment, the period of time is about 15 minutes. The white blood cells in the sample in each of the test and control samples can then be lysed with a lysing agent (step (b)(i)), thereby exposing the intracellular contents of the white blood cells, in particular LC3-I I. One example of a lysing agent is a radioimmunoprecipitation assay (RIPA) lysis buffer, but other lysing agents could also be used.

[0236] Optionally, unwanted particulates such as red blood cells can be removed from the sample. A variety of methods can be used to achieve this, including the use of red blood cell lysis buffers, membrane filtration, density gradient separation, separation by size and phoresis. This step can be performed at any stage, e.g. before step (a), after step (a) and before step (b), or after step (b) and before step (c). Alternatively, this step may not be performed at all.

[0237] A microfluidic device as described above can be used to perform one or more of steps (a), (b) and (b)(i), i.e. to split the sample into the test and control samples, treat the test sample with an autophagosome-lysosome fusion inhibitor and lyse the white blood cells in each sample.

[0238] In one embodiment, outlined in Figure 12, the sample is mixed with a running buffer and placed on a sample receiving zone of the microfluidic device. The device splits the sample into two portions, one of which (the test sample) is directed into a test channel and the other (the control sample) into a control channel. In a first section of the test channel, the test sample is exposed to the autophagosome-lysosome fusion inhibitor. For example, the first section of the test channel could have a well containing lyophilised BAF or chloroquine. When the sample reaches this well, the BAF or chloroquine dissolves and a serpentine or tortuous channel follows to allow exposure of the sample to this autophagosome-lysosome fusion inhibitor for about 15 minutes. This allows the autophagosome pool size to accumulate, and as a result, the LC3-II levels in the test sample will also increase. The white blood cells are separated (by removal of the red blood cells) in a second section of the channel. A third section incorporates the lysing agent. A fourth, optional, section of the channel deposits the test sample on a sample pad of a lateral flow immunoassay (LFIA) in a controlled fashion.

[0239] The control channel of the microfluidic device is similar to the test channel, except that the first section does not contain an autophagosome-lysosome fusion inhibitor.

[0240] The microfluidic device is designed in such a way that the control and test samples take the same time to flow through the device and are therefore deposited on the lateral flow test strip sample pads at the same time. In an alternative embodiment, the sample can be manually divided into two portions and then added to test and control channels of a microfluidic device as described above.

[0241] In yet a further embodiment, the steps of splitting the sample into two portions, treating the test sample with an autophagosome-lysosome fusion inhibitor, separating the white blood cells, lysing these cells and placing the samples on the sample pads of the lateral flow test strips can be performed manually.

[0242] A sandwich lateral flow immunoassay can be used to perform at least part of step (c). Two identical lateral flow test strips or channels are used, i.e. one for the test sample and the other for the control sample. The magnetically labelled detection antibodies and the immobilised captured antibodies are both specific for LC3 proteins (i.e. LC3-I and LC3-II) or for LC3-II proteins only.

[0243] The amount of LC3 (i.e. LC3-I and LC3-II) or LC3-II only (e.g. the number, concentration or relative magnetic intensity) can be quantified by measuring the magnetic signal intensity produced by the magnetic nanoparticles captured on the test line of each test strip (Figure 13). This can be done using a magnetic reader or sensor as described herein. The biomarker measurements can then be used to quantify autophagosome flux.

[0244] Figure 14 shows how the microfluidics device (A), lateral flow test strips (B) and magnetic reader (C) can be integrated. In summary, the microfluidic device (A) treats a whole blood sample after which a lateral flow immunoassay as discussed above (B) is performed. The sample from the test channel is expected to have a higher concentration of LC3-II than the sample from the control channel. The magnetic reader (C) reads the magnetic signals produced by the test lines of the lateral flow test strips and performs a differential measurement according to Equation 26: where J is the rate of autophagy flux, and Vtestand Vcontroiare the voltage readouts of the sensor for the test and control lateral flow test strips, respectively.

[0245] To the applicant’s knowledge, this is the first description of a method, device or system for measuring autophagy flux that is quantitative and also rapid and cost effective. It is also the first point of care or hand-held device to measure autophagy flux, i.e. to generate a differential autophagy measurement with time. It will provide people the ability to take “wellness” into their own hands by monitoring their own autophagy flux levels with changes in lifestyle patterns such as exercise, a change in diet, health drinks or caloric restriction / intermittent fasting regimen.

[0246] The device and system could be used for regular (e.g. daily, weekly or monthly) monitoring of autophagy flux, and could play a pivotal role in regulating healthy basal autophagy levels, thus preventing the onset of non-communicable diseases like cancer and Alzheimer’s, as well as managing these ailments, controlling healthy ageing, and assessing overall wellness. Such a device would prove useful to individuals predisposed to neurodegenerative and autoimmune diseases or cancers, elite athletes trying to manage skeletal muscle injury, those who prioritise longevity and a healthy lifestyle, and others. The device could also be used for efficacy screening of autophagy modulators associated with anti-aging, such as immunosuppressant drugs, antidiabetic drugs, caffeine, metformin, spermidine and the like.

[0247] The ability to monitor autophagy flux levels will allow a user (e.g. a clinician or nutritionist) to monitor a subject’s response (or an individual to monitor their own response) to a treatment or intervention on a daily basis, as one would monitor blood sugar levels with a glucometer or blood pressure with a blood pressure monitor. By utilizing real-time data on autophagy flux, healthcare professionals could move towards a more precision medicine approach, offering interventions that are finely tuned to the subject’s cellular health status and specific needs. For example, clinicians could tailor drug therapies and manage diseases by modulating autophagy; nutritionists might recommend specific fasting protocols, nutrient timing, and supplements; sports scientists could design exercise and recovery regimens to optimize cellular health; healthcare professionals could use autophagy data for early disease detection and preventive strategies; oncologists could optimize cancer treatments by monitoring autophagy; experts in ageing could develop anti-ageing interventions; metabolic health specialists could enhance weight management and diabetes care; neurologists could support neuroprotection and cognitive function through targeted interventions; or individuals could choose to alter their lifestyle (e.g. exercise or eating habits).

[0248] The above description therefore provides for the novel application of a miniaturised lock-in amplifier architecture to portable biosensing, specifically adapted for magnetic nanoparticle detection in lateral flow assays. By implementing the mixer stage using CMOS multiplexers, the design achieves significant reductions in size, cost, and power consumption compared to traditional lock-in amplifiers, while retaining the essential sensitivity and noise resilience. Coupled with differential measurement strategies, the device, system, and method described herein provide enhanced specificity and robustness, enabling applications in clinical diagnostics, wellness monitoring, and biomedical research that were previously impractical with conventional technology.

[0249] The invention will now be described in more detail by way of the following non-limiting examples. Persons skilled in the art will appreciate that numerous modifications and variations will fall within the scope of the invention. Carbodiimide conjugation of antibodies to magnetic nanoparticles

[0250] Carbodiimide chemistry was used to immobilise an anti-LC3B monoclonal antibody (mAb) produced in rabbit (purchased from Merck Life Science (Pty) Ltd, LC3B (D11 ) XP® Rabbit mAb #3868 (Cell Signaling)) onto the surface of 30 nm iron oxide (II, III) carboxyl-functionalised magnetic nanoparticles (purchased from Merck Life Science (Pty) Ltd). The cross-linking reaction is shown in scheme 1 .

[0251] Scheme 1 - Carbodiimide cross-linking reaction between carboxyl functionalised MNPs and antibody amine groups [2],

[0252] 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a zero-length cross-linking agent, was used to activate the carboxyl groups on the surface of magnetic nanoparticles to react with amine groups on antibodies to form an amide bond. N-hydroxysulfoxuccinimide (sulfo-NHS) was added to enhance EDC-mediated coupling efficiency, preventing undesirable intra- and intermolecular cross-linking of antibodies. Sulfo-NHS retains the negative charge of nanoparticles, thereby facilitate binding to positively charged antibodies.

[0253] The conjugation protocol was adapted from a method determined by Smith etal. [3]. The empirical and molecular formulae in Equations 27 - 29 were used to prepare the reagents required by the cross-linking protocol: where n is the number of moles (mol), m is mass (g), and M is molecular weight (g / mol); where c is the solution concentration (mol / L), and V is the volume of solution (L); and ■ > ; - (29) where Ci and Vi are the initial concentration and volume of a solution respectively, and C2and 1 are the final concentration and volume of a solution, respectively.

[0254] A 2-morpholinoethanesulphonic acid (MES) reaction buffer (10 mL, 0.1 M, pH = 5.0) was prepared by dissolving 0.1933g MES in dH2O, and adjusting the pH to 5.0 with sodium hydroxide (NaOH). An MES washing buffer (10 mL, 0.5 mM, pH = 5.0) was prepared by diluting the 0.1 M buffer in a 1 :199 ratio with dH2O. Sulfo-NHS solution (5 mL) was made up by dissolving 10.857 mg in dH2O. Glycine quenching solution (20 mL, 0.1 M, pH = 2,2) was prepared by dissolving 150 mg glycine in 1 x phosphate buffered saline (PBS). The pH of the solution was adjusted to 2,2 using hydrochloric acid (HCL). A storage buffer (10 mL, pH = 7,4) containing 0.05 % Tween 20 and 0.1 % BSA was prepared by adding 5 IA. Tween 20 and 1 mL BSA to 8.995 mL PBS. A 1 mL, 0,1 M activation buffer was prepared by dissolving 0.2 ml Sulfo-NHS in 0.8 ml 0.1 M MES buffer (pH = 5.0), yielding a 1 :4 ratio.

[0255] The storage solution of the magnetic nanoparticles was removed to get rid of any remnants that may lead to non-specific binding and interfere with conjugation. 20 / A. of 30 nm magnetic nanoparticles were aliquoted in a low protein binding centrifuge tube and washed three times with 250 / A. washing buffer, centrifuging at 3620 RCF between washing steps. The centrifuge tube was placed in a magnetic separator device to retain the magnetic nanoparticles whilst removing the supernatant. A 10 mg / mL EDC solution was prepared by adding 2,5 mg EDC to the activation buffer, mixing well to dissolve the solids. The carboxyl functional groups on the magnetic nanoparticles were activated for two hours on an agitator by adding the EDC solution in a 1 :1 volume ratio with the initial MNP aliquot, and vortexing every 30 min. The solution was sonicated if the magnetic nanoparticles started to aggregate.

[0256] The pAb was incubated with activated magnetic nanoparticles at a concentration of 5 mg / ml, achieved by adding 4,50 / A. pAbs. 39.83 mg polyethylene glycol (PEG) was added in a 5:1 molar equivalent to the mAbs to prevent aggregation of the MNP-Ab conjugates. The solution was shaken for 2 hours, vortexing every 30 min. The unbound antibodies were removed, following the same washing procedure as before. Unbound carboxyl groups on the magnetic nanoparticles were blocked by quenching the conjugate solution with 10 / A. glycine for 30 min on an agitator, and the solution was sonicated afterwards to disperse any aggregated magnetic nanoparticles and washed as before. The conjugates were resuspended in storage buffer to the desired concentration and stored in a fridge at 4 °C. LFIA development

[0257] Lateral flow test strips (LFTSs) with glass fibre sample and conjugate pads were purchased from Medical Diagnostech. A 3 / . volume of ab-MNP conjugate solution was spotted onto the conjugate pad and 1 / . of a 3 mg / ml pAb solution was striped in the test line region on the nitrocellulose (NC) membrane. The strips were air dried overnight at room temperature in a closed container and protected from light.

[0258] 1 x Purified PBS (pH = 7,4) was used as a sample running buffer. The sample buffer was spiked with mCherry LC3-II purified protein, starting at a control concentration of 1 jt / g / mL to test the upper limit. Positive and negative controls in 100 / . running buffer were run concurrently. The visual appearance of a brown line, due to the presence of magnetic nanoparticles, is expected to appear on the NC membrane in the case of a positive control. The absence of this line is expected in the case of a negative control. The protein concentration in the positive control was lowered until the visual detection limit was reached.

[0259] Fluorescence microscopy was used to validate (i) successful conjugation of the pAb to the magnetic nanoparticles, and (ii) specific detection of mCherry LC3-II protein. The mCherry protein exhibits fluorescence in the red spectral range, typically emitting light around 561 nm. The pAb was tagged with an anti-rabbit secondary antibody coated with Alexa Fluor® 488, which fluoresces in the green spectral range, emitting light around 488 nm. A Zeiss LSM780 Confocal Microscope was used to visualise the control groups listed in Table 2 with 40 x magnification. A 561 nm laser and 488 nm argon laser were used to excite the mCherry and Alexa Fluor® 488 fluorophores, respectively.

[0260] Table 2: Control groups for validation of Ab-MNP conjugation and LC3-II detection.

[0261] No. Control Group Expected Confirmation

[0262] Signal

[0263] 1 conjugates + mCherry protein red + green colocalization of mAbs and LC3-II on MNPs + Alexa Fluor® 488

[0264] 2 conjugates + mCherry red colocalization LC3-II on Ab-MNP protein conjugates

[0265] 3 conjugates + Alexa Fluor® green colocalization of mAbs on MNPs

[0266] 488

[0267] 4 conjugates none autofluorescence of conjugates

[0268] 5 unwashed MNPs none autofluorescence of MNPs

[0269] 6 washed MNPs none autofluorescence of MNPs

[0270] 7 washed MNPs + mCherry none non-specific binding of mCherry protein to MNPs protein

[0271] 8 washed MNPs + Alexa none non-specific binding of Alexa Fluor®

[0272] Fluor® 488 488 to MNPs

[0273] Preparation was done in a dark room to prevent photobleaching of the fluorophore. The control groups were prepared by pipetting 2 / . of sample per group in 8 plate wells, and magnetic nanoparticles were kept in an aggregated state to enable visual detection. This was achieved by excluding sonication steps from the carbodiimide conjugation protocol. The mCherry protein was diluted 1 :100 with PBS, resulting in a 1 ,21 jug / mL concentration. The Alexa Fluor® 488 was diluted 1 :200 with PBS. 2 / . of Alexa Fluor® 488 or mCherry protein was added to the wells where necessary.

[0274] Successful labelling of anti-LC3B antibody produced in rabbit with 30 nm iron oxide (II, III) carboxyl functionalised magnetic nanoparticles, as well as mCherry-LC3ll purified protein detection by the conjugates was confirmed using confocal microscopy. The conjugates were imaged in an aggregated state, as the 30nm nanoparticles are too small to be visualised otherwise. For application to a lateral flow test strip, aggregation presents a problem as the conjugate aggregates are too big to flow through the porous membrane. To prevent aggregation, sonication and the addition of a suitable surfactant (5000 Da PEG) were used.

[0275] Results from imaging the experimental control groups using the Zeiss LSM780 Confocal Microscope are shown in Table 3 and a selection of fluorescence microscopy images for the control groups is shown in Figure 15. The control groups showed colocalization of mCherry protein, antibodies (indicated by signal from Alexa Fluor® 488) and magnetic nanoparticles. This confirmed binding of the mAb to the magnetic nanoparticles and detection of mCherry LC3-II by the conjugate complex. The second and third control groups showed no unexpected green or red signal in the respective control groups. A low red signal was detected in control group five. It was hypothesised that this was autofluorescence stemming from the MNP storage buffer. However, control groups four and six did not exhibit this autofluorescence, indicating that the washing step removed these matrix components that interfere with the signal. A slight red signal was seen in control group seven, possibly indicating non-specific binding of mCherry protein to the magnetic nanoparticles, which can be eliminated with suitable blocking agents. No signal in control group eight indicated that Alexa Fluor® 488 does not bind non-specifically to the magnetic nanoparticles and therefore signal in the green channel can be trusted as an indication of the presence of pAbs. Table 3: Results for validation of Ab-MNP conjugation and LC3-II detection.

[0276] No. Control Group Expected Signal Actual Signal

[0277] 1 conjugates + mCherry protein + red + green red + green

[0278] Alexa Fluor® 488

[0279] 2 conjugates + mCherry protein red red

[0280] 3 conjugates + Alexa Fluor® 488 green green

[0281] 4 conjugates none none

[0282] 5 unwashed MNPs none red (minimal)

[0283] 6 washed MNPs none none

[0284] 7 washed MNPs + mCherry protein none red (minimal)

[0285] 8 washed MNPs + Alexa Fluor® 488 none none

[0286] A study was conducted to demonstrate formation and functionality of a sandwich assay for quantitative detection of LC3 using magnetic nanoparticle-based detection complexes and fluorescently labelled capture complexes.

[0287] The detection complex comprised magnetic nanoparticles (MNPs) conjugated to a LC3-specific antibody and tagged with a green-fluorescent probe to enable visualisation. Capture complexes consisted of three alternative antibodies, each labelled with a blue-fluorescent probe, to evaluate different epitope pairings. Three test groups were assessed:

[0288] 1 . No-antigen control — detection complex with capture complexes, no target protein.

[0289] 2. Non-fluorescent antigen (hisLC3) — detection complex, non-fluorescent LC3, and capture complexes.

[0290] 3. Fluorescent antigen (mLC3) — detection complex, fluorescent LC3 (red emission), and capture complexes.

[0291] Colocalisation analysis was performed to assess overlap of the green (detection), blue (capture), and red (antigen, where present) signals. Minimal background overlap was observed in the noantigen control. In both antigen-containing groups, marked increases in blue-green colocalisation were observed. In the fluorescent LC3 group, three-colour colocalisation (blue-green-red) confirmed simultaneous binding of both detection and capture complexes to the target protein.

[0292] Some non-specific overlap was detected in the no-antigen control but was significantly lower than that in antigen-containing groups, indicating specific signal amplification in the presence of target. Further optimisation of blocking conditions, surface chemistry, and washing protocols can minimise residual non-specific binding.

[0293] Fluorescence images were analysed in colocalisation plots, as shown in Figure 16, in which white regions indicate high signal overlap, grey regions partial overlap, and black regions no overlap. Bright clumps corresponded to aggregated 30 nm magnetic nanoparticles, which were used intentionally to aid confocal visualisation. Under operational LFA conditions the nanoparticles would be dispersed on the membrane, enabling single-particle level detection.

[0294] These results confirm that a sandwich assay design can discriminate between samples with and without target LC3 protein.

[0295] MNP Detection with a magnetic sensor

[0296] A 1 L and 5 / . sample of 5 mg / mL 30 nm magnetic nanoparticles were spotted on the NC membrane of two LFTSs, and the sensor response was tested by measuring the output voltage with an Agilent 34401 A digital multimeter. The results are tabulated in Table 4.

[0297] Table 4: GMR sensor readings from a digital multimeter with changing MNP volumes spotted on the NC membrane of an LFTS and the relative change in sensor response from no load condition.

[0298] Volume of MNPs [ / JL] Voltage output from Relative change in sensor sensor [V] response from no load [AmV]

[0299] 0 2.706 0

[0300] 1 2.703 -3

[0301] 5 2.701 -5

[0302] The relative change in sensor response from the steady state reading at no load (i.e. no nanoparticles are present under the sensor) was correlated to the number of nanoparticles under the sensor. A larger number of magnetic nanoparticles resulted in a larger relative change in sensor response.

[0303] The foregoing description has been presented for the purpose of illustration; it is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above disclosure.

[0304] The language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. It is therefore intended that the scope of the invention be limited not by this detailed description, but rather by any claims that issue on an application based hereon. Accordingly, the disclosure of the embodiments of the invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the following claims.

[0305] Finally, throughout the specification and accompanying claims, unless the context requires otherwise, the word ‘comprise’ or variations such as ‘comprises’ or ‘comprising’ will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0306] References

[0307] [1] NVE Corporation, GMR Sensors Catalog, https: / / www.nve.com / Downloads / catalog.pdf, Accessed: 21 Jan 2024, 2012.

[0308] [2] M. Kumari et al., “Antibody-conjugated nanoparticles for target-specific drug de- livery of chemotherapeutics”, Beilstein Journal of Nanotechnology, vol. 14, pp. 912- 926, 2023, issn: 21904286. doi: 10.3762 / bjnano.14.75.

[0309] [3] J. E. Smith et al., “Optimization of antibody-conjugated magnetic nanoparticles for target preconcentration and immunoassays”, 2010. doi: 10.1016 / j.ab.2O10. 11.005.

[0310] [4] B. Loos et al., “Defining and measuring autophagosome flux — concept and reality”, Autophagy, vol. 10(1 1 ), 99. 2087-2096, 2010.

Claims

CLAIMS:1 . A device for simultaneously and quantitatively assessing one or more magnetically-labelled analytes captured on at least two lateral flow immunoassay test strip test lines, the device including a magnetic reader comprising: a) means for receiving at least one lateral flow test strip having at least one test line; b) at least one magnetic sensor; c) one or more magnets which bias the magnetic sensor; and d) one or more magnets which magnetise magnetic particles on each test line.

2. A device according to claim 1 , wherein the magnetic reader includes a lock-in amplifier electrically connected to the magnetic sensor for enhancing a signal-to-noise ratio of magnetic readings from each test line.

3. A device according to claim 2, wherein the lock-in amplifier includes one or more multiplexers configured to operate as mixers for multiplying one or more signals from the at least one magnetic sensor with a reference waveform of the lock-in amplifier.

4. A device according to claim 2 or claim 3, wherein the magnetic reader is configured so that each test line of the at least one lateral flow test strip is positioned in line with a magnetic sensor when the at least one lateral flow test strip is received by the magnetic reader.

5. A device according to any one of claims 2 to 4, wherein the lock-in amplifier is configured to perform phase-sensitive detection of the magnetic readings obtained from the magnetic sensor, such that only readings having a defined phase relationship with the applied magnetic field used to magnetise the magnetic particles are detected.

6. A device according to any one of claims 2 to 5, wherein the lock-in amplifier is configured to enable detection of low concentrations of magnetically-labelled biorecognition molecules on each test line by amplifying magnetic readings from the magnetic sensor corresponding to the magnetised magnetic particles and rejecting broadband electronic noise.

7. A device according to any one of claims 2 to 6, wherein the lock-in amplifier is configured to reduce the effect of environmental magnetic interference on the magnetic readings obtained from the magnetic sensor by selectively filtering readings at the frequency of the applied magnetisation.

8. A device according to any one of claims 2 to 7, wherein the lock-in amplifier is configured to detect magnetic readings corresponding to magnetic particles located on multiple test lines of the same lateral flow test strip or on multiple lateral flow test strips.

9. A device according to any one of the preceding claims, wherein the magnetic sensor is a giant magnetoresistive (GMR) sensor.

10. A device according to any one of the preceding claims, wherein the one or more magnets which bias the magnetic sensor is an electromagnet and the electromagnet is a Helmholtz coil.

11. A device accordingly to any one of the previous claims, wherein the device includes a processing means for quantitatively assessing or comparing analyte levels from magnetic readings obtained by the magnetic reader and an output means for communicating the assessment or comparison to a user.

12. A device according to claim 1 1 , wherein the output means is a digital display.

13. A device according to claim 1 1 or claim 12, including a housing configured to at least house the processing means and the magnetic reader.

14. A device according to any one of claims 1 1 to 13 when depending on claim 2, wherein the lock-in amplifier may be implemented as an analog or digital lock-in amplifier within the magnetic reader or the processing means.

15. A device accordingly to any one of the previous claims, which is an autophagy flux measurement device.

16. A point-of-care device for simultaneously and quantitatively assessing one or more magnetically-labelled analytes captured on at least two lateral flow immunoassay test strip test lines, the device comprising: a housing; a magnetic reader including: a) means for receiving at least one lateral flow test strip having one or more test lines; b) at least one magnetic sensor configured to sense a magnetic signal from each of the at least two test lines, the two test lines being on the same or different lateral flow immune assay test strips; c) one or more magnets which bias the magnetic sensor;d) one or more magnets which magnetise magnetic particles on each test line of each lateral flow test strip; and e) a lock-in amplifier electrically connected to the magnetic sensor for enhancing a signal- to-noise ratio of magnetic readings from each test line; a processing means for processing the readings from each test line to provide a differential assessment of analyte levels on the at least two test lines; and an output means for communicating the differential assessment to a user.

17. A device according to claim 16, wherein the lock-in amplifier includes one or more multiplexers configured to operate as mixers for multiplying one or more signals from the at least one magnetic sensor with a reference waveform of the lock-in amplifier18. A system comprising: a) a magnetic reader according to any one of the previous claims; b) a processing means for processing one or more outputs received from the magnetic reader; and c) an output in data communication with the processing means for outputting the processed outputs.

19. A system according to claim 18, wherein processing includes processing the one or more outputs into readable results and outputting the processed outputs include outputting the readable results.

20. A method of simultaneously and quantitatively assessing one or more magnetically-labelled analytes captured on at least two lateral flow immunoassay test strip test line regions, the method comprising the steps of: a) sensing a magnetic signal from the magnetically-labelled analyte on each test line region and converting the magnetic signal for each test line region into an electric signal; b) processing the electric signal to obtain a quantitative value of the amount of magnetically-labelled analyte on each test line region; c) processing the values obtained in step (b) to provide a differential assessment between the magnetically-labelled analyte on each test line, wherein the differential assessment is obtained by subtracting one value from the other or by comparing one value to the other.21 . A method according to claim 20, wherein the electric signal in step (b) is processed by: amplifying the electric signal;filtering the amplified signal with a band-pass filter; splitting the filtered signal into two paths and: multiplying the signal of one path with a sine reference; multiplying the signal of the other path with a cosine reference; low-pass filtering each multiplied signal to attenuate frequency components and noise above a baseband and obtain demodulated baseband signals; amplifying each demodulated baseband signal to obtain output signals; and digitising the output signals for processing.

22. A method according to claim 20 or claim 21 , wherein each test line region is on a different lateral flow immunoassay test strip.

23. A method according to claim 20 or claim 21 , wherein the test line regions are on the same lateral flow immunoassay test strip.

24. A method according to any one of claims 20 to 23, wherein the magnetically-labelled analytes captured on each test line region is different or the same analyte but treated differently.

25. A method according to any one of claims 20 to 24, wherein the analyte is a biomarker, pathogen, antigen, toxin, allergen or other compound.

26. A method according to any one of claims 20 to 25, wherein the magnetic labels are superparamagnetic nanoparticles (SMNPs).

27. A method according to any one of claims 20 to 26, wherein the method is performed using a magnetic reader or system substantially as claimed in any one of claims 1 to 15, and 18 and 19, respectively.

28. A method of quantitatively assessing an analyte in a sample, the method comprising the steps of: a) dividing a sample from a subject into at least first and second portions; b) treating at least one of the portions with a treatment, wherein if both the portions are treated, the treatments of the first and second portions are different; c) applying the first and second samples to separate lateral flow test strips, each lateral flow test strip having a conjugate pad comprising magnetically-labelled biorecognition molecules which can specifically bind to the analyte and a test line region comprising biorecognition molecules which can specifically bind to the analyte, wherein the biorecognition molecules are immobilized on the test line region;d) magnetically sensing the magnetically-labelled biorecognition molecules on the test line for each portion and obtaining a quantitative value for each portion; e) processing the values obtained in step (d) to provide a differential and quantitative assessment of the quantity of analyte in the portions.

29. A method according to claim 28, wherein the analyte is a biomarker, pathogen, antigen, toxin, allergen or other compound.

30. A method according to claim 28 or claim 29, wherein the biorecognition molecule is an antibody.

31. A method according to any one of claims 28 to 30, wherein the magnetic labels are superparamagnetic nanoparticles (SMNPs).

32. A method according to any one of claims 28 to 31 , wherein the method is performed using a magnetic reader or system substantially as claimed in any one of claims 1 to 15, and 18 to 19, respectively.

33. A method of measuring autophagy flux, the method comprising the steps of: a) dividing a sample from a subject into first and second portions; b) contacting only the first portion with a sufficient amount of an autophagosomelysosome fusion inhibitor to inhibit fusion of autophagosomes and lysosomes in the first portion for a period of time; c) determining the level of LC3-I and LC3-II in each portion; and d) calculating the autophagy flux by comparing the level of LC3-I and LC3-II in the first portion with the level of LC3-I and LC3-II in the second portion with respect to time.

34. A method according to claim 33, wherein the autophagosome-lysosome fusion inhibitor is bafilomycin or chloroquine.

35. A method according to either one of claims 33 or 34, wherein the period of time is from about 5-30 minutes, preferably from about 10-20 minutes, more preferably about 15 minutes.

36. A method according to any one of claims 33 to 35, which further comprises a step of removing red blood cells from the sample.

37. A method according to any one of claims 33 to 36, which further comprises a step of contacting the white blood cells in the sample with a lysing agent.

38. A method according to any one of claims 33 to 37, wherein the level of LC3 in each portion is determined by contacting each portion with magnetically labelled antibodies which bind specifically to LC3 proteins, so as to form labelled-antibody-LC3 complexes, and magnetically sensing the amount of labelled-antibody-LC3 complex in each portion.

39. A method according to claim 38, wherein the magnetic labels are superparamagnetic nanoparticles (SMNPs).

40. A method according to claim 38 or 39, wherein a lateral flow immunoassay (LFIA) and magnetic reader are used to determine the number, concentration or relative magnetic intensity of labelled-antibody-LC3 complexes, and hence the level of LC3, in each portion.41 . A lateral flow immunoassay test strip for use in measuring autophagy flux, comprising: a) a sample pad for receiving a fluid sample; b) a conjugate pad comprising magnetically-labelled biorecognition molecules which can specifically bind to LC3-I and LC3-II proteins; c) a test line region comprising biorecognition molecules which can specifically bind to LC3-I NS LC3-II proteins, wherein the biorecognition molecules are immobilized on the test line region.

42. A lateral flow test strip according to claim 41 , wherein the biorecognition molecules are antibodies.

43. A microfluidic device for separating a fluid sample into at least two portions and differentially treating the portions, the device comprising: a) a zone for receiving a fluid sample; b) two passages; and c) means for dividing the fluid sample into two portions and directing one portion into one passage and the other portion into the other passage; wherein at least one of the passages has a reagent which will come into contact with the sample in that passage, and wherein the other passage does not have the same reagent.

44. A microfluidic device for measuring autophagy flux, the device comprising: a) a zone for receiving a fluid sample; b) a test passage; c) a control passage; andd) means for dividing the fluid sample into two substantially equal portions and directing one portion into the test passage and the other portion into the control passage; wherein the test passage comprises:(i) a zone containing an autophagosome-lysosome fusion inhibitor; and(ii) a zone containing a lysing agent for lysing white blood cells; and wherein the control passage comprises:(iii) a zone which does not contain an autophagosome-lysosome fusion inhibitor; and(iv) a zone containing a lysing agent for lysing white blood cells.

45. A device according to claim 44, comprising one or more zones in which red blood cells are removed from the sample, wherein one of the zones is located between the sample receiving zone and the means for dividing the sample into two portions.

46. A device according to claim 44 or claim 45, wherein the test and control passages comprise yet a further zone for depositing the respective portion of the sample onto a sample pad of a lateral flow immunoassay (LFIA).

47. A device according to any one of claims 44 to 46, which further comprises a lateral flow immunoassay (LFIA) leading from each of the test and control passages, each lateral flow test strip comprising: a) a sample pad for receiving the sample from the test or control passage; b) a conjugate pad comprising magnetically-labelled biorecognition molecules which can specifically bind to LC3-1 and LC3-II proteins; c) a test line region comprising biorecognition molecules which can specifically bind to LC3-I and LC3-II proteins, wherein the biorecognition molecules are immobilized on the test line region.

48. A kit comprising: one or more lateral flow immunoassay test strips; one or more microfluidic devices according to any one of claims 43 to 47; a device according to any one of claims 1 to 15; one or more cartridges for housing a lateral flow test strip and configured to engage with the device; a fusion inhibitor which inhibits fusion of autophagosomes and lysosomes; a white blood cell lysing composition; one or means for collecting a sample from a subject; and / or instructions for performing the method according to any one of claims 20 to 40.

Images