Method of detecting and quantifying a paramagnetic metal ion using micromagnetic resonance relaxometry (MRR)

Abstract

Disclosed is a method of detecting and / or quantifying a paramagnetic metal ion in a sample, comprising: (a) preparing the sample containing the paramagnetic metal ion in a hydrochloric acid, or a nitric acid, or a perchloric acid solution; and (b) establishing a standard curve between a relaxation rate R1 (or R2) and a standard paramagnetic metal ion concentration, using a Micromagnetic Resonance Relaxometry (MRR) device; (c) determining the R1 (or R2) of the sample and quantifying the paramagnetic metal ion according to the standard curve; wherein the concentration of the paramagnetic metal ion is at least 0.1 µM. Also disclosed is a method of identifying a risk of a disease, diagnosing a disease, or monitoring status of a disease in a patient, wherein the disease is associated with an amount of a paramagnetic metal ion in the patient, comprising the paramagnetic metal ion detecting and / or quantifying method as disclosed herein.

Description

METHOD OF DETECTING AND QUANTIFYING A PARAMAGNETIC METAL ION USING MICROMAGNETIC RESONANCE RELAXOMETRY (MRR)FIELD OF THE INVENTION

[0001] The present invention generally relates to a method of detecting and / or quantifying a paramagnetic metal ion in a sample. In particular, the present invention relates to a method of detecting and / or quantifying a paramagnetic metal ion in a sample using a Micromagnctic Resonance Rclaxomctry (MRR) device.BACKGROUND

[0002] The spin-lattice relaxation (Ti) and spin-spin relaxation (T2) of the aqueous samples (largely driven by water protons) can be shortened by the presence of paramagnetic ions. Paramagnetic ions are transition metal ions that have at least one unpaired electron in their electron configuration, such as Fe+(S=5 / 2), Mn2+(S=5 / 2), Cu2+(S=l / 2), Cr3+(S=3 / 2), Co2+(S=3 / 2), and Ni2+(S=2 / 2). An unpaired electron has an electron magnetic dipole moment generated by the electron's intrinsic spin property. Under the external magnetic field, the magnetic dipole moment of paramagnetic ions aligns with the Bo, leading to an enhanced local magnetic field [1 , 2]. The dipolar interaction between the unpaired electrons of these metal ions and water proton nuclear spins induces a much faster relaxation than proton-proton interactions, reflected by shortened Ti and T2 [1, 2]. Such a shortening of Ti and T2, or enhanced relaxation rate Ri (1 / Ti) and R2 (I / T2), is known to be dependent on the local composition and concentration of these paramagnetic ions.

[0003] Fe3+, Mn2+, Cu2+, Cr3+, Co2+, and Ni2+are the paramagnetic metal ions present in the human body. Among them, Fe3+and Mn2+have the most unpaired electrons numbers (S=5 / 2), while Fe3+and Cu2+are the most abundant paramagnetic ions in serum. Previous report of 1CP-MS analysis of human serum showed that Fe3+(1043 pg / L) and Cu2+(927 pg / L) were far higher than Mn2+(2.738 pg / L), Cr3+(1.323 pg / L), Ni2+(3.599 pg / L), and Co2+(0.139 pg / L)[3]. Iron as the most abundant transition metal in the human body, participates in many critical biological processes in almost all organisms, including electron transport in the citric acid cycle [4], deoxyribonucleic acid (DNA) synthesis and repair[5], oxygen transport[6J, neurotransmitter synthesis[7J, and collagen formation[8J.Iron is absorbed from the intestine and lost through bleeding and desquamation. In the body, approximately 60% of iron is bound to hemoglobin, 25% is stored in ferritin and hemisoderin, 5% is associated with myoglobin for muscle function, and the remainder is found in functional enzymes and transferring]. In the serum, iron is primarily present as Fe3+, either chelated to circulating transferrin or stored within the ferritin complex. For decades, numerous studies have reported the correlation between serum or plasma iron levels and various diseases. In Alzheimer’s disease (AD) patients, plasma iron levels are decreased and associated with a reduced loading of iron into transferrinflO], although an increase of brain iron deposition has been obscrvcd[l l]. In inflammatory diseases, hepcidin upregulation leads to the closure of the cellular iron export gate, ferroportin, resulting in decreased plasma iron levels, commonly referred to as inflammatory anemia

[0012] . A retrospective study reported that most COVID-19 patients (90%) had abnormally low serum iron, and the disease severity was inversely correlated with serum iron levels before and after treatment[13J.

[0004] Current serum or plasma iron measurement methods leave much to be desired in terms of cost, speed, and accuracy. In clinical laboratories, colorimetric assay is the most common method as it is easy to operate and relatively inexpensive. Fe2+can react with various spectrophotometric reagents (phenanthroline, ferrozine, salicylic acid, potassium thiocyanate) to form a color complexfl 4]. However, in clinical practice, a patient’s blood sample often contains ethylenediaminetetraacetic acid (EDTA) for anticoagulation. Iron is chelated by EDTA and interferes with its reaction with colorimetric regents, resulting in lowered iron recovery

[0015] . Moreover, the accuracy of colorimetric iron measurement was also influenced by lipcmic, even at a mild degree

[0016] . The most sensitive and accurate iron measurement method is mass spectrometry, specifically Inductively Coupled Argon Plasma Mass Spectrometry (ICP-MS) techniques

[0017] , However, the detection dynamic range of ICP-MS is narrow, generally between 0.1 ppb to 10 ppb, to ensure the accuracy of the result. Thus, a preliminary estimation of the iron concentration of an unknown concentration sample remains necessary, while this procedure can cause significant sample loss and is time-consuming. Besides, mass spectrometry is usually unavailable in routine biological laboratories due to the requirement of expensive and bulky equipment and professional operators for complex procedures. In addition to traditional iron measurement methods, the use of T2 relaxation times in magnetic resonance imaging (MR1) techniques to estimate tissue ironconcentration has been studied for decades[18, 19]. To date, some MRI[20, 21] or NMR

[0022] studies for organ or tissue samples have reported that the relaxation rate R2 (the reciprocal of T2) was correlated with the iron concentration but are limited to relatively low linear correlation (R2<0.9) and high LOD (> 5pM). Moreover, the bulky and expensive MR1 or NMR instruments present significant barriers for conventional laboratories.LOOO5J Previously, the inventors have developed a low-cost, benchtop MRR device that can measure the T2 relaxation time of microliter aqueous samples within a few minutes, and has found that T2 is an important marker for many discascs[23-25] and is correlated with cellular senescence

[0026] . However, direct MRR measurements of biological samples in earlier works can be of limited quantitative values. Iron solutions in physiological pH typically exhibit magnetic moments significantly lower than the spin- only value of 5.92 BM expected for S = 5 / 2 systems, while it could be restored upon the addition of the acids[27J. In 1955, Mulay and Seiwood showed that the magnetic susceptibilities of Fel+solution decreased with increasing pH

[0028] . In addition, acidic pH can release iron from carrier proteins in biological samplcs

[0029] .

[0006] hr this disclosure, a rapid, sensitive, and accurate method was developed for iron determination in one minute utilizing an inexpensive benchtop 0.5-Tesla Micromagnetic Resonance Relaxometry (MRR) system with acidification treatment of the samples. Hydrochloric acid or nitric acid solution was used to create a low pH environment (pH<l) and achieve quantitative measurements of iron. The method yielded a highly linear calibration curve (R2> 0.999) between the relaxation rate R2 and iron concentration from 0.5 to 1000 pM, with a limit of detection (LOD) of 0.28 pM and a minimal assay volume of 5 pL. The accuracy of this method was validated by ICP-MS with serum and EDTA plasma samples. The present method offers a rapid and convenient alternative for serum and plasma iron measurement, which can substantially reduce clinical diagnostic time and support real-time iron monitoring for patients.SUMMARY

[0007] In one aspect, the present disclosure refers to a method of detecting and / or quantifying a paramagnetic metal ion in a sample, comprising:(a) preparing the sample containing the paramagnetic metal ion in a hydrochloric acid, or a nitric acid, or a perchloric acid solution; and(b) establishing a standaid curve between a relaxation rate Ri (or Ro) and a standard paramagnetic metal ion concentration, using a Micromagnctic Resonance Rclaxomctry (MRR) device;(c) determining the Ri (or Rj) of the sample and quantifying the paramagnetic metal ion according to the standard curve, wherein the concentration of the paramagnetic metal ion is at least 0.1 pM.L0008] In another aspect, the present disclosure refers to a method of identifying a risk of a disease, diagnosing a disease, or monitoring status of a disease in a patient, wherein the disease is associated with an amount of a paramagnetic metal ion in the patient, the method comprising:(a) obtaining a sample containing the paramagnetic metal ion from the patient;(b) preparing the sample containing the paramagnetic metal ion in a hydrochloric acid, or a nitric acid, or a perchloric acid solution; and(c) establishing a standard curve between a relaxation rate Ri (or R2) and a standard paramagnetic metal ion concentration, using a Micromagnetic Resonance Relaxometry (MRR) device;(d) determining the Ri (or R2) of the sample and quantifying the paramagnetic metal ion according to the standard curve, wherein the concentration of the paramagnetic metal ion is at least 0.1 pM.

[0009] The MRR method yielded a highly linear calibration curve (R2> 0.999) between the relaxation rate Ri (or R2) and iron concentration from 0.5 to 1000 pM, with a minimal assay volume of 5 pL. The accuracy of this method was validated by Inductively Coupled Argon Plasma Mass Spectrometry (ICPMS) with iron-binding proteins, serum and EDTA plasma samples. The disclosed method offers a rapid and convenient alternative for serum and plasma iron measurement, which can substantially reduce clinical diagnostic time and support real-time iron monitoring for patients.

[0010] The MRR method also yielded a highly linear calibration curve (R2> 0.999) between the relaxation rate Ri (or R2) and manganese concentration from 0.5 to 1000 pM, with a minimal assay volume of 5 pL. However, a distinct n and 12 relaxivity of iron and manganese has been found, that n FC is higher than n MU, but r? i <- is lower than T2_Mn. For samples containing both high levels of iron and manganese, the amounts of iron and manganese can be calculated by the following simultaneous equations:(■Rl(sample) = Rl(solvent) + rlFe[M]Fe + rlMn[M]Mn (R2(sample) = R2(solvent) + r2Fe[M]Fe + r2Mn[M]MnFor cells that have high level of manganese, the AR2 / AR1 can serve as an indicator of the cellular' Mn / Fe ratio, offering potential application for monitoring changes in cell metabolism.AR2= Rlfsample) ~ R2(«o / vem)AR1= R| (sample) Rl (solvent)BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0012] Figure 1 is a schematic diagram for MRR device.

[0013] Figure 2 shows calibration curve of relaxation rate Rl and R2 versus Fe3+, Mn2+, and Cu2+concentration. Error bars were added (error bars are too small to be visible).

[0014] Figure 3 is a calibration curve of relaxation rate R2 versus (A) Fe3+and (B) Mn2+in different acid solutions (HNO3 and HC1).

[0015] Figure 4 shows the concentrations of paramagnetic metal ions in cell, FBS, and plasma samples analysed by ICP-MS. SD was estimated by measuring three replicates of each sample, and error bars were added (some error bars arc too small to be visible).

[0016] Figure 5 shows the results of validation of the accuracy of iron measurement by MRR. Comparison of the iron measured by MRR with ICP-MS. SD was estimated by measuring three replicates of each sample, and error bars were added (some error bars are too small to be visible).

[0017] Figure 6 shows the results of application of MRR iron measurement method in clinical plasma samples. (A) Daily monitoring of plasma iron levels in seven patients receiving CAR T-cell therapy. (B) Comparison of the daily plasma iron levels and ferritin levels in a representative patient with CRS grades (G1-G4). Anti-inflammation medicine, Dexamethasone and Anakinra, were administrated to this patient on day 7 and day 10. (C) Correlation analysis of the plasma iron change ratio with platelet count in samplesmarked with CRS (n=30). The iron change ratio is calculated as the change of daily iron level compared to the pre-infusion iron level.

[0018] Figure 7 is a schematic graph showing rapid serum / plasma iron measurement by MRR.

[0019] Figure 8 shows the results of application of MRR iron measurement method in monitoring cellular iron uptake. (A) Illustration of MRR as a rapid iron monitoring platform for iron overload study utilizing minimal cell numbers and medium volume in a 96-well plate culture system. (B) Iron level measured by MRR hourly in both the culture medium and cell lysate, and the sum of both was regarded as the total iron in the culture system. SD was estimated by measuring three replicates of each sample, and error bars were added.

[0020] Figure 9 is a plot of Mn / Fe ratio versus AR2 / AR1 .DETAILED DESCRIPTION

[0021] In one aspect, the present disclosure refers to a method of detecting and / or quantifying a paramagnetic metal ion in a sample, comprising:(a) preparing the sample containing the paramagnetic metal ion in a hydrochloric acid, or a nitric acid, or a perchloric acid solution; and(b) establishing a standard curve between a relaxation rate Ri (or R2) and a standard paramagnetic metal ion concentration, using a Micromagnetic Resonance Relaxometry (MRR) device;(c) determining the Ri (or R2) of the sample and quantifying the paramagnetic metal ion according to the standard curve, wherein the concentration of the paramagnetic metal ion is at least 0.1 pM.

[0022] In another aspect, the present disclosure refers to a method of identifying a risk of a disease, diagnosing a disease, or monitoring status of a disease in a patient, wherein the disease is associated with an amount of a paramagnetic metal ion in the patient, the method comprising:(a) obtaining a sample containing the paramagnetic metal ion from the patient;(b) preparing the sample containing the paramagnetic metal ion in a hydrochloric acid, or a nitric acid, or a perchloric acid solution; and(c) establishing a standard curve between a relaxation rate Ri (or R ) and a standard paramagnetic metal ion concentration, using a Micromagnctic Resonance Rclaxomctry (MRR) device;(d) determining the Ri (or R2) of the sample and quantifying the paramagnetic metal ion according to the standard curve, wherein the concentration of the paramagnetic metal ion is at least 0.1 pM.L0023] As disclosed herein, the MRR device is described in US10429467B2, tilted "Biosensor, palm-sized device and method based on magnetic resonance relaxometry", the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0024] The MRR device as disclosed herein comprises: a radio-frequency spectrometer comprising at least one field programmable gate array chip; a power amplifier electrically connected with the radio -frequency spectrometer and amplifying an electrical output of the radio-frequency spectrometer, thereby producing an amplified electrical signal comprising between about 0.1 Watts and about 10 Watts power; a duplexer configured to isolate the radio -frequency spectrometer from the amplified electrical signal during a receiving mode of the device wherein the duplexer comprises a passive duplexer without a quarter wavelength transmission cable; a radio-frequency detection probe configured to transmit radiofrequency electromagnetic radiation to excite nuclei under resonance during a transmission mode of the device, the radio-frcqucncy detection probe comprising a detection microcoil comprising an inner diameter of less than about 1 millimeter; and at least one magnet supplying an external magnetic field to a detection region of the radiofrequency detection probe, the external magnetic field being less than about 3 Tesla.

[0025] In further, related embodiments, the at least one magnet may comprise at least two permanent magnets separated by a gap of less than about 5 millimeters. The device may weigh less than about 0.5 kilograms. The radio -frequency spectrometer, power amplifier and the at least one magnet may be mounted on a single circuit board of less than about 500 square centimeters size. The device may be capable of detecting a nuclear magnetic resonance electromagnetic signal for at least a substantial portion of a blood sample of volume less than about 100 nanoliters in less than about 1 minute. The radio-frequency spectrometer may comprise a pulse programmer, a direct digital synthesis module, the transmitter and a receiver. The power amplifier may be mounted on a single circuit board of less than about 20 square centimeter area. The at least one magnet may fit within a volume of less than about 30 cubic centimeters. The duplexer may comprise a passive duplexer without a quarter wavelength transmission cable, the passive duplexer comprising: a chip inductor in parallel with a fixed capacitor that is in series with a pair of crossed diodes; a trimmer capacitor in series with the parallel combination of the chip inductor, fixed capacitor and pair of crossed diodes; and at least one surface mount radio frequency switch diode. The device may be configured to receive a centrifuge tube, which same centrifuge tube is configured to be received in a centrifuge. The device may further comprise a centrifuge tube, at least a portion of the centrifuge tube being inserted into the detection region of the radio-frequency detection probe. The centrifuge tube may comprise an outside diameter of less than about 1 millimeter, and may comprise a blood sample including blood from an animal body or other samples. The blood sample may comprise a volume of less than about 100 nanoliters. The power amplifier may comprise at least one surface mount power amplifier module. The device may be configured to perform at least one of Nuclear Magnetic Resonance (NMR) and Magnetic Resonance Imaging (MRI).

[0026] Tn one example, the detection region of the MRR device includes a volume of less than 1 pL of the sample. In another example, the detection region of the MRR device includes a sample with a volume of less than 1 pL, less than 2 pL, less than 3 pL, less than 4 pL, less than 5 pL, less than 10 pL, less than 15 pL, less than 20 pL, less than 30 pL, about 0.1 pL, about 0.5 pL, about 1 pL, about 2 pL, about 3 pL, about 4 pL, about 5 pL, about 6 pL, about 10 pL, about 20 pL, about 30 pL, or more. In another example, the detection region of the MRR device includes a volume of at least 5 pL of the sample.

[0027] As used herein, a standard paramagnetic metal ion concentration refers to a specific, controlled amount of paramagnetic metal ions in an acid solution, used as a reference or baseline for experiments and measurements. A standard paramagnetic metal ion concentration is used herein to establish a standard curve between a relaxation rate (R1 or R2) and a standard paramagnetic metal ion concentration, i.c. a graphical representation used to determine the concentration of paramagnetic metal ions in a sample.

[0028] In one example, the paramagnetic metal ion contained in the sample is iron (111). In another example, the iron (111) has a concentration of 0.1-1000 pM. In another, , , , , pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, and about 1000 pM.

[0029] The acid used to treat the sample containing a paramagnetic metal ion of the method as disclosed herein is selected from the group consisting of a hydrochloric acid, or a nitric acid, or a perchloric acid. The acid solution treatment was used to create an extremely low pH environment (pH<3, preferably pH<l) and achieve quantitative measurements of a paramagnetic metal ion such as iron.

[0030] In one example, the hydrochloric acid, or the nitric acid, or the perchloric acid solution used to add to the sample containing the paramagnetic metal ion has a concentration of 0.1-3 M, selected from the group consisting of 0.1-0.5 M, 0.5-1 M, 1-2 M, 2-3 M, about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.5 M, about 2 M, and about 3 M.

[0031] In another example, the sample in the hydrochloric acid solution has a pH <3. In another sample, the sample in the acid solution has a pH < 1.

[0032] In another example, the paramagnetic metal ion contained in the sample is iron (II), and wherein the sample is treated with the nitric acid or the perchloric acid to oxidize iron (II) (Fe2+) to iron (III) (Fe3+). This process involves the loss of an electron by the iron (II) ion. For the oxidization reaction, the concentration of the nitric acid or the perchloric acid is 0.1-3 M. In some examples, the concentration of the nitric acid or the perchloric acid is selected from the group consisting of 0.1-0.5 M, 0.5-1 M, 1-2 M, 2-3 M, about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.5 M, about 2 M, and about 3 M.

[0033] In another example, the paramagnetic metal ion contained in the sample is selected from the group consisting of titanium ion, chromium ion, manganese ion, iron ion, cobalt ion, nickel ion, copper ion, and gadolinium ion. As used herein, paramagnetic metal ions are transition metal ions that have at least one unpaired electron in their electron configuration. An unpaired electron has an electron magnetic dipole momentgenerated by the electron's intrinsic spin property. Under the external magnetic field, the magnetic dipole moment of paramagnetic ions aligns with the Bo, which create small magnetic moments. As used herein, the terms of titanium ion, chromium ion, manganese ion, iron ion, cobalt ion, nickel ion, copper ion, and gadolinium ion are the same and used interchangeably as terms of titanium, chromium, manganese, iron, cobalt, nickel, copper, and gadolinium, respectively.L0034] In another example, the paramagnetic metal ion contained in the sample is chromium. In another example, the concentration of the chromium is 0.1-1000 pM. In another example, the concentration of the chromium is selected from the group consisting of 0.1-0.5 pM, 0.5-1 pM, 1-10 pM, 10-100 pM, 100-300 pM, 200-400 pM, 300-500 pM, 400-600 pM, 500-700 pM, 600-800 pM, 700-900 pM, 800-1000 pM, 1-900 pM, 10-800 pM, 100-700 pM, 200-600 pM, 300-500 pM, about 0.1 pM, about 0.5 pM, about 1 pM, about 10 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, and about 1000 pM.

[0035] In another example, the paramagnetic metal ion contained in the sample is copper. In another example, the concentration of the copper is 1-1000 pM. In another example, the concentration of the copper is selected from the group consisting of 1-10 pM, 10-100 pM, 100-300 pM, 200-400 pM, 300-500 pM, 400-600 pM, 500-700 pM, 600-800 pM, 700-900 pM, 800-1000 pM, 1-900 pM, 10-800 pM, 100-700 pM, 200-600 pM, 300-500 pM, about 1 pM, about 10 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, and about 1000 pM.

[0036] In another example, the paramagnetic metal ion contained in the sample is manganese. In another example, the concentration of the manganese is 0.1-1000 pM. In another example, the concentration of the manganese is selected from the group consisting of 0.1-0.5 pM, 0.5-1 pM, 1-10 pM, 10-100 pM, 100-300 pM, 200-400 pM, 300-500 pM, 400-600 pM, 500-700 pM, 600-800 pM, 700-900 pM, 800-1000 pM, 1- 900 pM, 10-800 pM, 100-700 pM, 200-600 pM, 300-500 pM, about 0.1 pM, about 0.5 pM, about 1 pM, about 10 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, and about 1000 pM.

[0037] In another example, the paramagnetic metal ion contained in the sample is cobalt. In another example, the concentration of the cobalt is 10-1000 pM. In another, , , , , 1000 pM.LOO38] In another example, the paramagnetic metal ion contained in the sample is nickel, hr another example, the concentration of the nickel is 10-1000 pM. In another example, the concentration of the nickel is selected from the group consisting of 10-100 pM, 100-300 pM, 200-400 pM, 300-500 pM, 400-600 pM, 500-700 pM, 600-800 pM, 700-900 pM, 800-1000 pM, 10-900 pM, 100-800 pM, 200-700 pM, 300-600 pM, 400- 500 pM, about 10 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, and about 1000 pM.

[0039] The sample containing the paramagnetic metal ion as disclosed herein, to be detected and / or quantified using the method as disclosed herein, is a biological, chemical or environmental sample, indicating the method's wide applications.

[0040] In one example, the biological sample is obtained from a cell or tissue sample, saliva, blood, plasma, sera, stool, urine, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, or swab of skin or a mucosal membrane surface, a lysate of a cell or tissue sample, or a combination thereof.

[0041] hr another example, the chemical sample is obtained from a pharmaceutical substance, a chemical reagent, a culture medium, a cosmetic product, an industrial effluent, a pollutant, a hazardous material, a polymer, an adhesive, a solvent, an oil, a gas, a metal alloy, a nanomaterial, a fuel, a cleaning agent, a dye, or a combination thereof.

[0042] In another example, the environmental sample is obtained from a food, a beverage, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a waste water sample, a saline water sample, exposure to atmospheric air or other gas sample, or a combination thereof.

[0043] In another example, the biological, chemical or environmental sample is a crude sample which has not undergone any processing or purification to remove impurities or unwanted components. In another example, the biological, chemical orenvironmental sample is a crude sample wherein the paramagnetic metal ion is not purified from the sample.

[0044] hr another example, the biological, chemical or environmental sample is a pretreated sample which has undergone processing to release the paramagnetic metal ion from metal-binding proteins. In another example, the paramagnetic metal ion release process is selected from the group consisting of permeabilization, heating, proteinase incubation, or a combination thereof.

[0045] hi another example, determining the T2 value includes supplying a train of Carr-Purccll-Mciboom-Gill (CPMG) pulses over a period of less than one minute, selected from the group consisting of about 5 s, about 10 s, about 15 s, about 20 s, about 25 s, about 30 s, about 35 s, about 40 s, about 45 s, about 50 s, about 55 s, and about 60 s.

[0046] In another example, a strong linear relationship (R2> 0.999) exists between the R2 value (the reciprocal of T2) and the concentration of the paramagnetic metal ion, meaning that the concentration of the paramagnetic metal ion can be very accurately predicted from the relaxation rate R2 value.

[0047] hi another example, a strong linear relationship (R2> 0.999) exists between the Ri value (the reciprocal of Ti) and the concentration of the paramagnetic metal ion, meaning that the concentration of the paramagnetic metal ion can be very accurately predicted from the relaxation rate R2 value.

[0048] In another example, the method of detecting and / or quantifying a paramagnetic metal ion in a sample as disclosed herein can simultaneously detect and / or quantify two types of paramagnetic metal ions, such as iron and manganese in the sample, based on simultaneous Ri (reciprocal of Ti) and R2 (reciprocal of T2) equations (Equation 1):(■Rl(sample) = Rl(solvent) + rlFe[M]Fe + rlMn[M]Mn (R2(sample) = R2(solvent) + r2Fe[M]Fe + r2Mn[M]Mn

[0049] In another example, the sample for simultaneous iron and manganese detection and / or quantification is a biological sample as disclosed herein, for example a cell sample or a tissue sample.

[0050] As used herein, Ri (Longitudinal Relaxation Rate) is the reciprocal of the longitudinal relaxation time, Ti (Ri=l / Ti). Ti is the time constant that describes the rate at which the net magnetization vector returns to its equilibrium state along the longitudinal (z) axis. This process involves the exchange of energy between the nuclearspins and their surrounding lattice (environment), hence it is also called spin-lattice relaxation. Ri is measured using an inversion recovery sequence, where the recovery of longitudinal magnetization is monitored over time.

[0051] As used herein, R2 (Transverse Relaxation Rate) is the reciprocal of the transverse relaxation time, T2 (R2=l / T2). z is the time constant that describes the rate at which the net magnetization vector decays in the transverse (xy) plane. This process involves the loss of phase coherence among the spins due to interactions between them, hence it's also called spin-spin relaxation. R2 is measured using a Carr-Purcell-Meiboom- Gill (CPMG) pulse sequence, which helps to refocus the spins and measure the decay of transverse magnetization.

[0052] Iron and manganese have different R1 and R2 relaxivity. With the obtained relaxivity values, the relaxation rates of a sample containing both iron and manganese can be calculated by the simultaneous equations (Equation 1) as disclosed herein. In addition, ratio of Mn / Fe or Fe / Mn can also be calculated by Equation 1.

[0053] The method of identifying risk of a disease, diagnosing a disease, or monitoring status of a disease in a patient involves detecting and quantifying a paramagnetic metal ion in a biological sample of the patient, which can be obtained from a cell or tissue sample, saliva, blood, plasma, sera, stool, urine, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, or swab of skin or a mucosal membrane surface, a lysate of a cell or tissue sample, or a combination thereof. The amount of the paramagnetic metal ion in the sample as disclosed herein would assist identifying risk of a disease, diagnosing a disease, or monitoring status of a disease.

[0054] In one example, the disease is one that is associated with overload of the paramagnetic metal ion. In another example, tire disease is one that is associated with deficiency of the paramagnetic metal ion. In some examples, the disease associated with overload or deficiency of the paramagnetic metal ion is selected from the group consisting of anaemia, hemochromatosis, Wilson disease, Menkes disease, Alzheimer's disease, Parkinson's disease, multiple system atrophy, Huntington’s disease, Amyotrophic Lateral Sclerosis, Hyperpigmentation, Cirrhosis, Hepatocellular carcinoma. Diabetes Mellitus, Colon carcinoma, Arthritis, Hypogonadism, Diabetic Retinopathy, Age-related Macular Degeneration, Friedreich’s ataxia, Chronic Obstructive Pulmonary disease, Cardiomyopathy, Arrhythmia, Diabetic Nephropathy, Atherosclerosis, and Hypertension. In another example, the disease is associated with overload or deficiency of one type ofparamagnetic metal ion in a sample such as iron. In another example, the disease is associated with overload or deficiency of more than one type of paramagnetic metal ion in a sample, such as iron and manganese. Simultaneously detection and / or quantification of two types of paramagnetic metal ions, such as iron and manganese in the sample, in order to identify risk of a disease, diagnose a disease, or monitor status of a disease associated therewith, is based on simultaneous Ri (reciprocal of Ti) and R2 (reciprocal of T2) equations (Equation 1): tRl(sample) = Rl(solvent) + rlFe[M]Fe + rlMn[M]Mn (R2(sample) = R2(solvent) + r2Fe[M]Fe + r2Mn[M]Mn

[0055] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a paramagnetic metal ion” includes a plurality of paramagnetic metal ions, including mixtures and combinations thereof.

[0056] As used herein, the term “comprising” means “including.” Variations of the word "comprising", such as “comprise” and “comprises,” have correspondingly varied meanings. Thus, for example, a composition “comprising” X may consist exclusively of X or may include one or more additional unrecited components.

[0057] As used herein, the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means + / - 5% of the stated value, or + / - 4% of the stated value, or + / - 3% of the stated value, or + / - 2% of the stated value, or + / - 1% of the stated value, or + / - 0.5% of the stated value.

[0058] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0059] The disclosure illustratively described herein may suitably be practiced in the absence of any clement or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0060] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form pail of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0061] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0062] Other embodiments are within the following claims and non-limiting examples.EXAMPLES

[0063] Non-limiting examples of the disclosure will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the disclosure.

[0064] Example 1- Experimental Section

[0065] Chemicals and Material

[0066] 1000 mg / L Fc(NO3)3, 1000 mg / L Mn(N03)2,1000 mg / L Cu(NO3)2standard solution, and IN nitric acid solution were purchased from MERCK. 0.5 mol / l HC1 was prepared from IN Hydrochloric acid (Fisher). Fetal Bovine Serum (FBS) was purchased from Thermo Fisher Scientific. Human plasmas were collected from patients receivingCAR T-cell therapy for the treatment of refractory / relapsed diffuse large B-cell lymphoma in Singapore General Hospital, following informed consent under the approval of the ethics committee according to a protocol permitted by the SingHealth Centralised Institutional Review Board (CIRB ref.: 2022 / 2322). Briefly, 3mL of peripheral whole blood was drawn into an EDTA tube, then centrifuged at 1000 g for 10 minutes at ambient temperature. 25uL plasma was aliquoted from the plasma layer used for downstream analysis.

[0067] MRR measurement

[0068] The MRR device consists of a portable, permanent magnet (Mctrolab Instruments Ltd., Switzerland) with B0 = 0.5 T, a bench-top proton (1H) Nuclear Magnetic Resonance (NMR) console (Kea Magritek Ltd., New Zealand), and a temperature controller (RS component Ltd., UK) to maintain 28 °C inside the magnet. (Figure 1). No additional shimming or field-gradient control is used in the disclosed MRR system, making it simple and straightforward to build and use. To operate the measurement, 5pL of the aqueous sample was filled in a micro -capillar y tube (22-260-950, Fisherbrand Ltd., USA), then the tube was scaled with critoscal (Leica Microsystems Ltd., German) and positioned into the coil. T; of the sample was measured by the standard Carr-Purcell-Meiboom-Gill (CPMG) pulse program. Ti was measured using an inversion recovery sequence.

[0069] Ri and R2 measurement of Fe. Mn. Cu standard solutions for the calibration curve

[0070] Fe(NC>3)3, Mn(NOs)2, and Cu / NChh standard solutions were diluted in 0.4 mol / L HN03 to prepare different concentration samples from 0 to 1000 pM and subsequently analyzed with MRR for calibration curves. The limit of detection (LOD) is estimated by measuring replicates of a low -concentration sample (n=10) and calculating the standard deviation (SD). LOD = 3 xSD.

[0071] Measurement of paramagnetic ions in serum and plasma with ICP-MS

[0072] FBS was 1: 400 diluted with 0.4 mol / L HNO3. Human plasma was 1: 250 diluted with 0.4 mol / L HNO3. Diluted sampled were heated at 90 °C for 30 mins then centrifuged at 10,000 g for 10 mins to remove precipitate. Iron (Fc), manganese (Mn), copper (Cu), chromium (Cr), cobalt (Co), and nickel (Ni) concentrations were determined by ICP-MS (Agilent 8800). Same samples were also used for MRR T2 measurement.

[0073] Example 2- Results and Discussion

[0074] Analytical performance of MRR for Fe3+, Mn2+, and Cu2+measurement

[0075] To verify that MRR can be used as a quantification method, the serial-diluted paramagnetic ion standard solutions were measured for Ti and T2 relaxation time. Fe3+, Mn2+, and Cu2+were selected due to their relative abundance in biological samples26. Figure 2 shows the good linear calibration curves of relaxation rate Ri (1 / Ti) and R2 (I / T2) versus Fe3+, Mn2+, and Cu2+concentrations from 0 to 100 pM.

[0076] The calibration linear range, standard curve equation, R2 relaxivity of paramagnetic ions, and LOD were determined to evaluate the analytical performance of the method (Table 1). The relaxivity (n) was obtained from the standard curve equation, Ri( sample) — R?( sol-vent) + r2[MJ. Resample) and Ri(solvent) are the measured relaxation rates of the paramagnetic ions solutions, and pure solvent in s-1,[M] is the concentration of paramagnetic ions (pM). Table 1 shows that the relaxivity of Mn2+(, 2Mn) was 2 times higher than Fe3+(r2Fe), while the relaxivity of Fe3+(r2Fc) was about 16 times higher than Cu2+(,2c«). With the obtained relaxivity values, the relaxation rates of a mixture solution containing Fe3+, Mn2+, and Cu2+can be calculated by the equation, R2( sample) = R2(solvent) +r2Fe[M]Fe +r2Mn[MjMn +r2Cu[Mjcu The measured R2 is the net effect of all the paramagnetic ions in the sample and is dependent on their concentration and relaxivity.

[0077] Table 1. Analytical performance of MRR for Fe3+, Mn2+, and Cu2+measurement.

[0078] Interestingly, it was found that different acid solutions (HNO3 and HC1) contributed to different effects on Fc3+rclaxivity, but not affected Mn2+relativity (Figure 3). The relaxivity of Fe3+was about 1.5 times higher in HNO3 solutions than in HC1 solution. In a previous study using NMR analysis17, the author suggested that the higher relaxivity of Fe3+in HNO3 was due to the larger size of NO3’ compared to CT ions that reduced translational diffusion of water surrounding Fe3+. However, the present study indicated the size of NO3" and Cl" ions might not be the critical factors, since the relaxivity of Mn2+was not affected.

[0079] Accuracy of using MRR for iron quantification in serum and plasma samples

[0080] ICP-MS analysis of paramagnetic metal ions in serum and plasma samples revealed that iron is the most abundant element (Figure 4), which is consistent with the previous reports26. In FBS, Fe is 14 times higher than Cu and 80 times higher than Mn. In human plasma, Fe is 3 times higher than Cu and 729 times higher than Mn in human plasma. Given the low concentration of Mn2+(< 0.05pM) and the low relaxivity of Cu2+( <0.001), the equation Resample) = Resolvent) + r2Fe[M]Fe + mfo[M]Mu+ OGJ MJCU can be simplified to FG sample) = Resolvent) + r2Fe[M]Fe. It suggests that MRR can be effectively utilized for rapid iron quantification in serum and plasma samples, with negligible interference from other paramagnetic ions. As shown in Figure 2 and Table 1, a good linearity (R2> 0.999) between the relaxation rate R2 and iron concentration was achieved over the range of 0.5 to lOOOpM, and the LOD concentration is 0.28 pM. Compared with the previous study using commercial NMR scanners (LOD at 5 pM)1', significantly higher sensitivities with much simpler, less sophisticated scanning equipment is reported herein. To demonstrate the accuracy of MRR for iron measurement, the iron concentrations measured by MRR were compared to the results from ICP-MS utilizing cells, serum, plasma, and ferritin samples. Figure 5 shows that there is no statistically significant difference between the iron concentration measured by MRR and ICP-MS, validating the accuracy of the MRR iron measurement method.

[0081] Monitoring plasma iron levels in patients receiving CAR T-cell therapy

[0082] Transferrin saturation (TSAT) and serum ferritin levels are widely utilized in clinical settings to assess a patient's iron status. While scrum iron level is the key parameter that is believed to be correlated with many important conditions such as anaemia, local and systemic inflammation, and infections, the accuracy of these assays (using iron binding proteins as surrogate markers) in quantifying iron levels is limited27. Theserum levels of iron transport and storage proteins, transferrin and ferritin can be affected by factors unrelated to actual iron concentration. For example, a single ferritin protein can store from 0 to 4,500 iron atoms in its core28, limiting the quantitative correlation with iron levels. Moreover, evidence showed that the elevation of ferritin level is an acutephase reactant in response to inflammation, which complicates its use in iron deficiency patients29.LOO83] Serum iron concentration, necessary for calculating TSAT, is typically measured by colorimetric methods or spectrophotom-etry. However, the limitations of these traditional iron determination techniques hinder the use of TSAT for daily monitoring in hospitals. In this study, MRR method was applied to measure the plasma iron levels of seven patients receiving CAR T -cell therapy daily (Figure 6A), and six out of seven patients showed abnormally low plasma iron (< 5pM). This result is in accordance with previous reports that anaemia is prevailing in cancer patients30(40%) and patients treated with chemotherapy31(90%), mainly caused by impaired haemogenesis. The extremely low plasma iron level in cancer patients can cause difficulties in iron determination using traditional colorimetric kits, which could lead to inaccurate results for clinical diagnosis. The sample processing flow was shown in Figure 7.

[0084] More importantly, plasma iron levels can serve as an earlier indicator of cytokine release syndrome (CRS) compared to ferritin, as iron levels doubled at day 7, grade 3, whereas ferritin only showed a significant increase at day 9, grade 4 (Figure 6B). Additionally, the iron level showed an immediate response following the administration of anti-inflammatory medications, Dexamethasone and Anakinra, on days 7 and 10, whereas the ferritin level continued to rise steadily (Figure 6B). This lack of temporal alignment between ferritin and iron levels suggests that ferritin may not accurately reflect real-time changes in iron levels and may not be a reliable indicator for monitoring the effects of medication administration.

[0085] CRS is a potentially life-threatening side effect that has been observed after the administration of T-cell therapies for cancer, which is characterized by elevated levels of multiple cytokines, accompanied by fever and multiple organ dysfunction32. Up to date, many scrum cytokines or biochemical markers have been investigated as a prognostic indicator of CRS. Ferritin has been reported to have a significant association with CRS but failed to predict severe CRS33. Baseline blood platelet level was proposed as a predictive biomarker for severe neurotoxicity following CAR T cell therapy34. In ourstudy, the plasma iron change ratio, which is the daily iron level compared to the preinfusion iron level, showed a moderate correlation with platelet count (R2= 0.799) in samples marked with CRS (Figure 6C).

[0086] Monitoring iron level in culture media during the cell expansion

[0087] As a demonstrative example of using MRR as a convenient iron detection tool for biological studies, we carried out iron uptake measurements on mesenchymal stromal cells (MSC) cultures, using cell lysate and cell culture supernatants as the sample. MSCs at late passages are known to become senescent, correlated with intracellular iron overloading, which is a challenge in therapeutic MSC production. Cell-based iron overload models have been extensively utilized in investigating the pathogenesis of different diseases, as well as assessing the efficacy of various therapeutic strategies. Our MRR iron measurement method offers a rapid iron monitoring platform for iron overload study utilizing minimal cell numbers and medium volume in a 96-well plate culture system (Figure 8A). Figure 8B shows that the decrease in the spent medium iron levels, measured every hour, accurately reflected the iron uptake level in the cell since the total iron in the system remained consistent. For the first time, the iron uptake speed of MSC was directly measured with an hourly detection frequency, and this iron uptake dynamics could be significant for MSC-based cell therapy. In addition, MRR measurement can be extended to serve as a non-destructive and real-time iron monitoring method in a closed culture system, such as a bioreactor, by utilizing the spent medium.

[0088] Simultaneous equations of R i and Rifor iron and manganese determination by acid-MRR

[0089] During the initial development of the acid-MRR method for quantifying iron, it was observed that the R2-based acid-MRR technique demonstrated comparable accuracy to ICP-MS when applied to serum, plasma, and culture media samples. However, in analyzing cell samples, higher iron levels were frequently estimated from the R2 equation compared to ICP-MS results. ICP-MS assays of these cell samples revealed high manganese levels, in contrast to the exceptionally low manganese levels found in other biological samples. To address manganese's potential influence, an advanced measurement method based on simultaneous Ri and R2 was developed. The accuracy of this new approach was further validated using standard iron and manganese mixture solutions.

[0090] To perform the advanced measurement method, Fe(NC>3)3, Mn(NOs)2, and Cu(NO3)2 standard solutions were diluted in 0.4 mol / L HNO3 to prepare different concentration samples from 0 to 1000 pM and subsequently analyzed with MRR for Ri and R2 calibration curves. Ri (1 / Ti) is measured by inversion recovery program, and R2 (I / T2) is measured by Carr-Purcell-Meiboom-Gill (CPMG) pulse program.

[0091] Result 1: Iron and manganese have different ri and r2 relaxivity

[0092] Figure 2 shows the good linear calibration curves of relaxation rate Ri and R2 versus Fe3+, Mn2+, and Cu2+concentrations (R2>0.999). Interestingly, distinct n and n rclaxivity of iron and manganese were noticed, that n Fe (0.0117) is higher than ri_Mn (0.0073), but r2_Fe (0.015) is lower than r2_Mn (0.0397). And the extreme low relaxivity of Cu2+, ri. Cu (0.0009) and r2_cu (0.0008), suggested a negligible effect of copper content on sample relaxation.

[0093] Result 2: Iron and manganese determination in a mixture solution

[0094] With the obtained relaxivity values, the relaxation rates of a sample containing both iron and manganese can be calculated by the following simultaneous equations (Equation 1):Rl(sample) = Rl(solvent) + rlFe[M]Fe + rlMn[M]MnR2(sample) = R2(solvent) + r2Fe[M]Fe + r2Mn[M]Mn

[0095] Using FelNChh and Mn(NCh)2 mixed solutions, the difference between the real and calculated Mn and Fe concentration was validated to be within 5% (Table 2).

[0096] Table 2. Comparison of the real and calculated Mn and Fe concentration utilizing a mixture of Fe(NC>3)3 and Mn(N0s)2 solutions.

[0097] Notably, when sample only containing iron, the iron concentration can be measured by cither of the following equations (Equation 2):

[0098] Result 3: Mn / Re ratio in biological samples can be reflected by \R2 / 3sRl

[0099] In samples with relative high level of manganese, the Mn / Fe ratio can be calculated by simultaneous equations (Equation 1), with obtained constants from Figure 2. The plot of Mn / Fe ratio versus AR2 / AR1 was shown in Figure 9. The proportional trend observed in the curve suggests that AR2 / AR1 can serve as an indicator of the cellular Mn / Fe ratio, offering potential application for monitoring changes in cell metabolism.

[0100] Example 3- Conclusion

[0101] Tn this study, an excellent linear relationship between the R2 measured by MRR and iron concentration from 0.5 to 1000 pM (R2> 0.999) was experimentally validated, and the LOD of this method was 0.28 pM. Meanwhile, it was demonstrated that the accuracy of this rapid and quantitative iron measurement is compar able to that of ICP-MS with serum and plasma samples, using an acid treatment before MRR measurement. The method disclosed herein can be used to measure iron in various biological sample matrices, such as culture medium, serum, and cell lysate, which could be instrumental in advancing iron-specific rapid disease diagnostics.

[0102] The fact that acid inhibited Fe3+hydrolysis can be applied to enhance the R2 value of the iron solutions is the background of this invention. However, no one has applied this fact in real biological samples such as serum, cell culture supernatants, and cell lysates for iron determination, probably due to the low amount of iron and the complex sample components.

[0103] With the present MRR method combined with acid treatment, one can achieve a highly linear relationship that is critical for quantification of irons. Even in the earlier inventions on MRR

[0026] , only qualitative correlations were shown, not the quantification of irons in this manner.

[0104] Such a quantification of minute amounts of iron in real-world biological samples can be achieved in such a small sample volume (a few microliter) and a simple, low-cost systems, without incurring complex sample preparation methods.

[0105] Industrial Applicability

[0106] This invention is suitable for paramagnetic metal ion (such as iron) determination in both environmental and biological samples. The possible industrial applications including detection of paramagnetic metal ion (such as iron) contamination in environmental water, measurement of paramagnetic metal ion (such as iron) during culture media or serum manufacturing for quality control, measurement of paramagnetic metal ion (such as iron) in human body fluid, cell, and tissue for disease diagnostics.

[0107] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.References[1] D. Hao, T. Ai, F. Gocrncr, X. Hu, V.M. Runge, M. Tweedie, MRI contrast agents: basic chemistry and safety, J. Magn. Rcson. Imaging 36(5) (2012) 1060-1071.[2] S.H. Koenig, R.D. Brown III, Relaxation of solvent protons by paramagnetic ions and its dependence on magnetic field and chemical environment: implications for NMR imaging, Magn. Reson. Med. 1(4) (1984) 478-495.[3] G. Zhang, F. Zhang, W. Liu, C. Liu, I. You, M. Tian, T. Cao, J. Jiang, Z. Yang, H. Wu, W. Wu, A simple, rapid method for simultaneous determination of multiple elements in serum by using an ICP-MS equipped with collision cell, BMC Chemistry 17(1) (2023) 34.[4] H. Oexle, E. Gnaiger, G. Weiss, Iron-dependent changes in cellular energy metabolism: influence on citric acid cycle and oxidative phosphorylation, Biochim Biophys Acta Bioenerg 1413(3) (1999) 99-107.[5] S. Puig, L. Ramos-Alonso, A.M. Romero, M.T. Martmez-Pastor, The elemental role of iron in DNA synthesis and repair, Metallomics 9(1 1) (2017) 1483-1500.[6] I.M. Klotz, G.L. Klippenstein, W.A. Hendrickson, Hemerythrin: Alternative Oxygen Carrier: Nature has developed an effective transport protein with a binuclear iron center in place of a heme, Science 192(4237) (1976) 335-344.[7] T. Moos, E.H. Morgan, The metabolism of neuronal iron and its pathogenic role in neurological disease, Ann. N. Y. Acad. Sci. 1012(1) (2004) 14-26.[8] L.R. Weintraub, A. Goral, J. Grasso, C. Franzblau, A. Sullivan, S. Sullivan, Collagen biosynthesis in iron overload, Ann. N. Y. Acad. Sci. 526(1) (1988) 179-184.[9] P. Benito, D. Miller, Iron absorption and bioavailability: an updated review, Nutr. Res. 18(3) (1998) 581-603.

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Claims

Claims1. A method of detecting and / or quantifying a paramagnetic metal ion in a sample, comprising:(a) preparing the sample containing the paramagnetic metal ion in a hydrochloric acid, or a nitric acid, or a perchloric acid solution; and(b) establishing a standard curve between a relaxation rate Ri (or R2) and a standard paramagnetic metal ion concentration, using a Micromagnetic Resonance Relaxometry (MRR) device;(c) determining the Ri (or R2) of the sample and quantifying the paramagnetic metal ion according to the standard curve, wherein the concentration of the paramagnetic metal ion is at least 0.1 pM.

2. The method of claim 1 , wherein a detection region of the MRR device includes a volume of less than 1 pL of the sample.

3. The method of claim 1, wherein the paramagnetic metal ion is iron (111), and wherein the iron (III) has a concentration of 0.1-1000 pM.

4. The method of claim 1, wherein the hydrochloric acid, or the nitric acid, or the perchloric acid solution has a concentration of 0.1-3 M.

5. The method of claim 1, wherein the sample in the hydrochloric acid solution has a pH <3.

6. The method of claim 1, wherein the paramagnetic metal ion is iron (II), and wherein the sample is treated with the nitric acid or the perchloric acid.

7. The method of claim 6, wherein the concentration of the nitric acid or the perchloric acid is 0.1-3 M.

8. The method of claim 1, wherein the paramagnetic metal ion is selected from the group consisting of titanium ion, chromium ion, manganese ion, iron ion, cobalt ion, nickel ion, copper ion, and gadolinium ion.

9. The method of claim 8, wherein the paramagnetic metal ion is copper, and wherein the concentration of the copper is 1-1000 pM.

10. The method of claim 8, wherein the paramagnetic metal ion is manganese, and wherein the concentration of the manganese is 0.1-1000 pM.1 1 . The method of claim 8, wherein the paramagnetic metal ion is cobalt, and wherein the concentration of the cobalt is 10-1000 pM.

12. The method of claim 1, wherein the sample is a biological, chemical or environmental sample.

13. The method of claim 12, wherein the biological sample is obtained from a cell or tissue sample, saliva, blood, plasma, sera, stool, urine, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, or swab of skin or a mucosal membrane surface, a lysate of a cell or tissue sample, or a combination thereof.

14. The method of claim 12, wherein the chemical sample is obtained from a pharmaceutical substance, a chemical reagent, a culture medium, a cosmetic product, an industrial effluent, a pollutant, a hazardous material, a polymer, an adhesive, a solvent, an oil, a gas, a metal alloy, a nanomaterial, a fuel, a cleaning agent, a dye, or a combination thereof.

15. The method of claim 12, wherein the environmental sample is obtained from a food, a beverage, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a waste water sample, a saline water sample, exposure to atmospheric air or other gas sample, or a combination thereof.

16. The method of claim 12, wherein the biological, chemical or environmental sample is a crude sample and / or wherein the paramagnetic metal ion is not purified from the sample.

17. The method of claim 12, wherein the biological, chemical or environmental sample has undergone processing to release the paramagnetic metal ion from metal-binding proteins by a process selected from the group consisting of permeabilization, heating, and proteinase incubation.

18. The method of any one of claims 1-17, wherein determining the T2 value includes supplying a train of Carr-Purcell-Meiboom-Gill (CPMG) pulses over a period of less than one minute.

19. The method of any one of claims 1-18, wherein a linear relationship (r2> 0.999) exists between the R2 value (the reciprocal of T2) and the concentration of the paramagnetic metal ion.

20. The method of claim 1, wherein the amount of the paramagnetic metal ion in the sample is a biomarker of a disease.

21. The method of claim 1, wherein the MRR device comprises:a radio-frequency spectrometer comprising at least one field programmable gate array chip; a power amplifier electrically connected with the radio-frequency spectrometer and amplifying an electrical output of the radio -frequency spectrometer, thereby producing an amplified electrical signal comprising between about 0.1 Watts and about 10 Watts power; a duplexer configured to isolate the radio-frequency spectrometer from the amplified electrical signal during a receiving mode of the device wherein the duplexer comprises a passive duplexer without a quarter wavelength transmission cable; a radio-frequency detection probe configured to transmit radiofrequency electromagnetic radiation to excite nuclei under resonance during a transmission mode of the device, the radio-frequency detection probe comprising a detection microcoil comprising an inner diameter of less than about 1 millimeter; and at least one magnet supplying an external magnetic field to a detection region of the radio-frequency detection probe, the external magnetic field being less than about 3 Tesla.

22. The method of claim 1, wherein the method simultaneously detects and / or quantifies iron and manganese in the sample based on simultaneous R1 (reciprocal of Tl) and R2 (reciprocal of T2) equations (Equation 1 ): tRl(sample) — Rl(solvent) + rlFe[M]Fe + rlMn[M]Mn (R2(sample) = R2(solvent) + r2Fe[M]Fe + r2Mn[M]Mn '23. A method of identifying a risk of a disease, diagnosing a disease, or monitoring status of a disease in a patient, wherein the disease is associated with an amount of a paramagnetic metal ion in the patient, the method comprising:(a) obtaining a sample containing the paramagnetic metal ion from the patient;(b) preparing the sample containing the paramagnetic metal ion in a hydrochloric acid, or a nitric acid, or a perchloric acid solution; and(c) establishing a standard curve between a relaxation rate R1 (or R2) and a standard paramagnetic metal ion concentration, using a Micromagnetic Resonance Relaxometry (MRR) device;(d) determining the R1 (or R2) of the sample and quantifying the paramagnetic metal ion according to the standard curve; wherein the concentration of the paramagnetic metal ion is at least 0.1 pM.

24. The method of claim 23, wherein a detection region of the MRR device includes a volume of less than 1 pL of the sample.

25. The method of claim 23, wherein the paramagnetic metal ion is iron (III), and wherein the iron (III) has a concentration of 0.1-1000 pM.

26. The method of claim 23, wherein the hydrochloric acid, the nitric acid, or the perchloric acid solution has a concentration of 0.1-3 M.

27. The method of claim 23, wherein the sample in the hydrochloric acid solution has a pH <3.

28. The method of claim 23, wherein the paramagnetic metal ion is iron (II), and wherein the sample is treated with the nitric acid or the perchloric acid.

29. The method of claim 28, wherein the concentration of the nitric acid or the perchloric is 0.1 -3 M.

30. The method of claim 23, wherein the paramagnetic metal ion is selected from the group consisting of titanium ion, chromium ion, manganese ion, iron ion, cobalt ion, nickel ion, copper ion, and gadolinium ion.

31. The method of claim 30, wherein the paramagnetic metal ion is copper, and wherein the concentration of the copper is 1-1000 pM.

32. The method of claim 30, wherein the paramagnetic metal ion is manganese, and wherein the concentration of the manganese is 0.1 -1000 pM.

33. The method of claim 30, wherein the paramagnetic metal ion is cobalt, and wherein the concentration of the cobalt is 10-1000 pM.

34. The method of claim 23, wherein the sample is a cell or tissue sample, saliva, blood, plasma, sera, stool, urine, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, or swab of skin or a mucosal membrane surface, a lysate of a cell or tissue sample, or a combination thereof.

35. The method of claim 23, wherein the disease is a disease associated with overload or deficiency of the paramagnetic metal ion.

36. The method of claim 23, wherein the disease is selected from the group consisting of anaemia, hemochromatosis, Wilson disease, Menkes disease, Alzheimer's disease, Parkinson's disease, multiple system atrophy, Huntington’s disease, Amyotrophic Lateral Sclerosis, Hyperpigmentation, Cirrhosis, Hepatocellular carcinoma, Diabetes Mellitus, Colon carcinoma, Arthritis, Hypogonadism, Diabetic Retinopathy, Age- related Macular Degeneration, Friedreich’s ataxia, Chronic Obstructive Pulmonarydisease. Cardiomyopathy, Arrhythmia, Diabetic Nephropathy, Atherosclerosis, and Hypertension.

37. The method of any one of claims 23-36, wherein determining the T2 value includes supplying a train of Carr-Purcell-Meiboom-Gill (CPMG) pulses over a period of less than one minute.

38. The method of any one of claims 23-37, wherein a linear relationship (r2> 0.9999) exists between the R2 value (the reciprocal of T2) and the concentration of the paramagnetic metal ion.

39. The method of claim 23, wherein the MRR device comprises: a radio-frequency spectrometer comprising at least one field programmable gate array chip; a power amplifier electrically connected with the radio-frequency spectrometer and amplifying an electrical output of the radio -frequency spectrometer, thereby producing an amplified electrical signal comprising between about 0.1 Watts and about 10 Watts power; a duplexer configured to isolate the radio-frequency spectrometer from the amplified electrical signal during a receiving mode of the device wherein the duplexer comprises a passive duplexer without a quarter wavelength transmission cable; a radio-frequency detection probe configured to transmit radiofrequency electromagnetic radiation to excite nuclei under resonance during a transmission mode of the device, the radio-frequency detection probe comprising a detection microcoil comprising an inner diameter of less than about 1 millimeter; and at least one magnet supplying an external magnetic field to a detection region of the radio-frequency detection probe, the external magnetic field being less than about 3 Tesla.

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