WO2023064633A1 - Diamond magnetometry detection of biological targets - Google Patents

Abstract

A composition includes a fluorescent nitrogen-vacancy nanodiamond conjugated to a probe. The probe specifically binds to a target material when present in a biological sample. A device includes a flow cell containing a plurality of fluorescent nitrogen-vacancy nanodiamonds immobilized on a surface of the flow cell. Each of the plurality of fluorescent nitrogen-vacancy nanodiamonds is conjugated to a probe. A diamond magnetometry system includes an ODMR measurement system, a spin-lattice relaxation time (T1) system, a spin-spin relaxation time (T2) measurement system, and a flow cell.

Description

DIAMOND MAGNETOMETRY DETECTION OF BIOLOGICAL TARGETS

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63/256,093 filed October 15, 2021, which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present disclosure is directed to compositions, devices, and systems that employ diamond magnetometry to provide improved detection of specific target materials in samples. More specifically, the present disclosure is directed to compositions, devices, and systems that detect target biological materials in biological samples.

BACKGROUND

[0003] Specific target materials in biological samples can help diagnose diseases by detection of their presence, absence, or concentration. These target materials include, but are not limited to, nucleic acids (including oligonucleotides), proteins (including oligopeptides), electrolytes, and other substances that result from, or are increased or depleted due to, disease.

[0004] Detection and measurement of target materials can also track patient response to treatment, and facilitate high throughput screening to evaluate efficacy of potential treatments for such diseases by measuring changes in levels of the target.

[0005] Due to generally having the lowest time and sample size requirements, the most widely employed methods for detection of nucleotide target materials, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are polymerase chain reaction (PCR)-based methods, such as, for example, beads, emulsion, amplification, including, for example, magnetics (BEAMing) and droplet digital PCR (ddPCR), which have limits of detection of 0.01 and 0.001%, respectively.

[0006] PCR amplifies pieces of nucleotide target materials in a sample, for example, DNA, by several orders of magnitude to improve detection. A PCR procedure typically requires about 25 to 40 temperature cycles, with three 2-minute steps required per cycle, for a total of about 2.5 to 4 hours.

[0007] For detection of specific proteins, the most widely used tests are spectrometry-based and antibody -based methods. These methods require several hours to complete and require equipment that is not considered portable.

[0008] Generally, the existing technologies are relatively slow, creating practical timing challenges to rapidly diagnosing a disease state and to rapidly and efficiently screening for treatment response. Compositions, devices, and systems not suffering from these drawbacks are desirable in the art.

SUMMARY

[0009] In the case of in vitro diagnostics, there is a need for affordable point-of-care (POC) target detection that provides quicker test results with sensitivity that is equal to, or better than, that currently attainable from PCR, spectrometry, antibody-dependent assays (immunoassays), and other methods currently in use. In addition, there is a need for methods/technologies for rapid, high-throughput screening of drugs/genetic therapies for diseases that are detected by the above methods, as well as the means by which to better define therapeutic windows for such treatments.

[0010] In accordance with various embodiments, provided are compositions/reagents, systems, devices and methods for detecting a target material in a sample. In some exemplary embodiments, the sample is a biological sample taken from an organism, cell culture or other material that may include biological material from an organism. In some embodiments, the target material is a biomaterial, such as a polynucleotide (double or single stranded), a polypeptide (a protein or oligopeptides) or a combination thereof. In various embodiments, one or more reagents are used to detect the binding (typically non covenant) between the target material in a sample and a probe that has affinity for the target material wherein the systems include probes that are bound with one or both FNDs and MPs such that binding between the target and the probe results in a change of relative distance between the FND and MP as bound to the probe to produce a detectable change according to the interrogation and detection methods disclosed herein.

[0011] In exemplary embodiments, diamond magnetometry detection of target DNA, RNA, proteins, and/or other specific target materials in samples, including but not limited to biological samples, can reduce or eliminate the need for DNA amplification, immunoassays, and spectrometric identification, thereby reducing the time required for target detection from hours to minutes. In exemplary embodiments, diamond magnetometry provides sensitivity equal to that of the alternative methods at a lower cost. A composition according to the instant disclosure includes a fluorescent nitrogen-vacancy nanodiamond conjugated to at least one probe that includes at least one magnetic particle and one or more features specific for recognizing and binding to a target material in a test sample. A device may include a flow cell containing a plurality of fluorescent nitrogen-vacancy nanodiamonds immobilized on a surface of the flow cell. Each of the plurality of fluorescent nitrogen-vacancy nanodiamonds is conjugated to a probe. The probe specifically binds to a target material when present in a biological sample. A diamond magnetometry system includes one or more of an optically detected magnetic resonance (ODMR) measurement system, a spin-lattice relaxation time (Tl) system, a spin-spin relaxation time (T2) measurement system, and a flow cell containing a plurality of fluorescent nitrogen-vacancy nanodiamonds immobilized on a surface of the flow cell. Each of the plurality of fluorescent nitrogen -vacancy nanodiamonds is conjugated to a probe that is also bound to a magnetic nanoparticle. The probe specifically binds to a target material when present in a biological sample and a conformational change in the probe results in a change in the distance between the probe-bound fluorescent nitrogen-vacancy nanodiamonds and the probe-bound magnetic nanoparticle that is detectible evidencing the presence of the target material in the sample.

[0012] In a first embodiment, provided is a target detection system, comprising: at least a first reagent comprising a plurality of fluorescent nitrogen-vacancy nanodiamond-probe-magnetic nanoparticle conjugates (MND-probe conjugates), each MND-probe conjugate comprising at least one fluorescent nitrogen-vacancy nanodiamond (FND); at least one magnetic nanoparticle (MP); and at least one polymeric probe comprising a biomolecule selected from the group consisting of a polynucleotide, a polypeptide, or a combination thereof, wherein the FND portion of the MND- probe conjugate is chemically linked to the polymeric probe, and wherein the MP portion of the and the MP portion of the MND-probe conjugate is chemically linked to the polymeric probe.

[0013] In some particular embodiments, each of the plurality of MND-probe conjugates includes one FND, one MP and one polymeric probe.

[0014] In some particular embodiments, each the polymeric probe is characterized as having binding specificity to a target material, and wherein a distance between the FND and the MP in the MND-probe conjugate changes when the at least one polymeric probe contacts and binds to a target material to which the polymeric probe has binding specificity.

[0015] In some particular embodiments, each the binding specificity is not characterized by covalent bonding.

[0016] In some particular embodiments, each the at least one FND and the at least one MP comprises a surface comprising a chemical functional group linker that chemically links it to the at least one polymeric probe, the chemical functional group linker selected from carboxyl moieties, amine moieties, alcohol moieties, or combinations thereof.

[0017] In some particular embodiments, each the at least one FND is coated with a plurality of chemical functional group linkers comprising glycidol, and wherein the at least one MP is coated with a plurality of functional group linkers comprising a carboxyl moiety.

[0018] In some particular embodiments, each the at least one MP has a particle size in the range of about 10 nm to about 100 nm, and comprises a structure selected from the group consisting an iron oxide nanoparticle, a maghemite nanoparticle, a magnetite nanoparticle, a diamagnetic nanoparticle, a supermagnetic nanoparticle, a ferromagnetic nanoparticle, a ferrimagnetic nanoparticle, a quantum dot, an upconverting nanoparticle, a ferritin nanoparticle, a ferritin-like protein, a heme-containing protein, an oligonucleotide-containing magnetic nanoparticle, and a core-shell nanoparticle comprising a magnetic core and an outer shell of a component selected from the group consisting of silane, polysaccharide, gold, polymer, and dendrimer, and

[0019] wherein the at least one FND has a particle size in the range of about 20 nm to about 100 nm, and includes 1 to about 100 nitrogen-vacancy centers, and wherein the nitrogen -vacancy centers are planted about 5 to about 15 nm below the surface of the fluorescent nitrogen-vacancy nanodiamond.

[0020] In some particular embodiments, each the polymeric probe is selected from the group consisting of an oligonucleotide having a sequence configured to form a stem-loop structure, a polypeptide that comprises an antibody, or a polypeptide that comprises a protein subunit selected from the group consisting of an intein, an extein, and a combination thereof.

[0021] In another embodiment, provided is a target detection system, comprising: a first reagent comprising a plurality of fluorescent nitrogen-vacancy nanodiamond-probe conjugates (FND- probe conjugates), each FND-probe conjugate comprising at least one fluorescent nitrogenvacancy nanodiamond (FND); and at least one polymeric probe comprising a biomolecule selected from the group consisting of a polynucleotide, a polypeptide, or a combination thereof; and a second reagent comprising a plurality of magnetic nanoparticle-probe conjugates (MP-probe conjugates), each MP-probe conjugate comprising at least one magnetic nanoparticle (MP); and at least one polymeric probe comprising a biomolecule selected from the group consisting of a polynucleotide, a polypeptide, or a combination thereof, wherein the FND portion of the FND- probe conjugate is chemically linked to the polymeric probe, and wherein the MP portion of the MP-probe conjugate is chemically linked to the polymeric probe.

[0022] In some particular embodiments, the polymeric probe is characterized as having binding specificity to a target material wherein the FND-probe conjugate and the MP-probe conjugate each bind to the same target material, and wherein a distance between the FND and the MP in the MND- probe conjugate changes when the at least one polymeric probe in each of the FND-probe conjugate and the MP -probe conjugate contacts and binds to the target material to which the polymeric probe has binding specificity.

[0023] In some particular embodiments, the binding specificity is not characterized by covalent bonding.

[0024] In some particular embodiments, each of the plurality of FND-probe conjugates includes one FND and one polymeric probe, and each of the plurality of MP-probe conjugates includes one MP and one polymeric probe.

[0025] In some particular embodiments, the at least one FND and the at least one MP comprises a surface comprising a chemical functional group linker that chemically links it to the at least one polymeric probe, the chemical functional group linker selected from carboxyl moieties, amine moieties, alcohol moieties, or combinations thereof.

[0026] In some particular embodiments, the at least one FND is coated with a plurality of chemical functional group linkers comprising glycidol, and wherein the at least one MP is coated with a plurality of functional group linkers comprising a carboxyl moiety.

[0027] In some particular embodiments, the at least one MP has a particle size in the range of about 10 nm to about 100 nm, and comprises a structure selected from the group consisting an iron oxide nanoparticle, a maghemite nanoparticle, a magnetite nanoparticle, a diamagnetic nanoparticle, a supermagnetic nanoparticle, a ferromagnetic nanoparticle, a ferrimagnetic nanoparticle, a quantum dot, an upconverting nanoparticle, a ferritin nanoparticle, a ferritin-like protein, a heme-containing protein, an oligonucleotide-containing magnetic nanoparticle, and a core-shell nanoparticle comprising a magnetic core and an outer shell of a component selected from the group consisting of silane, polysaccharide, gold, polymer, and dendrimer, and

[0028] wherein the at least one FND has a particle size in the range of about 20 nm to about 100 nm, and includes 1 to about 100 nitrogen-vacancy centers, and wherein the nitrogen -vacancy centers are planted about 5 to about 15 nm below the surface of the fluorescent nitrogen-vacancy nanodiamond.

[0029] In some particular embodiments, the polymeric probe is selected from the group consisting of an oligonucleotide having a sequence configured to form a stem-loop structure, a polypeptide that comprises an antibody, or a polypeptide that comprises a protein subunit selected from the group consisting of an intein, an extein, and a combination thereof.

[0030] In some particular embodiments, a distance between the FND and the MP in the changes when the at least one polymeric probe contacts and binds to a target material to which the polymeric probe has binding specificity.

[0031] In another embodiment, provided is a process for detecting the presence of a target material in a sample, the system comprising: providing a target detection system according to the foregoing; providing an interrogation system capable of detecting at least one measurable change relating to the displacement of an MP toward or away from a FND; introducing the selected reagent into the detection system in contact with a sample suspected of containing the target material; and interrogating the reagent-sample combination to detect a measurable change.

[0032] In some particular embodiments, the process includes providing a flow cell and fluid medium for receiving the sample comprising a biological sample from one of cell, tissue or a combination thereof, wherein one or more reagents of the target detection system are immobilized on a surface of the flow cell and the flow cell is configured to flow the sample over the immobilized reagents.

[0033] In some particular embodiments, the interrogation system comprising optics configured to generate and measure one or more of optically detected magnetic resonance (ODMR), a spinlattice relaxation time (Tl), a spin-spin relaxation time (T2) of the plurality of magnetic nanoparticles, or a combination thereof.

[0034] In some particular embodiments, measuring an optically detected magnetic resonance (ODMR) of the plurality of fluorescent nitrogen-vacancy nanodiamonds after exposure to the biological sample determines a presence or an absence of the target material in the biological sample based on the ODMR.

[0035] In some particular embodiments, measuring a spin-lattice relaxation time (Tl) or a spinspin relaxation time (T2) of the plurality of fluorescent nitrogen-vacancy nanodiamonds after exposure to the biological sample determines a presence or an absence of the target material in the biological sample based on values of Tl or T2.

[0036] In some particular embodiments, the process is used in a biomedical application.

[0037] In another embodiment, provided is reagent for detection of target materials, the reagent comprising: a plurality of fluorescent NV-center nanodiamonds (FNDs), each of the plurality of FNDs having chemically bound to its surface at least one polymeric probe, the at least one polymeric probe comprising an oligonucleotide comprising a stem loop structure and a chemically bound magnetic particle.

[0038] In some particular embodiments, the oligonucleotide comprising a stem loop structure is single stranded DNA, and wherein hybridization of a target polynucleotide to a portion of a loop region of the stem loop structure causes the loop to open, thereby linearizing the stem-loop structure and moving the MP away from the FND whereby there is a change in a magnetic field property of the FND causing a change that is detectable by interrogation according to one or more of optically detected magnetic resonance (ODMR), a spin-lattice relaxation time (Tl), a spin-spin relaxation time (T2) of the plurality of magnetic nanoparticles, or a combination thereof.

[0039] Other features and advantages of the present invention will be apparent from the following more detailed description of the preferred embodiment, taken in conjunction with the accompanying drawings which illustrate, by way of example, the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1 schematically shows optics for ODMR of a diamond magnetometry device in an embodiment of the present disclosure. [0041] FIG. 2 schematically shows a flow cell for the diamond magnetometry device of FIG. 1.

[0042] FIG. 3 schematically shows expected outcomes for ODMR in the diamond magnetometry device of FIG. 1.

[0043] FIG. 4 schematically shows expected outcomes for spin-lattice relaxation time (Tl) and spin-spin relaxation time (T2) in a diamond magnetometry device.

[0044] FIG. 5 A schematically shows a magnetic particle (MP) held near a nitrogen-vacancy (NV) diamond surface by attaching either an NV diamond or MP to the intein and the other to the extein.

[0045] FIG. 5B schematically shows an NV diamond attached to one extein bound to an intein fragment and the MP to the other.

[0046] FIG. 5C schematically shows detection in an intein/extein system via cis-splicing.

[0047] FIG. 6A schematically shows antibody-antigen-antibody binding to bring the NV and MP together.

[0048] FIG. 6B schematically shows detecting antibody-secondary antibody binding to bring the NV and MP together.

[0049] FIG. 7 schematically shows a process for forming a fluorescent nitrogen-vacancy nanodiamond (FND)-probe-magnetic nanoparticle (MP) conjugate, also referred to as a “MND- probe conjugate” comprising and least one FND and at least one MP.

[0050] FIG. 8A shows ODMR of a thiolated FND.

[0051] FIG. 8B shows ODMR of an MND-probe conjugate.

[0052] FIG. 9A shows a streptavidin (SA)-biotin binding system.

[0053] FIG. 9B shows a fluorescence heat map of the FNDs for the binding system of FIG. 9 A.

[0054] FIG. 9C shows the ODMR spectra for the binding system of FIG. 9 A. [0055] FIG. 10 shows ODMR of an FND-DNA strand conjugate and an DNA-MP conjugate.

[0056] FIG. 11 schematically shows a process for forming an MND-probe conjugate stem-loop nucleic acid conjugate.

[0057] FIG. 12 shows ODMR of an MND-probe conjugate stem-loop nucleic acid conjugate in the presence and in the absence of a target material.

[0058] Wherever possible, the same reference numbers will be used throughout the drawings to represent the same parts.

DETAILED DESCRIPTION

[0059] Provided are compositions, processes, and devices for detection of target materials in biological samples.

[0060] Embodiments of the present disclosure, for example, in comparison to concepts failing to include one or more of the features disclosed herein, provide decreased detection time requirements, lower cost, equivalent or lower limits of detection, or combinations thereof.

[0061] A magnetometer device senses a magnetic field, measuring its strength and direction. A diamond magnetometry device can measure Tl, T2, and ODMR of fluorescent nitrogen-vacancy (NV) diamonds, and measure nanoscale magnetic fields, such as from magnetic particles, magnetic nanoparticles, or other magnetic materials close to the NV diamonds.

[0062] FIG. 1 schematically shows optical components for ODMR and Tl measurements in a diamond magnetometry device. Green (532 nm) laser light 1 passes through an acousto-optic modulator (AOM) 2 and then through an iris 3, a dichroic mirror 4, a galvo scanner 5, and various lenses 6 and mirrors 7 before being focused on an objective 8, such as, for example, a lOOx objective. To achieve a Gaussian laser beam, a spatial filter is placed at the beginning of the beam path. In short, the laser coming directly from the head is focused to a point with a 20-cm lens, and a 100-pm pinhole is located at the focus point followed by the iris 3. The iris 3 is closed down to block all but the central diffracted spot of the beam, which is then collimated by a second 20-cm lens. This results in a much more uniform beam going to the objective and a more stable reading from the sample.

[0063] The beam excites NV nanodiamonds that are affixed to a glass coverslip bonded to the top of a flow cell 11 that is mounted on a microscope stage. The flow cell 11 is positioned close to a thin microwave (MW) wire. The resulting emission fluorescence passes through the same set of mirrors and lenses and the emission fluorescence is filtered from the excitation laser by a 560-nm longpass (LP) filter 12 and passes through a 75-pm pinhole 13 to isolate the focal plane. The fluorescence is then split by a beam splitter 14 and sent either to a charge-coupled device (CCD) camera 15 for spectral analysis or to a photon counter 16. The image is processed by a computer 17 to produce the ODMR spectrum at a selectable region of interest. The computer also controls the MW source 18. The MW power amplifier 19 supplies about 1 Watt through a thin wire placed near the diamonds 10 and is analyzed by a MW analyzer 20.

[0064] The optical system for detecting and monitoring changes in NV-center Tl, T2, and ODMR is built on an optical table with vibration isolation supports. It consists of a few major components which can be re-positioned and replaced with other components depending on whether Tl, T2, or ODMR are being measured. A data acquisition card is used to record the experimental data. Tl and T2 measurements do not require the microwave components.

[0065] Referring to FIG. 2, the flow cell 11 includes a chamber body 21 with a sample inlet 22 leading to a hollow test chamber 23. A ridge 24 in the test chamber 23 formed into the chamber body 21 forces a sample fluid into close proximity with the cover slip 25 on the center of which an array of microdiamonds 26 is affixed. A depression 27 surrounding the test chamber 23 contains a gasket, which confines the sample fluid to the test chamber 23. A heating pad 28 applies heat, as needed, to increase reaction rates. The sample fluid is injected, such as, for example, by a sample syringe pump 29, into a mixing chamber 30, where it mixes with buffer also injected, such as, for example, by a buffer syringe pump 31, into the mixing chamber before flowing through the sample inlet 22 and into the test chamber 23. The buffer may be any appropriate pH buffer solution, such as, for example, phosphate-buffered saline (PBS) of a pH of about 7.5. [0066] In some embodiments, a fluorescent nitrogen-vacancy nanodiamond (FND) has a size in the range of about 20 nm to about 100 nm, alternatively in the range of about 20 nm to about 80 nm, alternatively in the range of about 30 nm to about 60 nm, alternatively in the range of about 40 nm to about 50 nm, or any value, range, or sub-range therebetween.

[0067] In some embodiments, the FND has 1 to about 100 nitrogen-vacancy centers planted at a depth of about 5 to about 15 nm below the surface of the FND. The nitrogen-vacancy (NV) color center of fluorescent diamond, an impurity of a nitrogen atom adjacent to a vacant lattice site, is paramagnetic, making it responsive to other magnetic species. In exemplary embodiments, the surface of the FND is free or substantially free of scavengers of diamond fluorescence. In exemplary embodiments, the surface of the FND is modified to include functional groups that permit coupling of probes to the FNDs, including nucleic acids or amino acids or combinations thereof to the surface. In some embodiments, the surface chemistry includes amines, triple bonding moieties such as for example propargyl group, or azides for click chemistry.

[0068] Excitation of an NV-center with green light results in a broadband photoluminescence emission with a zero phonon line (ZPL) at 575 nm and 637 nm with longer wavelength emissions extending into the infrared region. Diamonds containing NV-centers exhibit very long spin coherence times, making them extremely sensitive to magnetic fields, and this can be followed optically via ODMR and T1 and T2. Diamond magnetometry encompasses the Tl, T2, and ODMR of NV diamond and can detect minute changes in magnetic field.

[0069] The NV center has two charge states: neutral, NV° and negative, NV, of which the negative charge state is relevant for magnetometry. The negative charge state (NV) forms a spin triplet ground state having three spin sublevels (m_s = -1, 0, or +1). Excitation with green light optically polarizes the NV center into the m_s =0 sublevel (bright state) which scatters about 30% more photons than the m_s = ±1 states. When a resonant microwave field induces magnetic dipole transitions between these electronic spin sublevels, it disrupts the optically pumped spin polarization, resulting in a significant decrease of the nitrogen-vacancy center fluorescence, as shown in FIG. 3 at Bo), which is the ODMR. Due to symmetry, the m_s = ±1 sublevels of the nitrogen-vacancy defect are degenerate at zero magnetic field (B= 0), resulting in a single resonance line appearing in the ODMR spectrum at Bo and at 2,870 MHz, as shown in FIG. 3. If an external magnetic field is encountered, from a magnetic particle for instance, it lifts the degeneracy of m_s = ±1, leading to the appearance of two lines from which the external magnetic field can be measured by the distance between the two lines. As the magnetic field decreases, by increasing distance between the diamond and the MP for instance, the separation between the ODMR lines decreases. The splitting of the ODMR spectrum into separate components by a static magnetic field is called Zeeman splitting. In general, micron-sized NV-center diamonds can detect magnetic fields below 1 nT.

[0070] T1 and T2 of NV-centers are also sensitive to fluctuating magnetic fields, as shown in FIG. 4. T1 represents the decay lifetime for a population of excited NV-centers to return to the groundstate, and T2 represents the cumulative loss of at least 63% of original phase coherence. Unlike ODMR, T1 and T2 relaxation can be measured without the need for microwave excitation, which makes relaxometry faster to perform.

[0071] Briefly, a T1 relaxometry study involves applying a pulse probe sequence, as shown in FIG. 4, in which the NV-center diamond is given a millisecond laser pulse (532 nm) to preferentially populate the m_s = 0 sublevel, as described above. The laser is then shut off, and during this dark time (T), the system relaxes back towards the equilibrium condition. After a specified dark time, the diamonds are given a short, microsecond laser probe and the population remaining in the m_s = 0 sublevel is read out by monitoring the intensity of the emitted red light.

[0072] A T2 relaxation study is performed similarly, but the cumulative loss of fluorescence is monitored, rather than recovery of the equilibrium condition. FIG. 3 shows a typical relaxation curve for an NV-center diamond in the absence of a magnetic field. In the presence of strong magnetic noise, such as paramagnetic gadolinium ion (Gd⁺³), relaxation time becomes much shorter. This is because the magnetic noise from Gd⁺³ interferes with the optically pumped spin polarization, which resets the equilibrium condition of T1 and the net loss of spin coherence of T2.

[0073] In exemplary embodiments, diamond magnetometry detects specific materials in biological samples. In exemplary embodiments, the distance between NV diamonds and magnetic particles is controlled, and the response of the ODMR, Tl, and T2 NV diamonds is measured as the distance is changed. Examples 1 and 2 and FIG. 8A and FIG. 8B show how distance between and NV diamond and magnetic particles affects ODMR, Tl, and T2.

[0074] In exemplary embodiments, the magnetic particles are magnetic nanoparticles having a size in the range of about 10 nm to about 100 nm, alternatively in the range of about 10 nm to about 80 nm, alternatively in the range of about 20 nm to about 60 nm, alternatively in the range of about 30 nm to about 50 nm, or any value, range, or sub-range therebetween.

[0075] Appropriate materials for the magnetic nanoparticles may include, but are not limited to, iron oxide, maghemite, magnetite, a diamagnetic material, a supermagnetic material, a ferromagnetic material, a ferrimagnetic material, a quantum dot, an upconverting material, ferritin, a ferritin-like protein, a heme-containing protein, an oligonucleotide-containing magnetic material, and a core-shell including a magnetic core and an outer shell of a component such as silane, polysaccharide, gold, polymer, or dendrimer.

[0076] In exemplary embodiments, a diamond magnetometry system measures magnetic properties of fluorescent nitrogen-vacancy (NV) nanodiamonds conjugated with a probe for a targeted material that is exposed to a biological sample to determine the presence, identity, and/or amount of the target material in the sample.

[0077] The NV center of the diamond includes a point defect in the diamond crystal lattice that emits photostable red fluorescence following excitation with green light. The ODMR of an NV diamond, which is the decrease of fluorescence in the presence of a resonant microwave field, exhibits measurable variation based upon the proximity and strength of a magnetic field. The Tl and T2 of NV diamonds are also measurably responsive to the presence of, strength of, and proximity to, magnetic fields.

[0078] In exemplary embodiments, a composition includes NV microdiamonds with tethered probes that can control the distance of MPs from their surfaces. [0079] In some embodiments, the presence of a target material alters the conformation of the probe, changing the distance between the MP and NV diamond, such as, for example, moving them further from each other.

[0080] In some embodiments, the presence of a target material activates the probe’s attachment to, or release from, other materials that are connected to the MP, changing the distance between the MP and NV diamond, such as, for example, moving them either closer to or further from each other.

[0081] In some embodiments, the changes in Tl, T2, and ODMR of the NV diamond that results from the change in distance from the MP indicate the presence of a target of interest in a sample.

[0082] To control the distance between the NV diamonds and MP, a probe is attached to the NV diamond surface that can capture and hold the MP near the NV diamond surface, move the MP a predetermined distance from the surface, or release the MP from the NV diamond completely. In some embodiments, the probe includes a nucleic acid or an amino acid. In some embodiments, the nucleic acid includes a stem-loop structure. In some embodiments, the nucleic acid includes an aptamer. In some embodiments, the amino acid is part of an intein-extein system. In some embodiments, the amino acid is part of an antibody.

[0083] In some embodiments, the probe includes a nucleic acid conjugated to the fluorescent nanodiamonds. In some embodiments, the probe nucleic acid is a single stranded oligonucleotide up to 50 bases in length. In other embodiments, larger DNA fragments may be used. In some embodiments, the conjugation is accomplished with amines. In some embodiments, the conjugation is accomplished with azides. In other embodiments, other functional groups may be used to accomplish the conjugation, depending on the availability of functional groups from nucleic acid manufacturers. In some embodiments, the conjugation is at the 5’ and/or the 3’ end of the oligonucleotide. In other embodiments, the conjugation may be through a functional group on an internal base of an oligonucleotide. In other embodiments, other functional groups and chemistries may be used. The oligonucleotides may be DNA oligonucleotides, RNA oligonucleotides, or other oligonucleotides of DNA and/or RNA containing modified bases. Appropriate modified bases may include, but are not limited to, 2’-o-methoxyethyl bases (2’- MOE), which are used for antisense oligos (ASO), aptamers, and small interfering RNA (siRNA); 2'-o-methyl RNA bases; 2-aminopurine; 5-bromo deoxyuridine; deoxyuridine; 2,6-diaminopurine; dideoxy cytidine; deoxyinosine; 5-methyl deoxy cytidine; 5-nitroindole; 5-hydroxybutynl-2’- deoxyuridine; or 8-aza-7-deazaguanosine.

[0084] In one embodiment, the method for controlling distance includes linking fluorescent NV- center nanodiamonds (NV diamonds) to magnetic particles, magnetic nanoparticles, or other magnetic materials (MPs) through single-stranded DNA having a stem-loop structure, as shown schematically in FIG. 11 and described in more detail in Example 4. The stem-loop includes a single-stranded loop region that is flanked by self-complementary termini which hybridize to form the stem. One terminus of the stem region is attached to the NV diamond, and the other end is attached to the MP. The stem region of the stem-loop structure positions the MP close to the NV diamond, and thereby disrupts polarization of the NV-center. This produces Zeeman shifts which are observed as at least two dips in the ODMR spectra.

[0085] The DNA in the loop region is complementary to, and will hybridize with, the target DNA being detected. Hybridization of target polynucleotide to the loop region DNA causes the loop to open, thereby linearizing the stem-loop structure and moving the MP away from the nanodiamond surface. Since the distance (r) over which the magnetic field of the MP influences the NV-center is extremely short (1/r³), and the DNA sequence is long, the system returns to a zero magnetic field state and the resulting microwave sweep should produce only the zero-field resonance dip at 2,870 kHz in the presence of the target material.

[0086] In some embodiments, a stem-loop design of a nucleic acid probe is used in detecting nucleic acids target materials in a sample, as shown schematically in FIG. 11. As depicted, the stem-loop is a single-stranded DNA containing complementary DNA sequences at the 5’ and 3’ ends. The ends form a short double strand structure with the single-stranded loop region positioned between the two complementary sequences. Hybridization of a DNA/RNA sequence in the loop region destabilizes the stem structure and thus linearizes the DNA. [0087] In another embodiment, the same flow cell may be used, but the ODMR analysis is done in an optical system that creates a biased magnetic field. The presence of the magnetic field produces a Zeeman shift in both the unhybridized and hybridized states of the DNA probe thereby affecting the distance between the FND and the MP. In this method, the close approach of the magnetic nanoparticle further broadens the Zeeman splitting, by an amount equal to the size of the magnetic nanoparticle field strength.

[0088] The field strength of the MP affects Zeeman splitting and this is influenced by its composition. The MP may be composed of a metal core including FeO, Fe2O3, FesCfi, Co, Fe, Ni, CoFe, NiFe, CoO, NiO, or ferrites including MFe2O3, where M includes either Co or Ni. For biological applications MPs are coated with biocompatible molecules or polymers such that functional groups are available for conjugation. Under most conditions a commercial MP can be used, consisting of Fe3O4 and having carboxyl groups on the surface. Another magnetic entity that could also be used is the gadolinium +3 ion.

[0089] One method of creating a stem-loop DNA for quantum-based sensing is illustrated in FIG. 11. The alcohol groups on glycidol-coated nanodiamonds are converted into NHS esters using DSC. These are then reacted with an azido-PEGl l -amine (Broadpharm, San Diego, CA). The result of this procedure is the creation of a FND with azide groups on the surface. This group can be confirmed by FTIR. A stem-loop DNA sequence may be designed and tested for stable structures using the manufacturer’s software (Integrated DNA Technologies). The single-strand DNA may be designed with a propargyl group at the 5’ ends and an amine group at the 3’ end. The DNA is validated by mass spectrometry. The surface functionalized FNDs are contacted with the 5’ propargyl modified DNA strand to accomplish conjugation and form the DNA-FND conjugate.

[0090] In some embodiments, the nucleic acid of the probe is part of an aptamer. DNA aptamers are short single-stranded stretches of nucleic acids that form unique hairpin-like DNA structures that selectively bind specific protein target materials. Thus, they behave as antibodies. A feature of aptamers that can be exploited for ODMR is that they undergo conformational changes upon binding a target protein. The binding event translates into changes in spatial distance between the NV-center diamond and a magnetic nanoparticle, which affects ODMR spectra.

[0091] For example, cortisol is a glucocorticoid hormone produced in the adrenal gland and secreted as part of the flight or fight impulse in response to fear or stress but has other roles in mental health (stress and depression) and emotional events. In situations of chronic stress and disrupted sleep-wake cycles, the adrenal glands secrete an abnormal amount of cortisol in an abnormal rhythm. Monitoring cortisol levels is a useful measure of normal or abnormal conditions.

[0092] The DNA sequence (38-mer) for the cortisol aptamer is 5’- GCCCGCATGTTCCATGGATAGTCTTGACTAGTCGTCCC- -3’ and has a limit of detection of less than 1 ng/mL. Importantly, this aptamer sequence undergoes a significant conformational change upon binding cortisol, which makes it useful for ODMR. This aptamer is available as an oligonucleotide with an azide group on the 5’ end and a terminal amine group on the 3’ end. The same stem-loop approach described above may be used here, meaning that binding of the ligand, (cortisol) causes a conformational change in the DNA, which produces positional changes in the MP-FND spatial relationship in the MND-probe conjugate.

[0093] In some embodiments, a nanodiamond is spotted on the surface of a glass coverslip, which is then mounted on a flow cell. The flow cell is positioned over the microscope objective and the nanodiamonds are excited with a green laser. ODMR spectra are recorded as fluid is pumped across the flow cell. A test sample is then passed across the flow cell, where it interacts with the nanodiamond. A positive detection, representing capture of the cortisol molecule, is either a change in the magnitude of the Zeeman shift in a biased magnetic field or a change in the contrast of the resonance peaks in a non-biased magnetic field. The cortisol may be then stripped from the aptamer-FND conjugate using a series of laser pulses to heat the DNA such that it denatures and releases the cortisol molecule. The aptamer may be renatured to its original conformation by slowly cooling the aptamer conjugate.

[0094] In other embodiments, the aptamer is a split aptamer. In such embodiments, one of the ND and the MP may be attached to one half of the split aptamer with the other of the ND and the MP being attached to the other half of the split aptamer. Hybridization of the split aptamer indicating the presence of a target material brings the ND and MP close together as determined by T1 and T2 and/or ODMR measurements.

[0095] In some embodiments, the probe includes an amino acid conjugated to the fluorescent nanodiamonds. In some embodiments, the amino acid includes an intein or an extein. In some embodiments, the amino acid includes an antibody or an active portion of an antibody.

[0096] In some embodiments, the method of controlling distance between NV diamond and MP is their attachment to inteins, with their proximity being controlled using protein cis- or transsplicing. An intein is an internal protein segment removed from between two exteins, an N-extein and a C-extein, during protein splicing.

[0097] In protein trans splicing (PTS), a intein has been split into two fragments and reassociation of the fragments is required prior to splicing of the protein. Trans-splicing inteins have been engineered to undergo conditional protein splicing, or CPS. CPS requires the addition of a trigger to initiate splicing of a precursor fusion protein. For CPS of trans-splicing inteins, reassociation can be conditional on presence of a small molecule, such as a target material. FIG. 5 A, FIG. 5B, and FIG. 5C show some options for intein-extein probes.

[0098] Referring to FIG. 5 A, an MP 52 is held near an NV diamond 50 surface by attaching either an NV diamond or MP to the intein 42, 44 and the other to the extein 40, 46. When the target material 58 is encountered, it is bound by the receptors 54, 56, and the intein 42, 44 is excised from the extein 40, 46, and the MP 52 is removed from the NV diamond 50 surface. The resulting changes in the NV diamond's Tl, T2, and ODMR measurements indicate the presence of the target material 58 in a sample.

[0099] Referring to FIG. 5B, an NV diamond 50 is attached to one extein 40 bound to an intein fragment 42 and the MP 52 to the other extein 46 in another configuration using PTS. Upon binding of the target material 58 by the receptors 54, 56, and subsequent reassociation of intein fragments 42, 44, resulting in excision of the intein 40,46, the MP 52 is held near the NV diamond 50 surface, again affecting the Tl, T2, and ODMR of the NV diamond 50. [00100] Referring to FIG. 5C, detection via cis-splicing is similar to that of trans-splicing, however the intein 48 is intact instead of split. In this case, the target 58 binding to the receptor 54 causes excision of the MP -bound intein 48 that is positioned between two exteins 40, 46, again removing the MP 52 from the NV diamond 50 surface.

[00101] In some embodiments, the probe includes an antibody. In such embodiments, the method of controlling distance between NV diamond and MP is by their attachment to antibodies and the selective binding between antibodies and their antigens.

[00102] Referring to FIG. 6A, the probe on the plated-coated NV diamond 50 includes an antibody 60. The coated plate is then exposed to a biological sample. If the target material 58, antigen in this case, is present, it binds to the antibody 60. Antibody-bound MP is then added to the system and binding between the antibody 64 on the MP 52 and the antigen 58 bound to the antibody 60 on the NV diamond 50 brings the NV diamond 50 and MP 52 into close proximity for detection by Tl, T2, and/or ODMR.

[00103] Referring to FIG. 6B, a plate is coated with a capture antibody 70. The coated plate is then exposed to a biological sample. If the target material 58, antigen in this case, is present, it binds to the capture antibody 70. NV diamond 50 with a detecting antibody 72 probe is then added, and the detecting antibody 72 binds to the antigen 58, if present. Finally, MP 52 with a bound secondary antibody 74 is added, and the secondary antibody 74 binds to the detecting antibody 72, if antigen 58 is present, to bring NV diamond 50 and MP 52 into close proximity for detection by Tl, T2, and/or ODMR.

[00104] Detection of a target material in a biological sample by the compositions, devices, and systems disclosed herein may have many different applications. In some embodiments, the compositions, devices, and systems described herein are used for monitoring in a biomedical application. Such applications may include, but are not limited to, the detection and measurement of health-related biological indicators in body fluids, such as, for example, for cancer diagnosis or cardiovascular applications, monitoring for presence of environmental pathogens and chemicals, or monitoring of medical treatment efficacy or decontamination effectiveness. [00105] Such monitoring of human performance may include detection of stress indicators, such as, for example, cortisol, lactate, or urea in sweat. Monitoring may additionally or alternatively include detection of other health indicators, such as, for example, protein biomarkers, DNA, RNA, or microRNA that would be evidence of health concerns, such as, for example, pathogen exposure, stress, adverse cardiac events, or diseases such as cancer or diabetes. Some embodiments include single molecule detection to identify indicators that are present in minute quantities, making early detection and treatment possible. Indicators may be identified in body fluids, such as, for example, blood, sweat, saliva, or urine.

[00106] In some embodiments, monitoring of medical treatment efficacy includes measuring indicator levels following treatment, which permits the development of new treatments by tracking indicator levels following sample treatment with various potential therapies. By use of an array of therapy-activated fluorescent diamonds, multiple therapies may be analyzed for efficacy simultaneously.

[00107] Although the compositions, devices, and systems have been described for detection of a target material in a biological sample, the sample may alternatively be a non-biological sample.

[00108] In some embodiments, the presence of environmental concerns, such as, for example, hazardous chemicals and viral and bacterial pathogens, is monitored. This permits detection and monitoring of threats such as bio- or chemical warfare agents, accidental contamination, persistent environmental contamination, and the effectiveness of decontamination processes.

EXAMPLES

[00109] The invention is further described in the context of the following examples which are presented by way of illustration, not of limitation.

Nanodiamond Magnetometry System

[00110] Referring again to the drawings, a layout as schematically shown in FIG. 1 was built on a 6-ft x 4-ft x 12-in thick active-support table (Thorlabs Inc., Newton, NJ). A hanging shelf was installed above the table for the placement of electronic components. The optical design was for a confocal system having a pinhole to eliminate stray light and a galvanometer for scanning in the x-y plane. Additional components of this system included a 200-mW, 532-nm continuous wave diode-pumped solid-state (DPSS) laser representing the excitation source, and a 12-V acoustooptic modulator (AOM) that used sound waves to diffract and shift the frequency of light. The system included one iris and several lenses, mirrors, and filters, including a dichroic mirror and a 532-nm notch filter. The system used a lOOx microscope objective (Mitutoyo Corporation, Kawasaki, Japan), a CCD camera, a photon counter, a microwave generator, a microwave analyzer, and a thin wire placed near the diamonds to create the microwave field. The last component of the optical system was a flow cell containing fluorescent nanodiamonds. The flow cell was fitted with a microwave wire and positioned above a lOOx objective.

[00111] Flow Cell

[00112] As schematically shown in FIG. 2, a flow cell assembly in an optical set-up for a nanodiamond magnetometry system included a polymeric chamber body approximately 3 cm wide, 3 cm long, and 5 mm deep with a sample inlet leading to a hollow test chamber into which the sample/buffer mixture flowed. Encircling the lip of the test chamber, which was flush with the surrounding surface of the flow cell body, was an indentation holding a rubber gasket, or O-ring, which kept the sample fluid confined to the sample chamber. At the edge of the flow cell was a raised lip, which created an indentation for holding a coverslip in place atop the surface of the cell. In the center of the test chamber was a ridge formed into the flow cell body and extending the width of the hollow chamber. The ridge was perpendicular to the flow of sample fluid, with a height that reached approximately 1 mm below the coverslip. This forced the sample fluid into close proximity with the coverslip, on the center of which an array of nanodiamonds was affixed. A heating pad was attached to the bottom of the flow cell and was used to increase the sample temperature for enhanced reaction rates. The sample fluid was prepared prior to injection into the chamber by controlled mixing of the test sample with a biologically relevant buffer such as phosphate buffered saline (PBS) at pH 7.5. This was accomplished by using two separate syringe pumps, one for the sample and the other for the buffer, to inject the liquids into a mixing chamber. After mixing, the sample/buffer mixture flowed through the sample inlet of the flow cell and into the test chamber. [00113] Relaxometry Measurements

[00114] T1 and T2 of NV centers were recorded on glass slides placed above a 10X objective lens of the optical set-up. T1 and T2 relaxation were determined by measuring fluorescence intensity as a function of time following initialization of the NV spin states with a 200-millisecond pulse using a 532-nm laser light source. Pulsed optical excitation from the 532-nm laser was controlled by an acousto-optic modulator (AOM), in combination with a PulseBlaster acquisition card. The photoluminescent emission was filtered by a 560-nm long pass filter in combination with a 650-750 nm bandpass filter and recorded using a single photon counting avalanche photodiode, and the time-correlated photons captured with a multiple-event time digitizer card. Data acquisition was controlled via custom Lab VIEW code.

[00115] ODMR demonstration

[00116] The optical setup was modified to incorporate microwave field generation and magnets. Permanent magnets produced a magnetic field (Bo) of a few hundred gauss at a distance of a few centimeters from their surface and were preferrable over electromagnets. Testing included mixing a sample with buffer and flowing the mixture through a flow cell containing NV diamonds affixed to a glass coverslip. The NV diamonds on the coverslip were positioned over a 100X objective with surrounding magnet and microwave coils. In a typical ODMR measurement, the sample was illuminated with green light and the intensity of the red fluorescence was monitored while slowly sweeping an auxiliary microwave field into resonance. At resonance, a detectable reduction in fluorescence of 8% to 11% of fluorescence intensity was observed.

[00117] For ODMR studies, fluorescence at 637-nm was collected on an avalanche photo diode (APD) as the microwave frequency was swept from 2700 to 3000 MHz. The fluorescence intensity was plotted as a function of MW frequency, giving the ODMR spectrum. Since NV-centers are almost infinitely photostable, ODMR spectra were repetitively collectable without concern about quenching issues. The data was then averaged using statistical software and a zero-field resonance (dip) at 2,870 MHz (at zero magnetic field) was observed. The confocal setup used a single photon avalanche diode (SPAD) counter but alternatively could incorporate a high sensitivity Electron Multiplying CCD (EMCCD) camera, which can probe multiple individual nanodiamonds in parallel.

Example 1

[00118] Referring again to the drawings, FIG. 7 shows a process for forming MND-probe conjugates. Glycerol, reagents, and organic solvents were obtained from MilliporeSigma (St. Louis , MO). Briefly, 20 mg of 40-nm fluorescent nanodiamonds were cleaned by refluxing for three days in a sulfuric acidmitric acid (at a 9: 1 ratio) solution. Twenty mg of cleaned nanodiamonds were resuspended in 2 mL of glycidol and placed in a 150-mL jacketed glass beaker. Approximately 60 mL of glycidol was added and the reactor was heated to 116 ⁰ C for 6 hours with continuous low power sonication (Misonix Sonicator 3000, Cole-Parmer, Vernon Hills, IL). Following cooling, unreacted glycidol was removed by extensive dialysis against 5 L of pure water for three days, with two complete exchanges each day. The nanodiamonds were centrifuged and the pellet suspended in pure water at 1 mg/mL. This coating step introduces reactive alcohol groups for subsequent conjugation steps.

[00119] Approximately one mg of glycidol-coated FND was suspended in a 1 : 1 mixture of dimethylacetamide (DMAC) and tetrahydrofuran (THF), centrifuged, and then resuspended in a mixture containing 0.1 mL of DMAC and 0.9 mL of dimethyl formamide (DMF). The nanodiamonds were activated with 0.2 mmole of N,N'-disuccinimidyl carbonate (DSC). After the final rinse in DMAC/THF solvent, the nanodiamonds were quickly resuspended in 10 mM 4-(2- hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES)-O.Ol %Tween buffer, and 0.1 mL of a 0.1% polylysine solution was added. After shaking for 4 hours at room temperature, the aminated diamonds were rinsed and resuspended in DMAC/THF solvent.

[00120] The polylysine-coated FNDs had a high density of reactive amines on their surface. These were converted to reactive thiol groups by reaction with 2-iminothiolane (Traut’s reagent). The imidoester group of the cyclic imidothioester reagent reacted with the amines to form a stable, charged linkage, having a sulfhydryl group. The product of this reaction, thiolated FNDs, was then reacted with a commercially available, 100-nm magnetic nanoparticle having reactive mal eimide group on the surface. The terminal -SH group of the FND reacted with the maleimide group to create the final product, an MND-probe conjugate.

[00121] Referring again to the drawings, FIG. 8A shows the ODMR spectra for the thiolated FNDs. FIG. 8B shows the ODMR spectra for the MND-probe conjugates. Each graph shows time- averaged spectra for the FNDs under two different applied external fields. In both sets of graphs a Zeeman shift is clearly observed for the FND. The Zeeman shift is much smaller under the weak magnetic field than the strong external magnetic field. However, the MND-probe conjugate shows hardly any resonance peaks and a clear absence in contrast can be seen in both magnetic fields. This demonstrates the change in contrast as well as a shift in the resonance peaks of the ODMR in the presence and absence of magnetic nanoparticles.

Example 2

[00122] Referring again to the drawings, FIG. 9A shows a method of performing ODMR studies where the FND and MPs are attached to different target materials that come together through binding interactions. In this example, the binding interaction is between the biotin/ streptavidin pair in which streptavidin is a protein that binds the small molecule biotin with extremely high affinity. Standard carbodiimide chemistry was used to link these proteins to FNDs and MPs. Streptavidin was conjugated to an NHS-FND to create a SA-FND conjugate. Amino-PEG-biotin (Broadpharm, San Diego, CA) was conjugated to a carboxylated MP using l-Ethyl-3-(3- dimethylaminopropyljcarbodiimide (EDC) to create a biotin-MP.

[00123] Referring again to the drawings, FIG. 9A shows the overall design. Briefly, the SA- FNDs were spotted on an aminated glass coverslip then fitted to a flow cell. Phosphate-buffered saline was flowed across the flow cell using a syringe pump and ODMR spectra were collected. Next the biotin-MP was added to the PBS solution and allowed to flow across the SA-FND. The streptavidin recognized the biotin, thus bringing the MP close to the FND.

[00124] Referring again to the drawings, FIG. 9B shows a fluorescence heat map of the FNDs spotted onto the coverslip. The bright dots represent individual nanodiamonds, which allowed several spots to be chosen to monitor as the biotin-MP was flowed. In FIG. 9B, spot 8 was chosen to be followed. FIG. 9C shows the ODMR spectra for the SA-FNDs before and after the biotinylated-MP was passed across the flow cell. There was significant change in the NV-center contrast upon streptavidin binding the biotin-MP. This represents the expected results for using FNDs to detect the presence of a binding event between two molecules.

Example 3

[00125] DNA-FND conjugates were formed using a 5’ or 3’ amine on the DNA. In these studies, a 50 base single-strand DNA was designed with limited, if any, secondary structure at room temperature. The oligonucleotide included a 6-carbon spacer and a terminal amine at the 5 ’end. A short 17-mer single strand DNA strand complementary to the 50-mer was also purchased with the same amine group at the 5’ end.

[00126] The 50mer DNA strand was conjugated to glycidol coated FNDs via DSC activation as in Example 1. 50 pg of glycidol-coated FND was suspended in a 1 : 1 mixture of DMAC and THF, centrifuged and then resuspended in a mixture containing 0.1 mL of DMAC and 0.9 mL of DMF. The nanodiamonds were activated with 0.2 mmole of DSC by adding 50 mg DSC to the glycidol FND suspension and incubating for 4 hours. After the final rinse in DMAC/THF solvent, the nanodiamonds were quickly resuspended in PBS with 0.05% Tween-20, and 0.2 nanomoles of amine DNA 50mer was added. After shaking for 4 hours at room temperature, the diamonds were rinsed 3x in the same buffer.

[00127] The 17-mer DNA strand, complementary to the 50mer, was conjugated to a commercially available, 100 nm magnetic nanoparticle having a reactive mal eimide group on the surface. The amine group on the cDNA was converted to reactive thiol groups by reaction with Traut’s reagent. The terminal -SH group of the cDNA reacted with the mal eimide group to create the final product, an MP -DNA composite. This was done as a one-pot reaction including 50 pg maleimide MPs in 200 pL PBS with 0.05% tween-20, 0.2 nanomoles of amine cDNA, and 2 nanomoles of Traut’s reagent. After shaking for 2 hours at room temperature, the MPs were magnetically separated and rinsed 3x in the same buffer. [00128] The detection of DNA is an important biomedical application of quantum-based sensing using FNDs. A 50 base DNA oligonucleotide was designed with an amine group at the 5’- end, as shown in FIG. 10. 40-nm FNDs having NHS group were prepared as described above and conjugated to the 5 ’amine of the 50 base oligonucleotide (DNA-FND). A short complementary DNA sequence was coupled at the 5’ end to a 100-nm MP (DNA-MP). The DNA sequences are shown in the bottom panel of FIG. 10.

[00129] Referring again to the drawings, FIG. 10 shows ODMR results for the DNA-FND by itself and when it is hybridized with the DNA-MP. These results were collected in an optical setup having a biased magnetic field. Thus, both unhybridized and hybridized DNA-FNDs show a Zeeman shift. However, the ODMR spectrum for the hybridized DNA-FND shows broadening of the resonance peaks and this is different from that of the hybridized conjugate. These results demonstrate that simple hybridization of DNA can be detected by magnetometry.

Example 4

[00130] Referring again to the drawings, FIG. 11 schematically shows a procedure for forming a stem-loop MND-probe conjugate. Approximately 50 pg of 40-nm FND was coated with glycidol then activated by DSC treatment as described above, washed, then resuspended in 200 pL PBS with 0.05% Tween-20 (PBST). Approximately 1 pL of 100 mg/mL amino propyl azide was added and the suspension was gently shaken for 4 hours at room temperature. The FNDs, now with azide groups on the surface, were then rinsed 3x in PBST and resuspended, and approximately 0.7 nanomoles of the functionalized stem-loop DNA was added. A copper-dependent click reaction was then performed to link the DNA to the azide on the nanodiamond surface. The reaction included tris((l-hydroxy-propyl-lH-l,2,3-triazol-4-yl)methyl)amine (THPTA), copper sulfate, and ascorbic acid at final concentrations of 1.25 mM, 0.25 mM, 0.84 mM, respectively. The sample reacted on a shaker overnight at 4 °C. The DNA-FND conjugate was then rinsed lx in PBST with 1 mM EDTA, then 2x PBST, then resuspended in 200 pL PBST. Commercially-available polymer coated MPs with carboxyl functional groups were used in the following steps. MPs were EDC- activated using 2-step EDC activations where 50 pg of the MPs were suspended in 200 pl PBST and 2 mg of EDC and 2 mg of NHS were added. The reaction proceeded for 15 min at room temperature to convert the carboxyl group to an amine reactive NHS ester. The activated MPs were then rinsed 2X PBST and resuspended in 200 pL PBST. The DNA-FND suspension was added to the MP suspension and reacted for 4 hours at room temperature. The resulting FND-DNA-MP, or MND-probe conjugate was then separated by magnetic separation and rinsed in PBST. The final product was then very gently resuspended in 500 pL PBST.

[00131] Referring again to the drawings, FIG. 12 shows the stem-loop DNA sequence that was designed to detect the single strand microRNA species, miR21. The DNA-FND composite was created in which an FND was attached at one end of the stem-loop sequence and a MP is attached at the other end of the DNA. Under room temperature conditions, base-pairing at the stem region brings the MP near the FND. This affects the fluorescence of the FND and leads to a Zeeman shift in the ODMR spectra as shown in FIG. 12.

[00132] The nanodiamonds are attached to a glass cover slip and then sealed within a flow cell. Fluid, containing single strand DNA or RNA, is then flowed through the flow cell with hybridization of complementary DNA/RNA occurring in the loop region. This destabilizes the stem region and results in a linear structure that moves the MP away from the FND. In the linearized configuration, the MP does not affect FND fluorescence, and a zero-field resonance peak is observed at 2.87 mHz. The linearized DNA can be returned to its original structure by a short series of laser pulses to rapidly denature the hybridized DNA. This approach is illustrated in FIG. 12.

Example 5

[00133] Cleaned fluorescent nanodiamonds were coated with glycidol as described above. Approximately 1 mg of coated nanodiamonds are washed with a 50/50 mixture of N,N- dimethylacetamide (DMAC) and tetrahydrofuran (THF), then resuspended in N,N- dimethylformamide (DMF) containing 10% DMAC. Solid N,N'-disuccinimidyl carbonate (DSC) powder was added to 0.2 M DSC and vortexed, and the sample was allowed to react with shaking for 4-hours at room temperature. The activated gFND were then washed in DMAC and stored at -20 °C until use. Working quickly, NHS activated gFND were resuspended in 50 mM HEPES pH 7.4, 0.05% Tween-20 (HEPES-T) at a concentration of 10 mg/mL. Human IgG, rabbit IgG, goat anti-human IgG, or mouse IgG antibodies (Southern Biotech, Birmingham, AL) were quickly added to a final concentration of 1 mg/mL and incubated for 2-hours on an orbital shaker. Ethanolamine (10 pL) was added to quench the reaction and incubated for 10 minutes before rinsing three times with HEPES-T. The final volume was 1 mg/mL in the same buffer. Based on depletion studies, it was estimated that the antibody binding was between 1.5 to 3 ng IgG/mL per pg FND.

[00134] While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims

What is claimed is:

1. A target detection system, comprising: at least a first reagent comprising a plurality of fluorescent nitrogen-vacancy nanodiamond-probe-magnetic nanoparticle conjugates (MND-probe conjugates), each MND-probe conjugate comprising at least one fluorescent nitrogen-vacancy nanodiamond (FND); at least one magnetic nanoparticle (MP); and at least one polymeric probe comprising a biomolecule selected from the group consisting of a polynucleotide, a polypeptide, or a combination thereof wherein the FND portion of the MND-probe conjugate is chemically linked to the polymeric probe, and wherein the MP portion of the and the MP portion of the MND-probe conjugate is chemically linked to the polymeric probe.

2. The target detection system according to claim 1, where each of the plurality of MND- probe conjugates includes one FND, one MP and one polymeric probe.

3. The target detection system according to claim 1, wherein the polymeric probe is characterized as having binding specificity to a target material, and wherein a distance between the FND and the MP in the MND-probe conjugate changes when the at least one polymeric probe contacts and binds to a target material to which the polymeric probe has binding specificity.

4. The target detection system according to claim 1, wherein the binding specificity is not characterized by covalent bonding.

5. The target detection system according to claim 1, wherein the at least one FND and the at least one MP comprises a surface comprising a chemical functional group linker that chemically links it to the at least one polymeric probe, the chemical functional group linker selected from carboxyl moieties, amine moieties, alcohol moieties, or combinations thereof.

6. The target detection system according to claim 1, wherein the at least one FND is coated with a plurality of chemical functional group linkers comprising glycidol, and wherein the at least one MP is coated with a plurality of functional group linkers comprising a carboxyl moiety.

7. The target detection system according to claim 1, wherein the at least one MP has a particle size in the range of about 10 nm to about 100 nm, and comprises a structure selected from the group consisting an iron oxide nanoparticle, a maghemite nanoparticle, a magnetite nanoparticle, a diamagnetic nanoparticle, a supermagnetic nanoparticle, a ferromagnetic nanoparticle, a ferrimagnetic nanoparticle, a quantum dot, an upconverting nanoparticle, a ferritin nanoparticle, a ferritin-like protein, a heme-containing protein, an oligonucleotide- containing magnetic nanoparticle, and a core-shell nanoparticle comprising a magnetic core and an outer shell of a component selected from the group consisting of silane, polysaccharide, gold, polymer, and dendrimer, and wherein the at least one FND has a particle size in the range of about 20 nm to about 100 nm, and includes 1 to about 100 nitrogen-vacancy centers, and wherein the nitrogen-vacancy centers are planted about 5 to about 15 nm below the surface of the fluorescent nitrogen-vacancy nanodiamond.

8. The target detection system according to claim 1, wherein the polymeric probe is selected from the group consisting of an oligonucleotide having a sequence configured to form a stem-loop structure, a polypeptide that comprises an antibody, or a polypeptide that comprises a protein subunit selected from the group consisting of an intein, an extein, and a combination thereof.

9. A target detection system, comprising: a first reagent comprising a plurality of fluorescent nitrogen-vacancy nanodiamond-probe conjugates (FND-probe conjugates), each FND-probe conjugate comprising at least one fluorescent nitrogen-vacancy nanodiamond (FND); and at least one polymeric probe comprising a biomolecule selected from the group consisting of a polynucleotide, a polypeptide, or a combination thereof; and a second reagent comprising a plurality of magnetic nanoparticle-probe conjugates (MP-probe conjugates), each MP -probe conjugate comprising at least one magnetic nanoparticle (MP); and at least one polymeric probe comprising a biomolecule selected from the group consisting of a polynucleotide, a polypeptide, or a combination thereof wherein the FND portion of the FND-probe conjugate is chemically linked to the polymeric probe, and wherein the MP portion of the MP -probe conjugate is chemically linked to the polymeric probe.

10. The target detection system according to claim 9, wherein the polymeric probe is characterized as having binding specificity to a target material wherein the FND-probe conjugate and the MP-probe conjugate each bind to the same target material, and wherein a distance between the FND and the MP in the MND-probe conjugate changes when the at least one polymeric probe in each of the FND-probe conjugate and the MP-probe conjugate contacts and binds to the target material to which the polymeric probe has binding specificity.

11. The target detection system according to claim 9, wherein the binding specificity is not characterized by covalent bonding.

12. The target detection system according to claim 9, where each of the plurality of FND-probe conjugates includes one FND and one polymeric probe, and each of the plurality of MP- probe conjugates includes one MP and one polymeric probe.

13. The target detection system according to claim 9, wherein the at least one FND and the at least one MP comprises a surface comprising a chemical functional group linker that chemically links it to the at least one polymeric probe, the chemical functional group linker selected from carboxyl moieties, amine moieties, alcohol moieties, or combinations thereof.

14. The target detection system according to claim 9, wherein the at least one FND is coated with a plurality of chemical functional group linkers comprising glycidol, and wherein the at least one MP is coated with a plurality of functional group linkers comprising a carboxyl moiety.

15. The target detection system according to claim 9, wherein the at least one MP has a particle size in the range of about 10 nm to about 100 nm, and comprises a structure selected from the group consisting an iron oxide nanoparticle, a maghemite nanoparticle, a magnetite nanoparticle, a diamagnetic nanoparticle, a supermagnetic nanoparticle, a ferromagnetic nanoparticle, a ferrimagnetic nanoparticle, a quantum dot, an upconverting nanoparticle, a ferritin nanoparticle, a ferritin-like protein, a heme-containing protein, an oligonucleotide- containing magnetic nanoparticle, and a core-shell nanoparticle comprising a magnetic core and an outer shell of a component selected from the group consisting of silane, polysaccharide, gold, polymer, and dendrimer, and wherein the at least one FND has a particle size in the range of about 20 nm to about 100 nm, and includes 1 to about 100 nitrogen-vacancy centers, and wherein the nitrogen-vacancy centers are planted about 5 to about 15 nm below the surface of the fluorescent nitrogen-vacancy nanodiamond. The target detection system according to claim 9, wherein the polymeric probe is selected from the group consisting of an oligonucleotide having a sequence configured to form a stem-loop structure, a polypeptide that comprises an antibody, or a polypeptide that comprises a protein subunit selected from the group consisting of an intein, an extein, and a combination thereof. The target detection system according to claim 9, wherein a distance between the FND and the MP in the changes when the at least one polymeric probe contacts and binds to a target material to which the polymeric probe has binding specificity. A process for detecting the presence of a target material in a sample, the system comprising: i. providing a target detection system according to one of claims 1 and 2: ii. providing an interrogation system capable of detecting at least one measurable change relating to the displacement of an MP toward or away from a FND; iii. introducing the selected reagent into the detection system in contact with a sample suspected of containing the target material; iv. interrogating the reagent- sample combination to detect a measurable change. The process for detecting the presence of a target material in a sample according to claim 18, further comprising providing a flow cell and fluid medium for receiving the sample comprising a biological sample from one of cell, tissue or a combination thereof, wherein one or more reagents of the target detection system are immobilized on a surface of the flow cell and the flow cell is configured to flow the sample over the immobilized reagents. The process for detecting the presence of a target material in a sample according to claim 18, the interrogation system comprising optics configured to generate and measure one or more of optically detected magnetic resonance (ODMR), a spin-lattice relaxation time (Tl), a spin-spin relaxation time (T2) of the plurality of magnetic nanoparticles, or a combination thereof. The process for detecting the presence of a target material in a sample according to claim 20, wherein measuring ODMR of the plurality of fluorescent nitrogen-vacancy nanodiamonds after exposure to the biological sample determines a presence or an absence of the target material in the biological sample based on the ODMR. The process for detecting the presence of a target material in a sample according to claim 20, wherein measuring a spin-lattice relaxation time (Tl) or a spin-spin relaxation time (T2) of the plurality of fluorescent nitrogen-vacancy nanodiamonds after exposure to the biological sample determines a presence or an absence of the target material in the biological sample based on values of Tl or T2. The process for detecting the presence of a target material in a sample according to claim 18, wherein the process is used in a biomedical application. A reagent for detection of target materials, the reagent comprising: a plurality of fluorescent NV-center nanodiamonds (FNDs), each of the plurality of FNDs having chemically bound to its surface at least one polymeric probe, the at least one polymeric probe comprising an oligonucleotide comprising a stem loop structure and a chemically bound magnetic particle. A reagent for detection of target materials according to claim 24, wherein the oligonucleotide comprising a stem loop structure is single stranded DNA, and wherein hybridization of a target polynucleotide to a portion of a loop region of the stem loop structure causes the loop to open, thereby linearizing the stem-loop structure and moving the MP away from the FND whereby there is a change in a magnetic field property of the FND causing a change that is detectable by interrogation according to one or more of optically detected magnetic resonance (ODMR), a spin-lattice relaxation time (Tl), a spinspin relaxation time (T2) of the plurality of magnetic nanoparticles, or a combination thereof.