Patent Application
20240328944
A portion of the disclosure of this patent contains material that is subject to copyright protection. The copyright owner has no objection to the reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever.
The present invention relates to a device and method of detection of paramagnetic chemical species by analyzing changes in a magnetically induced fluorescence contrast of fluorescent nanodiamond particles introduced into a sample.
Nanodiamond (ND) particles containing nitrogen-vacancy (NV) centers (NDNV) exhibit uniquely coupled magneto-optical properties. The intensity of ND fluorescence depends on the NV centers' electronic states, which can be manipulated with external magnetic or electromagnetic fields. This quantum control and optical readout of the electronic state of the NV center in a combination with exceptional photostability and biocompatibility make NDNV a uniquely sensitive probe with applications from physics to biomedicine. One particular application of NDNV particles is detection of paramagnetic species including, but not limited to, reactive oxygen species (ROS), ferritin, gadolinium, manganese, and other species using the approach of NV T1 relaxometry. NV T1 relaxometry relates changes in NV relaxation time T1 (spin depolarization time) to the presence of magnetic noise (e.g., paramagnetic species) external to NV. Use of NV T1 relaxometry addresses problems with current optical reagents for ROS detection, such as organic fluorescent probes struggling with variability in their reporting primarily attributed to photobleaching and autocatalytic activation. This means that the traditional ROS probes do not accurately report on their targets and as such wastes time and resources. In practical implementation, NV T1 relaxation technology is based on application of microsecond light pulses to probe fluorescence during the spin relaxation of individual NDNV particles distributed in intra-cellular or extra-cellular environments and is most suitable for high-resolution microscopy. One limitation of NV T1 relaxometry is that it does not inherently address the problem of high fluorescence background which is often present in biological samples (e.g., in whole blood). Previously, it was shown that application of a periodically modulated magnetic field inducing periodically modulated ND-NV fluorescence is an effective way to separate the NDNV fluorescence signal from background autofluorescence. The use of phase-sensitive detection, such as that by lock-in amplifiers—a widely used signal processing method to extract small periodic signals present below noise levels—resulted in a further significant increase in the signal-to-noise ratio (SNR). Magnetically-modulated fluorescence has been used to perform NDNV imaging in the presence of high fluorescent background in different imaging scenarios, and, for example, demonstrated an impressive 100-fold improvement in SNR for NDNV labeling of sentinel lymph node. The readout of lateral flow assay (LFA) was recently improved by modulating fluorescence of ND-NV reporters associated (bound) with targeted analytes using microwaves and magnetic field in combination with using lock-in/narrow bandwidth signal amplification. All these applications of periodic magnetic modulation of NDNV fluorescence in combination with phase-sensitive detection were aimed to either image NDNV particles or enhance quantification of NDNV in the presence of fluorescence background.
The innovation of the present invention is in using magnetically induced fluorescence contrast ideally in combination with phase-sensitive detection to detect surrounding paramagnetic species. Moreover, to further improve the sensitivity of phase-sensitive detection, applicants implemented a double lock-in by adding amplitude modulation of the excitation light to the magnetic field modulation. This further reduced noise arising from instability in the light intensity, detector, or environmental noise.
In one embodiment, present invention relates to a method of monitoring of the fluorescence intensity of a diamond particle comprising:
In another embodiment the invention relates to a method of analyzing a paramagnetic chemical species comprising:
In another embodiment, it relates to a method of detection of a chemical species comprising:
In another embodiment it relates to a method of detection of a chemical species comprising:
In yet another embodiment it relates to a device for detection of a chemical species comprising:
While this invention is susceptible to embodiment in many different forms, there is shown in the drawings, and will herein be described in detail, specific embodiments with the understanding that the present disclosure of such embodiments is to be considered as an example of the principles and not intended to limit the invention to the specific embodiments shown and described. In the description below, like reference numerals are used to describe the same, similar, or corresponding parts in the several views of the drawings. This detailed description defines the meaning of the terms used herein and specifically describes embodiments in order for those skilled in the art to practice the invention.
The terms “about” and “essentially” mean±10 percent.
The terms “a” or “an”, as used herein, are defined as one or as more than one. The term “plurality”, as used herein, is defined as two or as more than two. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language). The term “coupled”, as used herein, is defined as connected, although not necessarily directly, and not necessarily mechanically.
The term “comprising” is not intended to limit inventions to only claiming the present invention with such comprising language. Any invention using the term comprising could be separated into one or more claims using “consisting” or “consisting of” claim language and is so intended.
Reference throughout this document to “one embodiment”, “certain embodiments”, “an embodiment”, or similar terms means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments without limitation.
The term “or”, as used herein, is to be interpreted as an inclusive or meaning any one or any combination. Therefore, “A, B, or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B, and C”. An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in some way inherently mutually exclusive.
It is further noted that the claims may be drafted to exclude any element which may be optional. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent such publication may set out definitions of a term that conflict with the explicit or implicit definition of the present disclosure, the definition of the present disclosure controls.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
The drawings featured in the figures are for the purpose of illustrating certain convenient embodiments of the present invention and are not to be considered as limitation thereto. The term “means” preceding a present participle of an operation indicates a desired function for which there is one or more embodiments, i.e., one or more methods, devices, or apparatuses for achieving the desired function and that one skilled in the art could select from these or their equivalent in view of the disclosure herein, and use of the term “means” is not intended to be limiting.
As used herein, the term “nanodiamond or diamond nanoparticles” refers to submicron sized particles. More specifically, the term nanodiamond particles refers to discrete diamond particles exhibiting at least one spatial dimension having a size of less than about 1000 nm. More commonly, the nanodiamond particles exhibit multiple spatial dimensions having a size of less than about 1000 nm.
As used herein, the term “micron-diamond or micron diamond particle” refers to discrete diamond particles exhibiting at least one spatial dimension having a size ranging from about 1 micron (μm) to 500 μm, while other dimensions are more than about 1 micron. More commonly, a micron diamond particle exhibits multiple spatial dimensions having a size of about 1 μm to 500 μm (0.5 mm).
As used herein, the term “FND” refers to fluorescent nanodiamond, a diamond nanoparticle exhibiting fluorescence.
As used herein, the term “luminescence” refers to the emission of electromagnetic radiation from crystallographic defects within the diamond lattice upon absorption of radiative energy from an appropriate excitation source of electromagnetic radiation.
As used herein, the term “electromagnetic radiation” refers to the propagating waves of electric and magnetic waves carrying radiative energy through space. More specifically, this term refers to the radiative energy necessary to promote luminescence emission from crystallographic defects centers within the diamond lattice, and may consist of ultraviolet, visible, infrared, or X-rays.
As used herein, the term “H3 color centers” refers to specific crystallographic defect centers within the diamond lattice which can exhibit luminescence. More specifically, the H3 center refers to the specific defect center consisting of an atomic arrangement of two nitrogen atoms surrounding a vacancy which can exhibit green luminescence upon excitation with an appropriate source of electromagnetic radiation.
As used herein, the term “quantum properties” of diamond particles is related to spin characteristics of the NV centers including, but not limited to, the spin coherence time (T2), spin dephasing time, spin-lattice relaxation time (T1), optically detected magnetic resonance (ODMR) spectroscopic characteristics (full width at half maximum, FWHM), ODMR contrast, and the difference in luminescence intensity of NV centers under applied magnetic field as compared to the luminescence intensity measurements without magnetic field. These characteristics can also include 13C dynamic nuclear polarization capability of P1 centers (single N dopants). Improvement in spin electronic and nuclear characteristics of color centers and spin-containing lattice elements is based on decrease of the lattice distortions and elimination of parasitic defects following high temperature annealing.
As used herein, the term “fluorescence intensity” means the rate of delivery of energy of photons emitted by analyzed mixture and measured, for example, by the signal from a photodiode.
As used herein, the term “diamond particle capable of exhibiting luminescence” means any discrete carbon-based material with a diamond cubic lattice which emits light via electronic excited transitions.
As used herein, the term “spectral range of 640 nm to 800 nm” means range of wavelengths of electromagnetic radiation emitted by NV centers in diamond.
As used herein, the term “optical radiation” means the optical radiation comprising wavelengths from the range about 190 nm to 2000 nm used for excitation of color centers in diamond.
As used herein, the term “magnetic field” means the vector magnetic field which exerts force on a magnetic body and moving electric charges.
As used herein, the term “sensing changes in the environment” means reporting or providing a means of observing the presence or change in the quantity of paramagnetic species, other species, temperature, and the like near the diamond particle.
As used herein, the term “paramagnetic chemical species” means a chemical analyte containing chemical species with one or more unpaired electrons. Where chemical species comprise: species, structures and molecules with unpaired electrons; free radicals, hydrogen peroxide, peroxides, paramagnetic metal ions, paramagnetic metal ion chelates, reactive oxygen species, reactive nitrogen species, reactive sulfur species, ferritin, metalloprotein, oxygen molecules, gaseous molecules containing unpaired electrons, gaseous molecules dissolved in fluid, spin traps, chemical traps; free radicals in proteins, antibodies, antigens; free radicals in nucleic acids; products of enzymatic reactions, products of metabolic reactions; free radicals in polymers.
As used herein, the term “NV center” means nitrogen-vacancy point defect present within the diamond lattice consisting of replacement of two adjacent carbons with a nearest neighbor pair of a nitrogen atom and lattice vacancy (site where a carbon atom is absent from the crystallographic lattice). NV center can have a neutral charge state or a negative charge state. In the majority of embodiments, the NV center as used herein is NV center with negative charge state.
As used herein, the term “analyzed mixture” means a combination of fluorescent nanodiamond particles containing NV centers and analyte, for example, a suspension containing fluorescent nanodiamonds and freely dispersed analyte molecules.
As used herein, the term “fluorescence detector” means any device capable of reporting fluorescence intensity or changes in the fluorescence intensity.
As used herein, the term “fluorescence signal detector” means a detector of fluorescence intensity.
As used herein, the term “fluorescence contrast” means difference in fluorescence intensities detected with and without applying magnetic field.
As used herein, the term “periodically time-varied light intensity” means repeated changes in the intensity of generated light over time, where light intensity is varied between low intensity state (or no light) and maximum intensity state with a certain frequency.
As used herein, the term “light source” means a device capable of producing photons including but not limited to the combination of the photon-generating device with means to produce light with periodically time-varying intensity or continuous radiation.
As used herein, the term “alternating in time” means repeated changes in the magnitude of a physical parameter (e.g. applied magnetic field) over time.
As used herein, the term NDNV is an abbreviation for nanodiamond particles containing nitrogen-vacancy (NV) centers.
As used herein, the term “phase-sensitive detection” refers to a method (lock-in detection) and an electrical instrument (lock-in amplifier or computer providing digital analysis of a recorded waveform) capable of extracting signal amplitudes and phases. A lock-in measurement extracts signals in a defined frequency band around the reference frequency, efficiently rejecting all other frequency components to report the signal magnitude.
As used herein, the term “modulating” refers to changing a physical parameter over time; where, for example, changing of magnetic field on and off can be a single event, or can be repeated multiple times with different time intervals between magnetic field on and off, or repeated in a periodic manner over time with a certain frequency.
As used herein, the term “nutritious substance” refers to food, the substance consisting essentially of protein, carbohydrate, fat, and other nutrients used in the body of an organism.
According to certain embodiments consistent with the present invention, diamond particles are introduced to a sample that may contain a paramagnetic chemical species to be analyzed forming an analyzed mixture, further comprising applying magnetic field for modulating fluorescence of NV centers and providing a fluorescence contrast; and where using the fluorescence contrast comprises determining the presence of paramagnetic chemical species. In another embodiment, fluorescent diamond particles contain plurality of NV and H3 color centers, where fluorescence intensity of NV centers measured with and without applying magnetic field is calibrated to the unchanged fluorescence intensity produced by excitation of H3 centers. This calibration improves sensitivity of the paramagnetic species detection.
According to certain embodiments consistent with the present invention, concentration of the fluorescent diamond particles in the analyzed mixture is known. It can be selected, for example, including but not limited to, from a range between about 1 ug/ml to about 10 mg/ml of NDNV. In one embodiment, presence of paramagnetic chemical species is defined by comparison between values of a fluorescence contrast in the analyzed mixture and a control sample, where the paramagnetic chemical species are absent. In another embodiment, concentration of paramagnetic chemical species in the analyzed mixture is defined from a calibration curve obtained from samples with known concentration of the paramagnetic chemical species. In yet another embodiment the fluorescence contrast is normalized by the value of fluorescence intensity detected in the state of no applied magnetic field compared to applied magnetic field, providing a normalized fluorescence contrast, so that the presence of paramagnetic chemical species is defined by comparison of values of a normalized fluorescence contrast in the analyzed mixture and a control mixture where the paramagnetic chemical species are absent. In another embodiment, the concentration of paramagnetic chemical species changes over time and thus, the presence of paramagnetic chemical species is defined from changes in the fluorescence contrast over time. Diamond particles used in the present invention comprise sizes between approximately 5 nm and 1000 nm, preferably between 10 nm and 200 nm, and most preferably between 20 nm and 70 nm.
In certain embodiments of the present invention diamond particles comprise particles with modified surface providing change in the fluorescence contrast compared to particles with unmodified surface comprising addition or removal of surface groups, including but not limited to surface spins, surface dangling bonds, surface charges, dipole-dipole interactions, and addition of groups or shells of differing dielectric composition from diamond such as silica. Modifying the diamond particle surface comprises functionalizing the diamond particles with at least one functional surface group selected from the group consisting of carboxylic, hydroxyl, amino, hydrogen, epoxy, poly(ethylene glycol), poly(glycerol), hydrocarbon, aromatic, nucleophile, thiol, sulfur, acid, base, silane, aluminum, halogen, and fluoro-containing. The modified diamond particle surface can further comprise changes in at least one of surface spins, surface dangling bonds, and surface charges due to the presence of analyte paramagnetic chemical species and where the change in the spin properties of the modified surface impacts spin properties of the NV centers and resulting fluorescence contrast. Changes in surface spins and surface charges of the modified surface could cause changes in the T1 relaxation time of NV centers resulting in a change in magnetically modulated fluorescence contrast. In another embodiment consistent with the present invention, diamond particles are modified with chemical groups which convert a transient analyte into stable forms. In yet another embodiment the analyzed mixture is altered to adjust the fluorescence contrast comprising at least one from the group comprising addition of non-analyte calibrants, chemical species, oxygenation, deoxygenation, diluted gaseous species, pH altering species and pH stabilizing species, salts. Yet another embodiment comprises conjugating with the diamond particles or attaching to the diamond particles at least one material selected from the group consisting of biological molecules, a site-specific targeting ligand, a nucleic acid, a peptide, a protein, an antibody, an antigen, oligonucleotide, aptamer, RNA, DNA, a ligand, a dye, a fluorescent specie, a spin trap, a radioactive specie, an image contrast agent, an isotope, a drug molecule, a hormone, a carbohydrate, and a polymer.
In certain embodiments of the present invention the analyzed mixture is produced by comprising one of: pouring, mixing, shaking, vortexing, incorporation by sonication, ballistic delivery of fluorescent nanodiamonds, ballistic delivery using a gene gun, drying of a mixed suspension, exposing a sample with analyte to fluorescent nanodiamond immobilized on a substrate, capillary, wall of a well, optical fiber or inside an optical fiber. In another embodiment the analyzed mixture further comprises: whole blood, blood plasma, serum, body fluids, fluids, nutritious substance, waste-water, environmental liquids, buffer, cellular membranes, intracellular membranes, cell compartments, organelles, cytoplasm, animal cell, stem cell, eukaryotic cell, prokaryotic cell, cell culture, intracellular fluid, organism, organ, tissue, plant fluids, plant tissue, plant cell, microorganism, bacteria, yeast, yeast membrane, yeast cytoplasm, intercellular fluid in a bioreactor, animal cell in a bioreactor, biomass in a bioreactor, products of fermentation, fermentation biomass, marine bacteria, phytoplankton, seaweeds, corals, microfluidic chip, organ-on-a chip, polymers, plastics, nitrocellulose membrane, optical fiber, matrix or a substrate, where the substrate is electronic component, a tag, a tracer, a label, a polymer, and an optically transparent solid.
In certain embodiments of the present invention analyzed mixture comprises at least one of: a continuous flow in a capillary or a channel, flow in a microfluidic device, and content of a multi-well plate. In another embodiment method of detection of a chemical species using magnetically modulated fluorescence of NVND further comprises a microfluidic flow assay, a microplate reader, flow cytometry assay, fluorescence activated cell sorting, an anti-Brownian electrokinetic (ABEL) trap, an acoustofluidic device, an electrophoretic device, a lateral flow assay, a vertical flow assay, PCR, and ELISA. For example, a possible method of detection of paramagnetic species using a lateral flow assay would comprise a method where:
In one embodiment of the detection of paramagnetic species using a lateral flow assay the analyte further comprises ferritin or apoferritin.
In certain embodiments of the present invention applying a magnetic field to the analyzed mixture comprises applying a periodically time-varying magnetic field with known frequency, amplitude, phase, and waveform. In one embodiment, a magnetic modulation frequency comprises a frequency selected from a range between about 1 Hz to about 10,000 Hz, most preferably from about 10 Hz to about 1,000 Hz. The amplitude of the applied magnetic field, either static or periodically time-varying, can be varied between about zero Gauss to a maximum magnetic field or between a minimum and a maximum magnetic field. In one embodiment, maximum magnetic field comprises magnetic field strength between approximately 1 Gauss and 5,000 Gauss and most preferably between approximately 400 and 1000 Gauss.
In certain embodiments of the present invention the periodically time-varying light intensity is further characterized by a known frequency, amplitude, phase, and waveform. In one embodiment, an optical modulation frequency comprises frequency selected from about 10 Hz to about 10,000 Hz. The optical radiation has wavelength between approximately 480 nm and 1100 nm, preferably between 530 nm and 640 nm, and most preferably between 530 nm and 580 nm.
Innovation of the present invention is in using magnetically induced fluorescence contrast in combination with phase-sensitive detection to detect surrounding paramagnetic species. Moreover, to further improve the sensitivity of phase-sensitive detection, the method comprises a double lock-in by adding amplitude modulation of the excitation light to the magnetic field modulation. This further reduced noise arising from instability in the light intensity, detector, or environmental noise. One embodiment of the present invention comprises taking an analyzed mixture and applying optical radiation with periodically time-varying intensity providing optical modulation frequency and also applying a magnetic field with periodically time-varying magnitude providing magnetic modulation frequency. The method further comprises phase-sensitive measurement of the detected fluorescence signal originated from the analyzed mixture modulated at the optical and magnetic modulations frequencies, where the detected fluorescence signal is processed by at least one of: a dual-frequency lock-in amplifier, two individual lock-in amplifiers, digital lock-in, computational lock-in, and Fourier analysis. In one embodiment the method further comprises parallel processing of the signals from two lock-in amplifiers synchronized to the same oscillator. In another embodiment the detected resulting fluorescence intensity may be further analyzed by an analog-to-digital convertor, where the converter is selected from the group comprising of oscilloscope, data acquisition device, and lock-in amplifier.
Now referring to the drawings,
In this example, 40 nm fluorescent nanodiamond (NDNV40) containing approximately 1.5 ppm of NV centers were dispersed in distilled water at 0.5 mg/mL and flowed through a sample unit (41 in
The dependence of magnetically modulated fluorescence contrast on magnetic field strength was also investigated.
In this example, the specified analyzed mixtures flowed through a sample unit at a rate of 25 uL/min as described in Example 1. A time-varying (AC) magnetic field (61, f1=41 Hz) was generated with an electromagnet (Uxcell). The electromagnet was driven by a current supply modulated by transistor-to-transistor logic (TTL) creating a field of approximately 50 mT at the sample unit. The TTL reference was generated from the lock-in amplifier. The output of photodetector 51 was then split and read by two separate lock-in amplifiers (SRS 850 and SRS 830) simultaneously, to process optical modulation and magnetic modulation at related frequencies, correspondingly.
The output of each lock-in amplifier was read and averaged by a data acquisition device 81 (National Instruments USB-6218). Normalized fluorescent contrast was generated by the ratio of the lock-in outputs (magnetic modulation output divided to optical modulation output). The concentration dependence of two chemical species, specifically the stable radicals TEMPOL and TEMPOL-NH3, were then studied (
In this example, measurements were taken as described in example 2. In this case, the analyzed mixture consisted of 70 nm fluorescent nanodiamond with approximately 3 ppm of NV centers. The NDNV were either coated with TEMPOL covalently linked to the surface through (3-aminoproply)trimethoxysilane (APTMS) or functionalized with only APTMS without TEMPOL. Samples were alternated in the flow capillary over the course of 30 minutes with washing the capillary by DI water between alternated samples. The samples with TEMPOL were readily differentiable from unlabeled samples as illustrated in
In this example, measurements were taken as in example 2, with the modification that for this analysis the direct fluorescence contrast was taken from the lock-in amplifier measuring magnetic modulation, without normalization to optically modulated output. The diamond concentration was kept constant for each measurement. The analyzed samples consisted of mixtures of 40 nm fluorescent nanodiamond (0.5 mg/mL) with 1.5 ppm NV and gadolinium nitrate at concentrations from 5 uM to 0 uM. As shown in
In this example, measurements were taken as in example 4, utilizing direct measurement of magnetically modulated fluorescence contrast with constant diamond concentration in the analyzed samples. Ferritin is a spherical iron-containing protein that plays a major role in iron homeostasis in the animal and human body. Apoferritin is the iron-free version of this protein that is molecularly identical but does not contain iron. Solutions of ferritin or apoferritin in DI water were made at equal mass concentration (2.5 mg/mL) with 40 nm fluorescent nanodiamond containing 1.5 ppm NV at 0.5 mg/mL. Solutions of proteins without nanodiamond (at concentration of proteins 2.5 mg/mL), and additional water control were also analyzed. As shown in
Xanthine oxidase (XO) is an enzyme which catabolizes, among other compounds, xanthine (X) into uric acid, consuming oxygen and producing reactive oxygen species such as superoxide and hydrogen peroxide. The progress of xanthine oxidation over time as compared to a control were measured by monitoring changes in normalized fluorescence contrast, in a manner as described in example 2. Data was collected in real time over the course of 10 minutes. Components of the reaction included the enzyme XO and its substrate (X). Pentetic acid (DTPA) chelates metals to prevent enzyme deactivation. Superoxide dismutase (SOD) was used as a control to prevent superoxide formation. DEPMPO is a diamagnetic molecule that can react with radicals to form a stable, paramagnetic molecule. For both the xanthine oxidase sample and control, all components were added except XO. The reaction composition consisted of 25 uM pentetic acid (DTPA), 150 uM X, 7 mM DEPMPO spin trap, 24 mU/mL XO, and 0.5 mg/mL 40 nm fluorescent nanodiamond (1.5 ppm NV). The control reaction consisted of 25 uM DTPA, 300 U/mL SOD, 7 mM DEPMPO spin trap, 24 mU/mL XO, and 0.5 mg/mL 40 nm fluorescent nanodiamond (1.5 ppm NV). XO was added last and immediately before the measurement began to initiate oxidation. The control sample with SOD and no substrate X produced no significant change in normalized fluorescence contrast over the 10 min observation period (
Peroxide is a reactive species that can both generate ROS in solution as well as react with the surface of fluorescent nanodiamond, though peroxides themselves are not paramagnetic species. Hydrogen peroxide plays an important role in oxygen-involving metabolism in biological systems, may regulate a wide variety of biological processes and is produced internally and externally to cells.
In this example, magnetically modulated fluorescence contrast was measured in two configurations: in a static magnetic configuration, with a setup similar to example 1 (
As opposed to NV centers, fluorescence in diamond from NVN centers (H3 centers) is invariant to modulation by external fields. In this example, solutions of fluorescent nanodiamond containing a mixture of NV and NVN centers within individual particles were produced by high temperature annealing (1600° C.) of electron-irradiated (5E18 e/cm2 dose) 1 um nanodiamonds. Suspension in DI water of the fluorescent nanodiamond particles was dropped into a well-plate format and dried. Measurements were collected on an inverted fluorescence microscope with mercury arc lamp illumination passed through a 517/20 nm bandpass filter for excitation and emission light collected via 561 nm longpass filter. Data was collected on a spectrometer with a CCD detector (Ocean Optics HR2000). A static DC magnetic field was generated by a rare-earth neodymium magnetic (K&Js Magnets) placed over the well containing the sample to generate a field greater than approximately 150 mT with covering to prevent light reflections. When the field was applied to the sample, changes in the spectral intensity occur in the region of the negatively charged NV center, predominantly >570 nm. Because of variability in the light source intensity, replicate measurements of the same sample showed variability in fluorescence (
Oxygen is a paramagnetic molecule present in solution typically at equilibrium with the environment in solution. Oxygen content can change due to changes in the ambient environment or biological processes producing or consuming oxygen in solution, such as the oxidation of metabolites or the generation of ROS. To measure changes in dissolved oxygen content, 40 nm fluorescent nanodiamond with 1.5 ppm NV at 1 mg/ml were put into an amber glass vial with septum. The septum was pierced with a needle placed under the surface of the liquid near the bottom and an additional needle added as a vent. Argon was bubbled through the solution for 45 minutes. To transport for measurement, the vent needle was removed to generate positive pressure, and the vial was then sealed with parafilm after removing the argon line.
Samples were then analyzed in a manner similar to example 2 for measuring fluorescent contrast. The argon-purged nanodiamond sample was drawn directly from the vial as soon as the vial was opened. After baseline data was collected, some of the solution was moved to a microfuge tube and the sample was vigorously aspirated with a micropipetter for one minute. After measuring the aspirated sample, this process was repeated with further aspiration. Upon each aspiration event, where oxygen was added to the sample, the measured normalized fluorescence contrast was lowered.
The surface of fluorescent nanodiamonds can play the role of the ability for NV to respond to their environment, causing changing in T1 relaxation times of NV and impacting the magnetically modulated fluorescence contrast value. Samples of 50 nm fluorescent nanodiamond (approximately 1 ppm NV) were coated with layers of SiO2, including samples without silica layer, and layers produced by terminating the reaction at different time points. Layers were created by a variant of the Strober process, where layers were formed by base-catalyzed condensation of tetraethylorthosilicate onto the particle surface in a time dependent fashion. The coated (or control uncoated) samples in ethanol were then mixed with either DI water or 1 mM TEMPOL in water producing solutions of 0.5 mg/mL fluorescent nanodiamond with 500 uM TEMPOL in water or pure water (control), with a final solution mixture of 1:1 water and ethanol. The ability to discriminate the external chemical species such as TEMPOL from control was found to decrease as the shell thickness was increased (
In this example, measurements were taken as described in example 2. All measurements were performed with 40 nm NDNV (0.5 mg/mL) in 10 mM HEPES buffer. Analyte concentration was set at 500 uM. TEMPOL contains a stable radical whereas the diamagnetic analog (2,2,6,6-tetramethylpiperidin-4-ol) does not. As illustrated in
Reactive oxygen species (ROS) play an important role in cell cultures in bioreactors including, but not limited to cultures of mammalian cells, stem cells, bacteria and yeast. For example, ROS play a major role in yeast's metabolism and relate to the yield of desired fermentation end products either directly through the anabolic pathways themselves or indirectly through oxidative stress which reduces product output. In order to use NDNV as sensors of ROS in cell cultures, NDNV have to be associated with cells, being distributed internally or externally to cells and in certain embodiments, to be targeted to specific sites (for example, mitochondria) within cells or externally to cells. For intercellular delivery, NDNV can be internalized by cells directly (e.g. by mammalian cells), delivered using electroporation, ballistic approaches (e.g. a gene gun) or other methods known in the field. NDNV can be also associated with cell membranes for measurement of extracellular ROS production, where ROS can be produced, for example, through the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, or diffused through a cell membrane from intercellular region (e.g. hydrogen peroxide).
This example demonstrates how NDNV can be colocalized with the yeast cell membrane in order to be used for further detection of analyte in the vicinity of cell membrane externally to a cell. For this, applicant functionalized NDNV with concanavalin A (ConA), a carbohydrate binding protein which binds especially well to α-D-mannosyl and α-D-glucosyl on cell membrane. This lectin was chosen as a robust protein which is readily available. For labeling of 120 nm NDNV with ConA (NDNV-120 nm-ConA), functionalization was done via EDC/NHS mediated carbodiimide coupling, followed by sequential centrifugal washing steps. Yeast (S. cerevisiae) cells were co-incubated with commercially available fluorescein isothiocyanate (FITC) modified concanavalin A (FITC-ConA) and NDNV-120 nm-ConA and washed from the unbound labels. Labeling was done in the presence of 0.1% bovine serum albumin as a blocking agent to ensure specific labeling. The cells were imaged using confocal microscope (Zeiss LSM 880) and 561 nm laser excitation and 570-677 nm emission to visualize NDNV and 488 nm laser excitation and 490-561 nm emission to visualize FITC. Fluorescence microscopy revealed unambiguous association of NDNV120 nm-ConA with the yeast surface, correlating to the standard FITC fluorophore.
For sensing of metabolites, size of the NDNV particle sensors becomes an important factor. Smaller particles (less than about 70 nm) are more effective sensors because a larger number of NV centers are susceptible to adjacent analytes due to larger surface area per unit mass. However, smaller particles have fewer NV centers per particle making their fluorescent output lower. In this example, NDNV particles with about 40 nm size (NDNV-40 nm) containing about 1 ppm of negatively charged NV centers was used in experiments. To functionalize this size regime with ConA, our functionalization methods were altered in comparison with example 12 to reduce aggregation. Functionalization still occurs via EDC/NHS mediated carbodiimide coupling, however as opposed to reaction of 120 nm particles which involved sequential centrifugal washing steps, here all modifications occurred directly in suspension, with selective activation and quenching steps, providing functionalized NDNV-40 nm-ConA. The result was a lower degree of interparticle aggregation and/or bridging of particles by the functionalization protein. One group of yeast cells (S. cerevisiae) was co-incubated with NDNV40 nm-ConA and washed from unbound NDNV40 nm-ConA. Another, a control group was incubated with NDNV-40 nm (untargeted) and washed. Two cell groups were analyzed in fluorescent inverted microscope (Olympus IX71) using 10× objective. During imaging using 532 nm wavelength for excitation of NV fluorescence, it was noticed that the background autofluorescence of yeast is significant, such that NDNV cannot readily be differentiated from the background via conventional microscopy alone. Analysis of yeast incubated with targeted (NDNV-40 nm-ConA) or untargeted (NDNV-40 nm) nanodiamond to detect presence of NDNV based on spin properties of NV centers was done by two complementary methods of analysis, continuous-wave microwave optically detected magnetic resonance (CW ODMR) or magnetically-induced fluorescence contrast. The essential components of the setup for measurement of ODMR spectra in yeast cells incubated with NDNV comprised an inverted microscope for fluorescence imaging, a light source, a photodetector, an arbitrary wave generator (AWG), a signal generator, a power amplifier, microwave antenna, an oscilloscope, and a PC equipped with remote control software. Measurements of magnetically-induced fluorescence contrast in yeast cells incubated with NDNV were done using the setup illustrated in
Those skilled in the art to which the present invention pertains may make modifications resulting in other embodiments employing principles of the present invention without departing from its spirit or characteristics, particularly upon considering the foregoing teachings. Accordingly, the described embodiments are to be considered in all respects only as illustrative, and not restrictive, and the scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description or drawings. Consequently, while the present invention has been described with reference to particular embodiments, modifications of structure, sequence, materials, and the like apparent to those skilled in the art still fall within the scope of the invention as claimed by the applicant.
This application is a Continuation in Part of U.S. application Ser. No. 17/233,843 filed on Apr. 19, 2021, which claims priority to PCT application number PCT/US19/56986 filed on Oct. 18, 2019 and U.S. provisional application No. 62/747,700 filed on Oct. 19, 2018 and are all incorporated herein in their entirety by reference.
This invention was made with government support under DE-SC0022441 awarded by U.S. Department of Energy Office of Science and 1R43GM144026-01 awarded by Department of Health and Human Services, National Institutes of Health, National Institute of General Medical Sciences. The government has certain rights in the invention. A portion of the research relating to the present technology was not federally sponsored.
| Number | Date | Country | |
|---|---|---|---|
| 62747700 | Oct 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US19/56986 | Oct 2019 | WO |
| Child | 17233843 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17233843 | Apr 2021 | US |
| Child | 18583400 | US |
