Magnetic nanoparticles for sample separation

Abstract

Silica-coated magnetic nanoparticles with greater ability to remain dispersed, and methods of making and using silica-coated magnetic nanoparticles. The magnetic nanoparticles comprise a core and a coating, where the core comprises Fe.sub.3O.sub.4 or other magnetic material and the coating has a thickness of from about 1.5 nm to about 2 nm. The magnetic nanoparticles are useful for preparing nucleic acids for analysis, by separating nucleic acids from other components and by normalizing nucleic acid concentrations.

Claims

1. A method of making silica-coated magnetic nanoparticles comprising: preparing an aqueous solution of Fe.sup.+3 and Fe.sup.+2 ions in deionized, de-oxygenated water in a Fe.sup.3+/Fe.sup.2+ molar ratio of about 2/1; sonicating the aqueous solution; adding a base to the aqueous solution to precipitate Fe.sub.3O.sub.4 nanoparticles from the aqueous solution, thereby forming a mixture comprising Fe.sub.3O.sub.4 nanoparticles in an alkaline water; removing substantially all the alkaline water from the Fe.sub.3O.sub.4 nanoparticles to form a concentrated mixture; adding a de-oxygenated alcohol to the concentrated mixture to form an aqueous alcohol mixture; adding a silicate solution to the aqueous alcohol mixture to form a silica coating on the Fe.sub.3O.sub.4 nanoparticles; and separating the silica-coated Fe.sub.3O.sub.4 nanoparticles from the silicate-containing aqueous alcohol mixture.

2. The method of claim 1, wherein the base added to the aqueous solution comprises ammonia or sodium hydroxide.

3. The method of claim 1, wherein the silicate solution added to the aqueous alcohol mixture comprises tetraethyl orthosilicate (TEOS).

4. The method of claim 1, wherein the addition of the base to the aqueous solution or the addition of the silicate solution to the aqueous alcohol mixture is performed under an inert atmosphere.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A and 1B are illustrations of an embodiment of the preparation of silica-coated magnetic nanoparticles according to the present technology.

(2) FIGS. 2A and 2B are electron micrographs showing individual silica-coated Fe.sub.3O.sub.4 nanoparticles and typical cluster size formed by the particles in aqueous dispersions. FIG. 2C shows laser diffraction data for silica-coated magnetic nanoparticles according to the present technology. FIGS. 2D to 2I are TEM images for silica-coated magnetic nanoparticles prepared in Example 1.

(3) FIG. 3 is an analysis of an embodiment of the silica-coated magnetic nanoparticles by FT-IR spectroscopy. The analysis reveals a SiO to FeO peak ratio of 3.45, and the SiO to FeO peak ratio is proportional to the thickness of the outer silica coating.

(4) FIG. 4 is a series of IR spectra showing the increase of the SiO to FeO peak ratio as an increasing volume of TEOS was used for the silica coating process (0.5 ml to 2.5 ml). The higher SiO to FeO peak ratio signals the formation of thicker silica shells on the Fe.sub.3O.sub.4 nanoparticle core.

(5) FIG. 5 is a graph illustrating that the thickness of the silica coating increases linearly with increasing TEOS amounts.

(6) FIG. 6 is a scanning electron microscopy (SEM) image of silica-coated magnetic nanoparticle according to the present technology, at a magnification of 250,000. The particles were made with 1.5 ml TEOS and sonication.

(7) FIG. 7 is a transmission electron microscopy (TEM) image of silica-coated magnetic nanoparticle according to the present technology. Typical Fe.sub.3O.sub.4 cores range from 7 to 10 nm. The average silica coating thickness on the particles using 1.5 ml of TEOS was about 1.6 nm. Prominent Fe.sub.3O.sub.4 crystalline lattice visible inside core indicates the lack of oxidation during synthesis.

(8) FIGS. 8A through 8F show Diagnostic Quantitative Reverse Transcription Polymerase Chain Reaction (QRT-PCR) results. Using manual (FIGS. 8A to 8C) and automated (FIGS. 8D to 8F) protocols and multiple lots of magnetic silica nanoparticles, viral RNA was recovered from a sample and analyzed by diagnostic QRT-PCR.

(9) FIG. 9 shows Next Generation Sequence related Normalization results. The data presented is electrophoresis verification that normalization, as defined below, is accomplished.

(10) FIG. 10 provides photographs of the magnetic separation of silica-coated magnetic nanoparticles from a stable suspension using a small magnet.

(11) The present teachings are best understood from the following detailed description when read with the accompanying drawing figures. The features are not necessarily drawn to scale.

DETAILED DESCRIPTION

(12) The silica-coated magnetic nanoparticles (NP) described herein are surprisingly effective for separating nucleic acids from a sample. The silica coating provides a hydrophilic substrate, greatly reducing non-specific binding in important applications, such as purification of nucleic acids.

(13) In some embodiments, the present technology is used for enrichment of RNA obtained from cellular or viral samples as part of a quantitative reverse transcription polymerase chain reaction (QRT-PCR) detection assay. By way of example, the present technology can be employed to enrich RNA from SARS-CoV-2 or other viral pathogens prior to detecting the RNA by QRT-PCR. In some embodiments, the present technology is used for enrichment of human RNA and human and bacterial DNA. For example, the present technology may be used to enrich and detect DNA from respiratory pathogens such as Legionella (Legionnaires' disease pathogen). In some embodiments, the present technology is used for normalization of a DNA library prior to Next Generation Sequencing (NGS). In some embodiments, the present technology is used for normalization of a DNA or RNA sample prior to analysis of the sample.

(14) Magnetic Nanoparticles Referring to FIG. 1A, the present technology provides a magnetic nanoparticle 100 comprising a core 10 and a homogeneous conformal coating 20 on the core 10 that substantially encapsulates the core 10. In one example, the coating 20 completely encapsulates the core 10. The core 10 can be made of a metal, an alloy, or a metal oxide and the coating can be made of silica. In an example, the core 10 can be Fe.sub.3O.sub.4 and the coating comprises silica. In an example, the silica coating is substantially non-porous. The ratio of the core diameter to the coating thickness and the characteristics of the coating surface may be such that a plurality of the magnetic nanoparticles 100 aggregate in a manner that allows an analyte in a sample solution to bind on the surface of the coating of the magnetic nanoparticles 100. The magnetic nanoparticles 100 having the analytes binded on its coated surface can then be rapidly removed from the sample solution by applying a magnetic field to the sample solution having the magnetic nanoparticles 100.

(15) FIG. 1A illustrates an embodiment of the preparation of the present magnetic nanoparticles 100. The magnetic nanoparticle 100 can include a core diameter to coating thickness ratio of from about 60:1 to about 30:5. For example, the core diameter to coating thickness can be in a ratio of about 30:6, such as 10:1, 20:3, 10:2, 30:3, or 30:4. In one example the magnetic nanoparticle 100 can include a size of from about 9 nm to about 40 nm, such 40 nm, such as from about 20 nm to about 30 nm. The core 10 can include a diameter of from about 5 nm to about 20 nm, such as from about 7 nm to about 15 nm or less, for example from about 10 nm to about 15 nm or less, such as about 8.8 nm or less, about 8 nm or less, or about 7.9 nm. The coating 20 substantially encapsulating the core 10. In some examples, the coating 20 can include a thickness of from about 0.5 nm to about 7.5 nm, from about 1 nm to about 2 nm, for example, from about 1.5 nm to about 2 nm. FIG. 1B illustrates the magnetic nanoparticle 100 having a particle size of about 15 nm with a coating thickness of about 2.5 nm.

(16) The ratio of the core diameter to the thickness of the silica coating 20, in combination of the characteristics of the silica coating 20 allows the magnetic nanoparticles 100 to aggregate and create a cluster as shown in FIGS. 2A and 2B. In an example, the clusters can be from about 10 nm to about 1000 nm, such as from about 20 nm to about 500 nm, from about 30 nm to about 400 nn, from about 40 nm to about 300 nm, or from about 50 nm to about 190 nm. FIG. 2C shows laser diffraction data measured with a Mastersizer 3000 from Malvern Panalytical, showing the distribution of particle sizes in a typical aqueous dispersion of silica-coated Fe.sub.3O.sub.4 nanoparticles. Individual particles up to clusters approximately 1 micron in diameter are present in the dispersion with the average cluster size being 128-174 nm in diameter.

(17) The present nanoparticles show high magnetic susceptibility in manual and automated workflows. This is particularly desirable in automated platforms where the timing and efficiency of magnetic particle capture is important, requiring rapid and quantitative particle collection at capture, wash, and elution steps.

(18) In some embodiments, the core of the nanoparticle is mostly or entirely composed of a crystalline lattice of Fe.sub.3O.sub.4. In some embodiments, the core of the nanoparticle is mostly or entirely composed of a single nanoscale crystal of magnetite (Fe.sub.3O.sub.4) or single domain magnetic nanoparticle of Fe.sub.3O.sub.4 that displays paramagnetism.

(19) In some examples, the magnetic nanoparticle has a Brunauer-Emmett-Teller (BET) surface area of at least 105 m.sup.2/g, such as at least from about 110 to about 180 m.sup.2/g or at least from about 110 m.sup.2/g to about 130 m.sup.2/g. The BET surface area of the nanoparticle can be determined by the nitrogen adsorption technique. For example, nitrogen adsorption/desorption isotherms can be measured at liquid nitrogen temperature (196 C.) using a Micromeritics ASAP2020 volumetric adsorption analyzer for mesoporosity determination.

(20) The silica-coated magnetic nanoparticles described herein can be used directly or functionalized to suit specific application needs. Functional groups such as carboxylate, epoxide, and tosylate can be included in or added to the silica coating, and such functional groups can be a convenient method for covalent functionalization. As further examples, the silica coating can also be modified by incorporating one or more types of organic groups into the silica matrix. Organic groups can covalently functionalize the surface and/or the pores of the silica coating. As another example, polymers can also functionalize the silica surface either by a simple coating or covalently attached.

(21) The coatings of the silica magnetic nanoparticles described herein can include pores that extend to the surface of the magnetic nanoparticle. The coating can be modified with functional groups both inside and outside its pores. In some examples, the coating further comprises a reactive chemical moiety configured to bind an analyte, such as a nucleic acid.

(22) Compositions Comprising Magnetic Nanoparticles

(23) The present technology provides compositions that can include a plurality of the magnetic nanoparticles described herein. The plurality of magnetic nanoparticles have a mean particle size of about 15 nm or less, or about 12 nm or less, or about 11 nm or less. The composition is a stable suspension of the magnetic nanoparticles in a liquid medium, such as water. In some examples, the magnetic nanoparticles are present in the stable suspension at a concentration of from 3 to 30 g/L, or from 10 to 20 g/L, or about 13 g/L.

(24) In some examples, the nanoparticles remain suspended for at least 6 months at a temperature of 25 C., alternatively for at least 9 months.

(25) The present technology also includes packaged stable suspensions of magnetic nanoparticles. The magnetic particles or compositions described herein can be provided in a sealed package, wherein the interior volume of the sealed package is a suspension of nanoparticles in water.

(26) Methods of Making Magnetic Nanoparticles

(27) The present technology provides a method of making silica-coated magnetic nanoparticles. The present method for producing silica-coated magnetic nanoparticles includes synthesizing magnetic iron oxides from aqueous Fe.sup.3+/Fe.sup.2+ salt solutions through the addition of a base (such as ammonia or NaOH) under an inert atmosphere at room temperature. Fe.sub.3O.sub.4 particles may be made using coprecipitation. The size, shape, and composition of the magnetic nanoparticles depends on the type of salts used (e.g. chlorides, sulfates, nitrates), the Fe.sup.3+/Fe.sup.2+ ratio, the reaction temperature, and pH value. Once the synthetic conditions are fixed, the final characteristics of the magnetite nanoparticles synthesized become fully reproducible. The advantages of this technique in forming Fe.sub.3O.sub.4 nanoparticles include rapid formation of particles, use of inexpensive solvents, magnetic separation for isolation of product, controlled particle size and morphology, reproducible magnetic properties, and the ability to perform synthesis of nanoparticles on larger industrial scales.

(28) The methods include preparing an aqueous solution of Fe.sup.3+ and Fe.sup.2+ ions in deionized, de-oxygenated water in a Fe.sup.3+/Fe.sup.2+ molar ratio of about 2/1. In some examples, the concentration of Fe.sup.3+ in the aqueous solution is from about 2 to about 50 mmol, and the concentration of Fe.sup.2+ in the aqueous solution is from about 1 to about 25 mmol. The aqueous solution can be prepared by dissolving FeCl.sub.3 and FeCl.sub.2 in deionized, de-oxygenated water, though other iron salts may be used as well. The water can be de-oxygenated by any suitable technique, such as by sparging with an inert gas, sonicating, and/or stirring, for a sufficient de-oxygenating period before combining the water with iron salts. The de-oxygenating period will generally be at least two hours, though longer or shorter periods may be employed.

(29) The aqueous solution of Fe.sup.3+ and Fe.sup.2+ ions may then be sonicated, and optionally its temperature may be adjusted to about 35 C., such as by heating the aqueous solution from room temperature. A base may be added to the heated aqueous solution under an inert atmosphere. In some examples, suitable bases include ammonia, sodium hydroxide, and others. The base is added in an amount sufficient for formation of Fe.sub.3O.sub.4 cores from the aqueous solution. For example, the base may be added at a 2-fold or greater (e.g., 4-fold or 8-fold) molar excess. As a result of adding a sufficient quantity of base, a mixture is formed which includes Fe.sub.3O.sub.4 nanoparticles and alkaline water.

(30) The mixture is maintained under an inert atmosphere without stirring for a settling period, during which the Fe.sub.3O.sub.4 nanoparticles settle out of the alkaline water. In some examples, the settling period is overnight, or about 12 hours; in other examples, the settling period is at least 4, 6, 8, 12, or 18 hours, and/or no more than 60 hours, 48 hours, 36 hours, 24 hours or 20 hours; the foregoing values can be combined to form a settling period range. After the settling period, most of the alkaline water is removing from the mixture via aspiration. This provides a concentrated mixture of Fe.sub.3O.sub.4 nanoparticles in alkaline water.

(31) A de-oxygenated alcohol is added to the concentrated mixture to form an aqueous alcohol mixture. The aqueous alcohol mixture is stirred and sonicated to suspend the Fe.sub.3O.sub.4 nanoparticles. In some examples, additional base is added to the aqueous alcohol mixture before further processing.

(32) While sonicating and maintaining an inert atmosphere, a silicate solution is added to the aqueous alcohol mixture. The silica coating thickness can be controlled by the amount of the reactants used. The silicate solution may be added dropwise to the aqueous alcohol mixture while stirring and sonicating the mixture. The silicate solution includes tetraethyl orthosilicate (TEOS) and an alcohol. In some examples, the silicate solution is anhydrous.

(33) This mixture is stirred for a coating period under the inert atmosphere, during which a silica coating forms on the Fe.sub.3O.sub.4 nanoparticles. In some examples, the coating period is overnight, or about 12 hours; in other embodiments, the coating period is at least 4, 6, 8, 12, or 18 hours, and/or no more than 60 hours, 48 hours, 36 hours, 24 hours or 20 hours; the foregoing values can be combined to form a coating period range.

(34) The silica-coated Fe.sub.3O.sub.4 nanoparticles are separated from the alcohol aqueous mixture by any suitable separation technique. In some examples, the Fe.sub.3O.sub.4 nanoparticles are separated by magnetic capture from the aqueous alcohol mixture. The silica-coated Fe.sub.3O.sub.4 nanoparticles can be washed with methanol or other alcohol or solvent. The separated silica-coated Fe.sub.3O.sub.4 nanoparticles can be dried, such as through vacuum drying. A powder of the silica-coated Fe.sub.3O.sub.4 nanoparticles is formed by drying. In some examples, the powder comprising silica-coated Fe.sub.3O.sub.4 nanoparticles is formed without milling or mechanically separating the nanoparticles.

(35) The powder may be dispersed into de-oxygenated water to form a composition. In some examples, the composition is a stable suspension.

(36) Methods of Separating Nucleic Acids from Samples with Magnetic Nanoparticles

(37) The present technology also includes methods for preparing nucleic acids for analysis, by separating the nucleic acids from a sample. In some examples, the methods comprise the steps of combining a sample comprising nucleic acids with a binding medium and a magnetic nanoparticle composition as described herein in a vessel. The nucleic acids bind to the coating of the magnetic nanoparticles. After a binding period, the nucleic acid-bound magnetic nanoparticles are separated from the binding medium within the vessel. The nucleic acid-bound magnetic nanoparticles are contacted with a washing solution to remove unwanted contaminants and then contacted with an elution medium, thereby separating the bound nucleic acids from the magnetic nanoparticles. The magnetic nanoparticles are drawn by a magnet and the resulting clarified solution containing the desired nucleic acids are removed from the vessel, thereby providing a nucleic acid preparation.

(38) The magnetic nanoparticles and compositions described herein have many uses, such as for separation of analytes such as small molecules, protein, and/or nucleic acids from biological samples. The magnetic nanoparticles and compositions may be used as carriers for chemical or biological species, including, for example, noble metal particles, small organic or inorganic molecules, DNA, peptides or polypeptides (e.g. antibodies and other proteins), and whole cells. Applications for such carriers may include magnetic resonance imaging (MRI), optical imaging, targeted drug delivery, and cell delivery.

(39) The present technology provides a method for preparing nucleic acids for analysis. The method comprises the steps of combining a sample comprising nucleic acids with a binding medium and a magnetic nanoparticle composition as described herein in a vessel. The nucleic acids bind to the coating of the magnetic nanoparticles. After a binding period, the nucleic acid-bound magnetic nanoparticles are separated from the binding medium within the vessel. In some examples, the nucleic acid-bound magnetic nanoparticles are then washed with a washing medium comprising aqueous alcohol or a variety of water miscible organic solvents, for example acetonitrile, acetone and sulfolane. Most of the liquid (binding medium and/or washing medium) is then removed from the vessel while taking care to retain the nucleic acid-bound magnetic nanoparticles within the vessel. The nucleic acid-bound magnetic nanoparticles are contacted with an elution medium, thereby separating the bound nucleic acids from the magnetic nanoparticles. The nucleic acids are removed from the vessel, thereby providing a nucleic acid preparation.

(40) The coating of the magnetic nanoparticles may comprise a binding moiety configured to selectively bind nucleic acid or another analyte.

(41) The separation of nucleic acids from a sample is illustrative of separating other analytes, such as small molecules such as pharmaceutical agents, proteins or polypeptides, lipids, or other analytes.

(42) The present method may be employed to normalize the amount of nucleic acids obtained from a sample. In such methods, the sample includes an input amount of nucleic acids, and the magnetic nanoparticle composition has a binding capacity for an output amount of nucleic acids. The output amount is less than the input amount.

(43) The magnetic nanoparticle composition may have a normalization factor between 0.8 and 1.2, wherein the normalization factor refers to the concentration value of nucleic acids recovered from magnetic silica nanoparticles after exposure to a high concentration of nucleic acids divided by the concentration value of nucleic acids recovered from magnetic silica nanoparticles after exposure to a low concentration of nucleic acids. Ideally for compositions prepared for NGS, the normalization factor will be about 1.0.

(44) The nucleic acid preparation includes nucleic acids having lengths within a predetermined length range. In some examples, the minimum nucleotide length is 50 nucleotides (nt), or 70 nt, or 75 nt, or 80 nt, or 100 nt, or 150 nt, or 200 nt, or 500 nt, or 2,000 nt, or 5,000 nt, or 10,000 nt, or 50,000 nt, or 100,000 nt; the maximum nucleotide may be 2 million nt, or 1 million nt, or 500,000 nt, or 200,000 nt, or 75,000 nt, or 25,000 nt, or 12,500 nt, or 6,000 nt, or 3,000 nt, or 1,500 nt, or 750 nt, or 400 nt, or 250 nt; any of the foregoing minimum and maximum may be combined to form a desired nucleotide length range, so long as the minimum is smaller than the maximum.

(45) The present method may be employed with the input sample includes a low amount of nucleic acid, for example, less than 1,000 ng, or less than 500 ng, or less than 200 ng, or less than 100 g, or less than 50 ng of nucleic acid.

(46) The present technology provides a method of obtaining RNA from a biological sample and preparing the RNA for analysis. In the method, a biological sample comprising RNA is placed in a vessel. The biological sample may be a cell or a virus.

(47) The method includes contacting the biological sample with guanidine thiocyanate (GTC) and Proteinase K to form a sample mixture, which is incubated at an elevated temperature (such as about 60 C.) for a sample preparation period (such as 10 minutes or less, or about 5 minutes). The GTC and Proteinase K may be added separately or premixed and added together. After the sample preparation period, neat alcohol is added to the sample mixture in the vessel, and one of the magnetic nanoparticle compositions described herein is also added to the sample mixture in the vessel. The sample mixture and the magnetic nanoparticles are mixed and incubated for a second incubation period (such as 10 minutes or less, or about 5 minutes).

(48) After the second incubation period, a magnetic force is applied to separate the magnetic nanoparticles from a supernatant within the vessel. The supernatant is removed from the vessel without disturbing the separating magnetic nanoparticles, which are then washed one or more times with an alcohol solution. Suitable alcohol solutions may comprise 80% v/v or greater of a lower alcohol such as methanol, ethanol, n-propanonl, iso-propanol, or a mixture thereof. A substantial variety of non-alcohol organic solvents that are miscible with water are also useful for the washing purpose and are known to those skilled in the art. Several of these include, but are not limited to acetonitrile, acetone and sulfolane. The separated magnetic nanoparticles are dried, and an elution medium is added to the vessel. The magnetic nanoparticles are mixed with the elution medium for an elution period (e.g., less than 10 minutes), after which RNA is eluted from the magnetic nanoparticles to form an eluate. The eluate containing RNA is removed from the vessel, and the RNA may then be subjected to analysis such as quantitative reverse transcription polymerase chain reaction (QRT-PCR) for diagnostic and research purposes or can be modified to serve as a substrate for nucleotide sequencing such as Sanger and Next Generation [NGS] sequencing. Alternatively, the removed eluate may be placed in a second vessel which is sealed and stored at a reduced temperature, such as for shipping or later analysis.

(49) In some examples, one, several or all steps of the foregoing methods are performed by an automated instrument.

Terminology

(50) It is to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting. The defined terms are in addition to the technical and scientific meanings of the defined terms as commonly understood and accepted in the technical field of the present teachings.

(51) The term nucleic acid and polynucleotide are used interchangeably herein to describe a polymer of any length, e.g., greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, greater than 10,000 or more bases, composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically which may hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. Naturally occurring nucleotides include guanine, cytosine, adenine, thymine and uracil (G, C, A, T, and U respectively).

(52) The term complementary, complement, or complementary nucleic acid sequence refers to the nucleic acid strand that is related to the base sequence in another nucleic acid strand by the Watson-Crick base-pairing rules. In general, two sequences are complementary when the sequence of one can hybridize to the sequence of the other in an anti-parallel sense wherein the 3-end of each sequence hybridizes to the 5-end of the other sequence and each A, T/U, G, and C of one sequence is then aligned with a T/U, A, C, and G, respectively, of the other sequence.

(53) The term duplex means at least two sequences that are fully or partially complementary undergo Watson-Crick type base pairing among all or most of their nucleotides so that a stable complex is formed. The terms annealing and hybridization are used interchangeably to mean the formation of a stable duplex.

(54) The terms hybridization, and hybridizing, in the context of nucleotide sequences are used interchangeably herein. The ability of two nucleotide sequences to hybridize with each other is based on the degree of complementarity of the two nucleotide sequences, which in turn is based on the fraction of matched complementary nucleotide pairs. The more nucleotides in a given sequence that are complementary to another sequence, the more stringent the conditions can be for hybridization and the more specific will be the hybridization of the two sequences. Increased stringency can be achieved by elevating the temperature, increasing the ratio of co-solvents, lowering the salt concentration, and the like.

(55) As used in the specification and appended claims, and in addition to their ordinary meanings, the terms substantial or substantially mean to within acceptable limits or degree to one having ordinary skill in the art. For example, substantially cancelled means that one skilled in the art considers the cancellation to be acceptable.

(56) As used in the specification and the appended claims and in addition to its ordinary meaning, the terms approximately and about mean to within an acceptable limit or amount to one having ordinary skill in the art. The term about generally refers to plus or minus 15% of the indicated number. For example, about 10 may indicate a range of 8.5 to 11.5. For example, approximately the same means that one of ordinary skill in the art considers the items being compared to be the same.

(57) In the present disclosure, numeric ranges are inclusive of the numbers defining the range. It should be recognized that chemical structures and formula may be elongated or enlarged for illustrative purposes.

(58) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those working in the fields to which this disclosure pertain.

(59) Before the various embodiments are described, it is to be understood that the teachings of this disclosure are not limited to the particular embodiments described, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present teachings will be limited only by the appended claims.

(60) As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed.

(61) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

(62) All patents and publications referred to herein are expressly incorporated by reference.

(63) As used in the specification and appended claims, the terms a, an, and the include both singular and plural referents, unless the context clearly dictates otherwise. Thus, for example, a moiety includes one moiety and plural moieties.

EXAMPLES

Example 1

(64) In this example, silica-coated magnetic nanoparticles (NP) are made using the method described previously and below. An exemplary procedure to synthesize Fe.sub.3O.sub.4 nanoparticles on the 1 g scale was performed as follows: 400 ml of DI water was extensively de-oxygenated using hard N.sub.2 purging, mechanical stirring, and sonication for 2 hrs. Once de-oxygenated, 2.508 g of FeCl.sub.3 (9.24 mmol) and 0.919 g FeCl.sub.2 (4.62 mmol) were added under nitrogen and additionally purged/sonicated. Over the course of the sonication, the temperature of the water increased to 35 C. Afterwards, 10 ml of 28% ammonia solution was quickly added (0.18 mol, which is approximately a 4 molar excess) to the rapidly stirring and sonicating solution. The solution became instantly black with nanoparticle precipitate. Sonication was stopped and the mixture was stirred under nitrogen for another 4 hours. Afterwards, the particles were left to sit under nitrogen until the next day without stirring. The nanoparticles settled to the bottom of the flask yielding a clear upper aqueous layer and a black bottom layer consisting of the magnetic Fe.sub.3O.sub.4 nanoparticles.

(65) On the next day the settled Fe.sub.3O.sub.4 nanoparticles were isolated by removing via aspiration a clear upper layer of ammonia/water. The remaining solvent (100 ml) was left in place. Three-hundred ml of de-oxygenated ethanol was then added to the black slurry. The particles were stirred and sonicated for 1 hr. to resuspend the nanoparticles. While sonicating, 1.5 ml of TEOS was mixed with 80 ml of anhydrous ethanol and placed in an addition funnel under a nitrogen purge and fitted to the flask. The flask was purged, then sealed under flowing N.sub.2. To the black NP suspension was then added 5 ml of additional ammonia solution (28%). The mixture was again sonicated and mechanically stirred for 10 minutes under a nitrogen flow. The TEOS solution was then added dropwise over the period of about 60 min. to the stirring and sonicating nanoparticles. After addition of the TEOS solution, the stirring was continued slowly overnight under N.sub.2.

(66) On the next day, a magnet was used to isolate the nanoparticles to the bottom of the flask. The clear ethanol/water mixture was decanted off the NPs which were held in place by the magnet. The silica-coated Fe.sub.3O.sub.4 nanoparticles were washed with fresh methanol twice and magnetically separated. The nanoparticles were then dried under a stream of nitrogen to a powder, then dispersed into 100 ml of de-oxygenated water. The NPs were sparged extensively with nitrogen and sonicated before sealing under inert atmosphere yielding a 1.3 wt. % solution of nanoparticles in water.

(67) Samples of the silica-coated Fe.sub.3O.sub.4 nanoparticles were isolated as the dry particles in order to perform various analyses. Scanning electron microscopy (SEM) was used to image the particles to approximate the degree of aggregation and overall size of the individual particles. FIGS. 2A to 2B are scanning electron microscopy (SEM) images of silica-coated magnetic nanoparticle according to the present technology. FIG. 2C shows laser diffraction data for silica-coated magnetic nanoparticles according to the present technology.

(68) Use of the current technique, which includes sonication during the synthesis, aids in reducing the size of the nanoparticles and prevents aggregation of the particles. Estimates by SEM show the final silica-coated Fe.sub.3O.sub.4 nanoparticles are between 10 and 15 nm in diameter. This suggests the core/coating structure is composed of an internal Fe.sub.3O.sub.4 nanoparticle which is approximately 7-10 nm in diameter which incorporates an external shell of silica approximately 1.5 nm in thickness. These sizes have been confirmed using high resolution TEM.

(69) Examples 1-1, 1-2, and 1-3 were prepared using the technique described above, with differing amounts of the TEOS solution. The silica-coated Fe.sub.3O.sub.4 nanoparticles were analyzed by IR and TEM, and they were found to have highly desirable size characteristics and SiO:FeO ratios. FIGS. 2D and 2E are TEM images for Example 1-1, FIGS. 2F and 2G are TEM images for Example 1-2, and FIGS. 2H and 2I are TEM images for Example 1-3. The following table provides the average shell thickness and average core particle size for each of these examples.

(70) TABLE-US-00001 Coating Core particle TEOS ml thickness size Example # (Eq. ml) SiO:FeO (nm) (nm) 1-1 11.3 (1.5) 3.4 1.5 0.3 11.3 3 1-2 7.5 (1.0) 2.4 1.4 0.3 10.6 2.1 1-3 15.0 (2.0) 4.3 2.3 0.6 12.6 3.1
In the foregoing table, the amount of TEOS used for the reaction is provided in volumes and in equivalents. The ratio of SiO to FeO in the resulting silica-coated nanoparticles was determined by IR analysis. The shell thickness and the core particle size were determined based on the TEM analysis.

(71) Analysis of the dried silica-coated Fe.sub.3O.sub.4 nanoparticles using Brunauer-Emmett-Teller (BET) theory indicate the materials surface area exceeds 105 m.sup.2/g, such as 113-127 m2/g, which matches well with the estimated diameter of the particles. Analysis of the nanoparticles was also performed using FT-IR spectroscopy before and after functionalization with the silica shell. FT-IR analysis of the non-coated Fe.sub.3O.sub.4 nanoparticles shows two overlapping bands at 580 cm.sup.1 and 630 cm.sup.1 which are associated with the metal-oxygen FeO stretching vibration in the crystalline lattice of the Fe.sub.3O.sub.4 magnetic phase. After coating the nanoparticles with silica, these bands are joined by additional modes at 3400 cm.sup.1, 1094 cm.sup.1, and 467 cm.sup.1 which arise from silica OH stretching and SiO stretching of the SiO.sub.2 shell (See FIG. 3). The appearance of the new modes confirms encapsulation of the nanoparticles with a thin shell of silica.

(72) Two other batches of silica-coated nanoparticles were prepared in the same manner as Example 1-1, and the coating thickness was assessed. As shown in the following table, the procedure resulted in silica-coated nanoparticles having extremely consistent coating thicknesses, with little batch-to-batch variability.

(73) TABLE-US-00002 SiO.sub.2 Coating Example # thickness (nm) 1-1 1.5 1-1A 1.54 1-1B 1.56

Example 2

(74) In this example, silica-coated nanoparticles were prepared following the procedure set forth in Example 1, except that different amounts of silicate were used in the procedure. The following table summarizes the five different embodiments of magnetic nanoparticles prepared in this Example.

(75) TABLE-US-00003 Example # Fe(III)Cl.sub.3 Fe(II)Cl.sub.2 TEOS SiO2@Fe3O4 IR Ratio 2-1 2.50 g 0.93 g 0.5 ml 1.25 g 1.09 2-2 2.50 g 0.92 g 1.0 ml 1.33 g 1.94 2-3 2.50 g 0.94 g 1.5 ml 1.41 g 2.84 2-4 2.51 g 0.95 g 2.0 ml 1.63 g 3.45 2-5 2.50 g 0.96 g 2.5 ml 1.62 g 4.23

(76) The silica-coated magnetic nanoparticles were analyzed by FT-IR, and FIG. 4 shows the spectra. The particles of the various embodiments have different thicknesses of their silica coatings. The IR analysis demonstrated that the thickness of the silica coating increases linearly with increasing TEOS amounts.

(77) FIG. 5 is a graph illustrating that the thickness of the silica coating increases linearly with increasing TEOS amounts.

(78) FIG. 6 is a scanning electron microscopy (SEM) image of silica-coated magnetic nanoparticle according to the present technology, at a magnification of 250,000. The particles were made with 1.5 ml TEOS and sonication.

(79) FIG. 7 is a transmission electron microscopy (TEM) image of silica-coated magnetic nanoparticle according to the present technology. Individual silica-coated magnetic nanoparticles are visible. Aggregation of particles is also visible. Typical Fe.sub.3O.sub.4 cores range from 7 to 10 nm. The average silica coating thickness on the particles using 1.5 ml of TEOS was about 1.6 nm. A Fe.sub.3O.sub.4 crystalline lattice is visible inside core, and this indicates the nanoparticles were not oxidized during synthesis or shipping.

Example 3

(80) In this example, several lots of silica-coated magnetic nanoparticles were prepared following the procedure set forth in Example 1, thereby producing Examples 3-1 to 3-14. The surface areas of several of those examples were determined by Brunauer-Emmett-Teller (BET) surface area, and were found to have surface areas between about 113 m.sup.2/g and about 127 m.sup.2/g.

(81) TABLE-US-00004 Example # Surface Area (m2/g) 3-1 113 3-2 116 3-3 127 3-4 120 3-5 122 3-6 122 3-7 125 3-8 116 3-9 3-10 120 3-11 113

Example 4

(82) In this example, RNA was recovered from a sample using magnetic nanoparticles of the present disclosure in both manual and automated protocols. In these methods, 75 L aliquots of a biological sample containing RNA were pipetted into 0.2 ml strip tubes. 75 L guanidine thiocyanate (GTC) containing Lysis Buffer and 2 L of Proteinase K (20 mg/ml) were added to each tube. The tubes were capped, mixed by vortexing, and spun. The tubes were incubated at 60 C. for 5 minutes. After incubation, 75 L of 100% EtOH were added to each tube, along with 2 L of magnetic nanoparticles having a particle size of 10.38 nm, based on a core particle size of 8.82 nm and a coating thickness of 1.56 nm. The tubes were capped, mixed, spun briefly, and incubated for 5 minutes at room temperature. During this second incubation period, RNA from the sample bound to the silica-coated magnetic nanoparticles.

(83) After the second incubation period, the tubes were placed on a magnet for 5 minutes. Then, all supernatants were removed from the tubes without disturbing the NP pellets. 200 L of washing medium (80% EtOH) was added to the pellets in the tubes. The wash medium was removed without disturbing the NP pellet and the wash and removal steps were repeated. The NP pellet was incubated at room temperature for 3 minutes to dry the nanoparticles.

(84) 75 L of elution medium pre-heated to 65 C. was then added to the tubes and it pipetted up and down several times to mix the nanoparticles while avoiding production of air bubbles. The tubes were allowed to stand for 2 minutes, nanoparticles were captured by a magnet and the eluate containing the RNA was recovered and transferred to a fresh tube. The fresh tube was capped and placed on ice.

Example 5

(85) In this example, RNA was recovered from a sample using magnetic nanoparticles of the present disclosure in a manual protocol. FIG. 8A provides a comparison of six nanoparticles (NP) lots for viral RNA extraction using the manual workflow.

(86) Contrived samples were prepared by spiking-in SARS-CoV-2 armored synthetic RNA into Nasal Swab (NS) collected into VTM to the final concentrations of SARS-CoV-2 N1 and N2 targets 10.sup.5, 10.sup.4, 10.sup.3, 10.sup.2, 10, 1 and 0.5 copies per microliter. RNA extraction was performed using 75 ul of each dilution in duplicate following the NAP manual protocol. Shortly, 75 ul of each sample was mixed with 75 ul of the lysis buffer and 2 ul of Proteinase K, incubated at RT for 5 min and at 60 C. for 5 min, then 75 ul of 100% ethanol containing 2 ul of NP beads were added. The following 6 NP lots were used in this experiment: Example 3-3, Example 3-5, Example 3-6, Example 3-7, Example 3-8, and Example 3-9. Samples were mixed and incubated at RT for 5 min to allow nucleic acids bind to magnetic beads, then tubes were transferred to a magnet stand and beads were collected at the bottom side of the tubes (RNA remains bound to magnetic beads at this step). After aspirating the supernatant, beads were washed twice with 80% ethanol, dried for 3 min. at RT and RNA was eluted with 30 ul of Low TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0).

(87) 5 ul of each extracted RNA sample were used in multiplex QRT-PCR reactions with primers and probes specific to SARS-CoV-2 N1 and N2 targets and human RNase P gene. The assay was run on the Agilent AriaMx Real-Time PCR system. Amplification curves of N1, N2 and RP targets presented in FIGS. 8A, 8B and 8C and corresponding Cq's presented in Tables A, B and C. No significant difference was observed between six NP lots and QRT-PCR detection of SARS-CoV2 N1 and N2 RNA targets at 10e50.5 copies/ul and human RP were demonstrated.

(88) TABLE-US-00005 TABLE A SARS N1 Cq (Rn) (copies/uL) Ex. 3-3 Ex. 3-5 Ex. 3-6 Ex. 3-7 Ex. 3-8 Ex. 3-9 100,000.00 16.54 16.31 16.16 16.3 16.39 16.23 10,000.00 19.74 20.15 19.25 19.47 19.3 19.29 1,000.00 22.96 22.9 22.34 22.57 22.71 22.77 100 27.59 26.49 25.77 25.79 26.11 25.94 10 30.29 30.14 29.18 29.21 29.51 30.03 1 33.09 31.63 32.2 32.19 33.52 32.25 0.5 33.29 32.24 31.46 33.54 32.85 31.62 0 No Ct No Ct No Ct No Ct No Ct No Ct

(89) TABLE-US-00006 TABLE B SARS N2 Cq (Rn) (copies/uL) Ex. 3-3 Ex. 3-5 Ex. 3-6 Ex. 3-7 Ex. 3-8 Ex. 3-9 100,000.00 16.82 16.65 16.74 16.81 16.88 17 10,000.00 20.49 20.56 19.9 20.4 20.13 19.83 1,000.00 23.6 23.69 23.54 23.46 23.4 23.76 100 27.79 27.19 26.7 26.69 26.86 26.68 10 31.25 30.79 30.18 29.76 30.64 30.87 1 35.45 33.14 34.19 33.49 34.8 34.69 0.5 35.33 33.69 33.58 37.61 36.15 33.2 0 No Ct No Ct No Ct No Ct No Ct No Ct

(90) TABLE-US-00007 TABLE C SARS RP Cq (Rn) (copies/uL) Ex. 3-3 Ex. 3-5 Ex. 3-6 Ex. 3-7 Ex. 3-8 Ex. 3-9 100,000.00 23.14 22.6 22.91 22.91 23.34 23.18 10,000.00 23 23.38 22.95 23.22 23.12 22.96 1,000.00 23.71 23.77 23.61 23.56 23.69 23.83 100 25.49 24.72 23.92 23.64 24.14 24.05 10 24.23 24.51 24.36 24.13 23.86 23.78 1 23.63 24.25 24.15 23.96 23.85 23.47 0.5 24.32 24.19 23.95 24.37 24.17 23.83 0 24.37 23.77 23.88 23.84 24.55 23.66

Example 6

(91) In the following example, RNA was recovered from a sample using magnetic nanoparticles of the present disclosure in an automated protocol. FIGS. 8D, 8E and 8F shows a comparison of six nanoparticles lots for viral RNA extraction using an automation workflow.

(92) Contrived samples were prepared by spiking-in SARS-CoV-2 armored synthetic RNA into Nasal Swab (NS) collected into VTM to the final concentrations of SARS-CoV-2 N1 and N2 targets 10.sup.5, 10.sup.4, 10.sup.1, 10.sup.2, 10, 1 and 0.5 copies per microliter. RNA extraction was performed using 75 ul of each dilution. Shortly, 75 ul of each sample was mixed with 75 ul of the lysis buffer and 2 ul of Proteinase K, incubated at RT for 5 min and then transferred into the Bravo instrument for an automatic processing. The following NP lots were used in this experiment: Example 3-2, Example 3-3 (from two different vials, designated 3-3a and 3-3b), Example 3-4, Example 3-5, and Example 1-1B. RNA was eluted with 30 ul of Low TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0).

(93) 5 ul of each extracted RNA sample were used in multiplex QRT-PCR reaction with primers and probes specific to SARS-CoV-2 N1 and N2 targets and human RNase P gene. The assay was run on the Agilent AriaMx Real-Time PCR system. Amplification curves of N1, N2 and RP targets presented in FIGS. 8D, 8E and 8F and corresponding Cq's presented in Tables D, E and F. No significant differences were observed between six NP lots and QRT-PCR detection of SARS-CoV2 N1 and N2 RNA targets at 10.sup.5 0.5 copies/ul and human RP were demonstrated.

(94) TABLE-US-00008 TABLE D SARS N1 Cq (Rn) (copies/uL) Ex. 3-2 Ex. 3-3a Ex. 3-4 Ex. 3-5 Ex. 1-1B Ex. 3-3b 100,000.00 15.88 17.79 15.92 15.68 15.66 15.69 10,000.00 19.22 19.32 19.00 18.95 18.83 19.02 1,000.00 22.42 22.54 22.30 23.01 21.93 22.04 100.00 25.68 25.83 26.60 25.96 25.67 25.40 10.00 29.02 29.41 29.12 28.68 29.53 29.14 1.00 32.85 31.85 31.80 32.65 32.94 32.76 0.5 No Ct 33.36 34.03 33.18 33.37 32.72 0 No Ct No Ct No Ct No Ct No Ct No Ct

(95) TABLE-US-00009 TABLE E SARS N2 Cq (Rn) (copies/uL) Ex. 3-2 Ex. 3-3a Ex. 3-4 Ex. 3-5 Ex. 1-1B Ex. 3-3b 100,000.00 16.60 27.46 16.89 16.74 16.42 16.28 10,000.00 19.84 19.91 19.90 19.84 19.59 19.81 1,000.00 23.19 23.43 23.13 23.79 22.88 22.64 100.00 27.03 27.02 27.93 26.87 26.59 26.07 10.00 29.67 29.72 29.63 29.44 29.92 29.80 1.00 33.40 33.22 32.97 32.45 34.28 33.77 0.5 34.76 34.17 33.95 34.12 34.08 33.31 0 No Ct No Ct No Ct No Ct No Ct No Ct

(96) TABLE-US-00010 TABLE F SARS RP Cq (Rn) (copies/uL) Ex. 3-2 Ex. 3-3a Ex. 3-4 Ex. 3-5 Ex. 1-1B Ex. 3-3b 100,000.00 28.72 28.78 28.97 28.89 28.96 29.31 10,000.00 27.77 28.03 27.93 27.82 28.00 27.60 1,000.00 27.96 27.52 27.85 27.98 27.65 27.50 100.00 28.44 28.63 27.93 28.54 28.44 28.14 10.00 28.51 28.96 28.68 28.47 28.44 29.03 1.00 28.88 28.52 28.91 28.71 29.06 28.71 0.5 29.01 29.01 29.20 29.02 28.98 28.98 0 28.13 28.72 28.78 29.28 28.85 29.32

Example 7

(97) In the following example, DNA samples destined for Next Generation Sequencing, referred to as libraries, having different concentrations were normalized, meaning that regardless of the DNA concentration to which magnetic silica nanoparticles were exposed, all resulting DNA concentrations following normalization possessed approximately equal concentrations.

(98) The following steps were all performed at 4 C.

(99) Add 10 L (0.1 g/mL; 1 g) of magnetic nanoparticles and 60 L of DNA binding medium into tubes A1-H1 (nanoparticles lot CS-019A) and A2-H2 (nanoparticles CS-019B) and mix by pipetting.

(100) Add 20 L of 10 nM DNA libraries into tubes A1-D1 and A2-D2 and 20 L of 100 nM DNA libraries into tubes E1-H1 and E2-H2, mix by pipetting and immediately capture the particles.

(101) Wash the captured particles once with 150 L of 80% EtOH, remove supernatants, dry particles, add 10 L of 10 mM Tris-0.1 mM EDTA (pH 8.0), mix, immediately capture particles and perform electrophoretic analysis (HSD1000 TapeStation) on the supernatants.

(102) FIG. 9 shows the results of TapeStation analysis. Despite different concentrations of nucleic acid in the DNA libraries used as inputs for the normalization method, Normalization Factors were 1.1 for CS-019A and 1.0 for CS-019B. Average DNA concentrations from tubes A1 to D1 and E1-H1 (CS-019A) were 9.4 nM and 10.4 nM, respectively. 10.4 divided by 9.4 results in a normalization factor of essentially 1.1. Average DNA concentrations from tubes A2 to D2 and E2 to H2 (CS-019B) were 9.8 nM and 9.7 nM, respectively. 9.8 divided by 9.7 results in a normalization factor of essentially 1.0.

(103) In view of this disclosure it is noted that the present methods, compositions, and systems can be implemented in keeping with the present teachings. Further, the various components, materials, structures and parameters are included by way of illustration and example only and not in any limiting sense. In view of this disclosure, the present teachings can be implemented in other applications and components, materials, structures and equipment to implement these applications can be determined, while remaining within the scope of the appended claims.