RARE EARTH SPATIAL/SPECTRAL BARCODES FOR MULTIPLEXED BIOCHEMICAL TESTING
Hydrogel microparticles spatially and spectrally encoded using upconverting phosphor nanoparticles are described for use in biochemical testing. In each microparticle, upconversion nanocrystals having spectrally distinguishable emission spectra are disposed in different partions of an encoding region of the microparticle.
The present application claims benefit of, and priority to U.S. patent application Ser. No. 14/214,594, filed Mar. 14, 2014, which claims the benefit of U.S. Provisional Patent Application No. 61/801,351, filed Mar. 15, 2013, and U.S. Provisional Patent Application No. 61/800,995, filed Mar. 15, 2013, all of the above applications being herein incorporated by reference in their entirety.
STATEMENT OF GOVERNMENT INTEREST
This invention was made with government support under Contract no. FA8721-05-C-0002 awarded by the U.S. Air Force and under Grant Nos. DMR-1006147 and CMMI-1120724 awarded by the National Science Foundation. The government has certain rights in the invention.
There are many different approaches currently being employed for performing multiplexed assays (e.g., two dimensional surface adsorbed arrays, fluorophore-bead systems, spatially-labeled microparticle systems). The multiplexing capabilities of these techniques may be insufficient to simultaneously probe some complex, heterogeneous biological systems.
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
Additional features, functions and benefits of the disclosed methods, systems and media will be apparent from the description which follows, particularly when read in conjunction with the appended figures.
Embodiments include hydrogel microparticles for use in biochemical or chemical assays, methods of producing the microparticles, and methods of performing biochemical or chemical assays using the microparticles. Each hydrogel microparticle has a probe region including one or more molecular recognition elements and an encoded region. The encoded region includes multiple portions, with each portion including an associated plurality of upconversion nanocrystals (UCNs) with a distinct spectral signature. The multiple portions of the encoding region enable spatial encoding of the microparticle. The associated plurality of UCN for each region are selected from a set of spectrally distinguishable UCN, which enables spectral encoding for each portion of the microparticle. By combining spatial and spectral encoding, the microparticles have massive multiplexing capabilities with superior scaling capability.
The coding scales exponentially as CS for asymmetric particles and as CS/2 for symmetric particles, where C is the number of distinguishable spectral signatures (UCN ‘colors’) and S is the number of spatial features (e.g., microparticle ‘stripes’). For example, for a symmetric microparticle with S encoding portions and a set of C different spectrally distinguishable UCNs, the following equation lists the number of codes or unique identifiers that would be available:
For example, about 20,000 unique identifiers/codes can be generated for a system in which the encoding region of symmetric microparticles has six portions and each portion includes a plurality of UCN selected from a set of five different types of spectrally distinct UCNs. As another example, about 500,000 unique identifiers/codes can be generated for a system in which the encoding region of the symmetric microparticle has six portions and each portion includes a plurality of nanocrystals selected from a set of nine different types of spectrally distinct nanocrystals. Thus, a modest number of colors may be coupled with a similarly modest number of stripes to yield considerable encoding capacities that scale rapidly with incremental changes to either quantity. To increase the encoding capacity, asymmetric microparticles could be employed. For example, an asymmetric microparticle with six portions with each portion including one of nine different types of spectrally distinct nanocrystals would produce over a million unique identifiers/codes.
Some embodiments combine spatial patterning with rare-earth upconversion nanocrystals (UCNs), single wavelength near-infrared excitation and portable charge-coupled device (CCD)-based decoding to distinguish particles synthesized by means of flow lithography. Some embodiments exhibit large, exponentially scalable encoding capacities (>106), an ultralow decoding false-alarm rate (<10−9), the ability to manipulate particles by applying magnetic fields, and dramatic insensitivity to both particle chemistry.
Some embodiments employ a robust encoding method for compatibility with high-throughput particle synthesis and portable CCD-based decoding. In some embodiments, the resulting particles and decoding system exhibit dramatic insensitivity to particle chemistry—enabling tuning of encoding capacity and decoding error rate independently of particle material properties—as well as the capacity for straightforward magnetic manipulation. In the examples described below, the inventors demonstrate quantitatively predictable decoding of both biocompatible particles in challenging, realistic environments. With single-particle encoding capacities in excess of 1 million and error rates of less than 1 part per billion (ppb), some embodiments expand the practically accessible number of codes for applications like multiplexed bioassays by orders of magnitude.
For example, in some embodiments, a first plurality of UCNs with a first spectral signature is disposed in a first portion 31 of the encoded region. A second portion 32 of the encoded region includes a second plurality of UCNs with a second spectral signature different than the first spectral signature. In some embodiments, the encoded region of the microparticle also includes a third portion 33 having a third plurality of UCNs. In some embodiments, the encoded region of the microparticle also includes a fourth portion 34 having a fourth plurality of UCNs. In some embodiments, the encoded region of the microparticle also includes a fifth portion 35 having a fifth plurality of UCNs. The plurality of microparticles in each portion (31-35) of the encoded region is selected from a set of spectrally distinguishable UCNs.
One of ordinary skill in the art in view of the present disclosure would recognize that each microparticle may include an encoding region with fewer than five portions and associated pluralities of UCNs (e.g., four portions, three portions, two portions) or more than five portions and associated pluralities of UCNs (e.g., six portions, seven portions, eight portions, nine portions, ten portions, etc.).
The spectral signature associated with a plurality of UCN disposed in a portion of the encoded region is also referred to herein as the spectral signature of the portion of the encoded region. In some embodiments, two or more portions of the encoded region may have the same spectral signature. In some embodiments, two or more portions of the encoded region with the same spectral signature may be adjacent to each other. In some embodiments, any portions of the encoded region with the same spectral signature must be separated from each other by one or more portions of the encoded region having different spectral signature(s). In some embodiments, each portion of the encoded region must have a spectral signature different from that of every other portion of the encoded region. In some embodiments, one or more portions of the encoded region do not include UCNs so that the portion or portions is “blank” without a spectral signature.
The spectral signature of a UCN includes information associated with the emission spectrum of the UCN that distinguishes it from another type of UCN. In some embodiments, the spectral signature of a UCN or of a plurality UCNs of the same type may include the integrated intensity of emission of one spectral band (or emission in one spectral range) versus another spectral band (or emission in another spectral range). A spectral signature or information regarding a spectral signature may be referred to herein as a spectral code.
One of ordinary skill in the art in view of the present disclosure would recognize that the set of UCN may include fewer than nine (e.g., eight, seven, six, five, four, three, two) or more than nine (e.g., ten, nine, ten, eleven, twelve, etc.) different types of spectrally distinguishable UCNs. Further, one of skill in the art in view of the present disclosure would recognize that UCNs having different spectra than those shown, and UCNs than emit in different bands than those shown, also fall within the scope of embodiments. For example,
The spectral signature of a plurality of UCNs may include information related to the ratio or ratios of the integrated intensities emitted in various bands (e.g., the ratio of the red band to the green band or vice versa, the ratio of the red band to the blue band or vice versa, the ratio of the blue band to the green band or vice versa, or any combination of the aforementioned). These ratios can be defined with respect to the emission spectra of the UCNs. However, in some embodiments, the spectral signature of a plurality of UCNs may include both information regarding the intensity of light emitted in various bands and include information regarding the responsivity of the image sensor to be used. Any detector, image sensor, or imaging device may be employed. For example, the detector or imaging device may be a charge-coupled device (CCD), a photomultiplier tube-based device (PMT), a complementary metal-oxide-semiconductor (CMOS) imaging sensor, an avalanche photodiode array (APD) imaging device, etc In some embodiments, an imaging sensor with more than one color channel may be employed.
For example, the Table 1 below shows the expected spectral response of a CCD device to the emission spectra of the UCN3-UCN7 and UCN10 types of UCNs (see
The inventors have found that employing UCN for identifying each encoded region of a particle has many benefits when compared with other techniques currently used for encoding particles. For example, some other techniques employ one-dimensional or two-dimensional thickness variations or holes in a fluorescently labeled coded region of a microparticle for identification.
In contrast with UCNs having multiple narrow emission bands, commonly used fluorescent labeling molecules (e.g., fluorophores) each tend to emit in a single broad band (e.g., DAPI fluorescent dye has a single emission band that is about 100 nm wide FWHM). In microparticles using fluorophores for encoding, the broad emission bands of the fluorophores limits the number of different fluorophores that may be employed without having significant overlap between emission bands and resulting ambiguity in identification. In addition, the absence of multiple emission bands for a single fluorophore may require the use of an external calibration standard. In contrast, UCNs have multiple narrow emission bands in different portions of the visible spectrum (e.g., separated by tens to hundreds of nm). The ratio of intensity of emission in various bands can be used to distinguish between different UCNs, and also acts as an internal calibration standard, obviating the need for external calibration.
Microparticles using UCNs for encoding may experience less reduction of the signal to noise ratio due to autoluminescence than microparticles using fluorophores for encoding. Luminescent UCNs absorb light in one range of wavelengths and emit light in a shorter range of wavelengths (e.g., absorb in the NIR range and emit in the visible range). In contrast, commonly used fluorophores and quantum dots usually absorb light in a wavelength range and emit light in a longer wavelength range (e.g., absorbing in the ultraviolet range and emitting in the visible range). For example, the commonly used fluorophore 4′,6-diamidino-2-phenylindole (DAPI) has absorption maximum around 370 nm (UV) and an emission maximum around 450 nm (blue). Illumination of the fluorophores for identification (e.g., with UV light) may result in unintended autofluorescence of materials and solvents in the visible wavelengths that decreases the signal to noise ratio, which can be a significant problem with biological samples. Because the UCNs described herein are upconverting, the NIR light used to excite the UCNs generally does not cause autoluminescence in the shorter wavelengths of the visible range. Thus, the use of UCN may improve the signal to noise ratio for an encoded region.
Microparticles using different types of UCNs for encoding may require only a single narrow band excitation source as opposed to microparticles using different types of fluorophores, which may require multiple light sources to provide excitation in different wavelength bands. For example, a 980 nm light source with a power density of less than 10 W/cm2 (e.g., an near infra-red (NIR) laser diode) may be used as a single excitation source for multiple different types of UCNs. In contrast, microparticles using common fluorophores for parts of the visual light spectrum, such as DAPI (blue), Oregon green 500 (green) and ALEXA FLUOR 633 (red) with absorption maximums at 350 nm, 503 nm and 632 nm, respectively, may require multiple different excitation sources such as a UV laser, an argon-ion laser, and a red helium-neon laser.
In some embodiments, the UCNs are rare-earth nanocrystals, which are bright anti-Stokes emitters with tunable spectral properties. Individual UCNs absorb continuous-wave (CW) NIR light at a single wavelength and emit in multiple narrow bands of the visible spectrum. Large anti-Stokes shifts reduce spectral interference from sample autogluorescence and lead to enhanced signal-to-noise ratios. In contrast to M-ink (an optically active dye in which nanostructured magnetic materials reflect different wavelengths of light) or quantum dots, these benefits persist even in the presence of obscurants or a complex reflective background. Tuning of emission intensities in multiple bands by adjusting relative stoichiometries of lanthanide dopants permits ratiometrically unique spectral encoding, in which the ratio of integrated intensities in two or more bands serve as the code, rather than absolute intensity. In some embodiments, external spectral standards (e.g., as required by porous silicon crystals), precise dye loading (e.g., as used with quantum dots and luminex), sensitive instrumentations (e.g. as required by M-Ink), and extensive calibration may be unnecessary for readout, enabling the use of standard CCD imaging for decoding.
Example Synthesis of UCNs
Lanthanide-doped NaYF4 UCNs were made via a scalable batch hydrothermal synthesis, which is only one of numerous known protocols for synthesis of NaYF4 UCNs.
Aqueous rare-earth chloride salts, sodium hydroxide, ammonium fluoride, ethanol and oleic acid were heated in a TEFLON-coated stainless steel pressure vessel. Specifically, 2 ml of ReCl3 (0.4 M, RE=Y, Yb, Er, Gd, Tm) and 2 ml of NH4F (2 M) were added to a mixture of 3 ml of NaOH (0.6 M), 10 ml of ethanol and 10 ml of oleic acid. The solution was transferred to a 50 ml TEFLON-lined autoclave and heated at 200° C. for 2 hours. The resulting products were centrifuged to collect the UCNs, which were then repeatedly washed with ethanol and deionized water and then re-dispersed in cyclohexane.
During synthesis, the inventors used the concentration of various lanthanide dopants and the reaction time and temperature to improve the luminescence intensity of the UCNs and to alter the upconversion spectrum of the nanocrystals.
The synthesis procedure described above can produce NaYF4 UCNs in two different phases having different crystal structures: an α-phase with a cubic crystal structure and a β-phase with a hexagonal crystal structure. Generally speaking, luminescence intensity is significantly higher in β-phase crystals than in α-phase crystals due to the lower ratio of surface defects to crystal volume in the β-phase. Without high levels of gadolinium doping, relatively high temperatures must be maintained for relatively long times (e.g., 350° C. for 24 hours) to induce the α→β phase transition in the UCNs. In contrast, the inventors doped with 30 mol % gadolinium (Gd) to induce the α→β phase transition at a lower temperature (200° C.) held for a shorter time (2 hours). The Gd has little to no effect on the shape of the upconversion emission spectrum generated due to the presence of the other dopants.
Increasing reaction time and increasing reaction temperature tended to increase the luminescence intensity of the UCNs due to increased nanocrystal size. Increasing the UCN size decreases the ratio of surface area to volume for the UCNs, thereby decreasing the ratio of surface defects to crystal volume. Further, luminescence for larger UCNs was less likely to be red-shifted due to preferential quenching of high frequency emission, which can occur in smaller UCNs.
The concentrations of dopants other than Gd were used to change the upconversion emission spectrum. Spectrally distinct UCNs were produced by adjusting the relative stoichiometries of the lanthanide ions Yb3+, Er3+ and Tm3+ in the UCN reaction premix. The lanthanide dopant stoichiometries have relatively little impact on the UCN nanostructure and surface chemistry, decoupling control of the emission spectrum from the particle chemistry and resulting material properties. Ytterbium (Yb3+) is an important dopant for bright multicolor emission, because it acts as a high-NIR absorption cross-section and energy transfer agent for upconverting emission. Increasing the Yb percentage tends to ‘red-shift’ the upconversion spectrum, increasing the ratio of the emission intensity in the red band (640-670 nm) relative to the emission intensity in the green band (520-560 nm) in Erbium (Er3+) co-doped crystals.
The inventors produced ten different types of spectrally distinguishable lanthanide-doped NaYF4 UCNs labeled UCN1-UCN10, whose spectra appear in
The inventors made several different batches of the same type of UCNs to confirm that the emission spectra were consistent from batch to batch. Upconversion luminescence spectra of UCNs were measured in a poly (urethane acrylate) (PUA) prepolymer solution (9/1 PUA/PI (v/v)) with a fluorescence spectrometer with a 1 W CW diode laser (980 nm) used as the excitation source.
The high Gd content of UCN1-UCN10 makes the UCNs paramagnetic and subject to physical manipulation through external magnetic fields. The inventors confirmed this by manipulating the nanocrystals suspended in vials using external ferromagnets.
Example Surface Modifications of UCN
The synthesis process described above produced UCNs capped with oleic acid, a fatty acid with a 17-carbon hydrocarbon tail. As a result of the oleic acid capping, the resulting UCNs were insoluble in aqueous media, which created problems with dispersing the UCNs in aqueous or hydrophilic source materials. Furthermore, the UCNs with oleic acid tails luminesced brightly only in hydrophobic media. Exposure of the oleic acid capped UCNs to water caused significant aggregation and a high degree of reversible luminescence attenuation due to surface defect-mediated quenching.
The inventors utilized a method of modifying the oleic acid tail on the UCNs to improve their solubility in water and increase their luminescence in hydrophilic media. The oleic acid double bond was oxidized to form an alcohol, and then cleaved, thereby releasing the outward-facing hydrophobic part of the oleic acid chain and forming a carboxylic acid group.
The specific procedure employed to modify the oleic acid tail of the UCNs involved adding 0.1 gram of UCNs to a mixture of cyclohexane (100 mL), tert-butanol (70 mL), water (10 mL) and 5 wt % K2CO3 solution (5 mL) and stirring for about 20 minutes at room temperature. Then, 20 mL of Lemieux-von Rudloff reagent (5.7 mM KMnO4 and 0.1 M NaIO4 aqueous solution) was added dropwise to the solution. The resulting mixture was stirred for 48 hours. The product was centrifuged and washed with deionized water, acetone, and ethanol. Subsequently, the UCNs were dispersed in hydrochloric acid (50 mL) of pH 4, and stirred for 1 hour forming carboxyl-terminated UCNs, which were washed 5 times with deionized water and collected by centrifugation. The resulting carboxyl-terminated UCNs dispersed without aggregation in aqueous media and luminesced strongly in hydrophilic media.
The inventors developed a method for modifying the carboxyl-terminated UCN to form acrylate-terminated UCN that could be cross-linked with the hydrogel material of the microparticle. The method included mixing 200 μl of EDC (20 mg/ml) and 200 μl of sulfo-N-hydroxysuccinimide (sulfo-NHS) (20 mg/ml) with 200 μl of carboxy-terminated UCNs in 2-(N-morpholino) ethanesufonic acid (MES) buffer (0.1 M, pH 6.0, 40 mg/ml) and stirring for two hours at room temperature to activate the surface as carboxylic acid groups. The NHS-activated UCNs were centrifuged and washed with water. The precipitate was re-dispersed in 200 μl of PBS buffer (0.1 M, 5 ml, pH 7.2) containing 200 μl of 2-hydroxyethylacrylate (20 mg/ml). The mixture was then stirred for 24 hours at room temperature. The resulting acrylated UCN were purified by repeated centrifugation (3000 rpm, 5 min, 5 times) and resuspended in deionized water.
The first encoded region source material, the second encoded region source material, and the probe region source material are cross-linked forming the first portion of an encoded region 31, the second portion of the encoded region 32, and the probe region 20. The probe region 20 is cross-linked with one or both of the first portion 31 and the second portion 32 of the encoding region to form a contiguous microparticle. In embodiments with more than two portions of the encoded region, each portion is cross-linked with one or more other portions of the encoded region and/or with the probe region.
In some embodiments, the UCNs for at least some of the portions of the encoded region have a hydrophilic surface. In some embodiments, the UCNs for at least some of the portions of the encoded region have a hydrophilic ligand. In some embodiments, providing the first encoded region source material and providing the second encoded region source material may include modifying the first plurality of nanocrystals and the second plurality of nanocrystals to have a hydrophilic surface and/or a hydrophilic ligand. Having a hydrophilic surface and/or a hydrophilic ligand may aid in dispersing the UCNs in the respective source material.
In some embodiments, the UCNs for at least some of the portions of the encoded region have acrylated ligands for cross-linking with the polymers of the hydrogel matrix. In some embodiments, providing the first encoded region source material and providing the second encoded region source material may include modifying the first plurality of nanocrystals and the second plurality of nanocrystals to include acrylated ligands. In some embodiments, the plurality of UCNs is bound to the polymer material at the time of particle synthesis through an acrylate group.
In other embodiments, another type of covalent linkage could be made between the UCNs and the hydrogel matrix. The UCNs can be bound to the hydrogel matrix using any number of covalent attachment mechanisms (e.g., amide linkages, disulfides, esters, ethers, aldehydes/ketones, cycloadditions, click chemistry, azides, and carbamates).
In some embodiments, at least some of the UCNs are doped with rare-earth metals. In some embodiments, at least some of the UCNs are doped with a composition including at least 30 mol % Gd. In some embodiments, at least some of the UCNs are paramagnetic.
In some embodiments, the material for each portion of the encoded region and for the probe region is the same material. In some embodiments, the material for the portions of the encoded region is different than the material for the probe region.
As noted above, in some embodiments, the UCNs have a hydrophilic surface. In some embodiments, the UCNs have a hydrophilic ligand. Having a hydrophilic surface and/or a hydrophilic ligand may aid in dispersing the UCNs in the source material.
In some embodiments, the method also includes co-flowing the source material for each encoded region and the source material for the probe region to an area for cross-linking. For example, a stop-flow lithography (SFL) technique may be employed for forming the microparticles. In SFL, viscous UV-sensitive pre-polymer solutions (which may be referred to herein as source materials) undergo laminar co-flow into a small microfluidic device, which may be made of polydimethylsiloxane (PDMS). For organic synthesis, the microfluidic device may be made from perfluoropolyether (PFPE). The flow of the pre-polymer solutions is stopped for a brief period in which the pre-polymer solutions in the device are exposed to photomask-patterned ultraviolet light. The UV light causes cross-linking, polymerization, or both within milliseconds in the region delineated by the photomask forming micro-sized polymeric particles. The shape of each particle is defined by the photomask. The composition of each striped portion of the particle is determined by the composition of the laminar co-flowing streams (e.g., the source materials). The SFL technique is particularly well suited for spatial and spectral encoding of microparticles using nanocrystals because of the ability to control both overall microparticle particle shape and the composition of different striped portions of the microparticle.
Although photomask 162 is shown having a pattern that only forms one microparticle at a time, in some embodiments, the photomask may have a pattern for forming multiple microparticles simultaneously. In some embodiments, a photomask may have a pattern that produces microparticles having different shapes simultaneously. In some embodiments, the photomask may produce asymmetric particles and/or particles having nonrectangular shapes.
Although microparticle 170 is shown with five encoded regions, in other embodiments, there may be more or fewer than six encoded regions. For example,
For further details regarding the SFL technique for forming hydrogel microparticles, see U.S. Patent Application Publication No. US 2012/0316082 A1, published Dec. 13, 2012, and U.S. Patent Application Publication No. US 2012/0003755 A1, published Jan. 5, 2012, each of which is incorporated by reference herein in its entirety. An exemplary flow lithography system is described below with respect to
Example Production of PEG-DA Hydrogel Microparticles with UCNs
The inventors produced polyethylene glycol diacrylate (PEG-DA) polymer microparticles by stop flow lithography. Initially, the inventors made sets of microparticles, with each set including only one type of nanocrystal to determine whether incorporating the nanocrystals into microparticles changes the emission spectral of the nanocrystals. For each of the nanocrystal types UCN1-UCN10, fifty PEG-DA hydrogel microparticles were produced. A CCD device was used to obtain a three color image (red channel, green channel and blue channel) of each microparticle while illuminated by NIR light producing a red channel image, a green channel image and a blue channel image. For each channel image, the intensity (pixel value) within the boundaries of each microparticle was integrated yielding a “pixel value” for each channel for each microparticle.
The mean measured integrated intensity values from fifty microparticles for each type of UCNs were then compared with the expected integrated intensity data obtained from a convolution of the UCN emission data and the image sensor response curves. Table 3 below includes measured mean integrated intensity data, the standard deviation and the coefficient of variability for UCNs in microparticles. Expected integrated intensity data based on emission spectra from UCNs in solution are also included for comparison. As shown in the table, the mean integrated intensity and the expected integrated intensity values are consistent. The average coefficient of variation across all particles and UCN colors was 2%. This corresponds to an average standard deviation of 2.1 RGB units (on a scale of 255) for separately acquired images of separately synthesized particles, indicating outstanding particle-to-particle reproducibility. In addition, error ellipses are non-overlapping to better than 6 sigma, indicating that decoding error rates of less than 1 ppb are to be expected. Thus, if the emission spectrum of a type of nanocrystals is known, the integrated intensity for detection in a color channel can be reliably predicted.
Thus, the inventors demonstrated noise-robust spectral discrimination of six different types of UCNs in hydrogel particles illuminated using an NIR diode laser and imaged using a standard CCD camera. Further, as shown by the green channel vs. red channel plot, the red channel integrated intensity and the green channel integrated intensity are sufficient to distinguish between the six different types of nanocrystals. The
The inventors also compared different batches of hydrogel microparticles produced at different times to determine the reliability and the predictability of the integrated intensities of microparticles from different batches. Five separate batches of fifty microparticles were produced, each batch including the same UCN4 type nanocrystals. The microparticles were illuminated with an NIR light source and color images were obtained using a CCD camera. Integrated intensity data was generated for microparticles in all five batches and the average integrated intensity values for each batch were compared.
The inventors confirmed that the oxidation and acrylation process does not change an emission spectrum of the UCNs.
The inventors also confirmed that there was no attenuation of the luminescence response of the UCNs integrated into hydrogel microparticles upon prolonged intense NIR irradiation due to photobleaching.
The inventors also compared the stability of hydrogel microparticles made with carboxyl-terminated UCNs, in which the nanocrystals are trapped in pores in the hydrogel matrix, and hydrogel particles made with acrylated UCNs, in which the nanocrystals are bonded to the hydrogel matrix via acrylates.
Example Formation of PEG-DA Hydrogel Microparticles with Spectral and Spatial Encoding
After establishing the predictability and reproducibility of the method for forming UCNs and the predictability and reproducibility of the spectra from hydrogel particles that each include only one type of UCNs, the inventors produced PEG-DA hydrogel microparticles with both spectral and spatial encoding. PEG-DA microparticles are biocompatible and mesoporous allowing diffusion of large biological macromolecules. Stable integration of UCNs into microparticles involved use of hydrophilic surface chemistry with a UV-active functional group on the UCNs for strong, covalent incorporation as described above. In an embodiment in which the hydrogel is more densely cross-linked covalent incorporation of the UCNs may not be needed. In an embodiment in which the UCNs are disposed in a non-hydrogel portion of the particle, covalent incorporation of the UCNs may not be needed. For example, in microparticles with a hydrogel probe region and a PUA encoded region, the dense cross-linking of the PUA and the hydrophobic surface chemistry large, rod-like UCN nanostructure may enabled homogeneous and irreversible physical entrainment of the UCNs in the PUA portion.
Specifically, elongated hydrogel microparticles were produced that each included a probe region and an encoding region. The encoding region was divided into five portions, (e.g., five stripes) with each portion including a plurality of UCNs having distinguishable spectral signature. Although the microparticles produced included five portions of a encoded region, in some embodiments, each microparticle may have an encoded region with more than five portions or less than five portions. Although the hydrogel microparticles produced were rectangular and elongated, in some embodiments, the hydrogel microparticles may have a different aspect ratio and/or a different shape. Further, the microparticles produced may be symmetric or asymmetric.
The microparticles were produced by SFL using encoding region source materials and a probe region source material. Specifically, for each encoding region source material, acrylated UCN were dispersed in a PEG-DA premixture solution yielding a mixture of 45 vol % PEG-DA (Mn=700), 40 vol % UCNs (0.5 mg/μl) 10 vol % poly(styrenesulfonate) PSS and 5 vol % DAROCUR 1173 photoinitiator (PI)). For microparticles with a PUA encoded portion, the PUA microparticle source material comprised 150 mg of UCNs dispersed in 300 μl of a 9:1 volume ratio PUA/PI solution.
The probe region source material employed a similar PEG-DA premixture solution that also included molecular recognition element, specifically a nucleic acid probe for miRNA target molecules. The source materials were used to form microparticles using SFL as described above with respect to
A microfluidic device was fabricated from poly-dimethylsiloxane (PDMS) for the SFL system. PDMS was mixed with a curing agent in a 10:1 ratio and degassed under vacuum for 30 min. Degassed PDMS was poured onto an SU-8 master mold and cured overnight at 65° C. Channels were then cut out of the mold and bonded with a glass slide coated with partially-cured PDMS in order to assure oxygen permeability. The assembled device was fully cured overnight at 65° C. The microfluidic channel in the microfluidic device of the SFL system was 300 μm wide and 36 μm high.
A photomask for the SFL was designed using a computer added drafting program and printed with a high-resolution printer. The mask was placed in the field-stop of a microscope before synthesis. A microfluidic device was fabricated from poly-dimethylsiloxane (PDMS) for the SFL system. PDMS was mixed with a curing agent in a 10:1 ratio and degassed under vacuum for 30 min. Degassed PDMS was poured onto an SU-8 master mold.
The microfluidic channel of the SFL system was loaded with the composite monomer solution, aligned on a microscope stage, and subjected to a pressure-driven flow. In every synthesis cycle, the monomer flow was halted (350 ms) and particles were photo-polymerized in the device using UV light filtered through a dichroic filter set (365 nm wavelength light for 100 ns exposure tine). The polymerized particles were then covected into a collection tube for 500 ms. Synthesis occurred at a rate of ˜5 particles per second. After synthesis the particles were rinsed. The PEG particles were rinsed 3 times with 1×TET (1×TE with 0.05% (v/v) Tween 20).
Although PEG-DA and PUA were used for the hydrogel microparticles and partial hydrogel microparticles in the examples described herein, any di-acrylated monomers that have been used in stop-flow lithography may be used for the encoded region. Further, any di-acrylated monomers into which UCNs (either nanocrystals with modified surfaces or ligands or nanocrystals with unmodified surfaces or ligands) may be well-dispersed can be employed.
In an initial batch of encoded hydrogel microparticles used for testing, each portion of the encoded region included a plurality of nanocrystals selected from the set of types UCN3, UCN4, UCN5 and UCN7, whose characteristics are described above. As used herein, encoded microparticles refers to microparticles that each have one or more portions of the encoded region and that each have one or more types of spectrally distinguishable UCNs. Eight encoded microparticles were illuminated with the NIR diode laser and imaged using a standard CCD image sensor. The integrated intensity was calculated for the red and green channels of the image sensor.
PEG-DA particles with distinct coding and bioassay regions were synthesized, each including an encoding region with five encoding regions (i.e., 5 stripes) yielding an encoding capacity on the order of 105. One set of the synthesized particles contained a microRNA (MiRNA) probe for miR-210 and another contained a probe for mi-R221.
The inventors produced microparticles having two different codes for use in a multiplexed assay. Microparticles with the first code (UCN4, UCN5, UCN3, UCN7, and UCN4 or 45734) included a probe region with a molecular recognition element for 210 miRNA (miR-210). Microparticles with the second code (47534) included a molecular recognition element for 221 miRNA (miR-221). Images of the two encoded microparticles under NIR illumination are shown in
In order to compare performance of spectrally-encoded nanoparticle-containing hydrogel microparticles particles to hydrogel particles without UCNs particle, a batch of “standard” hydrogel particles including a miR-221 probe region flanked by two control regions were synthesized. The standard particles had no encoding region and no UCNs. Both the encoded hydrogel microparticles and the standard hydrogel microparticles had probe regions of identical dimensions to ensure similar mass transport and reaction inside the gel network.
The spectrally-encoded hydrogel particles functionalized with DNA capture probes for miR-210 and miR-221 were used in a microRNA assay to demonstrate specific and multiplexed detection of the two targets. In the multiplexed assay, microparticles with the two different codes were exposed to four different sample solutions: one containing 500 amol of miR-210, one containing 500 amol of miR-221, one containing 500 amol of both 221 miR-210 and miR-221, and one including neither. This enabled evaluation of encoded microparticles both with regard to standard particles and with regard to specificity. Post-target incubation, bound miRNA targets were labeled using a biotinylated universal linker sequence and a streptavidin-phycoerythrin (SA-PE) fluorophore, and imaged under fluorescence.
Assay reactions were carried out in a final volume of 50 μL inside a 0.65 mL Eppendorf tube. Each reaction contained a total of 75 particles (25 particles of each type: (standard miR-221, spectrally-encoded miR-221, spectrally-encoded miR-210)). Target incubations were carried out in microRNA hybridization buffer for 90 minutes at 55° C. using a thermoshaker (1500 RPM). Post-incubation, particles were rinsed with three 500 μl volumes of microRNA rinse buffer (RB) using centrifugation. After each rinse, supernatant was manually aspirated, leaving 50 μL of solution and particles in the reaction tube. A volume of 235 μL of a ligation mastermix, which was prepared using 100 μL 10×NEB2, 900 μL TET, 800 U/mL T4 DNA ligase, 40 nM biotinylated universal linker sequence, and 250 nM ATP, was then added to the reaction for a 30 minute incubation at 21.5° C. and 1500 RPM. Microparticles were rinsed three more times using microRNA RB and incubated with streptavidin-phycoerythrin at a final concentration of 2 μg/mL for 45 minutes at 21.5° C. and 1500 RPM. After three more rinses with microRNA RB, particles were exchanged into PTET (TET with 25% (v/v) PEG-200) for imaging. DNA sequences for the two probes and the universal linker appear in Table 5 below.
The composite images shown in
Although the microparticles produced included a nucleic acid probe for miRNA as a molecular recognition element, one of ordinary skill in the art would recognize that many different types of molecular recognition elements could be employed and incorporated into the probe region source material of various embodiments. For example, other types of molecular recognition elements that could be employed include, but are not limited to various nucleic acids, miRNA, ssDNA, proteins, receptor proteins, antibodies, enzymes, peptides, aptamer, avimers, Fc domain fragments, phage, carbon nanotube sensors, peptides, etc. Any existing molecular recognition element, biological or not, compatible with the particle synthesis process, may be incorporated.
Although the microparticles produced only included one probe region, in some embodiments, each microparticle may include more than one probe region. For embodiments with more than one probe region, the different probe regions may have different types of molecular recognition elements. In some embodiments, multiple types of molecular recognition elements may be incorporated into one probe region. Although the microparticles produced include a probe region that is distinct from the encoded region, in some embodiments, the probe region may partially or completely overlap with one or more portions of the encoded region.
Example Formation of Hydrogel Microparticles with PEG-DA Probe Region and PUA Encoded Region
As noted above, after the inventors selected polyethylene glycol diacrylate PEG-DA as a suitable biocompatible polymer for forming the hydrogel of the probe region, it was discovered that oleic-acid capped UCNs do not disperse in the PEG-DA. Instead the oleic-acid capped UCNs aggregated forming clumped distributions in the PEG-DA pre-polymer solution, which led to clumped distributions of UCNs in the microparticles. Before the inventors developed the method of modifying the oleic acid group to form carboxyl-terminated UCNs, the inventors initially employed a hydrophobic polymer, polyurethane acrylate (PUA) (specifically MINS-300 produced by Minuta Tech, Co. Ltd. of Gyeonggi-Do Korea) for the encoded region in an attempt to address the problem of UCN aggregation. The nanoparticles dispersed well in the MINS-300 hydrophobic PUA, but the MINS-300 hydrophobic PUA wouldn't form a robust cross-linked interface with the PEG-DA of the probe region. The inventors then selected another PUA that is only slightly hydrophilic (specifically MINS-0311 produced by Minuta Tech, Co. Ltd. of Gyeonggi-Do Korea) for the encoded region. The UCNs did not disperse as well in the slightly hydrophilic second PUA MINS-0311; however, the MINS-0311 second PUA formed a robust interface with the PEG-DA of the probe region upon cross-linking. Using the MINS-0311 second PUA as the polymer for the encoded region source materials, the inventors were able to achieve some dispersion of the UCNs in the portions of the encoded region and a robust interface with the PEG-DA probe region.
Any di-acrylated monomers that have been used in stop-flow lithography may be used for the encoded region. Further, any di-acrylated monomers into which UCN (either UCNs with modified surfaces or ligands or UCNs with unmodified surfaces or ligands) may be well-dispersed can be employed.
A method of reading out the spectral codes of a microparticle is described with respect to
Specifically, images of particles with 6 stripes were taken via a CCD decoder and loaded into image processing and analysis software (e.g., MATLAB by Mathworks of Natick, Mass.). Particle boundaries were defined using a grayscale intensity-based edge detection algorithm. Boundary pixel x and y values were averaged to determine the particle centroid. Boundary pixels with minimum and maximum x and y values (four points total) were noted, and distances between adjacent points used to determine the particle end point, or the pixel located on the 2nd shortest edge of the particle boundary and its longitudinal axis. The end pixel and centroid pixel were then used to determine both the code orientation and a director for the particle's longitudinal axis. The centroid of each striped region of the particle was determined by segmenting the particle into six regions (the number of stripes were presumed known a priori) along its longitudinal director. In other embodiments, k-means image segmentation algorithms may be employed to define regions of the particle based on color, without a priori knowledge of the number of particle stripes. RGB values were measured by averaging pixels within each of the six striped regions of particles under test were compared against training RGB values and standard deviations, as determined from a particle training set. If an average set of RGB values fell within 3.5 standard deviations of a training RGB value, the values were determined to match. In this way, ‘analog’ RGB sequences were translated into ‘digital’ sequences of spectral signatures.
To test the identification, multiple microparticles were generated with a “true code” and some with a different “false code” as shown in
Further details regarding an exemplary system of particle synthesis are provided below.
The versatile, high-performance stop-flow lithography (SFL) systems and techniques described herein are a high throughput process for synthesizing particles. In a semicontinuous process, multiple coflowing laminar streams—each containing a single optically active UCN moiety or probe molecule—are convected into a microchannel (e.g., formed from poly(dimethylsiloxane) (PDMS) or a non-swelling thiolene-based resin for use with organic solvents), stopped, and photopolymerized in place via mask-patterned ultraviolet light (365 nm) to form barcoded particles at a rate of 18,000 particles/hr, which are then displaced when flow resumes. This ˜104 particles/hr synthesis rate is by no means limiting; hydrodynamic flow focusing has been used to increase the synthesis rate for similar particles to over 105 particles/hr. The synthesis platform may also be constructed using commercial off-the-shelf parts and free-standing optics. Parallelization in an industrial setting, with no further optimization, could readily increase the facility-scale synthesis throughput by orders of magnitude to meet industrial demand.
In describing exemplary embodiments, specific terminology is used for the sake of clarity. For purposes of description, each specific term is intended to at least include all technical and functional equivalents that operate in a similar manner to accomplish a similar purpose. Additionally, in some instances where a particular exemplary embodiment includes a plurality of system elements, device components or method steps, those elements, components or steps may be replaced with a single element, component or step. Likewise, a single element, component or step may be replaced with a plurality of elements, components or steps that serve the same purpose. Moreover, while exemplary embodiments have been shown and described with references to particular embodiments thereof, those of ordinary skill in the art will understand that various substitutions and alterations in form and detail may be made therein without departing from the scope of the invention. Further still, other aspects, functions and advantages are also within the scope of the invention.
Exemplary flowcharts are provided herein for illustrative purposes and are non-limiting examples of methods. One of ordinary skill in the art will recognize that exemplary methods may include more or fewer steps than those illustrated in the exemplary flowcharts, and that the steps in the exemplary flowcharts may be performed in a different order than the order shown in the illustrative flowcharts.
1. A microparticle for use in a biochemical or chemical assay, the microparticle comprising:
- a body comprising a hydrogel, the body including a probe region and an encoded region;
- a first plurality of upconversion nanocrystals disposed in a first portion of the encoded region, the first plurality of upconversion nanocrystals having a first spectral signature; and
- a second plurality of upconversion nanocrystals disposed in a second portion of the encoded region spatially separated from the first portion of the encoded region, the second plurality of upconversion nanocrystals having a second spectral signature different than the first spectral signature.
2. The microparticle of claim 1, wherein the second spectral signature is different than the first spectral signature.
3. The microparticle of claim 1, wherein the first plurality of upconversion nanocrystals includes a first material doped with one or more rare earth elements and the second plurality of upconversion nanocrystals includes a second material doped with one or more rare earth elements.
4. The microparticle of claim 1, wherein the body is asymmetric in shape.
5. The microparticle of claim 1, wherein the upconversion nanocrystals are covalently bound to the hydrogel material.
6. The microparticle of claim 1, wherein the upconversion nanocrystals are bound to the hydrogel material at the time of particle synthesis through an acrylate group.
7. The microparticle of claim 1, wherein for each portion of the encoded region, the plurality of upconversion nanocrystals are dispersed without aggregation.
8. The microparticle of claim 1, further comprising a third plurality of upconversion nanocrystals disposed in a third portion of the encoded region spatially separated from the first portion of the encoded region and spatially separated from the second portion of the encoded region, the third plurality of upconversion nanocrystals having a third spectral signature different than the first spectral signature.
9. The microparticle of claim 8, wherein the encoded region includes at least five different portions.
10. The microparticle of claim 1, wherein each spectral signature includes luminescence in multiple distinct bands within a range of 400-800 nm.
11. The microparticle of claim 1, wherein the probe region comprises polyethylene glycol diacrylate (PEG-DA).
12. The microparticle of claim 1, wherein the encoded region comprises di-acrylated monomer.
13. The microparticle of claim 1, wherein the probe region comprises a first polymer material and the encoding region comprises a second polymer material different than the first polymer material.
14. The microparticle of claim 1, wherein the probe region includes one or more molecular recognition elements.
15. The microparticle of claim 1, wherein the upconversion nanocrystals are paramagnetic or ferromagnetic.
16. A method of making a hydrogel microparticle for use in a biochemical or chemical assay, the method comprising:
- providing a first encoded region source material including a hydrogel and a first plurality of upconversion nanocrystals having a first spectral signature;
- providing a second encoded region source material including a hydrogel and a second plurality of upconversion nanocrystals having a second spectral signature;
- providing a probe region source material including a hydrogel;
- cross-linking the first encoded region source material, the second encoded region source material and the probe region source material forming a first portion of an encoded region, a second portion of the encoded region and a probe region with the probe region crosslinked with one or both of the first portion and the second portion of the encoded region to form a contiguous microparticle.
17. The method of claim 16, wherein the second spectral signature is different than the first spectral signature.
18. The method of claim 16, wherein each of the first plurality of upconversion nanocrystals and each of the second plurality of upconversion nanocrystals has a hydrophilic surface.
19. The method of claim 16, wherein the probe region source material includes one or more molecular recognition elements.
26. A method of performing a biochemical or chemical assay comprising:
- exposing a sample to a plurality of microparticles, each microparticle comprising: a body comprising a hydrogel, the body including a probe region with one or more molecular recognition elements and an encoded region; a first plurality of upconversion nanocrystals disposed in a first portion of the encoded region, the first plurality of upconversion nanocrystals having a first spectral signature; and a second plurality of upconversion nanocrystals disposed in a second portion of the encoded region spatially separated from the first portion of the encoded region, the second plurality of upconversion nanocrystals having a second spectral signature different than the first spectral signature;
- for each microparticle, illuminating the microparticle with an excitation light source;
- for each microparticle, detecting light emitted from the illuminated microparticle, the detected light including upconverted luminescent light from the first plurality of upconversion nanocrystals and the second plurality of upconversion nanocrystals and light associated with the one or more molecular recognition elements; and
- identifying each microparticle based on the detected light.