Patent Application
20210338211
This application is in the field of medical devices.
In recent years, mechanisms have been developed that analyze tears for biomarkers of a variety of diseases through collection of basal tears followed by bioassays (for example, U.S. Patent Application Publication No. 2016/0003786 entitled “Methods of Detecting Cancer”). However, a fundamental issue preventing the effective use of tear fluid for diagnostics is the difficulty of collecting and analyzing tears from patients. The most commonly employed methods, Schirmer strips and microcapillary tubes have major flaws that prevent them from being broadly utilized in clinics. In particular, there are major issues with extraction of proteins from Schirmer strips, whereas microcapillary tubes require long collection times during which patients keep their eyes open while the clinician or technician must steadily hold the tube near the patient's eye. Both methods require relatively long chair times in order to extract relatively small volumes of tears (typically 1-10 μL), making detection of low concentration biomarkers exceedingly difficult. Furthermore, different methods of collection have been shown to yield different collected tear compositions, which, in conjunction with differences in chosen methods of tear biomarker analysis, have produced widely differing, often conflicting, reports of tear composition. Furthermore, these tests only show a snapshot of a patient's medical condition at the time at which the tears were collected, and therefore analysis results may be subject to potentially large fluctuations in tear composition throughout a given day. In order for tear biomarkers to become clinically useful, there is a critical need to establish a less invasive, convenient, standardized, repeatable method for their collection, as well as one that can yield information of a patient's medical condition over a continuous, extended period of time.
Recently, methods have been proposed and claimed for analyzing analytes in tear fluid by collecting tear fluid in a contact lens (for example, U.S. Pat. No. 7,429,465 B2 entitled “Process for Analyzing Tear Fluid” and U.S. Pat. No. 9,320,460 B2 entitled “In-Situ Tear Sample Collection and Testing Using a Contact Lens”) or by continuously monitoring analytes through an embedded sensor in a contact lens (for example, U.S. Pat. No. 6,312,393 B1 entitled “Contact Device for Placement in Direct Apposition to the Conjunctive of the Eye”). A contact lens, however, is not an ideal platform for detecting or capturing analytes in tear fluid, as not everyone can tolerate putting in and removing contact lenses. They may cause irritation to the eye, and they generally cannot be worn continuously for periods longer than a day. In particular, continuous wearing of contact lenses may lead to corneal hypoxia and eye infections. Furthermore, the efficiency of tear collection of contact lenses is imperfect because it is unlikely that all tear fluid impregnates a contact lens before draining through the lacrimal puncta. Another major issue is contact lenses alter the physiology of tears, and therefore they cannot act as a perfectly passive platform for evaluating the physiology of tears. Additionally, the form factor and types of materials used for analyte capture are limited in a contact lens, as any opaque material (for example, iron oxide or gold nanoparticles) would have to be limited to the periphery of the lens so as not to create an optical or aesthetic defect in the lens.
An ocular device designed to be inserted into the eye of a patient and continuously capture specific analytes from tear fluid may be described herein. The ocular device may be inserted into a lacrimal punctum, a conjunctival sac, or the like. A number of forms of the device may be described herein, including one that contains one or more open channels and/or pores that enable tears to drain naturally through the device while specific analytes are captured. A specific mechanism of detecting analytes in bodily fluids using materials comprised of DNAzymes and/or aptamers may be described herein. Aptamers may be oligonucleotide or peptide molecules that bind to a specific target molecule. DNAzymes are a class of catalytic oligonucleotides designed to catalyze a number of biological reactions such as RNA cleavage, DNA cleavage, ligation or phosphorylation reactions. A number of ways an ocular insert may capture analytes in tear fluid in vivo for subsequent in vitro analysis may be described herein. Additionally, a specific mechanism for colorimetric detection of analytes using aptamer- and/or DNAzyme-crosslinked hydrogels may be described herein.
An ocular device described herein may be inserted into a puncta (for example, tear ducts), a conjunctival sac, built into a contact lens, or the like. For ocular devices that may be inserted into the puncta at the upper and/or lower eyelids to prevent drainage of basal tears, helping to treat for dry eye syndrome may be described herein. These plugs are typically silicone-based, cause minimal-to-no irritation to the patient, and may be safely left in the puncta for years if not physically dislodged, providing a positive role in the treatment of patients suffering from dry eyes, with little to no negative side effects.
A person's tears may be used to monitor various physiological and/or biochemical states (for example, presence of vitamins, glucose level, and the like), and even to diagnose diseases based on distinct biomarkers present in the tear fluid. Similar to blood, tears are representative of the biochemical composition of the body. There are various kinds of tears including basal tears, reflex tears (for example, resulting from exposure to tear gas), and psychic tears (for example, released when crying). Basal tears are continually produced, and their components may include, but are not limited to, glucose, minerals (for example, iron), vitamins, neurotransmitters, metabolites, amino acids, urea, anti-oxidants, polynucleotides, and many proteins and/or their associated metabolites. Recently, the analysis of toxic heavy metals in tears (for example, lead and arsenic) was shown to be a promising diagnostic tool, as significantly different ranges of concentrations of these heavy metals were detected in representative rural versus urban populations.
An ocular insert with surface-bound capture agents (for example, as antibodies or aptamers) that capture specific analytes in tear fluid for subsequent in vitro analysis after removing the insert, may be advantageous to previously described ocular inserts that have built-in sensors that attempt to perform analysis in vivo. For example, they do not require the complex circuitry, reagents or calibration methods to be confined to the ocular insert and may instead rely on optimizing the actual analysis of captured tear analytes using a much broader range of available techniques and instruments. In addition an ocular insert with surface-bound capture agents that bind to specific tear analytes may be advantageous over ocular inserts that merely collect tears. For example, such methods may only be able to extract relatively small volumes of tears and making analysis of many of the most promising biomarkers of diseases in tears, which have extremely low concentrations (e.g. 1-1000 pg/ml) is exceedingly difficult.
A capture agent may be designed to specifically bind to one (or sometimes several) specific analytes. An example of a capture agent used in molecular biology is an antibody, which specifically binds to one protein out of often thousands typically present in a bodily fluid.
One advantage of surface-bound capture agents, described herein, are the ability to continuously concentrate targeted analytes. This is especially crucial for biomarkers that are present in tears at very low concentrations and thus very difficult to analyze. For example, many cytokines, which are promising diagnostic biomarkers, are present in tears at concentrations at the 1-10 pg/ml scale. For a typical 1-10 microliter tear sample collected by conventional methods, this means there are only 0.001-0.1 pg of cytokine to analyze. On the other hand, an ocular insert that continuously collects low concentration biomarkers such as cytokines, may collect 1-10 pg over the course of the day, making subsequent analysis of the targeted biomarker significantly more reliable.
Punctal inserts, in particular, may offer benefits as a platform for an embedded analyte or biomarker collector because they are low cost, easy to insert, less invasive and less cumbersome to wear and maintain than contact lenses, and, because virtually all tear fluid drains through the two approximately 500 μm wide lacrimal puncta in each eye, thereby having direct interaction with tear fluid without actually coming in contact with the eye. A variety of materials and methods may be used for analyte capture, including opaque materials, because ocular inserts may be designed to not obstruct the vision of the wearer and, with the exception of a contact lens inserts, are not as easily seen by an external observer.
A specific mechanism of detecting analytes in bodily fluids using materials comprised of DNAzymes and/or aptamers may be described herein. DNAzymes are a class of catalytic nucleic acids designed to catalyze a number of biological reactions such as RNA cleavage, DNA cleavage, ligation or phosphorylation reactions. As described herein, “DNAzyme” is meant to broadly encompass all types of systems containing DNAzymes. DNAzymes have been used as a mechanism for designing a variety of different types of sensors with both high sensitivity and high specificity. Their stability in non-controlled environments makes them particularly attractive platforms for sensors.
A number of ways an ocular insert may capture analytes in tear fluid in vivo for subsequent in vitro analysis may be described herein. In another embodiment, an indirect mechanism for detecting and/or quantifying analytes in biofluids by immersing a material comprised of DNAzymes in a biofluid (which may be tears, blood, urine, sweat, or saliva) may be described herein. However, instead of directly relying on analyte capture, the DNAzymes themselves may be analyzed.
Additionally, a specific mechanism for colorimetric detection of analytes using aptamer- and/or DNAzyme-crosslinked hydrogels may be described herein. Aptamers are similar to DNAzymes in that they are oligonucleotides that selectively bind to an analyte, but unlike DNAzymes, they do not catalyze a subsequent reaction. Both aptamer and DNAzyme crosslinked hydrogels have been demonstrated to undergo cleavage upon reacting with a target analyte, leading to dissociation of the hydrogel network. Aptamer and DNAzyme hydrogels have been used as colorimetric biosensors, however, the reported methods are not ideal for operation in vivo. For example, many of these reported methods rely on release of a cargo encapsulated by the DNA-crosslinked hydrogel. However, release of cargo is not ideal for colorimetric reporting in a dynamic and open biological environment (such as in the eye). Another clever device for colorimetric detection of analytes may be comprised of capillary tubes plugged with DNAzyme-crosslinked hydrogels. In the absence of the target analyte, the hydrogel may prevent flow of liquid into the capillary, but when the target analyte is present the DNAzymes may cleave, causing the hydrogel to dissolve and fluid to flow into the capillary, generating a visual change in the capillary that may be detected by the naked eye. This mechanism, however, may not enable easy visual detection in a sensor as compact as one built into an ocular insert.
A mechanism that solves these problems by using a DNAzyme hydrogel whose sole function is to visually conceal a visually distinctive material underneath may be described herein. Upon dissolution or washing away of the DNA-crosslinked hydrogel, the visually distinctive material may be revealed and thus observable by either a detector or the naked eye. For example, such a device may be comprised of fluorescent quantum dots embedded in a polymer (so as to remain mechanically stable), and above this fluorescent material, a DNAzyme-crosslinked hydrogel that may contain a dye that masks the fluorescent quantum dots from an observer and/or from the fluorescent excitation wavelengths of light. Upon dissolution of the opaque hydrogel, the fluorescent quantum dots may be revealed and thus optically detectable. This mechanism may provide a number of benefits over existing methods of DNAzyme and/or aptamer-based colorimetric sensing. For example, it may not rely on releasing any cargo, or on binding fluorescent dyes and quenchers to a DNAzyme (which can be expensive), and instead, may enable the device to be comprised of commercially available color-signaling materials (for example, commercial quantum dots) that have already been optimized for stability and reliability in biological environments.
One application for the methods described herein may be to provide a simple, inexpensive platform for continuously monitoring exposure to pathogens from the environment using easily removable porous lacrimal punctal inserts functionalized for viral capture. These punctal insertions may capture virus particles, bacteria or other pathogens that come in contact with the eye or that may be present in tears due to an existing infection. On a regular basis, the inserts may be removed for analysis and then replaced.
In an exemplary embodiment, a lacrimal punctal insert may be functionalized with a surface-bound capture agent designed to capture one or multiple target analyte(s) from tears. The analyte(s) may be specific organic compounds, pharmacological agents, metal ions, viruses, bacteria, fungi, enzymes, extracellular vesicles, allergens, RNA, DNA, proteins, peptides or other biomarkers.
The punctal insert may or may not be permeable. For example, a permeable punctal insert may be permeable to tears to enable a higher surface area for tears to interact with analyte capture agents. In an alternative example, the punctal insert may only have the capture agent immobilized on a top surface of a completely impermeable punctal plug. The punctal insert may be a specific kind of punctal insert that blocks tears from draining.
The punctal insert may contain a porous and/or permeable material for tear collection and analyte capture, but may not enable tears to continue flowing through the lacrimal puncta. This may, for example, help treat certain forms of dry eyes.
The punctal insert may be entirely comprised of porous and/or permeable materials or that contains one or more channels to enable tears to diffuse or flow through the insert into the lacrimal canaliculi.
The punctal insert may be comprised of one or more of the following scaffold materials that immobilize the capture agents : hydrogels, cellulose fibers, microparticles, porous filter membranes, nano- or microgels (for example, Nanotrap® particles), porous inorganic materials (for example, silicon, silica, ceramics, activated carbon, or graphite), magnetic particles (for example, Mag4C viral capture nanoparticles or Magnetofection™ nanoparticles), non-magnetic particles (for example, carbon, gold, silver, silica or alumina) or molecularly imprinted polymers (used to bind specific analytes based on a lock and key inspired model). The one or more of these materials for analyte capture may contain one or more functional groups. The functional groups may be comprised of one or more of the following: a charged organic molecule or chemical moeity, a charged polymer or oligomer, a hydrophobic polymer or oligomer, an aptamer, an antibody, a peptide, a protein, a bacteriophage, a DNAzyme, a nanozyme, a ligand or a chelator.
The punctal insert may be designed for size exclusion. A size exclusion material may include a porous polymer or inorganic material having a specific pore size, a hydrogel having a specific mesh size based on its swollen state in tear fluid, a size exclusion “gel” comprised of packed hydrophobic particles, or a hydrophobic polymer or inorganic material containing channels of a specific size and geometry (such as the herringbone channels).
The punctal insert may be designed for ion exclusion. For example, ion inclusion may include ion selective membranes.
The punctal insert may be combined with a colorimetric indication mechanism (for example, fluorescence, luminescence, optical absorbance, and/or reflectance) as described by US20180289326A1, which is incorporated herein by reference.
The punctal insert may include a surface-bound capture agent that specifically binds at least one target analyte from tear fluid. There may be several options for surface-bound capture agents. For example, capture agents may bind directly to the surface of the punctal insert, either the outer or inner punctal insert may contain a channel or pore. In another example, capture agents may bind to a separate scaffold material that is contained with an open channel within the punctal insert. An example of this may be an antibody-bound cellulose paper contained within a hollow channel of the punctal insert.
Using an antibody may allow for specific capture of 1 targeted protein of the approximately 1500 proteins that may be present in tears. The targeted proteins may be free proteins or proteins bound to the surface of a biological particle. For example, the biological particle may be an extracellular vesicle, a virus, a bacterium, or the like. Exosomes are a type of extracellular vesicle of particular interest because the exosomes of breast cancer patients have been found to contain specific breast cancer biomarkers
In another example, to determine if someone has COVID-19 antibodies in their tears, the spike protein of SARS-COV-2 may be incorporated into the punctal insert. The punctal insert may then scavenge any SARS-COV-2 antibodies that flow through it.
In another example, to capture a specific strand of DNA present in tears, the complementary DNA strand may be used in the punctal insert.
The capture agent may include a metal-binding ligand, an aptamer, or DNAzymes. The aptamer may be a polypeptide or polynucleotide. One way of synthesizing an aptamer or DNAzyme may be through the SELEX process (as well known in the art).
Importantly, in all cases, the capture agent is immobilized on the surface of a scaffold material to ensure the capture agent remains contained within or on the punctal insert in the presence of continuously draining tear fluid. In some embodiments the scaffold material may be embedded within an open channel that penetrates through the punctal insert. In other embodiments, the punctal insert itself may act as the scaffold material. For example, the punctal insert may be entirely made out of a microporous hydrogel or foam. Typical materials that may be used as the scaffold are silicones, silica, metal oxides, metal phosphates and biocompatible polymers such as poly(HEMA), polysaccharide based polymers (e.g. cellulose, agar, alginic acid, and chitosan), polypeptide based polymers or gels (e.g. gelatin or crosslinked proteins such as bovine serum albumin).
The scaffold material of the punctal insert may consist of labeled microparticles. For example, labeled microparticles may be fluorescently or colorimetrically tagged microspheres. An example of commercially available fluorescently-tagged beads may be Luminex MagPlex Microspheres. Labeled microparticles are particularly convenient for analysis as they can be easily purified via centrifugation, or magnetic separation in the case of magnetic microparticles, as well as aliquoted for multiple analyses from the same sample. To prevent microparticles from leaching out of the punctal insert during in vivo analyte capture, the labeled microparticles may be contained within the open channels of the insert by one or more membranes, which may be removed after removing the punctal insert from the patient to release the microparticles for in vitro analysis.
In addition to fluorescent or colorimetric dyes, microparticles may be labeled by a compound that is released and recognized by a chromatograph and/or mass spectrometer, an enzyme, fluorescent quantum dots, or a chemo-, electro- or photo-luminescent particle or dye.
The capture-agent-immobilized scaffold material may contain a surface-bound blocking agent that prevents non-specific binding of proteins in tears. This may also be known as biofouling. Typical fouling resistant blocking agents may be bovine serum albumin (BSA), polyethylene glycol, zwitterionic polymers, and the like.
In an example method for in vivo capture of an analyte and subsequent in vitro analysis of the analyte, the punctal insert may be inserted into one or more lacrimal puncta of a host enabling collection of target analyte(s) for a predetermined period of time (for example, 15 minutes for a quick analysis to days or weeks for a time-averaged analysis of a patient's tear biomarker profile). The punctal insert(s) may then be removed and the contents of the punctal insert(s) analyzed. The analysis method depends on the nature of the one or more capture agent(s). In cases where the capture agents consist of antibodies, the captured biomarkers may be directly analyzed via a colorimetric, fluorescence, electroluminescence, and photo-luminescence or chemoluminescence immunoassay. Alternatively, captured biomarkers may first be released from antibodies via an eluting solution (for example, 6M guanidine-HCl) and subsequently analyzed via liquid chromatography mass spectrometry (LC-MS), MALDI-TOF or gel electrophoresis.
A visible cue of a health condition, for example, a change in color or fluorescence, may be generated, after which the punctal plug may be removed and the contents of the punctal insert(s) analyzed.
The punctal inserts may be analyzed using polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), nicking enzyme amplification reaction (NEAR), loop mediated isothermal amplification (LAMP), RT-LAMP, enzyme-linked immunosorbant assay (ELISA), or genomic sequencing.
The punctal inserts may be analyzed using nuclear magnetic resonance (NMR), mass spectrometry, high performance liquid chromatography (HPLC), and/or elemental analysis.
In an example method of analyte detection and/or quantification in a bodily fluid (for example, tears, saliva, blood, sweat or urine), a sensor, specifically containing DNAzymes, may be put in contact with bodily fluid, removed, and the contents analyzed. The content may be analyzed to determine if either any of the DNAzymes reacted with an analyte (qualitatively) and/or the concentration or number of DNAzymes that reacted with an analyte (quantitatively), while the DNAzyme-sensor was in contact with the bodily fluid.
The sensor may be put into contact with a bodily fluid so as to undergo reaction in vivo in the presence of a specific analyte (if that analyte is present), and subsequently, the DNAzyme device may be removed for in vitro analysis.
In another example method of analyte detection, cleavage of a certain concentration of DNAzymes and/or aptamers in a DNAzyme-linked and/or aptamer-linked hydrogel sensor leads to a colorimetric signal may be detectable by the naked eye. After the colorimetric signal is detected, the contents of the sensor may be analyzed as previously described herein.
The material comprised of DNAzymes and/or aptamers may be analyzed using NMR, mass spectrometry, HPLC, PCR, RT-PCR, NEAR, LAMP, RT-LAMP, DNA sequencing, RNA sequencing, and/or elemental analysis.
In another example, a colorimetric device for optical detection of an analyte may be described herein. The colorimetric device may be comprised of a fluorescent or luminescent material or a material of an easily distinguishable pattern or color. Overlaying this optically distinctive material may be a hydrogel that conceals the visually distinctive material from a detector and/or the naked eye. The material may be crosslinked by DNAzymes and/or aptamers that cleave upon interaction with the analyte. Upon cleavage of a significant amount of DNAzyme (and/or aptamer) crosslinks, the concealing hydrogel may wash away, dissolve or be collected in a separate chamber of the device, thereby revealing the optically distinctive material to the optical detector and/or naked eye.
The colorimetric device may be embedded within a permeable lacrimal insert. The colorimetric device may be designed to display an optical signal in response to an analyte in tear fluid.
In an example method, once the ocular insert is removed from the patient, all loosely bound tear constituents may be washed out of the insert to yield a purified sample of concentrated targeted biomarkers ready for analysis. Specifically for protein capture, performing an immunoassay directly on the capture-agent immobilized scaffold material. An example of a sandwich-type immunoassay adapted for our system is may be as described herein.
For example, the lacrimal punctal insert may contain microparticles as the capture agent scaffold. After removing the lacrimal punctal insert from the patient, one end of the insert is either punctured or a membrane at one end is physically removed to release the particles for performing flow cytometry and/or a bioassay.
In some embodiments the detection antibody may already be tagged with the fluorescence, color or luminescence inducing moiety. Alternatively, the detection antibody may be labeled with a reactive functional group, in which case additional steps may be added for introducing a fluorescence/colorimetric/luminescence generating moiety that then binds with the functional group on the detection antibody (these variations are all well known in the art). For example, using a biotin-labeled detection antibody and a streptavidin labeled dye.
In another example, specific to aptamers or DNAzymes, attaching a FRET-based dye system (as is well known in the art) that produces a fluorescence signal upon binding of the target analyte may be used. In this example, a signal may be produced in vivo upon binding of the analyte with the capture agent. The signal may be observed both by eye qualitatively as well as quantitatively via in vitro analysis.
In another method, a bioassay may be performed directly on the punctal insert. This method may be particularly useful for inserts that are transparent (for example, silicone) but may also be done on light-scattering inserts. In one example of this method, the lacrimal punctal insert may be removed from the patient and inserted into a microfluidic device. The microfluidic device may be used to inject solutions to perform a subsequent bioassay. For example, the microfluidic device may be used to efficiently wash out any unbound tear constituents as well as flow in reagents for performing the bioassay. In specific cases where the captured analytes are meant to be removed prior to analysis (for example, for performing LC-MS) the microfluidic device could also inject an eluting buffer to release the captured analytes from the punctal insert.
In some embodiments, a microfluidic device may also contain the components necessary to perform the bioassay immediately after the reagents are introduced. For example, a microfluidic device with an integrated light source and photodetector could be used to perform a fluorescence immunoassay.
In another example method, the scaffold material may first be removed or exposed from the insert. A bioassay may then be performed on the removed/exposed scaffold.
The capture agent of the lacrimal punctal insert may be bound to a surface on or within the punctal via a cleavable bond. A cleavable bond may be any bond that can be cleaved under relatively mild conditions. For example, the capture agent may be bound to a surface via a disulfide bond, which is relatively stable in vivo. After removing the lacrimal punctal insert from the patient, the disulfide bond may be cleaved under reducing conditions (for example, using dithioreitol or TCEP) to release the capture agents along with any analytes bound to the capture agents.
In some embodiments, two or more types of capture agents may be bound to the ocular insert in order to simultaneously capture two more specific analytes in tear fluid. After removing the insert from the patient, the concentrations of the two or more specific analytes may be quantitated with respect to one another in order to yield a multiplex bioassay.
This application claims priority from U.S. Provisional Application No. 63/019,008 filed May 1, 2020, which is incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 63019008 | May 2020 | US |
