Patent Application
20240012002
This application was filed with a Sequence Listing XML in ST.26 XML format accordance with 37 C.F.R. § 1.831. The Sequence Listing XML file submitted in the USPTO Patent Center, “208192-9121-US02_sequence_listing_30-MAY-2023.xml,” was created on May 30, 2023, contains 2 sequences, has a file size of 2.93 Kbytes, and is incorporated by reference in its entirety into the specification.
Described herein are systems and methods for a microfluidic immunoassay for in situ mass spectrometry analysis of intracellular protein biomarkers in tissue. In some embodiments, the tissue may comprise human brain tissue. In some embodiments, the protein biomarkers may comprise Aβ species comprising monomers and oligomers of Aβ1-42, Aβ1-40, Aβ1-39, Aβ2-43, or combinations thereof. In some embodiments, the systems and methods may comprise laser capture microdissection (LCM) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.
Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by the aggregation of amyloid-β peptide (Aβ) and tau protein. Protein aggregation starts decades before most individuals present any dementia symptoms. Although the pathophysiology of AD is still not well understood, a well-supported hypothesis suggests that soluble Aβ oligomers (aggregate intermediates along the formation of plaques) are the main neurotoxic species involved in the pathological cascade of the disease, however, their role is yet to be fully established. An improved understanding of the Aβ species and their abundance in neurons will contribute to elucidating the role of Aβ in AD, developing therapeutic and diagnostic tools, and advancing the cure of this devastating disease.
Aβ peptides are the proteolytic products of the amyloid protein precursor (APP), cleaved into fragments with amino acid lengths ranging from 37 to 43 residues. It has been proposed that AD is caused by an imbalance between Aβ production and clearance. Aβ aggregates in the extracellular matrix into plaques. However, the soluble Aβ species that can localize intracellularly are among the likely culprits for the cognitive impairment and neurotoxicity in AD pathogenesis. A wealth of data is available about Aβ through in vitro studies and animal models; however, given the complexity of the human nervous system, assessment of oligomeric Aβ in the human brain can provide information more relevant to human AD. Clinically annotated human brain samples from tissue banks represent a golden opportunity to perform such studies.
Immunohistochemistry and immunofluorescence methods can effectively localize proteins in tissue, but they are semi-quantitative techniques and depend on the availability of tags for target molecules. Mass spectrometry (MS) approaches allow for quantitative unbiased studies and biomolecule identification. Brain homogenates, widely used in combination with immunoprecipitation (IP) and MS analysis, such as liquid chromatography (LC) MS/MS, electrospray ionization (ESI) MS, or matrix-assisted laser desorption ionization (MALDI) MS, do not provide information with spatial resolution. Fortunately, MS can be performed with microscopic amounts of tissue. MALDI MS Imaging (MALDI MSI) is a widely used technique for tissue protein profiling, which provides label-free detection and mapping of multiple analytes. Limitations in MALDI MSI may arise for low abundant analytes, which can be masked by other highly abundant species. In addition, MALDI MSI often requires confirmation by other techniques such as LC-MS.
Laser capture microdissection (LCM) allows for selectively excising single cells from specific tissue regions of interest. This method has been widely used in single-cell genomics. Yet, proteomics can provide information on phenotype, post-translational modifications, protein concentration, and protein-protein interactions. While there are technologies to address the genome and transcriptome of single cells, there is still a lack of technology for quantitative analysis of the intracellular protein content of specific subpopulations in tissues. The proteomic analysis of small cell populations must avoid sample-processing dilution and loss of precious minute analyte amounts. However, by assessing the entire proteome, low-abundance proteins, as is the case of intracellular Aβ oligomers, can be masked by high-abundance ones. A combination of nanoliter droplet arrays with MALDI-MS was used for the analysis of proteins secreted by encapsulated cells. This approach, however, is inadequate for the assessment of the intracellular content of individual tissue cells.
What is needed are novel systems and methods for a microfluidic immunoassay for in situ mass spectrometry analysis of intracellular Aβ species in tissue, such as human brain tissue.
One embodiment described herein is a method for analyzing tissue for the presence of Aβ-M and Aβ-O species, the method comprising: providing a sample of tissue comprising cells; microdissecting the cells and transferring the cells to an upper chamber of a manifold comprising a plurality of layered wells each comprising an upper chamber and a lower chamber, each chamber comprising independent fluidic connections and an adjustable valve separating the upper chambers and lower chambers; assembling the manifold on an indium-titanium oxide coated glass slide; introducing one or more anti-Aβ antibodies into the lower chamber of the layered well containing the cells in the upper chamber, incubating for a period of time, and washing the layered well; opening the adjustable valve separating the upper chamber and lower chamber to permit the cells in the upper chamber to contact the one or more anti-Aβ antibodies in the lower chamber, incubating for a period of time, and washing the layered well to remove non-captured material; introducing a matrix solution and allowing crystallization; and removing the manifold and analyzing a co-crystallized sample using mass spectrometry to identify the presence of the Aβ-M and Aβ-O species. In one aspect, the tissue is human brain tissue comprising human brain cells. In another aspect, the manifold is comprised of a polymeric material comprising poly(dimethylsiloxane) (PDMS), polycarbonate (PC), poly-methyl-meta-acrylate (PMMA), cyclic olefin copolymer (COC), polyimide, or combinations thereof. In another aspect, the manifold is comprised of PDMS. In another aspect, the one or more anti-Aβ antibodies comprises an Aβ specific antibody, an amyloid oligomer-specific antibody, or a combination thereof. In another aspect, the one or more anti-Aβ antibodies comprises an immunoglobulin G (IgG) 6E10 antibody. In another aspect, the matrix solution comprises α-cyano-4-hydroxycinnamic acid or sinapinic acid in acetonitrile and trifluoroacetic acid. In another aspect, the mass spectrometry comprises matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In another aspect, each layered well comprises a well area size ranging from about 50 μm×about 50 μm to about 500 μm×about 500 μm. In another aspect, each layered well comprises a well area size of about 500 μm×about 500 μm. In another aspect, microdissecting the cells comprises laser capture microdissection (LCM). In another aspect, each layered well comprises from about 1 to about 100 individual cells. In another aspect, each layered well comprises from about 1 to about 20 individual cells. In another aspect, the Aβ-M species comprise monomers of Aβ1-42, Aβ1-40, Aβ1-39, Aβ2-43, or combinations thereof. In another aspect, the Aβ-O species comprise oligomers of Aβ1-42, Aβ1-40, Aβ1-39, Aβ2-43, or combinations thereof. In another aspect, the oligomers of Aβ1-42, Aβ1-40, Aβ1-39, Aβ2-43, or combinations thereof comprise dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, 11-mers, 12-mers, 13-mers, 14-mers, 15-mers, 16-mers, 17-mers, 18-mers, 19-mers, 20-mers, or combinations thereof. In another aspect, the method further comprises a bovine serum albumin (BSA) blocking step in the layered well prior to opening the adjustable valve. In another aspect, the method has a limit of detection for the Aβ-M and Aβ-O species of about 1.60×108 to about 2.90×1011 Aβ molecules per layered well.
Another embodiment described herein is a system for analyzing tissue for the presence of Aβ-M and Aβ-O species, the system comprising: an apparatus for microdissection of cells from a sample of tissue; a manifold comprising a plurality of layered wells each comprising an upper chamber and a lower chamber, each chamber comprising independent fluidic connections and an adjustable valve separating the upper chambers and lower chambers, wherein the manifold is assembled on an indium-titanium oxide coated glass slide; one or more anti-Aβ antibodies positioned within the lower chamber of the layered well; a matrix solution; and a mass spectrometer. In one aspect, the sample of tissue is a sample of human brain tissue comprising human brain cells. In another aspect, the apparatus for microdissection comprises a laser capture microdissection (LCM) apparatus. In another aspect, the manifold is comprised of a polymeric material comprising poly(dimethylsiloxane) (PDMS). In another aspect, the one or more anti-Aβ antibodies comprises an Aβ-specific antibody, an amyloid oligomer-specific antibody, or a combination thereof. In another aspect, the one or more anti-Aβ antibodies comprises an immunoglobulin G (IgG) 6E10 antibody. In another aspect, the matrix solution comprises a cyano-4-hydroxycinnamic acid or sinapinic acid in acetonitrile and trifluoroacetic acid. In another aspect, the mass spectrometer comprises a mass spectrometer configured for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.
Another embodiment described herein is a method for analyzing tissue for the presence of one or more protein biomarkers, the method comprising: providing a sample of tissue comprising cells; microdissecting the cells and transferring the cells to an upper chamber of a manifold comprising a plurality of layered wells each comprising an upper chamber and a lower chamber, each chamber comprising independent fluidic connections and an adjustable valve separating the upper chambers and lower chambers; assembling the manifold on an indium-titanium oxide coated glass slide; introducing one or more antibodies into the lower chamber of the layered well containing the cells in the upper chamber, incubating for a period of time, and washing the layered well; opening the adjustable valve separating the upper chamber and lower chamber to permit the cells in the upper chamber to contact the one or more antibodies in the lower chamber, incubating for a period of time, and washing the layered well to remove non-captured material; introducing a matrix solution and allowing crystallization; and removing the manifold and analyzing a co-crystallized sample using mass spectrometry to identify the presence of the one or more protein biomarkers.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.
As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.
As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.
As used herein, the term “or” can be conjunctive or disjunctive.
As used herein, the term “substantially” means to a great or significant extent, but not completely.
As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ±10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol “˜” means “about” or “approximately.”
All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.
As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.
As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non human primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human. In one embodiment of the present invention, a tissue sample from a human subject is provided.
As used herein, the term “tissue” refers to any commonly known tissue of a mammal, such as a human. Tissue may include, but is not limited to, nervous tissue (e.g., brain tissue, spinal cord tissue, nerves, neuronal tissue), connective tissue (e.g., bone tissue, ligament tissue, tendon tissue, blood tissue, lymph tissue), epithelial tissue (e.g., skin surface tissue (epidermis), tissue lining of GI tract organs and other hollow organs), and muscle tissue (e.g., cardiac muscle tissue, smooth muscle tissue, skeletal muscle tissue). In some embodiments of the disclosed systems and methods, tissue may comprise human brain tissue.
As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
Described herein is a microfluidic assay for in situ mass spectrometry of immunocaptured Aβ species from archived human tissues. In this work, the previously reported microfluidic platform in tandem with MALDI mass spectrometry (MIMAS) was coupled with LCM to assess the Aβ protein content from tissue cells. See e.g., Cruz Villarreal et al., “MIMAS: microfluidic platform in tandem with MALDI mass spectrometry for protein quantification from small cell ensembles,” Anal. Bioanal. Chem. 414: 3945-3958 (2002); Yang et al., Quantitative Approach for Protein Analysis in Small Cell Ensembles by an Integrated Microfluidic Chip with MALDI Mass Spectrometry,” Anal. Chem. 93, 6053-6061 (2021), both of which are incorporated by reference herein in their entirety for their teachings. The LCM-MIMAS approach allows for selective dissection of individual cells from tissues, their transfer to the microfluidic platform for sample processing on-chip, and subsequent mass spectrometry identification and quantification. This work targets Aβ monomeric and oligomeric species from brain cells due to their relevance in the current amyloid hypothesis that soluble Aβ oligomers are responsible for neuronal dysfunction.
Also described herein is an approach that allows for the selective dissection of individual cells from tissues, their transfer to the microfluidic platform for sample processing on-chip, and mass spectrometric characterization. In one aspect, cells may be collected directly onto an integrated microfluidic assay in tandem with the MIMAS device. In another aspect, cells may be translocated to a device having a well area of about 500 μm×500 μm.
Three methods of cell collection were used in the described exemplary assays. In one aspect, cells were directly collected into milli-wells with 2 mm diameter wells. In another aspect, cells were directly collected into a MIMAS device. In another aspect, cells were collected into a collection layer fabricated in polydimethylsiloxane (PDMS) elastomer on top of the MIMAS device. Any cells or debris not translocated to the collection area were removed with the PDMS layer prior to further assembly steps. The misplaced dissected cells on device surfaces other than the MIMAS-wells could interfere with further assembly steps.
All experimental examples described herein with the MIMAS devices were performed using a removable PDMS collection layer in the cell collection step. In one non-limiting example, the MIMAS platform device assembly comprised an indium tin oxide (ITO) coated glass slide with a PDMS manifold. In this particular example, the PDMS manifold forming the MIMAS platform had two wells, one well with the protein of interest and one well with immobilized antibodies for capture. Each “fluidic line” contained five wells (i.e., 10 wells total), and pairs were formed from one well in each layer separated by a valve. Capture antibodies were then used for immunocapture of Aβ species and oligomers (e.g., IgG 6E10). The immunocapture steps involved adding the antibody, incubation, and washing steps. The captured Aβ species were removed from the chip and sent for MS analysis.
It should be understood that the described MIMAS platform is not to be restricted or limited to any particular number of wells. The example configurations described herein are only exemplary embodiments and are not meant to be limiting in any way. For example, the systems and methods described herein may comprise a manifold comprising a plurality of layered wells, wherein the plurality of layered wells may comprise from 2 wells to 1,000 wells or greater. In one embodiment, the manifold may comprise from 2 wells to 10 wells. In another embodiment, the manifold may comprise from 10 wells to 100 wells. In another embodiment, the manifold may comprise from 100 wells to 500 wells. In another embodiment, the manifold may comprise from 500 wells to 1,000 wells. In another embodiment, the manifold may comprise greater than 1,000 wells.
In some non-limiting aspects, the chip construction may comprise a fluidic layer with a thickness of about 25 μm, a PDMS mixture of about 15:1 w/w base to curing agent spin coated over a master wafer for the fluidic layer, creating a layer of about 63 μm thickness, double-layer PDMS slabs peeled-off, reservoirs punched using 2 mm biopsy punchers, and a removable collection layer added to facilitate the collection of cells.
One embodiment described herein is a method for analyzing tissue for the presence of Aβ-M and Aβ-O species, the method may comprise: providing a sample of tissue comprising cells; microdissecting the cells and transferring the cells to an upper chamber of a manifold comprising a plurality of layered wells each comprising an upper chamber and a lower chamber, each chamber comprising independent fluidic connections and an adjustable valve separating the upper chambers and lower chambers; assembling the manifold on an indium-titanium oxide coated glass slide; introducing one or more anti-Aβ antibodies into the lower chamber of the layered well containing the cells in the upper chamber, incubating for a period of time, and washing the layered well; opening the adjustable valve separating the upper chamber and lower chamber to permit the cells in the upper chamber to contact the one or more anti-Aβ antibodies in the lower chamber, incubating for a period of time, and washing the layered well to remove non-captured material; introducing a matrix solution and allowing crystallization; and removing the manifold and analyzing a co-crystallized sample using mass spectrometry to identify the presence of the Aβ-M and Aβ-O species.
In one aspect, the tissue may be human brain tissue comprising human brain cells.
In another aspect, the manifold may be comprised of a polymeric material comprising poly(dimethylsiloxane) (PDMS), polycarbonate (PC), poly-methyl-meta-acrylate (PMMA), cyclic olefin copolymer (COC), polyimide, or combinations thereof. In another aspect, the manifold may be comprised of PDMS.
In another aspect, the one or more anti-Aβ antibodies may comprise an Aβ-specific antibody, an amyloid oligomer-specific antibody, or a combination thereof. In another aspect, the one or more anti-Aβ antibodies may comprise an immunoglobulin G (IgG) 6E10 antibody. IgG 6E10 recognizes all species of Aβ without regard to conformation. Amyloid oligomer-specific antibodies may recognize all types of amyloid oligomers, but not monomers or fibrils. Any suitable Aβ-specific antibodies and/or amyloid oligomer-specific antibodies known in the art may be used in the systems and methods disclosed herein for analyzing tissue for the presence of Aβ-M and Aβ-O species.
In another aspect, the matrix solution may comprise α-cyano-4-hydroxycinnamic acid or sinapinic acid in acetonitrile and trifluoroacetic acid.
In another aspect, the mass spectrometry may comprise matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.
In another aspect, each layered well may comprise a well area size ranging from about 50 μm×about 50 μm to about 500 μm×about 500 μm. In another aspect, each layered well may comprise a well area size of about 500 μm×about 500 μm. In some aspects, the sensitivity of the disclosed systems and methods may increase when smaller well area sizes are used. For example, systems and methods comprising well area sizes less than about 500 μm×about 500 μm may have a greater sensitivity as compared to systems and methods comprising well area sizes of about 500 μm×about 500 μm.
In another aspect, microdissecting the cells may comprise laser capture microdissection (LCM).
In another aspect, each layered well may comprise from about 1 to about 100 individual cells. In another aspect, each layered well may comprise from about 1 to about 20 individual cells. In some aspects, the number of cells in each layered well may depend on the specific tissue type analyzed.
In another aspect, the Aβ-M species comprise monomers of Aβ1-42, Aβ1-40, Aβ1-39, Aβ2-43, or combinations thereof. In another aspect, the Aβ-O species may comprise oligomers of Aβ1-42, Aβ1-40, Aβ1-39, Aβ2-43, or combinations thereof. In another aspect, the oligomers of Aβ1-42, Aβ1-40, Aβ1-39, Aβ2-43, or combinations thereof may comprise dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, 11-mers, 12-mers, 13-mers, 14-mers, 15-mers, 16-mers, 17-mers, 18-mers, 19-mers, 20-mers, larger oligomeric species, or combinations thereof. For example, in some aspects, the oligomers of Aβ1-42, Aβ1-40, Aβ1-39, Aβ2-43, or combinations thereof may comprise oligomeric species up to 30-mers, 40-mers, 50-mers, or larger.
In another aspect, the method further may comprise a bovine serum albumin (BSA) blocking step in the layered well prior to opening the adjustable valve.
In another aspect, the method may have a limit of detection for the Aβ-M and Aβ-O species of about 1.60×108 to about 2.90×1011 Aβ molecules per layered well.
Another embodiment described herein is a system for analyzing tissue for the presence of Aβ-M and Aβ-O species, the system may comprise: an apparatus for microdissection of cells from a sample of tissue; a manifold comprising a plurality of layered wells each comprising an upper chamber and a lower chamber, each chamber comprising independent fluidic connections and an adjustable valve separating the upper chambers and lower chambers, wherein the manifold is assembled on an indium-titanium oxide coated glass slide; one or more anti-Aβ antibodies positioned within the lower chamber of the layered well; a matrix solution; and a mass spectrometer.
In one aspect, the sample of tissue may be a sample of human brain tissue comprising human brain cells.
In another aspect, the apparatus for microdissection may comprise a laser capture microdissection (LCM) apparatus.
In another aspect, the manifold may be comprised of a polymeric material comprising poly(dimethylsiloxane) (PDMS).
In another aspect, the one or more anti-Aβ antibodies may comprise an Aβ-specific antibody, an amyloid oligomer-specific antibody, or a combination thereof. In another aspect, the one or more anti-Aβ antibodies may comprise an immunoglobulin G (IgG) 6E10 antibody. IgG 6E10 recognizes all species of Aβ without regard to conformation. Amyloid oligomer-specific antibodies may recognize all types of amyloid oligomers, but not monomers or fibrils. Any suitable Aβ-specific antibodies and/or amyloid oligomer-specific antibodies known in the art may be used in the systems and methods disclosed herein for analyzing tissue for the presence of Aβ-M and Aβ-O species.
In another aspect, the matrix solution may comprise α-cyano-4-hydroxycinnamic acid or sinapinic acid in acetonitrile and trifluoroacetic acid.
In another aspect, the mass spectrometry may comprise matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.
Another embodiment described herein is a method for analyzing tissue for the presence of one or more protein biomarkers, the method comprising: providing a sample of tissue comprising cells; microdissecting the cells and transferring the cells to an upper chamber of a manifold comprising a plurality of layered wells each comprising an upper chamber and a lower chamber, each chamber comprising independent fluidic connections and an adjustable valve separating the upper chambers and lower chambers; assembling the manifold on an indium-titanium oxide coated glass slide; introducing one or more antibodies into the lower chamber of the layered well containing the cells in the upper chamber, incubating for a period of time, and washing the layered well; opening the adjustable valve separating the upper chamber and lower chamber to permit the cells in the upper chamber to contact the one or more antibodies in the lower chamber, incubating for a period of time, and washing the layered well to remove non-captured material; introducing a matrix solution and allowing crystallization; and removing the manifold and analyzing a co-crystallized sample using mass spectrometry to identify the presence of the one or more protein biomarkers.
It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.
Materials and Methods Aβ peptides, Aβ1-42 (
The MIMAS device (
To load the cells into the MIMAS wells, a collection layer was designed and fabricated (shown in
Aβ1-42 and Aβ1-40 monomers (Aβ-M) and oligomers (Aβ-O) were prepared as follows. Briefly, HFIP-treated Aβ peptide was aliquoted, lyophilized, and stored at −20° C. until used. For Aβ-M, the peptide was dissolved in 1 μL DMSO, sonicated for 10 min and diluted to 80 μM using ice-cold 10 mM sodium phosphate buffer. For Aβ-O, the peptide was dissolved in 5 μL DMSO, sonicated for 10 min, diluted to 150 μM using Ham's F-12 medium, and incubated at 4° C. for 24 h. After incubation, the solution was diluted to 80 μM using 10 mM sodium phosphate buffer. All further dilutions were performed with 10 mM sodium phosphate buffer. The composition of synthetic monomeric Aβ peptides was confirmed by MALDI-MS spotting the peptide solution mixed with matrix in a 1:1 ratio to the MALDI target plate, and by SDS-PAGE following standard procedures. For Aβ1-42, the monomeric band was extracted from the gel and analyzed LC-MS/MS.
Frozen human tissue (brain slices from the middle frontal gyrus from non-AD tissue sections, without signs of amyloid plaques) was obtained from the Banner Sun Health Research Institute (Sun City, USA). A phase-contrast Leica LMD6500 microscope with a universal holder for collection devices (Leica Microsystems, Germany) was used for cell laser capture microdissection (LCM). Brain sections were inspected under the microscope to identify pyramidal cells based on their morphology. LCM 6.6 software (Leica Microsystems, Germany) was used to draw and cut over an outline around the body of each of the pyramidal cells to ensure the sample contained only intracellular contents. Once the laser cuts around the outline, the selected pyramidal cell's bodies fell into the collection area by gravity. The dissected cells were loaded directly into (a) a MIMAS device with its wells facing up (
The immunocapture of Aβ was performed in milli-wells and MIMAS wells following an adapted, previously reported protocol for on-chip protein immunocapture and MS analysis. For the MIMAS platform, solution loading consists of valve opening by applying vacuum to the control layer inlets, allowing the solutions to fill up the wells by capillary action. The solutions were removed from the wells by opening the valves and applying a vacuum to the line outlets. For the MIMAS protocol, a solution of IgG 6E10 was loaded into the fluidic layer I by opening valve line A (
Amyloid-β Immunocapture from Brain Cells
The developed immunoassay workflow was then carried out with brain cells. The brain cell protocol was established first with milli-wells and then with the MIMAS platform. The collection layer was aligned over the MIMAS device and filled up with buffer. The body of a pyramidal cell from non-AD brain tissue was identified by its unique morphology, micro-dissected, and allowed to fall into the collection layer opening. After the buffer in the well was dried, the collection layer was removed. An ITO-coated glass slide was cleaned with acetone and isopropanol and treated with oxygen plasma for 1 min under medium RF conditions. The PDMS MIMAS manifold containing the dissected cells was then bonded on a treated ITO-coated glass slide (
MALDI-MS analysis was performed by placing the sample on the ITO-coated glass target in a Bruker Microflex LRF in linear mode. Crystals were identified on the ITO slide by visual inspection using the instrument's camera. The LOD of Aβ was determined with monomeric Aβ1-40 and Aβ1-42 at various concentrations, pre-mixed with matrix, loaded into the milli-wells and the MIMAS wells, and allowed to dry at RT. For MS analysis using milli-wells (n=3), 4,000 shots/milli-well were averaged. For MS analysis using the MIMAS device (3 devices=15 MIMAS wells), the crystals in a well were depleted and averaged (>2,000 shots/well). The LOD was determined using Origin (OriginLab, USA) by linear fitting the standard curve and LOD=(3.3 σ)/S; where σ is the y-intercept standard deviation and S the slope. The reported m/z values correspond to the average mass in the MS spectrum.
The immunocapture of Aβ was performed in milli-wells mounted on ITO-coated glass slides. First, 5 μL of 0.05 mg/mL immunoglobulin G (IgG) 6E10 in 10 mM phosphate buffer were loaded into each milli-well and incubated for 2 h at 36° C. in a humidity chamber. Then, milli-wells were washed with 20 mM sodium phosphate buffer three times. A blocking step was performed by loading 5 μL of 1% BSA in 10 mM phosphate buffer to each milli-well and incubating in a humidity chamber for 1 h at 36° C., followed by washing with 20 mM sodium phosphate buffer thrice. Then, 5 μL of Aβ solution was loaded into the milli-wells and incubated for 1 h at room temperature (RT), followed by a wash step with 50 mM ammonium bicarbonate which was repeated thrice. Finally, the matrix solution was loaded into the milli-wells and the contents dried at RT for co-crystallization. Once dried, the PDMS manifold was peeled-off from the glass slide. The ITO-coated glass slide was then used as the target in the MALDI-MS instrument. All experiments were performed in triplicates (three milli-wells per condition). A saturated solution of α-CHCA matrix in 40% acetonitrile and 0.1% TFA was used for Aβ-M. A 10 mg/mL solution of sinapinic acid in 50% acetonitrile and 0.05% TFA was used as the matrix for Aβ-O. Representative spectra of the Aβ-M and Aβ-O immunocapture using milli-wells are shown in FIG. 4 and
Amyloid-β Immunocapture from Brain Cells Using Milli-Wells
First, 5 μL of 0.05 mg/mL IgG 6E10 solution was loaded into the reservoir and incubated for 2 h at 36° C. in a humidity chamber. The reservoirs were washed three times using 20 mM sodium phosphate buffer. Next, 5 μL of 1% BSA blocking solution was loaded into the reservoir and incubated 1 h at 36° C., followed by a washing step with 20 mM sodium phosphate buffer. Then, the reservoirs were placed on the LCM instrument for brain cell dissection and loaded as described in the cell microdissection and collection section above. Then, the devices were incubated for 1 h at RT, followed by a washing step with 50 mM ammonium bicarbonate. Finally, 5 μL of matrix solution was added to the reservoirs. Once the solution was dried, the PDMS manifold was peeled-off from the glass slide, and the slide was then used as the target in the MALDI-MS instrument. All experiments were performed in triplicates. A 10 mg/mL matrix solution of sinapinic acid in 50% acetonitrile and 0.05% TFA was used for experiments with brain cells.
Developing the current assay for Aβ from brain cells entailed several important steps: optimization of cell transfer into the microfluidic platform, MS detection limit characterization, immunocapture, characterization of bound Aβ species with MALDI MS, and workflow implementation in the MIMAS platform. Along with the integration of all the functional microfluidic elements necessary for an entirely on-chip assay (
Collection directly into the MIMAS device wells implies gravity translocation of the cells to a 0.25 mm2 area, more than 60 times smaller than the collection area presented by the 4.5 mm-diameter microtube caps typically used with the LCM. The LCM capture efficiency of cells from tissue into such caps is around 90%; however, it can be as low as 20% to 50% for low humidity conditions or tissue sections with dimensions larger than a hundred micrometers. To study the transfer efficiency of this platform, three methods were tested, which are schematically represented in
The immunocapture assay was first developed and optimized in milli-wells using IgG 6E10, then developed in the MIMAS platform. The selected IgG is one of the most commonly used for AD research, known to bind both monomeric and oligomeric Aβ species. The IgG 6E10 binding epitope corresponds to the human Aβ amino acid residues 5 to 7. The affinity of IgG 6E10 for monomeric and oligomeric Aβ1-40 and Aβ1-42 was verified using synthetic Aβ solutions in both the milli-wells and the MIMAS platform. For Aβ1-40-M and Aβ1-42-M solutions incubated with immobilized IgG 6E10, MS spectra peaks [Aβ+H]+ and [Aβ+2H]2+ confirmed the affinity binding of the monomer peptides to the IgG (
It is worth comparing this assay based Aβ1-42 LOD with a standard curve for various concentrations of Aβ-M (see
In vitro synthesized Aβ1-40-O and Aβ1-42-O were used to assess oligomer IgG 6E10 capture and bound species by MS. Oligomeric species of Aβ1-40 up to 9-mers and Aβ1-42 up to 12-mers have been detected using MALDI-MS, which indicates MALDI-MS can be used to characterize Aβ-O IgG 6E10 capture. The spectra of immunocaptured Aβ1-40-O and Aβ1-42-O in milli-wells are included in
The IgG 6E10 affinity for Aβ monomers, oligomers, and fibrils has been well established, although without much detail on specific low molecular weight oligomeric species binding. Aβ monomers, dimers, and trimers have been found in human brain homogenate immunoblotting. Pham et al. reported the detection of up to heptamers using IgG 6E10 in brain homogenate immunoblots, with the caveat that the trimers and tetramers were not fully resolved (Pham et al., FEBS J. 277 (14): 3051-3067 (2010)). The immunoassay with MS analysis demonstrated here provides a distinctive advantage to further assess the 6E10 antibody binding to lower molecular weight oligomers. To our knowledge, there are no previous reports of in-vitro MS characterization of Aβ-O species immunocaptured with IgG 6E10. In this work, Aβ1-40-O up to the trimeric species were identified by MALDI-MS after on-chip immunocapture. This indicates that MS analysis allows sensitive identification of distinct immunocaptured oligomeric and other species that might not be identified by previously employed methods.
There are several possible reasons for larger oligomers not being found after the immunoassay, although in vitro-generated species can be detected by MALDI MS. Large oligomer binding by the 6E10 antibody might be inhibited. It is also possible that these species are either transient or in concentrations too low for MS. It also seems plausible that larger oligomers might dissociate into smaller species during MS. However, oligomers up to 12-mers were identified in in vitro preparations with MALDI-MS in preliminary work, and have also been reported by Wang et al., J. Am. Soc. Mass Spectr. 29 (4): 786-795 (2018). The peaks corresponding to the monomers and dimers exhibit a wide m/z distribution, which could originate from the dissociation of larger species or multi-charged larger species that cannot be resolved with the employed MALDI-MS instrument.
Control experiments without IgG were also performed to test for non-specific oligomer binding. In addition to the peak with m/z ˜3880 associated with the BSA blocking step often observed in the MIMAS assays, peaks at m/z ˜66,000, ˜33,000, ˜22,000, and ˜16,500 were also observed (see the representative spectrum in
It is interesting to note that the peaks not associated with oligomeric species that appear in the Aβ1-40-O spectra (U1/U2) and in the Aβ1-42-O spectra (U3/U4) have a lower m/z than expected monomeric or dimeric species (see Tables 1 and 2). The higher m/z peaks U2 and U4 may be considered a dimer version of U1 and U3, respectively. Alternatively, these peaks may also represent multi-charged species of larger oligomers. It is important to mention that MALDI MS was performed in linear mode, as otherwise (i.e., using the reflectron mode), the sensitivity to detect the Aβ-O was not reached. Additionally, the limited resolution of the instrument did not allow for identification of the observed peaks and further work to properly define the unknown species U1-U4 must be performed.
Analysis of Amyloid-β from Brain Cells in Milli-Wells
To demonstrate Aβ extraction and immunocapture from microdissected brain tissue cells, the immunoassay was first performed with brain cells in milli-wells. Pyramidal cells from frozen brain tissue sections were identified based on their morphology. The body of pyramidal cells was selected for dissection to avoid the use of extracellular material in the assay. Experiments were performed in milli-wells filled with 20 mM phosphate buffer and 100 microdissected cells per well. As a control, 100 cells were collected on wells without immobilized antibodies. A representative spectrum obtained after extraction and immunocapture is shown in
Furthermore, the peak assignment of expected Aβ species was compared with those obtained by MALDI-MS and other potential candidates (Table 3). Aβ species other than Aβ1-40 in brain homogenate have been identified by immunoprecipitation (IP) with the 6E10 antibody and MALDI-MS. The m/z 4270.3 peak was assigned to Aβ39 with either a K+ adduct ([Aβ39+K]+) or an acetonitrile adduct ([Aβ39+ACN+H]+). Potential adducts were considered based on common adducts present in MALDI-MS based on the Mass Spectrometry Adduct Calculator and are listed in Table 3 together with calculated masses. Since Aβ39 has been identified by analysis of bulk brain samples using IgG 6E10 via IP and MALDI-MS, 20, 62 and K+ is present in the cell lysis buffer while acetonitrile is present in the matrix solution, the two potential adducts seem likely. Correspondingly, the m/z of 8571.8 could be the Aβ39 dimer either as a K+ or acetonitrile adduct. Similarly, the m/z 4374.6 and m/z 8760.6 peaks match the m/z of Aβ1-40 monomers and dimers with potential adducts of Na+, K+, or acetonitrile. Lastly, m/z 4490.1 and m/z 8969.2 peaks can be matched to the Aβ2-43 monomer and dimer, respectively. Interestingly, peaks identified as [Aβ1-39+K]+ or [Aβ1-39+ACN+H]+ are close in m/z to the U1 and U2 peaks observed in the immunocapture of Aβ-O (see
Analysis of Amyloid-β from Brain Cells in the LCM-MIMAS Platform
The LCM-MIMAS workflow (
The minimum number of cells for the LCM-MIMAS approach was estimated based on reported Aβ content in neurons and reservoir size and shape limitations. Soluble Aβ ranges from about 1 to 104 pg per μg of total neuron protein content. According to this, a 15 μm-thick neuron slice can be expected to contain 1.5×104 to 1.5×108 Aβ molecules. The LOD in a MIMAS well (
Using the MIMAS assay, peaks from the brain cells were successfully identified (representative spectrum shown in
The extremely high sensitivity of the LCM-MIMAS assay for Aβ-species from as few as 20 cells is similar to our previously demonstrated MIMAS assay with MCF-7 breast cancer cells, where the apoptosis-related protein Bcl-2 was detected from as few as 10 cells. This extremely high sensitivity is attributed to a reduced cell lysate complexity that limits analyte masking as well as to the overall MIMAS workflow that minimizes sample loss and dilution effects.
This work demonstrates the LCM-MIMAS platform for the analysis of Aβ species in small cell populations from archived brain tissue. A workflow was developed entirely on a chip, starting from laser-microdissection of cells into a microfluidic platform and ending in MALDI MS identification of IgG 6E10 immunocaptured Aβ-M and Aβ-O species from healthy human brain tissue. A novel capture element for coupling LCM with the MIMAS platform has been demonstrated, allowing the collection of exclusively intracellular components in the LCM-MIMAS assay. In the milli-wells, detection of immunocaptured intracellular Aβ species was achieved with ˜100 dissected cells from archived brain tissue. In the MIMAS platform, Aβ species were identified with as few as 20 cells. Monomer and dimer adduct candidate species of Aβ1-39, Aβ1-40, and Aβ2-43 have been identified in the brain cell mass spectra when the assay was performed in the milli-wells and the MIMAS platform. However, in the MIMAS platform, an additional species was identified as Aβ1-42. The in situ identification of Aβ species from as few as 20 cells from archived brain tissue sections puts forward the LCM-MIMAS approach as a powerful tool to elucidate intracellular Aβ species further. The LCM-MIMAS approach is advantageous compared to in vitro and in vivo Aβ-O characterization through commonly used gel electrophoresis because sodium dodecyl sulfate (SDS) in gels compromises the oligomer structural integrity, as reported by multiple studies. Although alternatives to avoid SDS exist, gel electrophoresis lacks the appropriate resolution to identify individual oligomers. LCM-MIMAS is suitable for studying small cell subpopulations with a defined type and disease state as well as extracellular species, towards elucidating the origin of AD and other diseases through a novel, sensitive assay for crucial disease proteins.
This application claims priority to U.S. Provisional Patent Application No. 63/358,562, filed on Jul. 6, 2022, which is incorporated by reference herein in its entirety.
This invention was made with government support under R21 AG067488 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63358562 | Jul 2022 | US |
