Patent Application
20220291235
The present invention is related to the field of analytical chemistry. In particular, the invention defines a new and improved device and method to detect precise differences in protein secondary, tertiary or quaternary structures. By applying standing acoustic waves to a proteinaceous fluid, differences in protein unfolding characteristics (e.g., melting temperatures) are detected which are beyond the capabilities of conventional thermal shift assays. Such improvements are based upon concomitant protein aggregation that increases local protein concentrations permitting increased temperature shift detection sensitivity.
Proteins are essential components of organisms and participate in most biological processes. Studying protein dynamics and its interaction with other molecules impact almost every field from fundamental biology to clinical applications.
However, conventional measurement assays and sample preparation takes from many hours up to a whole day and/or requires bulky/expensive facility and experiences. This limitation is one of the main challenges that lead to high cost and long development cycles for therapeutics and drug development in pharmaceutical industry and particularly in the development of portable medical devices for fast and low cost diagnosis and healthcare.
What is needed in the art is a device and method providing a single step that rapidly and accurately identify protein characteristics for “point of care” analysis.
The present invention is related to the field of analytical chemistry. In particular, the invention defines a new and improved device and method to detect precise differences in protein secondary, tertiary or quaternary structures. By applying standing acoustic waves to a proteinaceous fluid, differences in protein unfolding characteristics (e.g., melting temperatures) are detected which are beyond the capabilities of conventional thermal shift assays. Such improvements are based upon concomitant protein aggregation that increases local protein concentrations permitting increased temperature shift detection sensitivity.
In one embodiment, the present invention contemplates a method, comprising: a) providing: i) an acousto-thermal device comprising a surface acoustic wave source and at least one microfluidic channel or chamber; and ii) a sample comprising at least one protein; b) introducing said sample into said at least one microfluidic channel or chamber; c) controlling the temperature of the said sample with said surface acoustic wave source to a plurality of precise temperatures within said microfluidic channel or chamber under conditions that create a precipitated protein; and d) aggregating said precipitated protein with said surface acoustic wave into a pattern. In one embodiment, said pattern comprises parallel lines or arrays. In one embodiment, said aggregating increases a local concentration of said precipitated protein. In one embodiment, said aggregating is performed simultaneously with said protein precipitation. In one embodiment, the method further comprises measuring protein gray intensity. In one embodiment, said protein gray intensity measurements determine a protein melting curve. In one embodiment, said sample comprises plurality of biological cells. In one embodiment, the method further comprises lysing at least a portion of said plurality of biological cells with said surface acoustic wave source. In one embodiment, the acousto-thermal device further comprises a piezoelectric substrate comprising at least one microchannel or chamber. In one embodiment, the acousto-thermal device further comprises at least two parallel interdigital transducers deposited longitudinally in said at least one microchannel or chamber. In one embodiment, the acousto-thermal device further comprises a fluid comprising a plurality of proteins disposed between said at least two parallel interdigital transducers. In one embodiment, each of said interdigital transducer comprises thirty (30) pairs of electrodes. In one embodiment, said electrode pairs comprise chromium and gold. In one embodiment, each of said electrode pairs have a thickness of approximately 5/100 nm. In one embodiment, wherein each of said electrode pairs comprise an electrode finger of 50 μm in length, a pitch of 100 μm, and an aperture of 10 mm. In one embodiment, said electrode pairs yield a standing acoustic wave having a frequency of approximately 20 MHz. In one embodiment, said piezoelectric substrate comprises a material selected from the group consisting of silicon, glass, plastic, quartz and polydimethylsiloxane (PDMS). In some embodiments, the method comprises an acoustic microfluidic device that can control protein precipitation. In other embodiments, the method comprises distinguishing the protein solubility difference upon a protein interaction with other molecules or the solubility change due to the protein configuration change. In other embodiments, the method measurements can be done on a microchip within a few minutes without peripheral systems. In some embodiments, the method comprises controlling, aggregating and characterizing a precipitate on a single microchip without any additional systems or steps. In some embodiments, the methods comprise a low cost and fast drug screening. In other embodiments, the method comprises a fast label-free diagnostic device to diagnose diseases including, but not limited to, sickle cell disease, malaria, hemoglobinopathies or many other diseases that is related to a protein disorder where modified proteins have a melting temperature shift. In other embodiments, the method comprises at least one microfluidic channel and/or chamber, or two or more chambers or channels to simultaneously measure multiple protein samples. In other embodiments, the method comprises protein aggregation, patterning and concentrating precipitated protein. In other embodiments, the method comprises measuring either gray intensity or fluorescence intensity. In other embodiments, the method comprises cell lysis before such protein aggregation, patterning and concentrating. In other embodiments, the method comprises using surface acoustic waves for protein precipitation, protein patterning and concentration. In other embodiments, the method comprises cell lysis, protein precipitation and protein aggregation or patterning. In other embodiments the method comprises simultaneous or almost simultaneous lysis, precipitation, and aggregation or patterning. In other embodiments, the method does not comprise cell lysis.
In one embodiment, the present invention contemplates an acousto-thermal device, comprising: i) a piezoelectric substrate comprising at least one microchannel or chamber; ii) at least two parallel interdigital transducers deposited longitudinally in said at least one microchannel or chamber; and iii) a fluid comprising a plurality of proteins disposed between said at least two parallel interdigital transducers. In one embodiment, each of said interdigital transducer comprises thirty (30) pairs of electrodes. In one embodiment, said electrode pairs comprise chromium and gold. In one embodiment, each of said electrode pairs have a thickness of approximately 5/100 nm. In one embodiment, wherein each of said electrode pairs comprise an electrode finger of 50 μm in length, a pitch of 100 μm, and an aperture of 10 mm. In one embodiment, said electrode pairs yield a standing acoustic wave having a frequency of approximately 20 MHz. In one embodiment, said piezoelectric substrate comprises a material selected from the group consisting of silicon, glass, plastic, quartz and polydimethylsiloxane (PDMS).
In one embodiment, the present invention contemplates a method, comprising: a) providing: i) an acousto-thermal device comprising a surface acoustic wave source and at least two microfluidic channels or chambers; ii) a first sample comprising at least one first protein disposed in a first microfluidic channel or chamber; and iii) a second sample comprising at least one second protein disposed in a second microfluidic channel or chamber;; b) controlling the temperature of said first and second sample with said surface acoustic wave source to a plurality of precise temperatures within said microfluidic channel or chamber under conditions that create a first and second precipitated protein; c) aggregating said first and second precipitated protein with said surface acoustic wave into a first and second pattern; d) measuring a gray intensity of said first and second precipitated protein; e) determining a first and second melting temperature of said first and second precipitated protein; and f) calculating a difference between said first and second melting temperature with a three to thirty-five-fold increased sensitivity as compared to conventional thermal shift assays. In one embodiment, said second protein is bound to a ligand. In one embodiment, the ligand is selected from the group consisting of a small organic molecule, an antibody and a protein. In one embodiment, the second protein comprises a mutation as compared to a wild type sequence. In one embodiment, said difference diagnoses a genetic disease. In one embodiment, said pattern comprises parallel lines or arrays. In one embodiment, said aggregating increases a local concentration of said precipitated protein. In one embodiment, said aggregating is performed simultaneously with said protein precipitation. In one embodiment, said sample comprises plurality of biological cells. In one embodiment, the method further comprises lysing at least a portion of said plurality of biological cells with said surface acoustic wave source. In one embodiment, the acousto-thermal device further comprises a piezoelectric substrate comprising at least one microchannel or chamber. In one embodiment, the acousto-thermal device further comprises at least two parallel interdigital transducers deposited longitudinally in said at least one microchannel or chamber. In one embodiment, the acousto-thermal device further comprises a fluid comprising a plurality of proteins disposed between said at least two parallel interdigital transducers. In one embodiment, each of said interdigital transducer comprises thirty (30) pairs of electrodes. In one embodiment, said electrode pairs comprise chromium and gold. In one embodiment, each of said electrode pairs have a thickness of approximately 5/100 nm. In one embodiment, wherein each of said electrode pairs comprise an electrode finger of 50 μm in length, a pitch of 100 μm, and an aperture of 10 mm. In one embodiment, said electrode pairs yield a standing acoustic wave having a frequency of approximately 20 MHz. In one embodiment, said piezoelectric substrate comprises a material selected from the group consisting of silicon, glass, plastic, quartz and polydimethylsiloxane (PDMS). In some embodiments, the method comprises an acoustic microfluidic device that can control protein precipitation. In other embodiments, the method comprises distinguishing the protein solubility difference upon a protein interaction with other molecules or the solubility change due to the protein configuration change. In other embodiments, the method measurements can be done on a microchip within a few minutes without peripheral systems. In some embodiments, the method comprises controlling, aggregating and characterizing a precipitate on a single microchip without any additional systems or steps. In some embodiments, the methods comprise a low cost and fast drug screening. In other embodiments, the method comprises a fast label-free diagnostic device to diagnose diseases including, but not limited to, sickle cell disease, malaria, hemoglobinopathies or many other diseases that is related to a protein disorder where modified proteins have a melting temperature shift. In other embodiments, the method comprises at least one microfluidic channel and/or chamber, or two or more chambers or channels to simultaneously measure multiple protein samples. In other embodiments, the method comprises protein aggregation, patterning and concentrating precipitated protein. In other embodiments, the method comprises measuring either gray intensity or fluorescence intensity. In other embodiments, the method comprises cell lysis before such protein aggregation, patterning and concentrating. In other embodiments, the method comprises using surface acoustic waves for protein precipitation, protein patterning and concentration. In other embodiments, the method comprises cell lysis, protein precipitation and protein aggregation or patterning. In other embodiments the method comprises simultaneous or almost simultaneous lysis, precipitation, and aggregation or patterning. In other embodiments, the method does not comprise cell lysis.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity but also plural entities and also includes the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
The term “about” or “approximately” as used herein, in the context of any of any assay measurements refers to +/−5% of a given measurement.
The term “surface acoustic wave source” or “surface acoustic wave generator” as used herein refers to a component of a device that emits a standing acoustic wave over the surface of a fluid. For example, a surface acoustic wave source/generator comprises a microchip having a pair of interdigitated transducers in parallel.
The term “interdigitated transducer” (IDT) as used herein, refers to two interlocking comb-shaped arrays of metallic electrodes that form pairs in the fashion of a zipper. These metallic electrodes are deposited on the surface of a piezoelectric substrate, such as quartz or lithium niobate, to form a periodic structure. IDTs primary function is to convert electric signals to surface acoustic waves (SAW) by generating periodically distributed mechanical forces via piezoelectric effect (an input transducer).
The term “microfluidic” as used herein relates to components where moving fluid is constrained in or directed through one or more channels wherein one or more dimensions are 1 mm or smaller (microscale). Microfluidic channels may be larger than microscale in one or more directions, though the channel(s) will be on the microscale in at least one direction. In some instances the geometry of a microfluidic channel may be configured to control the fluid flow rate through the channel (e.g. increase channel height to reduce shear). Microfluidic channels can be formed of various geometries to facilitate a wide range of flow rates through the channels.
The term “microfluidic device” refers to a substrate comprising at least one channel that is configured to support fluid flow. Such a device may be constructed out of a variety of materials including, but not limited to, silicon, quartz, glass, plastic and/or polydimethylsiloxane (PDMS) or other polymer(s). For example, some microfluidic devices may comprise a microchip.
The term “microchannels” refer to pathways (whether straight, curved, single, multiple, in a network, etc.) through a medium (e.g., silicon, glass, polymer, etc.) that allow for movement of liquids and gasses. Channels thus can connect other components, i.e., keep components “in communication” and more particularly, “in fluidic communication” and still more particularly, “in liquid communication.” Microchannels are channels with dimensions less than 1 millimeter and greater than 1 micron. It is not intended that the present invention be limited to only certain microchannel geometries. In one embodiment, a four-sided microchannel is contemplated. In another embodiment, the microchannel is circular (in the manner of a tube) with curved walls. In yet another embodiment, a combination of circular or straight walls is used.
The term “chamber” or “microfluidic chamber” refers to an enlarged section of a microfludic channel with a volume sufficient to allow mixing of various reagents and biological samples. A microfluidic chamber may also have windows or ports to permit analytical sampling or non-invasive data collection. A microfluidic chamber may also have an inlet microchannel and an outlet channel to permit continuous flow through the microfluidic chamber for serial data collection.
The term “disease” or “medical condition”, as used herein, refers to any impairment of the normal state of the living animal or plant body or one of its parts that interrupts or modifies the performance of the vital functions. Typically manifested by distinguishing signs and symptoms, it is usually a response to: i) environmental factors (as malnutrition, industrial hazards, or climate); ii) specific infective agents (as worms, bacteria, or viruses); iii) inherent defects of the organism (as genetic anomalies); and/or iv) combinations of these factors.
The term “ligand” as used herein, refers to any compound capable of interacting with (i.e., for example, attaching, binding etc) to a binding partner under conditions such that the binding partner alters its conformational shape. For example, if a binding partner is a protein, a conformation shape change may include, but is not limited to, changes in secondary, tertiary or quaternary structure. Ligands may include, but are not limited to, small organic molecules, antibodies, and proteins/peptides.
The term “patient” or “subject”, as used herein, is a human or animal and need not be hospitalized. For example, out-patients, persons in nursing homes are “patients.” A patient may comprise any age of a human or non-human animal and therefore includes both adult and juveniles (i.e., children). It is not intended that the term “patient” connote a need for medical treatment, therefore, a patient may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies.
The term “protein” as used herein, refers to any of numerous naturally occurring extremely complex substances (as an enzyme or antibody) that consist of amino acid residues joined by peptide bonds, contain the elements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general, a protein comprises amino acids having an order of magnitude within the hundreds. A protein may have a conformation shape described, in part, by secondary structure (e.g., twists), tertiary structure (e.g., turns) and quaternary structure (e.g., induced by binding with other proteins). When a protein binds to a ligand, changes in conformation shape may occur including changes in secondary, tertiary, quaternary structure or any combination thereof.
The term, “purified” or “isolated”, as used herein, may refer to a peptide composition that has been subjected to treatment (i.e., for example, fractionation) to remove various other components, and which composition substantially retains its expressed biological activity.
Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition (i.e., for example, weight/weight and/or weight/volume). The term “purified to homogeneity” is used to include compositions that have been purified to ‘apparent homogeneity” such that there is single protein species (i.e., for example, based upon SDS-PAGE or HPLC analysis). A purified composition is not intended to mean that all trace impurities have been removed.
As used herein, the term “substantially purified” refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and more preferably 90% free from other components with which they are naturally associated. An “isolated polynucleotide” is therefore a substantially purified polynucleotide.
The term “small organic molecule” as used herein, refers to any molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size from approximately 10 Da up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
The term “sample” or “biopsy” as used herein is used in its broadest sense and includes environmental and biological samples. Environmental samples include material from the environment such as soil and water. Biological samples may be animal, including, human, fluid (e.g., blood, plasma and serum), solid (e.g., stool), tissue, liquid foods (e.g., milk), and solid foods (e.g., vegetables). For example, a pulmonary sample may be collected by bronchoalveolar lavage (BAL) which comprises fluid and cells derived from lung tissues. A biological sample may comprise a cell, tissue extract, body fluid, chromosomes or extrachromosomal elements isolated from a cell, genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and the like.
The term “bind” as used herein, includes any physical attachment or close association, which may be permanent or temporary. Generally, an interaction of hydrogen bonding, hydrophobic forces, van der Waals forces, covalent and ionic bonding etc., facilitates physical attachment between the molecule of interest and the analyte being measuring. The “binding” interaction may be brief as in the situation where binding causes a chemical reaction to occur. That is typical when the binding component is an enzyme and the analyte is a substrate for the enzyme. Reactions resulting from contact between the binding agent and the analyte are also within the definition of binding for the purposes of the present invention.
The term, “device” as used herein, describes components including, substrates, surfaces and points of contact between reagents.
The term, “substrate” as used herein, describes a material having a surface as well as solid phases which may comprise the array, microarray or microdevice. In some cases, the substrate is solid and may comprise PDMS.
The term “luminescence” and/or “fluorescence”, as used herein, refers to any process of emitting electromagnetic radiation (light) from an object, chemical and/or compound. Luminescence results from a system which is “relaxing” from an excited state to a lower state with a corresponding release of energy in the form of a photon. These states can be electronic, vibronic, rotational, or any combination of the three. The transition responsible for luminescence can be stimulated through the release of energy stored in the system chemically or added to the system from an external source. The external source of energy can be of a variety of types including chemical, thermal, electrical, magnetic, electromagnetic, physical or any other type capable of causing a system to be excited into a state higher than the ground state. For example, a system can be excited by absorbing a photon of light, by being placed in an electrical field, or through a chemical oxidation-reduction reaction. The energy of the photons emitted during luminescence can be in a range from low-energy microwave radiation to high-energy x-ray radiation. Typically, luminescence refers to photons in the range from UV to IR radiation.
The term “piezoelectric” as used herein, refers to an ability of certain crystalline materials to generate an electric charge in response to applied mechanical stress.
The present invention is related to the field of analytical chemistry. In particular, the invention defines a new and improved device and method to detect precise differences in protein secondary, tertiary or quaternary structures. By applying standing acoustic waves to a proteinaceous fluid, differences in protein unfolding characteristics (e.g., melting temperatures) are detected which are beyond the capabilities of conventional thermal shift assays. Such improvements are based upon concomitant protein aggregation that increases local protein concentrations permitting increased temperature shift detection sensitivity.
In one embodiment, the present invention contemplates an on-chip acousto=thermal shift assay (A-TSA) that represents a novel thermal shift assay strategy and demonstrates unprecedented speed and sensitivity for label-free analysis of protein thermodynamic stability in real time. A-TSA requires less sample volume and is faster than any conventional TSA in measuring single protein melting curve without any needs of molecular markers, allowing its broad potential applications in fast diagnosis. Data presented herein demonstrates a superior sensitivity to detect protein stability than conventional fluorescence-based TSA methods. Consequently, A-TSA has a superior advantage over all C-TSAs in having an improved quantitative protein measurement and precise binding affinity. A-TSA is also highly compatible with automatic processing techniques and cellular phone interface. A-TSA is able to benefit a plethora of applications in fundamental biomedical research, drug industry and fast diagnosis.
Although it is not necessary to understand the mechanism of an invention it is believed that although surface acoustic waves can pattern microparticles and cells, that ability does not necessarily lead to protein patterning. It is further believed that because protein precipitation and patterning involves small molecule aggregation, it is not obvious in comparison to SAW aggregation of larger objects such as biological cells or microbeads. Notably, the present invention makes the first disclosure of the ability of SAW to generate precipitated protein patterns subsequent to protein aggregation and concentration, in addition to cell lysis.
Protein-ligand interactions are not only involved in almost every process in biological systems, but are also play a role in the external modulation of protein function by drugs. Frederick et al. ,“Conformational entropy in molecular recognition by proteins” Nature 448:325-329 (2007). Protein thermal shift assays (TSAs) are a set of techniques to investigate protein-ligand interactions by detecting the changes in thermodynamic stability of the protein under varying conditions, including ligand binding. Huber et al., “Proteome-wide drug and metabolite interaction mapping by thermal stability profiling” Nat. Methods 12:1055-1057 (2015); and Vedadi et al., “Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination” Proc. Natl. Acad. Sci. USA 103: 15835-15840 (2006). Multiple TSA's methods have been implemented, however, they have a variety of drawbacks. For example, mass spectrum-based TSAs are very expensive and labor/time intensive while other fluorescence-based TSA approaches suffer from sensitivity issues. These disadvantages limit the capability of these techniques to detect small thermal shifts, particularly for low abundance or large, complex proteins. Other limitations include, but are not limited to, time-consuming workflow, expensive equipment, technical experience, a lack of reliant labels such as antibodies, and/or demand for large amounts of purified proteins. Huynh et al., “Analysis of protein stability and ligand interactions by thermal shift assay” Curr. Protoc. Protein Sci. 79:28-29 (2015); Jafari et al., “The cellular thermal shift assay for evaluating drug target interactions in cells” Nat. Protoc. 9:2100 (2014); and Dart et al., “Homogeneous Assay for Target Engagement Utilizing Bioluminescent Thermal Shift” ACS Med. Chem. Lett. 9:546-551 (2018). These limitations present a barrier to the broader applications of TSA in life science and medicine, particularly in the areas sensitive to efficiency and cost such as pharmaceutical industry and fast diagnosis.
As one of the primary analysis tools of life and chemistry scientists, protein thermal shift assays (also known as thermal denature assay, differential scanning fluorimetry, or ThermoFluor) are an effective assay in measuring purified protein-drug engagement in the drug industry and in detecting protein interaction in academia. Protein thermal stability is very sensitive to protein dynamics and will change accordingly when protein binds to other molecules such as ligand or other protein to form complexes. Such thermal stability change is called thermal shift and can be measured using fluorescence, western blot, or mass spectrometer analysis. Molina et al., “Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay” Science 341:84-87 (2013). Recently a modified thermal shift assay called cellular thermal shift assay (CETSA) can measure intracellular protein thermal shift and provide insight of protein interactions within living cells. Tan Tan, et al., “Thermal proximity coaggregation for system-wide profiling of protein complex dynamics in cells” Science 359:1170-1177 (2018).
Here is disclosed the first thermal shift assay enabled by acoustic mechanisms (an acousto-thermal shift assay (A-TSA)). In one embodiment, surface acoustic waves (SAWs) are employed to unfold proteins and concentrate precipitated proteins on a microfluidic chip by coupling acoustic heating with acoustic forces.
When an acoustic field is imposed on a fluid, it exerts acoustic forces on suspended particles (e.g., proteins or microparticles) induced by acoustic scattering and also on the suspending fluid thereby causing acoustic fluid streaming due to viscous attenuation of the acoustic energy in the fluid. Such acoustic forces have been reported to manipulate fluid, particles, and cells. Ding et al., “Surface acoustic wave microfluidics” Lab on a Chip 13:3626-3649 (2013); Friend et al., “Microscale acoustofluidics: Microfluidics driven via acoustics and ultrasonics” Reviews of Modern Physics 83:647 (2011); Ding et al., “On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves” Proc. Natl. Acad. Sci. USA 109:11105-11109 (2012); Collins et al., “Two-dimensional single-cell patterning with one cell per well driven by surface acoustic waves” Nat. Commun. 6:8686 (2015); Franke et al., “Surface acoustic wave actuated cell sorting (SAWACS)” Lab on a Chip 10:89-794 (2010); and Frommeltet al., “Microfluidic mixing via acoustically driven chaotic advection. “Physical review letters 100:034502 (2008).
Acoustic heating results from viscous attenuation of the acoustic energy into the fluid and was typically considered in the art as a major hurdle for biomedical applications because the temperature rise is usually not compatible with biological samples. Recent studies have shown that a well-controlled acoustic heating can be a valuable asset in driving chemical and biological reactions. Kulkarni et al., “Surface acoustic waves as an energy source for drop scale synthetic chemistry” Lab on a Chip 9:754-755 (2009); Reboud et al., “Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies” Proc. Natl. Acad. Sci. USA 109:15162-15167 (2012).; and Shilton et al., “Rapid and controllable digital microfluidic heating by surface acoustic waves” Adv. Funct. Mater. 25;5895-5901 (2015).
In one embodiment, the present invention contemplates a method comprising a standing acoustic wave (SAW) generator, wherein the SAW generator provides acoustic heating energy and acoustic force energy. In one embodiment, the acoustic heating energy provides fast and precisely controlled temperature ramping that unfolds and precipitates proteins without biological damage to the proteins. In one embodiment, the acoustic force energy drives an aggregation of precipitated proteins along the nodes and/or antinodes of the standing acoustic wave field. In one embodiment, the precipitated protein aggregation concomitantly results in a significantly enhanced local concentration and thereby increases a signal amplitude (e.g., increases the signal to noise ratio of the measured detection signal).
For example, one detection signal for monitoring protein unfolding comprises a gray intensity of precipitated proteins measured as a function of SAW time to analyze the thermal stability of proteins. In one embodiment, the A-TSA assay time is less than 2 minutes as compared to. tens of minutes or hours for conventional TSAs. In one embodiment, the A-TSA sensitivity is up to 34-fold higher than conventional TSAs. In one embodiment, the improved sensitivity of the A-TSA can provide detection of small thermal shifts upon protein-ligand bindings to diagnose mutational protein diseases (e.g. sickle cell disease (SCD)).
ps III. Acousto-Thermal Devices
In one embodiment, the present invention contemplates a protein acousto-thermal shift device comprising an acoustic manipulation element and an acoustic heating element. See,
Although it is not necessary to understand the mechanism of an invention, it is believed that such an acousto-thermal shift device performs an assay including, but not limited to, the steps of: (1) sample preparation; (2) sample characterization; and (3) data collection. In one embodiment, the device performs sample preparation, characterization and data collection in less than a few minutes. In one embodiment, the sample has a volume of ˜0.0001 mL. In one embodiment, the acuosto-thermal shift device is integrated into a single microchip. In one embodiment, the acuosto-thermal shift microchip device is operated by a cellular phone. In one embodiment, the sample comprises either purified protein or intracellular proteins.
A. A-TSA Device Design And Characterization
In one embodiment, the present A-TSA device comprises an acoustic element that generates surface acoustic waves (SAWs). Surface acoustic waves are believed to be a kind of sound and is a mechanical wave that propagates at the surface of solid substrate. In one embodiment an SAW generator applies an AC signal to a pair of interdigital transducers (IDTs) on top of a piezoelectric material. Although it is not necessary to understand the mechanism of an invention, it is believed that, once generated, an SAW propagates into the interface of a substrate and a liquid media. Further, the SAW may deflect into the liquid media as a leaky Rayleigh wave that can be used as an acoustic tweezer to: i) manipulate and pattern micro/nano particles/molecules (e.g., proteins: ii) generate fluid streaming; and iii) generate acoustic heating.
It has been reported that such acoustic tweezers have many demonstrated applications including: i) on-chip manipulation of C. elegans worms, cells, and nanoparticles; ii) tunable patterning of cells and molecules; iii) separation of label-free cancer cells; iv) cell lysis; and v) acoustic heating. Acoustic fields can produce rotational vortices that mechanically lyse both red blood cells and parasitic cells in a drop of blood as well as streaming at low powers. Although acoustic heating was described, cell lysis was not reported, nor was SAW-patterning of precipitated proteins in a microfluidic chamber. Reboud, J., et al., “Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies” PNAS USA 109(38):15162-15167, (2012). Mild sound wave intensities and frequencies have been disclosed to process cells in a manner that did not damage biological cells. Consequently, it is known that SAW preserves cell integrity during an acoustic separation process. While it is demonstrated that small radio frequency power can support a fully integrated cell separation and analysis system surface acoustic wave cell lysis is not shown. Ding, X., et al., “Cell separation using tilted-angle standing surface acoustic waves” PNAS USA 111:12992-12997 (2014). A noninvasive acoustic based method has been reported that manipulates single microparticles, cells or entire organisms in a microfluidic chip utilizing surface resonance to gently manipulate the sample. Cell viability tests were performed to verify the technologies compatibility with biological objects, but cell lysis with SAW resonance is not shown. Ding, X., et al., “On-Chip Manipulation of Single Microparticles, Cells, and Organisms Using Surface Acoustic Waves” PNAS USA 109:11105-11109 (2012). Acoustic radiation force providing a continuous flow of tunable surface acoustic waves has been reported to directly manipulate and sort cells, rather than using acoustic streaming to manipulate the fluids in which the cells are embedded but cell lysis and releasing intercellular proteins with surface acoustic waves was not shown. Ding, X., et al., “Surface acoustic wave (SAW) microfluidics” Lab on a Chip 13:3626-3649 (2012). The prior art fails to disclose that SAW results in protein precipitate and protein patterning.
However, a combination of multiple acoustic functions and effects into one device for protein thermal shift analysis has not been reported yet. Because of the great controllability of SAW, the present invention contemplates an acoustic thermal effect that precisely controls the temperature of a sample over a region of interest within a microfluidic channel or chamber. If proteins are present, a thermal induced protein unfolding results in the formation of a protein precipitate. Simultaneously, a standing SAW is formed that induces a pattern in the precipitated proteins taking the form of parallel lines or arrays between the acoustic pressure nodes or antinodes. Although it is not necessary to understand the mechanism of an invention, it is believed that such protein precipitate patterns dramatically enhances the local concentration of proteins, thus increasing measurement sensitivity.
Protein gray intensity can be measured by a camera (e.g., a cellular phone camera) at a series of time points through the whole precipitation process (about 10-50 seconds) to measure the melting curve and melting temperature. The melting temperature or melting curve shift provides immediate data regarding protein dynamics, interaction, or configuration. It is further belied that there is: i) a relationship between the gray density of protein precipitate and its concentration; ii) a protein response to acoustic waves over a broad range of frequency and amplitude; iii) an A-TSA application for diagnosis of diseases that is related to protein disorders (e.g., hemoglobinopathies) where modified proteins have melting temperature shifts; iv) an interaction between acoustic waves and protein dynamics that affects protein thermal stability in A-TSA.
Specifically, two identical SAWs were generated by applying an AC (alternating current) signal to a pair of interdigital transducers (IDTs) deposited on the surface of a lithium niobate piezoelectric substrate. A standing SAW was formed within a 1×10 mm2 polydimethylsiloxane (PDMS) microchannel. The microchannel was bonded on top of a substrate between these two IDTs. See,
Simultaneously, the standing SAW aggregates and concentrates precipitated protein along the acoustic pressure nodes and/or antinodes. See,
In one embodiment, the present invention contemplates a method for protein analysis, comprising: a) providing an acousto-thermal shift device comprising a microchannel and an acoustic element; b) loading a small amount of sample is loaded into the microchannel; c) heating the sample with said acoustic element to a first precise temperature; d) streaming and mixing the sample with said acoustic element, wherein cells in the sample are lysed and release intracellular proteins; and e) manipulating the intracellular proteins into specific patterns with said acoustic element under a second precise temperature, wherein the specific patterns are parallel lines or arrays within the microchannel which significantly enhance the local protein concentration and achieve a very high signal-noise ratio. In one embodiment, the acousto-thermal shift device performs steps a)-e) on a single microchip within a few minutes from loading sample to reading out the measured data.
Although it is not necessary to understand the mechanism of an invention, it is believed that low sample volumes and short assay times has not been previously reported for thermal shift assay devices. It is further believed that such advantages have great impact across multiple disciplines including, but not limited to: (1) drug industries by dramatically reducing the time cost for drug screening and drug discovery; (2) clinical applications by providing a low cost, fast, portable, and label-free tool for proteopathy diseases (protein disorder diseases) diagnosis such as Alzheimer's disease, prion diseases, amyloid, sickle disease etc., and for real-time monitoring about the efficacy of therapies; and (3) academia by providing a low cost and fast protein analysis device.
Most proteins are relatively soluble in aqueous solution at room temperature and can be denatured and/or unfolded at a certain temperature to form a precipitate. Each protein has a precise denaturation temperature that is generally known in the art as a melting temperature (Tm). Variations in melting temperature is very sensitive to protein configuration and its interaction with a binding ligand or other proteins. Consequently, a protein melting temperature shifts when the protein binds to a ligand or other proteins to form a complex. As a result, the observed melting temperature shift upon formation of these complexes can be used to study and determine protein dynamics, interactions, or status. In one embodiment, the present invention contemplates an acousto thermal shift device and assay (A-TSA) that utilizes surface acoustic wave-induced: i) acoustic heating; ii) acoustic streaming; and iii) acoustic patterning/manipulation to integrate cell lysis, heating, protein concentration, and data measurement all in one single step within one single microchip.
The data presented herein demonstrate protein unfolding and concentration of a purified protein of human hemoglobin (Hb) under SAW actuation. See,
A. Assessment of Protein-Ligand Binding
In some embodiments, SAW-driven protein unfolding and concentration can be used for analysis of protein-ligand binding to analyze the melting temperature (Tm) and its shift (Δtm) upon protein-ligand binding. Gray intensity of the precipitated and concentrated proteins was measured and plotted as a function of SAW time in A-TSA. See,
Two compounds, palmatine chloride (Pal) and oxaloacetic acid (OAA), were selected due to interact with (e.g., bind to) Hb and CS, respectively. Liu et al., “Studies on the interaction of palmatine hydrochloride with bovine hemoglobin” Luminescence 29:211-218 (2014); and Niesen et al., “The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability” Nat. Protoc. 2:2212 (2007). Under SAW actuation (e.g., 19.6 MHz, 3 Watts), a typical sigmoidal melting curves of Hb and Hb-Pal complexes reveals that the Tm of Hb (46.85 s) was reduced by 3.52 s and 7.30 s in the presence of 0.62 mM and 1.24 mM Pal, indicating destabilization of Hb by Pal. See,
Exemplifying the superiority of the presently disclosed A-TSA, the data shows that DSF-TSA could not successfully produce such typical sigmoidal melting curves for Hb and Hb-Pal complexes due to the inference from the intrinsic fluorescence of both Hb and Pal. See,
Likewise, the Tm of CS (60.3° C.) was increased by only 2.0° C. and 2.9° C. in the presence of 1 mM and 2 mM OAA as detected in the BCA assay. See,
To make a direct comparison between the presently disclosed A-TSA and C-TSA methods, their relative sensitivity was evaluated by defining the relative shift (Δtm/tm0 or ΔTm/Tm0) and sensitivity (tangential slope). The results showed that the presently disclosed A-TSA showed superior and enhanced sensitivity in monitoring the protein-ligand binding compared to the C-TSAs DSF-TSA and BCA-TSA. See,
Precision, as defined by the ratio between Δtm (or ΔTm) and standard deviation (s.d.) of tm (or Tm), i.e. Δtm s.d. (or ΔTm s.d.), was evaluated. See,
In addition, the present data shows that the magnitude of melting time shift Δtm in A-TSA could be facilely tuned by varying the SAW power. See,
B. Assessment of Protein Mutation
These promising results encouraged us to further investigate the potential of our ATSA method for diagnosis of mutational protein diseases, which are associated with protein misfolding and subsequent thermal stability changes. Cohen et al., “Therapeutic approaches to protein-misfolding diseases” Nature 426:905-909 (2003). Sickle cell disease (SCD) is one example which affects millions worldwide and is caused by polymerization of sickle Hb in individual RBCs. Piel et al., “Sickle cell disease” New Engl. J. Med. 376:1561-1573 (2017). However, the lack of practical diagnostic approach leads to an inability to early treatment and high childhood mortality especially in resource-limited areas. Ilyas et al., “Emerging Point-of-Care Technologies for Sickle Cell Disease Diagnostics” Clin. Chim. Acta (2019). Thermodynamic instability of sickle Hb is reported to be characteristic of SCD. Tam et al., “Sickle cell hemoglobin with mutation at αHis-50 has improved solubility. J. Biol. Chem. 290:21762-21772 (2015); and Meng et al., “Substitutions in the β subunits of sickle-cell hemoglobin improve oxidative stability and increase the delay time of sickle-cell fiber formation” J. Biol. Chem. 294:4145-4159 (2019).
We examined whether our technique could distinguish the Hb stability profiles with red blood cell (RBC) lysate from healthy (Ctrl) and SCD human donors. See,
In addition, under SAW actuation, the parallel-aligned protein patterns were formed in the microfluidic channels and no visible difference in their morphology was observed between healthy and SCD Hb. See,
Two proteins, i.e. human hemoglobin (Hb; Millipore Sigma, St. Louis, Mo., USA) and citrate synthase (CS) from porcine heart (Millipore Sigma), and two corresponding compounds, i.e. palmatine chloride (Pal; Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and oxaloacetic acid (OAA; Millipore Sigma) were primarily used. Human whole blood samples with K2EDTA as an anticoagulant were purchased from Zen-Bio Inc. (Research Triangle Park, N.C., USA) and stored in 4° C. (always used within 3-14 days after collection). Human blood plasma was obtained by centrifuging the human whole blood samples at 500 g for 10 min in 4° C. Sickle cell diseased (SCD) red blood cell lysate were obtained from patients with SCD, upon receiving written informed consent and in conformity with the declaration of Helsinki under a protocol approved by the Duke University Medical Center (no. NCT02731157) as described previously. Culp-Hill et al., “Effects of red blood cell (RBC) transfusion on sickle cell disease recipient plasma and RBC metabolism” Transfusion 58:2797-2806 (2018). All abovementioned chemicals were dissolved or diluted using Dulbecco's Phosphate-Buffered Saline (DPBS; Thermal Fisher Scientific, Hampton, N.H., USA) except Pal that was first dissolved in dimethyl sulfoxide (DMSO; Millipore Sigma) but then further diluted in DPBS. The DMSO concentration is 2% (vol/vol) in final Hb-Pal complex solution in order to prevent the potential damage to the protein by contact with high concentrations of DMSO. Fluorescent microspheres (polystyrene, 7.5 μm diameter) were obtained from Bangs Laboratories Inc. (Fishers, Ind., USA).
The SAW was generated and propagated on piezoelectric 128° Y-cut X-propagating lithium niobate (LiNbO3) wafer (500 μm thick). The device consisted of a pair of interdigitated transducers (IDTs) in parallel in order to generate two series of identical SAWs propagating in the opposite direction, producing a standing SAW. Each IDT consists of 30 pairs of electrodes (Cr/Au, 5/100 nm) with the width of electrode finger of 50 μm, pitch of 100 μm, and an aperture of 10 mm, yielding a frequency of approximately 20 MHz for the propagating SAW. Although different IDTs can be used, the resonance frequencies of most IDTs are in the range between 19.5 and 19.6 MHz. A PDMS microchannel with height of 100 μm and width of 2 mm was then fabricated through a standard soft-lithography and model-replica procedure. Lastly, both the PDMS channel and the IDT substrate were treated with oxygen plasma and bonded together to form the final SAW device. See,
The A-TSA SAW device was mounted on the stage of an inverted microscope (ECLIPSE Ti-U, Nikon, Japan). A radio frequency (RF) signal was generated by a function generator (EXG Analog Signal Generator, Keysight, Santa Rosa, Calif., USA) and amplified by an amplifier (403LA, Electronics & Innovation, Rochester, N.Y., USA). Five microliters of protein, plasma, red blood cell lysate or protein-compound mix solutions were injected into the channel before the RF signals were applied. A fast camera (ORCA-Flash4.0LT, Hamamatsu, Japan) was connected to the microscope to capture the process, and all the videos were recorded in 4 frames per second.
All image and videos processing were performed in ImageJ (National Institute of Health, Bethesda, Md., USA) in the same way as described below. The same sized regions of interest (ROIs) were traced around the perimeter of each pattered protein fiber in order to monitor the gray intensity and its change during the course of protein melting and aggregation. At least five ROIs were selected and characterized for each video. Melting time was defined as the time point when there is 50% gray intensity change Im=(Imax+Imin)/2.
Two conventional methods were adopted for thermal shift assay: i) SYPRO differential scanning fluorimetry (DSF) assay; and ii) bicinchoninic acid (BCA) assay.
SYPRO DSF assay: SYPRO Orange melting curves were collected using the 7900HT Fast Real-Time PCR System. The SYPRO Orange fluorescent signal is detectable using the calibration setting for the ROX filter. Melting curves were performed using 1 mg/mL of protein with a 1:2500 dilution of SYPRO Orange (Molecular Probes Inc #S-6651) in 100 mM PBS, pH 7.4, using a minimum of 4 replicates. A 1% ramp rate from 25° C. to 95° C. was utilized during data collection. Drug concentrations are as indicated. To analyze melting curves, the fluorescence was normalized to the starting temperature and to no protein controls. The data was then scaled to interval (0,1) and then the replicates were averaged, and standard deviation calculated.
BCA assay: For thermal gradient profiling, a gradient program was created using a PTC-200 thermal cycler (MJ Research, Reno, N.V., USA) to cover the temperature points indicated in each figure. A PCR plate was prepared with 25 μL per well of recombinant protein or lysate and sealed (4titude Random Access, PN 4ti-0960/RA 96-well plate). The plates were spun at 1200 g for two minutes at 4° C., and then kept at 4° C. prior to use. The plate was placed in the thermal cycler with the heated lid closed for 3 minutes and was then spun at 1200 g for two minutes to remove any condensation. The PCR tubes were removed from the PCR plate, carefully placed in 1.5 mL tubes, and spun at 21,000 g for 30 min at 4° C. to pellet the aggregate protein. Supernatant was carefully removed from each tube and placed in a clean, low-retention, 1.5 mL tubes. 10 ul of solution was removed and a Pierce BCA protein assay kit (PN 23225) was used for the determination of the total protein in each sample.
All data were expressed as means±SD. The data (melting time and melting temperature) from multiple runs (n≥3) were plotted using Graphpad Prism 8.0 software (GraphPad Software Inc., La Jolla, Calif., USA). The BCA-TSA data were fitted using a Sigmoidal dose-response (variable slope) curve fit. Unpaired t-test or ordinary one-way ANOVA with Tukey's multiple comparison test was used to analyze statistical significance. A p-value<0.05 was considered statistically significant. Within the figures, the significance was denoted by the following marks: * or # for p<0.05; ** or ## for p<0.01; and *** or ### p<0.001.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/48451 | 8/28/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62894423 | Aug 2019 | US |
