Acoustic-based microfluidic amplification

Abstract

Systems, devices, and techniques are configured to leverage acoustic-based microfluidic amplification for the detection of a biological substance, such as misfolded proteins associated with a protein-misfolding disease. In one example, a system (100) includes a housing defining a main channel (104) configured to contain a fluid solution containing a sample including a plurality of proteins and one or more side channels (106) in fluid communication with the main channel, wherein each side channel of the one or more side channels are configured to establish a liquid-gas interface (108) between the fluid solution in the main channel and air contained in the one or more side channels. The systems may also include at least one acoustic generation element configured to generate acoustic waves within the air of the one or more side channels that causes vibration of the liquid-gas interface and mixing of the fluid solution within the main channel.

Description

ACOUSTIC-BASED MICROFLUIDIC AMPLIFICATION

[0001] This application is a PCT application that claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 497,907, filed April 24, 2023, the entire contents of which is incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure relates to fluid devices, and, more specifically, microfluidic devices for acoustic-based amplification of biological substances.BACKGROUND

[0003] Prion diseases (PDs) are a class of fatal neurodegenerative diseases, collectively known as transmissible spongiform encephalopathies (TSEs), which exist in several forms; Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in cervids. These diseases are caused by the accumulation in the brain of an abnormally folded and protease-resistant form (PrPSc) of cellular prion protein (PrPc). Once formed, PrPSccan bind to PrPcand act as a seed to induce misfolding and the creation of additional PrPSc, which eventually leads to subsequent aggregation into fibrils and plaques. After onset, aggregated PrPScpropagates throughout the central nervous system, damaging tissues and eventually causing death. PDs have a long incubation time, at least 16 months, before an infected animal develops signs of infection. However, throughout the incubation period, PD-infected individuals spread the PrP&cwithin the population through both direct and indirect contact. Since the first reported case in 1967, CWD has spread across North America, infecting both captive and free-ranging cervid populations in the United States and Canada. Spontaneous cases of CWD have now been identified in Scandinavia and the disease was introduced to South Korea in 1997 through tire shipment of infected elk from Canada, Infected animals with CWD release infectious prions through saliva, urine, and feces, which results in rapid spread among cervids and potentially to other animals.SUMMARY

[0004] This disclosure describes systems, devices, and techniques configured to leverage acoustic -based microfluidic amplification for the detection of a biological substance, such as misfolded proteins associated with a protein-misfolding disease. A system may include anacoustic generation element, such as a piezoelectric element, photoacoustic mixer, or a speaker, in contact with a housing of the device to cause vibration of a liquid-gas interface or thin membranes within a chamber or channel of the device. The gas may include multiple types of modules (e.g., atmospheric air that includes molecules such as nitrogen, oxygen, carbon dioxide, etc.) or only a single type of molecule (e.g., nitrogen). Vibration of this liquid-to-gas (or air-to-fluid) interface or thin membrane can cause mixing within the fluid within the device. This mixing can cause certain biological substances to contact each other and amplify those biological substances. In the case of chronic wasting disease, which is a misfolded protein disease, misfolded proteins associated with the disorder can cause normal proteins to also misfold when subject to acoustic-based mixing and effectively amplify the number of misfolded proteins in a sample. By amplifying the amount of misfolded proteins within a sample, the system may be able to detect the presence of the misfolded proteins and determine whether the sample is positive for the disease in question.

[0005] In one example, a system includes a housing defining: a mam channel configured to contain a fluid solution containing a sample including a plurality of proteins; and one or more side channels in fluid communication with the main channel, wherein each side channel of the one or more side channels are configured to establish a liquid-gas interface between the fluid solution in the main channel and air contained in the one or more side channels; and at least one acoustic generation element configured to generate acoustic waves within at least one of the gas of the one or more side channels or tire fluid sol ution m the main channel that causes vibration of the liquid-gas interface and mixing of the fluid solution within the main channel. Alternatively, or additionally, one or more thin membrane structures configured to extrude into the main channel; and at least one acoustic generation element configured to generate acoustic waves which induces vibration of thin membrane and mixing of the fluid solution within the main channel.

[0006] In one example, a method includes containing, within a main channel of a housing, a fluid solution containing a sample including a plurality of proteins, wherein the housing comprises one or more side channels in fluid communication with the main channel or one or more thin membranes, and wherein each side channel of the one or more side channels are configured to establish a liquid-gas interface between the fluid solution in the main channel and air contained in the one or more side channels; and generating, via at least one acoustic generation element, acoustic waves within at least one of the gas of the one or more side channels or the fluid solution in the main channel that causes vibration of the liquid-gas interface and mixing of the fluid solution within the main channel.

[0007] The details of one or more examples of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1A is a conceptual illustration of protein amplification caused by acoustic amplification of sample proteins within a fluid, as described herein.

[0009] FIG. 1B is a side view cross-sectional conceptual and schematic diagram illustrating the example acoustic-based amplification device of FIG. 1A.

[0010] FIG. 1C is a perspective view of an example acoustic -based amplification device of FIG. 1A.

[0011] FIGS. 2A and 2B are images of example fluorescent microparticles moving using acoustic vibration as described herein.

[0012] FIG. 2C is a graph of particle velocity for different driving voltages.

[0013] FIG. 2D is a graph of particle veloci ty for different frequencies of piezoelectric transducers.

[0014] FIG. 3 includes images of fluorescence after different amplification times.

[0015] FIGS. 4A and 4B are graphs of fluorescence intensity after different amplification times.

[0016] FIGS. 4C and 4D are transmission electron microscope (TEM) images of prion fibrils amplified as described herein.

[0017] FIG. 5A is a graph of fluorescence intensity for positive and negative samples.

[0018] FIG. 5B includes images of positive and negative stunpies with differing colors using gold nanoparticle aggregation assays.

[0019] FIG. 6 is a flow diagram illustrating an example technique for amplifying a biological substance using an example acoustic-based amplification device described herein.DETAILED DESCRIPTION

[0020] This disclosure describes systems, devices, and techniques configured to amplify proteins of a sample using acoustic -based amplification for the detection of one or more types of proteins, such as misfolding prions associated with various diseases. This acoustic-based amplification may leverage a liquid-gas interface to induce this amplification. Various diseases can be caused by the accumulations of abnormally folded or mis-fokied proteins. One example protein is a mis-fokling prion protein associated with CWD. Recent studieshave shown that infected herd populations decrease by approximately 10.4% in white-tailed deer and 21% in sympatric mule deer populations in southeastern Wyoming. Moreover, the social cost for CWD is tremendous as Wisconsin has reported that over $32.3 million has been spent for CWD surveillance and management in 2001 and 2006. Therefore, there is an urgent need for a reliable in situ diagnostic platform for the management of CWD in wildlife.

[0021] Current gold standards for the diagnosis of TSE's rely heavily on antibody-based enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) technologies that are expensive, time consuming, and require substantial training and expertise to operate. Moreover, diagnostic sensitivity is significantly hampered due to the inability of antibodies to distinguish between PrPC and PrPSc and the need for enzymatic, chemical, and / or heat digestion to enrich PrPSc. Therefore, a definitive diagnosis of TSE often calls for post-mortem histopathological examination. Recently, remarkable improvements in the diagnosis of CWD have been made by a novel ultrasensitive seeding assay that is based on in vitro amplification of PrPCWD. Real-time quaking -induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) both exploit the ability of PrPSc to induce PrPC to misfold in a cyclical fashion to form aggregates of PrPSc fibrils. The former utilizes shear force while the latter utilizes sonication to mechanically fragment the PrPSc fibrils to smaller nucleation sites. This results in exponential growth of PrPSc which can be monitored in real-time by their ability to bind a fluorescent dye, namely thioflavin T. This process can take up to 30 to 90 hours and requires periodic shaking to induce fibril formation6. While RT-QuIC boasts high sensitivity, its application in in-situ diagnosis is limited by several factors. For example, it requires bulky and expensive equipment. Moreover, manual handling of reagents increases the risk of contamination as well as the reagent cost. Thus, there is a critical need to develop an in-situ assay platform that confers ultrafast diagnosis of CWD in a fully automated manner.

[0022] Microfluidics offers multitudes of advantages in diagnostic application over macroscopic platforms including, low' reagent requirement, high specific surface area-to- volume ratio, biohazard containment and high heat and mass transfer rate. However, conventional microfluidic devices suffer from an inherently low Reynolds number, which limits the mass transfer to a diffusion-limited regime. Moreover, the use of elaborate pumps limits their application in on-site diagnosis. Both active and passive micromixers have been developed including, inertial, acoustofluidic, electrokmetic and magneto-hydrodynamics micromixers. Among these, acoustofluidic micromixers can be a powerful tool due to thehigh mixhig index, low-cost of operation, biocompatibility, portability and the contact-free nature of the technology.

[0023] As described herein, devices, systems, and techniques involve leveraging acoustic amplification of biological elements such as proteins. While a number of acoustofluidic micromixers can be used, lateral cavity acoustic transducers (liquid-gas interfaces) can be used for biological application. For example, shear stress induced by liquid-gas interfaces can be enough to fragment 50 kbp DNA into 5 kbp fragments. Acoustofluidic microstreaming could provide enough shear stress to fragment non-covalent interaction between amyloid fibril subunits. It is important to note that the operational mixing frequencies of these devices are versatile, allowing for effective mixing at frequencies that may or may not coincide with the acoustic resonance of the liquid-gas interface, the mechanical structure, or the fluidic channels. This flexibility significantly enhances the device's utility across a variety of experimental conditions and sample types.

[0024] A liquid-gas interface as described herein may refer to any liquid phase of a substance, e.g., as a fluid that may include one or more biological components, and a gas phase of a substance, e.g., atmospheric air that may include one or more different types of gas molecules. A device may be configured to establish a liquid-gas interface that transfers energy generally in two dimensions perpendicular from the liquid-gas interface. This two dimensional interface may be present in a channel that has a width substantially greater than a height, for example. Such a channel may establish a relatively planar liquid-gas interface or a curved liquid-gas interface with the curve generally confined to two dimensions. In other examples, the device may be configured to establish a liquid-gas interface that transfers energy in three dimensions. This three dimensional liquid-gas interface may be established with a channel that has a relatively large cross-sectional area that facilitates a liquid-gas interface that is curved substantially in three dimensions. In some examples, a three- dimensional liquid-gas interface may even be a partial or complete bubble (e.g., an enclosed liquid-gas interface). In this manner, a three dimensional liquid-gas interface may transfer energy in substantially three dimensions.

[0025] FIG. 1A is a conceptual illustration of protein amplification caused by acoustic amplification of sample proteins within a fluid, as described herein. As described herein, system 100 can include integrated RT-QulC on a PDMS-coated glass slide with an acoustic microstreaming-based microfluidic device to mix the PrPSc and PrPC. Mixing of PrPSc and PrPC is described as one example of misfolding proteins or other biological substance amplification for which system 100 can be utilized. The sample, including proteins such asPrPSc and PrPC, can be contained within fluid of main channel 104. Air (or some gas) is contained within air cavity 110 of one or more side channels 106. The liquid-gas interface 108 is provided to transfer vibrations to the fluid. Substrate 102 may be at least a portion of housing that contains mam channel 104 and one or more side channels 106.

[0026] In some examples, the reagent mixing (e.g., within the fluid contained within system 100, is achieved with lateral cavity acoustic transducers (liquid-gas interfaces) where arrays of dead-end side channels (e.g,, one or more of side channels 106) capture air bubbles which function as a vibrative membrane. In some aspects, the reagent mixing can be performed on an acoustic resonance of the liquid-gas interface, or the mechanical structure, or the fluidic channel. Liquid-gas interfaces are one example of an acoustic generation element. In some aspects, the liquid-gas interface can be in a two dimensional (2D) or a three dimensional (3D) arrangement as described herein. For example, the liquid-gas interface can be the interface between the liquid and one or more air bubbles. This vibrative membrane may also be referred to as the liquid-gas or fluid-air interface. Upon the application of the high frequency soundwave (e.g., 4.6 kHz), one or more liquid-gas interfaces 108 resonate and the resulting acoustic field energizes the bulk liquid and produces a net force perpendicular to the bubble interface and out the end of the cavity (e.g., one or more of air cavities 110 within side channels 106). The vibration of the acoustic transducer may cause liquid-gas interface 108 and / or another structure of the device to resonate and energies the liquid in the channel. The resulting acoustic streaming enhances the collision between PrPSc and PrPC as well as fragmentation of PrPSc into smaller pieces, which in turn result, in the multiplication of active seeds for the PrPSc nucleation, liquid-gas interfaces can be well suited for this application due to the simplicity in fabrication, tunability of mixing rate, and high mixing index. In some examples, tire tunability of the mixing rate may refer to the amplitude of the applied energy, resonance frequency of one or more fluids or structures, or other selectable parameters.

[0027] By combining the reagent microstreaming induced by the acoustofluidic micromixer and the quaking based prion fibril amplification, this method could drastically reduce the amplification time from 48 hours using other techniques to 3 hours or less using the acoustic amplification described herein. Furthermore, this technique of amplification can be compatible with a gold-nanoparticle-based aggregation assay, which eliminates the need for bulky auxiliary detection modules. In some aspects, this technique of amplification can be compatible with capillary height measurements, a colorimetric assay (e.g., a gold nanoparticle colorimetric assay, a silver nanoparticle calorimetric assay, or another colorimetric assay using other particles or dyes such as one or more organic dyes), orfluorescence imaging. In some aspects, detection of a target biological substance that can be amplified as described can occur using the naked eye instead of microscopes or other equipment. In some aspects, detection of a target biological substance that can be amplified as described can be performed using a microscope or camera or other image capturing or viewing device. This ultrafast prion amplification in an acoustically-driven microfluidic platform can thus reduce amplification time and reduce cost associated with detecting the results of any amplification. The Micro-QuIC device, such as system 100, features advantages such as simplicity of use, automation, low cost, and portability. These advantages are ideal for future development of an automated all-in-one, on-chip prion amplification toolkit for in situ diagnosis.

[0028] As described herein, a microfluidic based RT-QuIC system can be used to amplify target biological substances to detect whether or not a particular biological substance is present in a sample. Misfolded proteins for CWD are described herein, but other misfolding protein diseases or other conditions may also be detected using this method. In one example, tissue is added to a solution containing a high concentration of healthy non-misfolded prion and injected to a microfluidic channel. The sample is then shaken for a period of time, such as 4 hours. Upon application of AC bias to the transducer, the acoustic wave generated by the piezoelectric transducer will cause vibration in liquid-gas interface. In some aspects, the liquid-gas interface can be in a 2 dimensional or a 3 dimensional arrangement. For example, tire liquid-gas interface can be the interface between the liquid and an air bubble. This can result in streaming of reagent solution which vigorously mixes prion proteins. If the sample had misfolded prion (As CWD positive animals do), the misfolded prions will cause the healthy prions to misfold, thus greatly increasing the number of misfolded prions. If the sample had no misfolded prions (as in CWD negative animals) there would not be any protein misfolding of the healthy prions. After the mixing, microfhiidic channel is observed under fluorescent microscope or via another visualization such as using gold nanoparticles. If tire solution exhibit high fluorescence intensity or specific color, the sample is CWD positive, if the solution has very low fluorescence intensity or specific color, the sample is CWD negative.

[0029] In some examples, system 100 may include a housing defining amain channel 104 configured to contain a fluid solution containing a sample including a plurality of proteins, one or more side channels 106 in fluid communication with the main channel, wherein each side channel of the one or more side channels are configured to establish a liquid-gas interface 108 between the fluid solution in the main channel and air contained in the one ormore side channels 106, and at least one acoustic generation element (not shown in FIG. 1 A) configured to generate acoustic waves within the air of the one or more side channels 106 or the fluid within a side channel or main channel 104 that causes vibration of the liquid-gas interface 108 and mixing of the fluid solution within the main channel 104. Liquid-gas interface 108 is shown in the example of FIG. 1 A as a two dimensional interface because energy may generally be transferred within the plane of substrate 102. However, if the thickness of substrate 102 may be increased such that the liquid-gas interface is similar to the width of side channels 106, liquid-gas interface 108 may form more of a three dimensional interface that transfers energy within the plane of substrate 102 and also in different angles within the height of substrate 102.

[0030] The main and side channels may have a variety of different dimensions. These dimensions may be selected according to one or more characteristics of a target sample, biological substance(s) within the sample, fluid viscosity, frequency, or any other characteristics. In some examples, the mam channel has a width from 100 micrometers to 500 micrometers. In the example of FIG. 1A, the width of main channel 104 is approximately 300 micrometers. In some examples, each of the side channels 106 has a width from 50 micrometers to 500 micrometers. In one example, the width of side channels 106 is approximately 100 micrometers. In some examples, the width each of the side channels 106 is less than a width of the main channel. Although side channels 106 are described for establishing liquid-gas interface 108, any structures or changes to mam channel 104 that can maintain an air bubble or interface between air and liquid may be used for conveying the acoustic waves to the fluid. For example, the channel may take the form of a bulge, gap, crack, or any other structure in fluid communication with main channel 104.

[0031] The housing that defines main channel 104 and one or more side channels 106 may include a portion of substrate 102 m some examples. In some examples, the housing may include a glass slide as substrate 102 and a material, such as a first polydimethylsiloxane (PDMS) layer. Polymethyl methacrylate (PMMA) or glass, disposed on the glass slide and at least partially defining the main channel between the glass slide and the first housing layer. In some examples, the system may include first tubing in fluid communication with an inlet of the main channel and second tubing in fluid communication with an outlet of the main channel through which a sample may be added to the main channel. In some examples, the housing includes a second PDMS PMMA or glass layer disposed around at least a portion of the first housing layer and defining a space between the first housing layer and the second housing layer and a hydrophobic fluid disposed in the space between the first housing layerand the second housing layer. In some examples, the housing may be constructed from any polymer. One example housing may be a CD-ROM material.

[0032] System 100 may include an acoustic generation element, or multiple such elements. In one example, the acoustic generation element is a piezoelectric transducer. In another example, the acoustic generation element is a lateral cavity acoustic transducer (liquid-gas interface). The acoustic generation element may be in contact with a portion of the housing and / or disposed at an open end of one or more of the side channels exposed to air. System 100 may also include a controller configured to energize the acoustic generation element for one or more periods of time. This controller may turn a voltage from a power source on or off, or, in some examples, the controller may adjust the current or voltage to adjust the frequency and / or amplitude of the acoustic generation element in order to generate the desired acoustic waves within the main chamber.

[0033] In some examples, the at least one acoustic generation element is configured to generate the acoustic waves within the air of the one or more side channels to cause misfolded proteins to contact other proteins within the plurality of proteins to amplify a quantity of the misfolded proteins to a detectable level. System 100 may also facilitate detection of any amplified biological substances. In one example, the amplification may be visualized with the naked eye via a transparent portion of the housing and a change in color of the sample. In some examples, at least a portion of the housing is transparent and enables fluorescence intensity measurement of at least some of the plurality of proteins of the sample within the mam channel after mixing. In some examples, outlet of the housing is connected to a capillary tubes to measure the change in viscosity. In some examples, an additional capillary channel is configured in conjunction with the main channel to measure change in viscosity of the amplified reagent. Although substrate 102 is generally shown as a fiat, or planar, substrate, device 100 may instead be configured to create liquid-gas interfaces that are three dimensional. In some examples, the three dimensional liquid-gas interface may form one or more partial or complete bubbles.

[0034] FIG. 1B is a side view cross-sectional conceptual and schematic diagram illustrating the example acoustic-based amplification system 120, which may be similar to system 100 of FIG. 1A. System 120 of FIG. 1B may also be representative of a cross-sectional diagram of system 100. System 120 may include a substrate 122 upon which first layer 126 partially rests to form channel 124. A second layer 130 surrounds at least some of first layer 126, and a substance 128, such as mineral oil, may be disposed between first layer 126 and second layer 130. Tubing 132Aand 132B (collectively "tubing 132”) enables a sample to be injectedinto and removed from channel 124, as tubing 132 is configured to travel through the first layer 126 and second layer 130. One or more piezoelectric transducer 134 can be placed in contact with a portion of system 120, such as in contact with an underside of substrate 120. Piezoelectric transducer 134 is just one example, element that may be used to generate and transfer vibration energy to a portion of system 120 at a target resonance frequency.

[0035] As shown in the example of FIG. 1B, a sample may be delivered through tubing 132 and into the main channel 124, which is defined by first layer 126 (e.g., PDMS, PMMA or glass) on substrate 122 coupled to piezoelectric transducer 134. Second layer 130 (e.g., PDMS, PMMA or glass) may define a channel that can contain another substance 122, such as mineral oil or other fluid, which may be provided to stabilize the sample and fluid within mam channel 122.

[0036] System 120 of FIG. 1B is an example of a microfluidic quaking induced conversion (Micro-QuIC) device. As described herein, this device can combine the active mixing benefits of acoustic technology with quaking induced prion amplification. To that end, FIG. 1A and 1B provide a schematic and photo of an example assembled Micro-QuIC device, respectively. In one example, the foundation (e.g., substrate 122) of the acoustofluidic device is a thin glass coverslip. This glass coverslip serves to transfer the vibration energy from acoustic transducer 134 (shown in FIG. 1B) into the PDMS chip via flexural waves that travel out from the transducer and along the glass coverslip. The primary design of the microfluidic channel geometry' may include lateral cavity structures (e.g., one or more side channels such as channels 106 shown in FIG. 1A) which capture air cavity upon injection of reagents. The resulting one or more liquid-gas interfaces serves as an oscillating membrane in response to vibration from the transducer, producing acoustic streaming in the channel to agitate the samples. As shown in the example of FIG. 1A, the lateral cavity structure (e.g., main channel 104) is designed with a channel width of 300 pm and a height of 100 pm,. In total, there are 16 sets of lateral cavity structures over the length of the main channel (e.g., channel 104 or channel 124) in these examples.

[0037] Second layer 130, which may by an outer channel and include PDMS, PMMA or glass channel, can be attached on top of first layer 126 (e.g., a microfluidic PDMS, PMMA or glass layer) to create a void which is filled with substance 128 (e.g., mineral oil) to prevent heat-mediated evaporation of the reagent. The sample reagents can be introduced into the mam channel 104 or 124 through tubing 132A or 132B (e.g., example inlets or outlets) while incubating the entire device at 45 °C. Piezoelectric transducer 143, a speaker, or other vibration generation device, can be attached at the back of substrate 122 (e.g., a glass slide) toprovide an acoustic field to induce vibration of the liquid-gas interface. This creates micro- vortex streaming in the main channel 104 or 124, which accelerates both collision between PrP particles and shear-induced fragmentation of PrPSc fibrils. The collision between PrP particles in turn accelerates conversion of PrPc to PrPSc. In other words, the misfolded proteins contact normal folding proteins and cause additional misfolding of proteins to amplify the misfolded proteins in the sample. The fragmented PrPSc serves as a nucleation site for further fibril formation. As the number of fibrils increases, a fluorescent dye, Thioflavin T, specifically binds to the amyloid aggregates which can be observed under a fluorescent microscope. Although fluorescence can be used in some examples, other types of visualization can be used. For example, gold nanoparticles can be used to bind to the aggregates that change the visible color of the fluid sample after amplification if sufficient misfolded proteins are present.

[0038] In some examples, misfolded chronic wasting disease (CWD) prion seeds originating from biological samples are added to rPrP solutions and injected into a microfluidic device. This fluid creates a liquid-gas interface between the main channels and side channels. The application of high frequency sound waves (e.g., 4.56 kHz) can induce vibration of the liquidgas interface to create a vortex current. These solutions can then be shaken and incubated for approximately 3 hours at 45 degrees Celsius, although shorter and longer times, or higher or lower temperatures are also contemplated. If present, PrPCWD induces conformational changes of the rPrP upon contact during mixing caused by the acoustic waves. Resulting products can be labeled with Thioflavin T which can be detected under fluorescent microscope (e.g., an excitation wavelength in a range from about 300 nanometers (nm) to about 480 nm and an emission wavelength in a range from about 395 nm to about 500 nm.). These excitation and emission wavelengths are merely examples, and other wavelengths may be used in other examples. As shown in FIG, 1B, double layers (e.g., first layer 126 and second layer 130) of PDMS devices can be bonded to a thin glass slide. Mineral oil (e.g., substance 128), or some other oil or hydrophobic fluid, is injected between the two layers of PDMS to prevent solution evaporation. Piezoelectric transducer 134 or other acoustic generation element can be attached on the back of substrate 122 (e.g., a glass slide). These materials described with respect to FIGS. 1A and 1B are merely examples, as other materials may be used to construct systems 100 and 120 in other examples.

[0039] FIG. 1C is a perspective view of an example acoustic-based amplification device 160 w'hich may be similar to system 100 of FIG. 1A and / or system 120 of FIG. 1B. As shown in FIG. 1C, device 160 includes housing 164 to which piezoelectric transducer 162 is atached.The fluid including the sample biological substances, such as proteins which may or may not have misfolded proteins, can be injected into the main channel within housing 164 via tubing 166. Fluid can be extracted from the main channel via exit tubing 168.

[0040] FIGS. 2A and 2B are images of example fluorescent, microparticles moving using acoustic vibration as described herein. The device (e.g., system 100, system 120, or device 160) may be characterized in different manners. 'The ability of the device to generate micro- vortex streaming was characterized using fluorescent microparticles as shown in FIGS. 2A and 2B. Fluorescent microparticles showed vigorous circular motion upon application of an input bias voltage of 10 Vpp at 4.6 kHz in FIG. 2B indicated by the circular arrow's. The streaming velocity largely depends on two parameters: frequency and voltage.

[0041] In one example, to determine the frequency at which the liquid-gas interface generated the strongest acoustic streaming effect, the AC frequency was swept from 1 kHz to 100 kHz with 50 Hz increments. This experimental result indicated that the strongest acoustic streaming effect was generated when the device was excited at 4.6 kHz, which coincided with the resonance frequency of the piezoelectric transducers. Outside the resonant frequency of the piezoelectric transducers, the streaming velocity drastically decayed, as shown in FIG.2D. Therefore, the AC bias was maintained at the resonant frequency for ail of our experiments. The device was further characterized by applying different driving voltages to tlie piezoelectric transducer. FIG. 2C shows the mixing performance with the different driving voltages at a frequency of 4.6 kHz. The results show that as the driving voltage of the piezoelectric transducer increased, the mixing efficiency was increased. These voltages and frequencies are specific to one specific device with specific materials and dimensions of the main and side channels. Other devices with one or more different characteristics may be operated at different frequencies and / or voltages. Although the resonance frequency of the piezoelectric transducers were used in this example, other resonances may be used m addition or as an alternative in other systems. For example, the energy may be transferred via resonance of the liquid-gas interface, the resonance of a channel, the resonance of a structure of the device, and / or the resonance of any other element that is part of the device containing the liquid-gas interface.

[0042] FIG. 3 includes images of fluorescence after different amplification times. Using the devices described herein, techniques can achieve on-chip Prion Amplification with Micro- QuIC. Next, we established the experimental condition for Micro-QuIC. In some examples, real-time prion amplification in a microfluidic channel can occur. Solution containing CWD positive sample were injected in one example, acoustically mixed with 30 second on / offinterval at 45 °C. Upon application of AC bias, acoustic wave cause vigorous vibration of liquid-gas interface which mixes prion proteins. As misfolded protein collide with non- misfolded protein, the conversion of non-misfolded protein to misfolded protein occurs. Misfolded proteins starts to form fibril structure which in turn allows binding of thioflavin T binding and hence fluoresce.

[0043] In this example, initially, a spontaneously misfolded rHaPrP (positive control) was spiked in a master mix consisting of IX PBS, ImM F.DTA, 170mM NaCl, 10 μM ThT and rHaPrP ranging from 0.1 mg / mL to 0.4 mg / mL. The use of a high concentration of the rHaPrP can accelerate the reaction kinetics and hence the rHaPrP concentration was maintained at 0.4 mg / mL at all times unless stated otherwise. Both positively and negatively seeded master mixes were injected in a Micro-QuIC device and amplified for 180 minute. Here, the 10 Vpp AC bias at the frequency of 4.6 kHz was applied in a 30 minute interval.

[0044] The fluorescent images were taken every 30 minutes to follow the amplification process over 180 minutes, as shown in FIG. 3. For the quantification of fluorescent intensity, fluorescent particles were analyzed in the field of view by thresholding. Both the number of fluorescent particles and their size increased from the positively seeded sample, indicating successful prion amplification, while there was no measurable change in fluorescence intensity in the negatively seeded sample. Based on this result, prion amplification can be achieved in 180 minutes, which is 16 times faster than conventional RT-QuIC. To validate tire amplified prion fibrils, transmission electron microscopy (TEM) to compare the dimensions and the morphology of amyloid fibrils amplified by both RT-QuIC and Micro- QuIC (FIG . 4C and 4D. As reported previously, the fibrils from both samples were helical in shape with an average width of 25 nm (but can be smaller or larger in other examples) which further validates that amplification products were equivalent in both methods.

[0045] FIGS. 4A and 4B are graphs of fluorescence intensity after different amplification times. These graphs are examples of real sample amplification and AuNP-based optical detection. Since Micro-QuIC can greatly increase rHaPrP misfolding and amplification kinetics, the device and techniques can be leveraged for potential Micro-QuIC for CWD diagnostics using PrPCWD positive and negative white-tailed deer lymphoid tissues, as one example. Homogenates of independently confirmed CWD positive and negative white-tailed deer medial retropharyngeal lymph nodes (RPLN) can be prepared. Micro-QuIC techniques could facilitate fast misfolding and amplification of rHaPrP solutions seeded with CWD positive tissue samples. In one example, microfluidic devices described herein were prepared and injected with mastermix seeded with positive (n=5) and negative (n=5) tissue samples.Each sample has beers incubated at 40 degrees Celsius with periodic acoustic mixing for 3 hours. As shown in FIG. 4A, end-point fluorescence measurement of the microfluidic channel showed that the technique was able to distinguish CWD positive and negative samples through the difference in fluorescence intensity. For comparison, FIG. 4B shows real-time prion amplification in a conventional RT-QuICreaction that can take 34 hours to reach maximum amplification for positive samples.

[0046] FIG. 5A is a graph of fluorescence intensity for positive and negative samples. As shown in the graph of FIG. 5A, fluorescence intensity of the positive samples that included misfolded proteins which were amplified by the acoustic amplification techniques w'ere delectably different than negative samples. The significant reduction in assay time (approximately 3 hours, in some examples) makes Micro-QuIC an attractive alternative to the current gold-standard for CWD diagnostics.

[0047] In some examples, an optical-based misfolded PrP detection system using gold nanoparticles (MN-QuIC) can eliminate the need for a bulky fluorescence detection module. In conjunction with Micro-QuIC, the AuNP based detection system would provide a valuable tool towards the field deployable CWD testing platform. In one example, Micro-QuIC and MN-QuIC were used together. Tissue samples were first amplified in a microfluidic channel at 40 degrees Celsius with periodic acoustic mixing for 3 hours as described herein. The post- amplified mixture was drawn from the microfluidic channel and incubated with a gold nanoparticle reagent for 10 minutes. Upon addition of positive samples, the absorbance peak remained unchanged at 515 nm while the addition of negative sample absorbance peaks were shifted to a longer wavelength of approximately 560 nm.

[0048] FIG. 5B includes images of positive and negative samples with differing colors using gold nanoparticle aggregation assays, which illustrates the longer wavelength of the negative tissue samples. This difference in detectable color demonstrates a portable detection method for field application. Both CWD positive and negative sample were amplified using the microfluidic channel as described herein. Gold nanoparticle aggregation assay are able to detect CWD positive / iiegative sample. Briefly, CWD negative samples allow aggregation of AuNPs which result in blue-shift in their absorbance spectrum while, CWD positive sample prevents AuNPs and hence the absorbance spectrum remains unaffected. This eliminates the need of bulky and expensive fluorescence detection system which is more adequate for field application.

[0049] CWD is just one example use case for the devices, systems, and techniques described herein. Given the continued spread of CWD among cervid populations throughout NorthAmerica and Northern Europe, there is an urgent need to develop novel diagnostic tools tor CWD. Here, RT-QuIC amplification of CWD prions can be combined with an acoustofluidic micromixer for ultrafast amplification for the positive and negative CWD samples. An acoustofluidic micromixer can exert high shear stress between PrPSc fibrils to actively fragment, which will then serve as a new nucleation site. This technique has significantly reduced the lag phase time to approximately 3 hours, which is at least one order of magnitude faster than the current gold standard such as RT-QuIC and PMCA, This amplification strategy is also compatible with other non-fluorescent based detection methods such as AuNP aggregation assay which proves its potential in situ diagnosis.

[0050] Micro-QuIC assays as described herein thus can be used for the visual identification of samples positive of certain biological substances, such as CWD positive and negative lymph tissues following QuIC amplification. In some examples, RPLN and palatine tonsils collected from white-tailed deer were the basis for the analyses conducted herein because these tissues are ideal for early and accurate identification of CWD infection. In other examples, the techniques may leverage Micro-QuIC for antemortem CWT) diagnostics. RT- QuIC amplification assays using samples acquired from living deer could readily be combined with Micro-QuIC to provide field-deployable ante-mortem tests of both wild and fanned cervids. Moreover, it is possible that Micro-QuIC has utility as a food-safety test given the recent documentation of RT-QuIC-based detection of CWD in white-tailed deer muscles that are used for human and animal consumption. More broadly, the Micro-QuIC assay has the potential to be a versatile platform for detecting a variety of TSEs and proteopathies where RT-QuIC and PMCA have been utilized, including scrapie in sheep, BSE in cattle, and CJD, Parkinson’s, and Alzheimer’s in humans.

[0051] FIG. 6 is a flow diagram illustrating an example technique for amplifying a biological substance using an example acoustic-based amplification device described herein. The technique of FIG. 6 will be described with respect to system 100, but can be performed using any of the acoustic amplification systems and devices described herein, such as system 120 and device 160.

[0052] As shown in the example of FIG. 6, a user can inject a sample that includes biological substances, such as a plurality of proteins, into main channel 104 of the acoustic-based amplification device of system 100 (600). For example, the sample may be in a fluid that is injected into main channel 104 via one or more tubes. System 100 then contains the fluid sample within main channel 104 (602). System 100 then generates, via at least one acoustic generation element, acoustic waves within air of one or more side channels 106 to causevibration of the liquid-gas interface and mixing of proteins within the fluid in main channel 104 (604). This mixing process may proceed for minutes or hours, usually 4 hours or less. In some examples, the mixing process may take three hours or less, two hours or less, or even less than one hour. Tire mixing during this period causes misfolded proteins to contact normal proteins and cause the normal proteins to also misfold.

[0053] After the amplification process is complete, the user may determine an image of the sample within the main channel 104 (606). In other examples, the sample may be extracted for visualization. Fluorescence imaging, gold nanoparticle visualization, other optical techniques or viscosity measurement may be leveraged to detect a change in the amplified proteins (if present in the sample). The user or automated optical detection system (e.g., a camera and visualization detection software) can then determine, from the image, a status of misfolded proteins within the sample that may be indicative of the positive or negative presence of misfolded proteins (608). For example, fluorescence intensity above a positive threshold or lack of blue shift in gold nanoparticle visualization may indicate that the sample was positive for misfolded proteins and the disease, such as CWD.

[0054] The folding methods and materials were used in the examples described, but other methods and materials may be used in other examples to achieve similar results. Materials included SU82050 (Microchem Inc.) silicon wafer (Siegert Wafers), SU8 developer solutions (Microchem Inc.) microscope cover glass (24 X 50 mm. Globe Scientific Inc.), Piezoelectric transducers (7BB-27-4L0, Mouser electronics). Thioflavin T (Till), 100 kDa Pall MWCO filter. Ethylenedi aminetetraacetic acid (EDTA), sodium chloride (NaCl), phosphate buffered silane (PBS), and trimethoxysilane were purchased from Sigma Milipore. Fluorescent microspheres (FCDG006) were purchased from Bangs Laboratories Inc., and Polydimethylsiloxane (PDMS) and curing agent were obtained as SYLGARD®184 silicone elastomer kit from Dow Coming.

[0055] The following are example techniques for constructing and using the acoustic amplification systems described herein.

[0056] Preparation of Recombinant Substrate: The synthesis and purification of recombinant hamster PrP (HaPrP90-231) followed the methods of Schwabenlander et al. In brief, a truncated form (amino acids 90-231) of the Syrian hamster PRNP gene was cloned into the pD431-SR expression vector (ATOM, Newark, CA, USA) and was expressed in Rosetta (DE3) E. coh to synthesize the substrates.

[0057] RT-QuIC for spontaneous misfolding of recombinant prion protein. For QuIC analysis, a master mix was prepared following specifications: IX PBS, ImM EDTA, 170mMNaCl, 10 μM thioflavin T (ThT), and 0,4 mg / mL rHaPrP. The 10% tissue homogenates were further diluted 100-fold in 0.1% SDS / PBS / N2 (final tissue dilution: 0.1%), and 2 μL of the diluent were added to each well containing 98 uL of RT-QuIC master mix. Spontaneous misfolding of recombinant prion protein was done similarly but with unfiltered recombinant proteins and reagents. For these reactions, no infectious seed was necessary. The spontaneously misfolded material was used to seed reactions for both Micro-QulC and RT- QuIC . Plates were amplified on a FLUOstar® Omega plate reader (BMG Labtech, Cary, North Carolina, USA) (42°C, 700 rpm, double orbital, shake for 57 s, rest for 83 s). Fluorescent readings were taken at -45 min increments.

[0058] Thermomixer-based Amplification: A standard benchtop shaking incubator (thermomixer) can be used to produce QuIC-based prion amplifications. Plates which were made for amplification on the thermomixer were prepared identical to those amplified on the plate reader. Reactions were performed on a ThermoMixer® C equipped with SmartBlock plate and Thermotop (Eppendorf, Enfield, Connecticut, USA) at 48°C for 24hrs at 600 RPM (60s shake and 60s rest). A 24 hour run time based on independent RT-QuIC results for lymph nodes and palatine tonsils from CWD+ white-tailed deer, including those examined herein, showing significant seeding activity withm 9 to 24 hours.

[0059] Preparation of Microfluidic device and operation: The example PDMS-based microfluidic device fabrication was performed using soft-lithography as described previously. Mater molds were prepared on 4” silicon wafers using a spin coating (Model CEE- 100, Brewer Science Inc.) SU8 2050 to a height of 50 um. Following the coating process, a pre- baking step was performed before UV-light exposure through a film mask with the requisite design, onto the SU8 coated silicon wafer for a duration of 12 seconds (MA6, Karl Suss). A post-baking step was performed before the SU8 development process. SU8 development was performed by gently washing the wafer in the developer solution for 3 minutes. Finally, the Si-wafer was silanized for 30 minutes with trimethoxy silane. PDMS-based microfluidic chips were produced by heat curing (90 degrees Celsius for 3 hours) PDMS with a curing agent mixture (10: 1) on the master mold Si-wafer. Cured PDMS was peeled and cut into individual chips. Holes were punched at respective inlets and outlets using a 1 mm biopsy puncher (Acuderm Inc.) before bonding the PDMS to microscope cover glass. The bonding procedure w'as performed by treating both cover glass and the PDMS with a high-frequency generator(BD-10A, Electro-Technic Inc.) for 1 minute. The microfluidic devices were heated for 2 hours at 65 degrees Celsius to help with the bonding process. For the fabrication of the outerPDMS casing, a master mold was fabricated by ataching a rectangular mold (3 cm x 1.5 cm x 0.5 cm) on 4” silicon wafer, followed by silanization with trimethoxy silane for 30 minutes.

[0060] Outer PDMS casing was fabricated by heat curing the PDMS master mix at 90 degrees C for 3 hours. Once cut into individual chip size, holes were punctured at respective inlets and outlets using a 1 mm biopsy puncher. The outer PDMS casing was bonded to a microfluidic device by aligning the inlets and outlets. Mineral oil was injected in between the microfluidic PDMS channel and the outer PDMS casing. Finally, piezoelectric transducers were attached at the back of the device using epoxy glue. The photograph of a final assembled device can be seen in FIG. 1C. Before conducting the amplification experiments with recombinant PrP samples, all of the devices were experimentally tested with 0.005% fluorescent polystyrene microspheres (1 pm) to determine the optimized working frequencies of piezoelectric transducers to achieve uniform mixing. Based on these tests, 4.6 kHz was determined to be the working frequency for all our micro-QuIC devices; all the amplification experiments were conducted at this frequency. To track aggregated PrP, fluorescent images were obtained every 30 minutes for approximately 3 hours using laser excitation at 445 nm. The fluorescence intensity was measured by modifying the corrected total cell fluorescence measurement.

[0061] Gold -nan oparticle based detection: Gold-n anoparticle based prion detection followed tlie methods of Christenson et al. In brief, 2.45 nM 15 nm citrate capped gold nanoparticles (usaNanopartz™, Loveland, Colorado, USA) were buffer exchanged to low concentration phosphate buffer (lOmM Na2HPO4(Anhydrous), 2,7mM KC1, 1 ,8mM KH2PO4 (Monobasic)). After the amplification, protein solutions were diluted by two fold in IX PBS with the addition of final concentrations of 1mM EDTA, 170mMNaCl, 1.266mM Sodium Phosphate. Finally, 40ul of the dilute protein solution was then added to the 360ul AuNP solution and left to react at room temperature (RT) for 30 minutes. The color changes were observed both by naked eye and colorimeter (FLUOstar® Omega plate reader, BMG Labtech, Cary, North Carolina, USA) at 400-800 nm wavelength.

[0062] Tissue preparation followed the methods of Christenson et al. (Christenson, P. R , Li, M., Rowden, G., Schwabenlander. M. D„ Wolf, T. M„ Oh, S. H„ & Larsen, P. A., A field- deployable diagnostic assay for the visual detection of misfolded prions, Scientific reports. J2( 1), 12246, 2022). In brief, eight white-tailed deer tissues (4 CWD-negative and 4 CWD-positive) were obtained from white-tailed deer through collaboration with the Minnesota Department of Natural Resources and their CWD status was identified using the Bio-Rad TeSeE Short Assay Protocol (SAP) Combo Kit (BioRad Laboratories Inc., CA,USA), White-tailed deer retropharyngeal lymph nodes (RPLNs) and palatine tonsils were homogenized in PBS (10 %w / v) with 1.5 mm zirconium beads with a BedBug Homogenizer (Benchmark Scientific, Sayreville, New Jersey, USA) on max speed for 90s.

[0063] GraphPad Prism version 9.0 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com) was used for conducting statistical analysis. Three technical replicates were used to demonstrate the potential application of AuNP on spontaneously misfolded rHaPrP. Three and four technical replicates were used fortesting CWD prions using AuNP and RT-QuIC, respectively for each animal. RPLN and / or palatine tonsil tissues from ten positive and fourteen negative animals were included in the examples described herein. The one-tailed Mann-Whitney unpaired u-test (a-0.05) was used to test the average difference for all parameters of interests between samples.

[0064] The following example process can be used to make an acoustic-based amplification device, such as any of systems 100 and 120 or device 160, and fluids fortesting a sample. These are merely examples, and other techniques may vary in materials, times, or order of steps, for example.

[0065] Purpose: Micro-QuIC is a method for amplifying and detecting prionogenic fragments in a sample. It may work by amplifying fibril formation through acoustically driven streaming mixing of a sensitive form of prion, usually HaPrP, inside a microfluidic channel. After mixing, microfluidic channels containing post-amplified solutions are observed under fluorescent microscope. Differences between samples with fibril formation vs no fibril formation can be readily distinguished via the fluorescence intensity measured from microfluidic channels. Moreover, the system can be compatible with previously reported MN-QuIC system where the presence of misfolded PrP can be distinguished using gold-nanoparticle aggregation assay.

[0066] Equipment Identifiers: Hotplate, AC-function generator, Vacuum chamber, MA6 Aligner, and BD-10A High frequency generator.

[0067] Materials:

[0068] Procedure:Mold Fabrication (1) -- Microfluidic PDMS channel1. Place a 10 mm silicon wafer in piranha solution (H2SO4: H2O2= 3:1) for 5 min and wash thoroughly with DI water,2. Place silicon wafer on top of the spin coater and dispense 5 mL of SU-8 photoresist.3. Spin at 500 rpm for 10 sec and 1250 rpm for 30 sec.4. Bake the silicon wafer at 65 °C for 5 min and 95 °C for 20 min5. Place photolithography mask and silicon wafer on an aligner and expose UV light (240 mJ / cm2)6. Bake the silicon wafer at 65 °C for 2 mm and 95 °C for 10 min7. Immerse the silicon wafer in SU-8 developer and gently shake for 10 min.8. Rinse with DI water and dry to finish mold fabrication.9. Place the mold m a sealed container with 100 μL trimethoxy silane.10. Wait for trimethoxy silane to evaporate.Mold Fabrication (2) - Outer PDMS casing1. Attach a 3 cm x 1.5 cm rectangular mold on silicon wafer using epoxy glue.2. Place the mold in a sealed container with 100 μL trimethoxy silane.3. Wait for trimethoxy silane to evaporate.Device Fabrication1. Mix 30 ml of SYLGARD-184 elastomer base with 3 mL ofctiring agent.2. Degas tinder vacuum for 20 min.3. Pour SYLGARD- 184 mix (PDMS) over the mold for microfluidic PDMS channel and outer PDMS casing, and bake at 65 °C for 2 hr.4. Cut off microfluidic PDMS channel and outer PDMS casing and puncture an inlet and outlet using 1 mm biopsy punch.5. Treat the surface of microfluidic PDMS channel and 20 mm X 50 mm microscope cover glass with high frequency generator for 5 min.6. Attach microfluidic PDMS channel on the microscope cover glass and bake for 30 min at 65 °C.7. Treat the surface of outer PDM S casing and the glass portion of the microfluidic channel with high frequency generator for 5 min.8. Attach outer PDMS casing on top of the microfluidic channel by aligning inlets and outlets.9. Bake for 30 mm at 65 °C.10. Attach piezoelectric transducer on the glass side of microfluidic chip using epoxy glue.11. Connect PTFE tubing to the inlets and outlets of the PDMS chip.12. Inject mineral oil into the chamber between the microfluidic PDMS channel and the outer PDMS casing.Sample Solution PreparationThis procedure includes dilutions for lymph and nerve tissue. Extraction protocols for other sample types include the correct dilutions forthose samples. For other sample types start at step 4.1. Take samples out of freezer and put in biosafety cabinet.2. Dissect tissues for subsampling,3. Add 100 mg of tissue to labeled bead tube with 900 μL 1 X PBS (1.5 mm Zirconium bead tubes).4. Homogenize using BeadBeater at max speed for 90 seconds.5. Put samples at -80°C until ready to am.6. (Optional) Weigh out ThT (0.3 mg) and add to 1 mL filtered water. This makes 1 mM ThT.● This may be made up to 5 days ahead of time Remove a sufficient number of tubes of HaPrP from -80°C, and allow to thaw.● DO NOT VORTEX. While the HaPrP is thawing, prepare the reaction cocktail as follows, but do not add the HaPrP yet● Reaction cocktail is for 1 full plate (scale down as needed for less than a full plate)● All components should be 0.22 μm filtered within 1 monthMaster Mix / Reaction Cocktail:● PrP: can vary between batches● Water: varies depending on PrP concentration● 5X PBS: 2,000 μL● 2M NaCl: 850 μL● 100mM EDTA: 100 μL● 1mM ThT: 100 μL (optional, if ThT not used just add 100 μL Water) Vortex the cocktail without the HaPrP.● This cocktad may be filtered with a 0.2 micron syringe filter if contamination is a problem Make the tissue dilution buffer 0.1% SOS in PBS. Once thawed, filter the HaPrP with a 100 kDa spin column, 3,000 x g, 15 minutes. Filter can be used twice. (Tubes are typically 500 μL aliquots. Max volume of filter is 500 μL). While the HaPrP is spinning down, make appropriate dilutions of tissue homogenate in the prepared solution (0.1% SDS in PBS) down to 10-10(or required dilution). The original homogenate is considered 10-1.● For lymph and brain tissue, 10-5is best.● Controls should be diluted to KF3(1 pl, control in 99 pl, prepared solution) Gently add the appropriate amount of PrP to reaction cocktail / master mix Mix by inversion. DO NOT VORTEX Gently inject 10 μL of the master mix to the microfluidic channel. Seal the PTFE tubing and place the microfluidic chip on a hot plate (45 °C). Connect piezoelectric transducer with AC function generator. Run function generator with 30 seconds on, 30 seconds off cycle.; tor 4 hours Remove gloves and dispose of in yellow barrel Clean up hood and RT-QulC bench● Wipe down hood and anything in contact with 50% bieach then 70% EtOH.● Ail potentially infectious items, including disposable lab coat and gloves, go into yellow waste barrel (if there is any question, put it in the yellow waste barrel)After the shaking protocol is complete:21. Remove microfluidic chip from hot plate22. Place the microfluidic chip on fluorescent microscope (excitation and emission wavelength = 427 nm / 450 nm)23. Take fluorescence intensity measurement at 20X magnification.Master Mix

[0069] As described herein, CWD is one example disease that can be amplified in an acoustiofluidic device. In comparison to tire traditional RT-QuIC, this described method leverages a microfluidic channel to accelerate amyloid aggregation kinetics by restricting shear-induced secondary' prion amplification in micrometer scale. Thi s method is also compatible with wade range of other acoustic actuators such as thin finger-like membrane (Sharp-edge structures) and acoustic cavitation instead ofjust prior amplification using liquid-gas cavity structures tor acoustic actuation. This method is also compatible with various detection methods such as fluorescence intensity measurement, gokl-nanoparticle based aggregation assay and capillary' action measurement.

[0070] The described examples include a functional prototype of a microfluidic device that leverages acoustics to effectively amplify misfolded proteins. The microfluidic device can detect misfolded CWD prions within a few hours. This device and technique will also be useful for detecting misfolded alpha-synuclein associated with Parkinson's disease. For thisreason, acoustic-based protein amplification will have broad utility tor detecting an array of human and animal protein misfolding diseases. The microfluidic device, the method of mixing recombinant protein with misfolded seeds, can amplify and detect protein misfolding diseases. The rapid detection of prion and protein misfolding diseases including CWD, BSE, scrapie, Creutzfeldt-Jakob Disease, Parkinson's disease, Alzheimer's disease, PPID, etc. Our device is also portable, thus opening the possibility' to rapid diagnostics in a wide variety of settings.

[0071] The following examples are described herein.

[0072] Example 1. A system comprising: a housing defining: amain channel configured to contain a fluid solution containing a sample including a plurality of proteins; one or more side channels in fluid communication with the main channel, wherein each side channel of tlie one or more side channels are configured to establish a liquid-gas interface between the fluid solution in tire main channel and air contained in the one or more side channels; and at least one acoustic generation element configured to generate acoustic waves within at least one of the gas of the one or more side channels or the fluid solution in the main channel that causes vibration of the liquid-gas interface and mixing of the fluid solution w'ithin the main channel.

[0073] Example 2. The system of example 1, wherein the main channel has a width from 100 micrometers to 500 micrometers.

[0074] Example 3. The system of example 2, wherein the width is approximately 300 micrometers.

[0075] Example 4. The system of any of examples 1 through 3, wherein each of the side channels has a width from 50 micrometers to 500 micrometers.

[0076] Example 5. The system of example 4, wherein tire width is approximately 100 micrometers.

[0077] Example 6. The system of any of examples 1 through 5, wherein a width each of the side channels is less than a width of the mam channel.

[0078] Example 7. The system of any of examples 1 through 6, wherein the acoustic generation element comprises a piezoelectric transducer or speaker.

[0079] Example 8. The system of any of examples 1 through 7, wherein the acoustic generation element is in contact with a portion of the housing.

[0080] Example 9, The system of any of examples 1 through 8, wherein the housing comprises: a glass slide; and a first layer comprising one of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), or glass disposed on the glass slide and at least partiallydefining the main channel between the glass slide and the first layer; and wherein the system further comprises: first tubing in fluid communication with an inlet of the main channel; and second tubing in fluid communication with an outlet of the main channel.

[0081] Example 10. The system of example 9, wherein the housing comprises: a second layer comprising PDMS, PMMA, or glass disposed around at least a portion of the first layer and defining a space between the first layer and the second layer; and a hydrophobic fluid disposed in the space between the first layer and the second layer.

[0082] Example 11. The system of any of examples 1 through 10, further comprising a controller configured to energize the acoustic generation element for one or more periods of time.

[0083] Example 12. The system of any of examples 1 through 11, wherein the at least one acoustic generation element is configured to generate the acoustic waves within the air or thin membrane of the one or more side channels to cause misfolded proteins to contact other proteins within the plurality of proteins to amplify a quantity of the misfolded proteins to a detectable level.

[0084] Example 13. The system of any of examples 1 through 12, wherein at least a portion of the housing is transparent and enables fluorescence intensity measurement of at least some of the plurality of proteins of the sample within the mam channel after mixing.

[0085] Example 14. The system of any of examples 1 through 13, further comprising one or more thm membrane protruding into the main channel, wherein each thin membrane of the one or more thm membranes are configured to establish a direct contact with the fluid solution in the main channel.

[0086] Example 15. A method comprising: containing, within a main channel of a housing, a fluid solution containing a sample including a plurality of proteins, wherein the housing comprises at least one of one or more side channels or one or more thin membranes m fluid communication with the mam channel, and wherein each side channel or thin membrane of the one or more side channels or thin membranes are configured to establish a liquid-gas interface between the fluid solution or direct contact in the main channel and air contained in the one or more side channels; and generating, via at least one acoustic generation element, acoustic waves within at least one of the gas of the one or more side channels or the fluid solution in tire mam channel that causes vibration of the liquid-gas interface and mixing of the fluid solution within the main channel.

[0087] Example 15. The method of example 14, wherein generating the acoustic waves causes amplification of any misfolded proteins within the plurality of proteins.

[0088] Example 16. The method of example 15, wherein generating the acoustic waves comprises generating the acoustic waves to generate a detectable quantity of the misfolded proteins present within the sample in less than two hours.

[0089] Example 17. The method of any of examples 14 through 16, wherein the plurality of proteins comprises one or more misfolded proteins associated with a protein-misfolding disease.

[0090] Example 18. The method of example 17, wherein the protein-misfolding disease comprises chronic wasting disease.

[0091] Example 19. The method of any of examples 15 through 18, further comprising detecting a characteristic of tire plurality of proteins using a gold-nanoparticle-based aggregation assay,

[0092] Example 20. The method of any of examples 15 through 19, further comprising detecting a characteristic of the plurality of proteins using at least one of externally inserted capillaries or integrated capillaries.

[0093] Various examples of the disclosure have been described. Any combination of the described systems, operations, or functions is contemplated. These and other examples are w'ithin the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A system comprising: a housing defining: a main channel configured to contain a fluid solution containing a sample including a plurality of proteins; one or more side channels in fluid communication with the mam channel, wherein each side channel of the one or more side channels are configured to establish a liquid-gas interface between the fluid solution in the main channel and gas contained in the one or more side channels; and at least one acoustic generation element configured to generate acoustic waves within at least one of the gas of the one or more side channels or the fluid solution in the main channel that causes vibration of the liquid-gas interface and mixing of the fluid solution within the main channel.

2. The system of claim 1, wherein the main channel has a width from 100 micrometers to 500 micrometers.The system of claim 2, wherein the width is approximately 300 micrometers.

4. The system of any of claims 1 through 3, w herein each of the side channels has a width from 50 micrometers to 500 micrometers.

5. The system of claim 4, wherein the width is approximately 100 micrometers.

6. The system of any of claims 1 through 5, wherein a width each of the side channels is less than a width of the main channel.The system of any of claims 1 through 6, wherein the acoustic generation element comprises a piezoelectric transducer or speaker.

8. The system of any of claims 1 through 7, wherein the acoustic generation element is in contact with a portion of the housing.

9. The system of any of claims 1 through 8, wherein the housing comprises: a glass slide; and a first layer comprising one of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), or glass disposed on the glass slide and at least partially defining the main channel between the glass slide and the first layer; and wherein the system further comprises: first tubing in fluid communication with an inlet of the mam channel; and second tubing in fluid communication with an outlet of the main channel.

10. The system of claim 9, wherein the housing comprises: a second layer comprising PDMS, PMMA, or glass disposed around at least a portion of the first layer and defining a space between the first layer and the second layer; and a hydrophobic fluid disposed in tire space between the first layer and the second layer.

11. The system of any of claims 1 through 10, further comprising a controller configured to energize the acoustic generation element for one or more periods of time.

12. The system of any of claims 1 through 11, wherein the at least one acoustic generation element is configured to generate the acoustic waves within the gas or thin membrane of the one or more side channels to cause misfolded proteins to contact other proteins within the plurality of proteins to amplify a quantify of the misfolded proteins to a detectable level .

13. The system of any of claims 1 through 12, wherein at least a portion of the housing is transparent and enables fluorescence intensity measurement of at least some of the plurality of proteins of the sample within the main channel after mixing.

14. The system of any of claims 1 through 13, further comprising one or more thin membrane protrading into the main channel, w herein each thin membrane of the one or more thin membranes are configured to establish a direct contact with the fluid solution in the main channel.

15. A method comprising : containing, within a. main channel of a housing, a fluid solution containing a sample including a plurality of proteins, wdierein the housing comprises at least one of: one or moreside channels or one or more thin membranes in fluid communication with the mam channel, and wherein each side channel or thin membrane of the one or more side channels or thin membranes are configured to establish a liquid-gas interface between the fluid solution or direct contact in the main channel and gas contained in the one or more side channels; and generating, via at least one acoustic generation element, acoustic waves within at least one of the gas of the one or more side channels or the fluid sol ution m the main channel that causes vibration of the liquid-gas interface and mixing of the fluid solution within the main channel.

15. The method of claim 14, wherein generating the acoustic waves causes amplification of any misfolded proteins within the plurality of proteins.

16. The method of claim 15, wherein generating the acoustic waves comprises generating the acoustic waves to generate a detectable quantity of the misfolded proteins present within the sample in less than two hours.

17. The method of any of claims 14 through 16, wherein the plurality of proteins comprises one or more misfolded proteins associated with a protein-misfolding disease.

18. lire method of claim 17, wherein the protein-misfolding disease comprises chronic wasting disease.

19. The method of any of claims 15 through 18, further comprising detecting a characteristic of the plurality of proteins using a gold-nanoparticle-based aggregation assay.

20. The method of any of claims 15 through 19, further comprising detecting a characteristic of tire plurality of proteins using at least one of externally inserted capillaries or integrated capillaries.

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